Changes in intestinal immunity and gut microbiota associated necrotic enteritis induced by Clostridium perfringens infection and exposure to heat stress in chicken

Changes in intestinal immunity and gut microbiota associated necrotic enteritis induced by Clostridium perfringens infection and exposure to heat stress in chicken | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Changes in intestinal immunity and gut microbiota associated necrotic enteritis induced by Clostridium perfringens infection and exposure to heat stress in chicken Abdelmotaleb Elokil, Li Jingdu, Zhili Qi, Yantao Lv, Md Touhiduzzaman Sarker, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6855798/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The present study investigated the effects of heat stress (HS) and Clostridium perfringens (C. perfringens) infection on the performance, jejunal morphometry, immunomodulatory responses of T cells, expression of cytokines and immune-related genes, and gut microbiota dynamics of male Qingyuan Patridge chickens. Healthy 20-day-old of male Qingyuan chicks (192) were randomly assigned to four groups of 12 replicates (n = 4 chicks/replicate). A 2 × 2 factorial arrangement experiment was performed, including two levels of oral C. perfringens dosages, without (CP0) and with 2×10 8 CFU/mL (CP2) under two ambient temperatures of 25°C (L) and 35°C (H) for 30 d. Results The results showed that exposure of chicks to HS and C. perfringens infection in the H CP 2 (chicks reared under H ambient temperature with an oral dosage of CP ) group decreased ( P < 0.05) feed intake, body weight, daily weight gain, feed conversion ratio, villus height, and villus-to-crypt depth ratio in the jejunum compared to the LCP0 group. The plasma concentrations of interleukin (IL)-6 and IL-10 were lower ( P < 0.05) in the H CP 2 group than in the L CP 0 group (chicks reared under L ambient temperature without an oral dosage of CP). The T lymphocyte subsets of peripheral blood CD4 + T cells, CD3 + CD4 + T cells, and the CD3 + CD4 + /CD3 + CD8 + ratio decreased ( P < 0.05) in the H CP 2 group compared with the other groups. Intestinal mRNA expression of IL-4 , IL-6 , IL-10 , IFN-γ was downregulated; however, IL-8, tumor necrosis factor-α ( TNF-α ), nuclear factor kappa B ( NF-κB ), zonula occludens-1 ( Zo-1 ), and Claudin-1 were upregulated in the H CP 2 group compared to the L CP0 group. The gut microbiota in the H CP 2 group had a higher relative abundance of Esherichia-Shigella , Staphylococcus, Clostridia, Cyanobacteria , and Listeriaceae compared to the other groups. Conclusions These results demonstrate the potentially harmful effects of exposure to HS and C. perfringens infection on necrotic enteritis-related disturbances, growth performance, jejunum morphometry, immune-modulatory responses of T cells, expression of cytokines and immune-related genes, and gut microbiota dynamics in Qingyuan Patridge chickens exposed to HS. Heat-stressed chickens Clostridium perfringens infection Necrotic enteritis Intestinal health Host microbiota composition Metagenomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Clostridium perfringens ( C. perfringens ) is a common pathogenic that can cause both of animal and human diseases, such as necrotic enteritis (NE) in poultry [ 1 ]. Heat stress (HS) is one of the most important stressors that compromise the immune system of birds and increases their susceptibility to enteric pathogens such as C. perfringens [ 2 , 3 ]. NE is caused by C. perfringens type A, which produces alpha toxin and has become increasingly prevalent in countries that have ceased using growth-promoting antibiotics [ 4 ]. In addition, abdominal hyperthermia by HS and infection with C. perfringens cause NE-associated disturbances, such as intestinal congestion, mucosal perturbation, barrier dysfunction, ischemia, hyperpermeability, and cholangiohepatitis, via the invasion of enteric pathogens from bacterial lipopolysaccharide (LPS) and opportunistic gut microbiota communities [ 5 ]. Under the predominance of NE, excessive production of both reactive oxygen species and reactive nitrogen species negatively affects enteric antioxidant defense and increases cellular lipid peroxidation (LP) and intestinal tissue congestion, thereby reducing feed intake (FI) and growth rate (GR) [ 6 , 7 ]. C. perfringens is a gram-positive, spore-forming, anaerobic bacterium classified based on its toxins into over 20 types, including seven toxin-producing strains predominant in poultry, known for alpha, beta, epsilon, and iota toxins [ 8 , 9 ]. Similarly, C. perfringens infection is a persistent threat to animal and public health and is associated with NE via the production of alpha- and beta-toxins that enter the bloodstream. C. perfringens infection and HS are key factors that induce NE in poultry, leading to huge economic losses in the broiler industry through performance degradation, inefficient feed use, and increased mortality. Reduced FI during the exposure of animals to HS and C. perfringens infection decreases systemic energy homeostasis. This reduces the panting rate to dissipate excess body heat, and the immune response against pathogenic infections [ 10 , 11 ]. Notably, HS reduces intestinal absorption owing to epithelial cell injury and hyperpermeability, negatively affecting cellular functions [ 12 ]. Host health efficiency regulates the pattern of microbiota interactions by determining the motility, proliferation, and lifestyle of the gut microbial communities. The resulting bacteria can produce essential amino acids and vitamins and regulate digestion to enhance host health and function [ 13 , 14 ]. Therefore, the composition and structure of the intestinal microbiota have been identified as novel molecular markers to investigate community diversity-regulated candidate performance, including GR [ 15 ], egg performance (Elokil, et al., 2020a), immune responses [ 16 ], and fertility [ 17 ]. Lymphoid tissues in the gastrointestinal tract (GIT) are well developed and form the primary defense against enteric pathogens. The T-cell-mediated immune response is important in the early process of cellular defense, including CD4 + as inducer T cells that stimulate the phagocytosis of macrophages and antibody production by B cells, whereas CD8 + cytotoxic T cells kill infected cells. Jiang et al. [ 18 ] reported that the percentage of T-cell subsets and the expression level of cytokine mRNA in the small intestine was reduced with contaminated chicks diet with 0.6 mg/kg aflatoxin B1, implying a negative effect on the immunity and function of the intestinal mucosa. In contrast, probiotic strains, such as Bacillus and Lactobacilli , have improved the activation and proliferation of T-helper populations of CD4 + T cells and stimulated cytotoxic T cells (CD8 + T cells), presumably through surface contact [ 19 , 20 ]. Collectively, avian NE induced by C. perfringens infection and exposure to HS is a serious disease causing huge losses in chicken farms. Therefore, the present study was performed to explore the impacts of C. perfringens and exposure to HS on performance and stability of gut health (i.e., jejunum morphometry, immune-modulatory responses of T cells, expression of cytokines and immune-related genes), together with the dynamic changes (composition and structure) of endogenous key microbial genera and functional groups in gastrointestinal tract of Qingyuan Patridge chickens. To the best of our knowledge, this is the first study to explore changes in intestinal immunity and gut microbiota associated NE induced by C. perfringens infection and exposure to HS in chicken. Material and methods Animals and experimental design A total of 192 male Qingyuan Patridge chicken at 3 weeks old were obtained from Guangdong Aijiankang Biotechnology Co., Ltd., Qingyuan City, China, and randomly assigned to a 2×2 factorial arrangement experiment, including four treatments, each comprising 12 replicates (n = 48 chicks/treatment; n = 4 chicks/replicate). A 2 × 2 factorial arrangement experiment was performed, including two levels of oral C. perfringens dosages without ( CP0 ) and with 2×10 8 CFU/mL ( CP2 ) under two ambient temperatures of 25°C (L) and 35°C (H) for 30 d. Four experimental groups were assigned as follows: L CP0 group, chicks reared under L of ambient temperature and without an oral dosage of C. perfringens ( CP 0); L CP2 group, chicks reared under L of ambient temperature and with an oral dosage of C. perfringens ( CP 2); H CP0 group, chicks reared under H of ambient temperature and without an oral dosage of C. perfringens ( CP 0); and H CP2 group, chicks reared under H of ambient temperature and with an oral dosage of C. perfringens ( CP 2). The HS application involved exposing birds to an ambient temperature of 35°C for 10 h (from 9:00 am to 7:00 pm), after which the ambient temperature returned to 25°C. All birds were housed in cages of the same size (40 × 50 × 70 cm), and the experimental period lasted for 15 days. The diet was formulated to meet or exceed the nutritional requirements of yellow chickens (Ministry of Agriculture of China), according to the national research council (NRC) recommendations [ 21 ]. The composition of the feed ingredients and the estimated nutrient levels are presented in Table 1 . Table 1 Composition and nutrition levels of the basal diet (%, as fed-basis). Ingredients Percentage (%) Nutrient content 2 Level (%) Corn 53.00 Metabolizable energy (MJ/kg) 12.13 Wheat bran 7.70 Crude protein 20.59 Soybean meal (CP 43%) 28.30 Calcium 0.96 Corn protein flour 3.00 Phosphorus 0.77 Soybean oil 3.00 Available phosphorus 0.50 L-lysine 0.21 Lysine 1.33 DL-Methionine 0.19 Methionine 0.50 Threonine 0.07 Stone powder 1.02 Calcium hydrogen phosphate 2.25 Salt 0.26 Premix 1 1.00 Total 100 1 Each kilogram of diet (including feed ingredients) provides VA 12,000 IU, VD3 600 IU, VE 45 IU, VK 2.5 mg, VB1 2.4 mg, VB2 5.0 mg, VB6 2.8 mg, niacin 42 mg, pantothenic acid 12 mg, Folic acid 1.0 mg, choline 1,300 mg, Fe 80 mg, Cu 7 mg, Mn 80 mg, Zn 85 mg, I 0.70 mg, Se 0.15 mg. 2 The crude protein, calcium, total phosphorus are analyzed values and metabolizable energy (MJ/kg) and others were calculated values. Inoculation protocol of C. perfringens The pathogenic strain of C. perfringens type A (No. ATCC13124) was provided from the China Veterinary Culture Collection Center (Beijing, China). The bacterial strain was kept in our laboratory Institute of Animal Health, Guangdong Academy of Agricultural Sciences in glycerol at -80°C and the bacterial culture protocol was aseptically inoculated into fluid thioglycollate medium overnight at 37°C in an anaerobic environment before being used for the inoculation [ 22 ]. Briefly, chicken in the L CP 2 and H CP 2 groups orally gavage 1 mL actively growing culture of C. perfringens type A with a concentration of 2×10 8 CFU/mL (Colony-forming units per milliliter) from 31d to 34d. Whereas at the same time, two no-inoculated groups (L CP 0 and H CP 0) of chickens received 1 dose of 1 mL PBS (phosphate buffered saline). Growth performance assessment The supplied and residual feed amounts were recorded daily for each replicate to calculate the average daily feed intake (ADFI). The number of dead birds was recorded daily to calculate the percentage of the surviving birds. Chicks in each replicate were weighed on days 20, 30, and 36 in the morning after 12 h of fasting without feeding to measure body weight and calculate the average daily gain (ADG) and feed-to-gain ratio (F/G). FI, daily gain (DG), and feed conversion rate (FCR) were calculated based on the following formulas: FI (g/d) = total food intake (g)/feed days (d); DG (g/d) = (final weight - initial weight) (g)/feed days (d); FCR (g/g) = ADFI (g/d)/ADG (g/d). On day 36, 12 birds, approximately the average body weight for each group, were selected to collect samples, including plasma, organs, and cecal digesta [ 7 ]. Hematoxylin − Eosin staining and intestinal morphometric analysis To analyze the jejunal morphometric traits, tissue sections were stained using the hematoxylin and eosin staining. Briefly, the jejunum segments were fixed using a 4% paraformaldehyde solution, dehydrated using xylene, and embedded in paraffin. The paraffin-embedded jejunal tissues were sliced and placed on glass slides. Two sections (100 µm) were obtained from each sample and stained with hematoxylin and eosin for 1 min and 10 s, respectively. All morphometric variables were measured using a camera (Olympus; TH4-200; Tokyo, Japan) coupled with computer-assisted digital Image-Pro Plus analysis software (Image-Pro Plus 4.5, Media Cybernetics, Silver Spring, MD, United States) to assess the maximum villus length, crypt depth, and submucosa/muscularis/serosa. Plasma biochemical variables The plasma concentration (n = 6) of different biochemical parameters, including interleukins (IL-1β, IL-6, IL-10, IL-22), tumour necrosis factor-α (TNF-α), interferon-γ (IFN- γ), immunoglobulins (IgG, IgA, and IgM) were measured by ELISA kits. The experimental kits were acquired from Jiangsu Enzyme Immunization Industrial Co., Ltd. (Yancheng, China), and the procedure was carried out according to the manufacturer's instructions. Preparation of target cells for detection of immune-related cytokines add 10 µL of sample with 40 µL of sample diluent placed in each well then seal and place in incubator 37°C for 30 min. The well with diluted was washed solution5 times, then added 60 µL of enzymes to each well, seal with film and place in an incubator 37°C for 30 min then wash it 5 times and let stand 30 s. Add chromogen A50 and B50 to each well, mix and place in the dark at 37°C for 10 min for colour development. To terminate the reaction, we added 50 µL stop solution to each and every well. The microplate reader was used to measure the absorbance value of each and every well at different wavelengths. Detection of blood and jejunal lamina propria T lymphocytes by flow cytometer Blood samples (2.5 mL) were collected for T lymphocyte analysis, and then phosphate-buffered saline (PBS) and 4 mL Ficoll (Paque TM Plus, Amersham) were added to separate peripheral blood mononuclear cells (PBMC). The samples were centrifuged at 2000 × g for 20 min at 20°C to separate the uppermost layer and transferred to a 5 mL tube for centrifuge again at 1500 × g for 10 min at room temperature. The procedure was repeated twice and washed 2–3 times with 2 mL of PBS. To determine the T lymphocyte of intestinal lamina propria, a 1 cm jejunum sample was taken, then digestion at 37°C in a water bath for 10 min by the addition of 5 mL of predigestion solution (5 mL PBS + 1 µM DTT) and 10 mL of predigestion solution (10 mL PBS + 2% FCS + 1 mM EDTA). The sample was washed 2–3 times with PBS, then 1 mL of digestion solution (5 mL RPMI 1640 cell culture medium + 2% FCS + 200 µg/mL Dnase 1 + 1 mg/mL Collagenase A) was added for digestion at 37°C in a water bath for 60 min. The samples were centrifuged at 1500 × g for 10 min at room temperature, and the supernatant was washed with 1 mL of PBS. Using the cell counting machine to measure the viable cell concentration, 10 µL of the sample was mixed with 0.4% trypan blue stain and dropped into the counting board for assessment. For the concentration calculation, 50 mL of suspended viable PBMC (1×10 6 cells) samples were transferred to a 96-well plate, followed by the addition of 50 mL of the appropriate antibody (Southern Biotech, Birmingham, USA). The PBMC cell suspensions were multi-stained with a cocktail of T lymphocyte CD marker antibodies (CD3–FITC, CD4-APC, and CD8-PE, Thermo Fisher Scientific). The well plate was shaken manually for 15 s and incubated at 4°C in the dark for 30 min for each step of antibody labeling. Subsequently, the plate was pipetted and washed three times with the gentle addition of 1 mL of PBS. The cells were resuspended with 500 µL of PBS solution. A total of 50,000 cells were acquired and analyzed using a Becton Dickinson flow cytometer (BD Accuri C6 plus, USA) and Cell Quest software (Becton Dickinson Immunocytometer System, San Jose, CA, USA). RNA extraction and real-time qPCR analysis RNA extraction, cDNA synthesis, and qPCR analyses were performed [ 23 ], approximately 0.05 g of a jejunal mucosal sample was obtained for total RNA extraction using TRIzol reagent (Tiangen Biochemical Technology, Beijing, China). The RNA samples were purified by incubation with RNase-free DNase (Dalian Takara Co. Ltd., Dalian, China) to clean the genomic DNA according to the manufacturer’s protocol. The integrity and purity were evaluated using 1.2% agarose gel electrophoresis, and a NanoDrop ND-1000 UV (NanoDrop Technologies, Wilmington, DE, USA), and isolated RNA was chosen for further reactions. After dilution, 1 µL was taken for reverse-transcribed into cDNA using a Primer Script RT reagent kit (Takara, Japan). Real-time fluorescence quantitative PCR was performed using an ABI 7500 PCR system (Applied Biosystems, USA). The amplification was performed in a total volume of 20 µL which comprised 2 µL of cDNA product, 10 µL SYBR primer mix (Takara, Biotechnology Co. Ltd. Dalian, China), 0.4 µL each of the forward and reverse primers, and 7.6 µL of dH2O. The primer sequence was based on the chicken gene sequence uploaded from GenBank, designed using Primer 3.0 software, and synthesized by Shanghai Sangon Biotechnology Company, China. The primer sequences are listed in Table 2 . For housekeeping gene, GAPDH was used to normalize target gene expression levels. The 2 −ΔΔCt technique [ 24 ] was used to calculate the expression results. Table 2 Primer sequences used for real-time PCR Gene 1 Primer sequences( 5'-3') Accession ID GAPDH F-GGTGAAAGTCGGAGTCAACGG NM_204305.2 R-TCGATGAAGGGATCATTGATGGC IL-1β F-CTTCACCCTCAGCTTTCACG NM_204524.2 R-TGTGGTGTGCTCAGAATCCA IL-2 F-ACCGGAAGTGAATGCAAGAT NM_204153.2 R-AGTGGTCCCAGAATGGACAG IL-4 F-GCCAGCACTGCCACAAGAAC NM_001398460.1 R-CGTGGGACATGGTGCCTTGAG IL-6 F-CTCCTCGCCAATCTGAAGTC NM_204628.2 R-GGATTGTGCCCGAACTAAAA IL-8 F-GCTCTGTCGCAAGGTAGGAC NM_205498.2 R-GGCCATAAGTGCCTTTACGA 1 L-10 F-GATGCTGCGCTTCTACACAG NM_001004414.4 R-TCCCGTTCTCATCCCATCTTC I L-17 F-ATGTTGTCAGCCAGCATTTC NM_204460.2 R-GGAGCCAGTGAGCGTTTG TNF-α F-CTGTTCTATGACCGCCCAGT NM_204267.2 R-TCAGAGCATCAACGCAAAAG NLRP-3 F-CTGGTGAACCCCAAGTCAGT NM_001348947.2 R-GGAAGGTGTCCAAATCCTCA IFN-γ F-TCAAAGCCGCACATCAAACA NM_205149.2 R-GAAGAGTTCATTCGCGGCTT TLR-4 F-CACAGCTCTGGATTTCAGCA NM_001030693.2 R-TTCCGCAGTAGATCCTGCTT TGF-β F-GGAGGAGGAGAAGGAGGAGA NM_205454.2 R-GGAACTCTGCTCGAAACAGG NF-κB F-AGAGGATGCTTCGTTGTGCT NM_001396396.1 R-TCCTGGACAGCAGTGAGATG MYLK-1 F-CATCATGCAGTGCTCAGGTC NM_001322361.3 R-TTCTTTCCCATTGTGCAGCC MUC-2 F-GCTACAGGATCTGCCTTTGC XM_040673077.2 R-AATGGGCCCTCTGAGTTTTT Occludin F-GTCTGTGGGTTCCTCATCGT NM_205128.1 R-TTCTTCACCCACTCCTCCAC ZO-1 F-ACCAGAGGTCAGAGCCTTCA XM_046925214.1 R-AGCGGTGGTGTTTGTTTTTC Claudin-1 F-GGTTGGTGTGTTTGTTGCTG NM_001013611.2 R-TCTGGTGTTAACGGGTGTGA 1 IL-1β = interleukin-1β; IL-2 = interleukin-2; IL-4 = interleukin-4; IL-6 = interleukin-6; IL-8 = interleukin-8; IL-10 = interleukin-10; IL-17 = interleukin-17; TNF-α = tumour necrosis factor- α; NLRP-3 = non-like receptor protein; IFN- γ = interferon- γ; TLR-4 = toll-like receptor-4; TFG-β = transforming growth factor –β; NF-κB = nuclear factor kappa B; MUC-2 = mucin-2; ZO = zonula occludens. Western blot analysis for jejunal lamina propria protein expressions The frozen specimens were powdered in liquid nitrogen and homogenized in a radioimmunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor. The supernatants samples was incubated on ice for 10 min and centrifuged at 12,500 × g for 6 min. BCA protein quantification kit (Beyotime, Jiangsu, China) was used to determine the total protein concentration. The denatured protein was separated on an 8–12% SDS-PAGE gel and transferred onto the nitrocellulose (NC) membrane in equal amounts. Then, the separated proteins were incubated for 1 h at room temperature in a blocking buffer (5% skimmed milk in Tris-buffered saline containing 1% Tween-20) and incubated overnight at 4 ℃ with target primary antibodies. All antibodies were obtained from Proteintech Group, Inc., Wuhan, China, with the catalog numbers for ZO-1:21773-1-AP, IL8:27095-1-AP, IL10:60269-1-AP, TNF-α: 17590-1-AP, and Claudin-1:28674-1-AP. After washing, the cells were incubated with a horseradish peroxidase-conjugated secondary antibody (1:10,000, Biosharp, BL003A) for 1 h at room temperature. Microbial genomic DNA extraction, sequencing quality assessment, and 16S rRNA gene sequencing To extract microbial genomic, total DNA of jejunum digesta samples ( n = 6) were extracted using the MagBind Soil DNA Kit (OmegaBiotec, Norcross, GA, USA) according to the manufacturer’s instructions. The cecal microbiota composition was analyzed by sequencing the V3–V4 hypervariable region of 16S rRNA genes, as described by [ 25 ]. PCR amplification of the bacterial hypervariable regions V3–V4 was performed using the forward primer 338F of 16S rRNA gene (Table 2 ). High-quality and pure clean reads were quantified using Qubit and qPCR (CFX 96Touch, Bio-Rad, Singapore) and merged using FLASH (Fast Length Adjustment of Short reads, v1.2.11) software. The sequencing data were analyzed and classified using RDP, and QIIME 2 software with operational taxonomic units with a 97% similarity level was set. Additionally, alpha diversity (observed species, coverage, Chao, OTUs, Simpson, and dominance) and beta diversity principal coordinate analysis (PCoA) were calculated in different groups. LEfSe analysis was performed by using LEfSe software. Statistical analysis Data from this study were statistically analyzed based on a 2 × 2 factorial design using the following model: Y ijk = µ + S i + D j + SD ij + e ijk , where Y ijk = an observation, µ = the overall mean, Si = effect of ambient temperature ( i = L, 25°C and H, 35°C), Dj = effect of inclusion of C. perfringens ( j = CP0 , 0 CFU/g feed and CP2 , 2 × 10 8 CFU/g feed), SD ij = the interaction between levels of Si and Dj, and e ijk = random error. Data were analyzed using SPSS software (IBM Statistics 26.0, Armonk, NY, USA). Replicates were used as experimental units. Duncan’s new multiple range test was used to calculate the differences among means within the same factor and significance are based on P ≤ 0.05. Data are presented as mean ± standard error of the mean, with different superscript letters indicating significant differences (P < 0.05). Gut microbial analysis was performed using the R program version 3.4.1 (R Foundation for Statistical Computing, Vienna, Austria). Results Growth performance decreases in response to exposure of chickens to HS and C. perfringens infection The effects of HS and C. perfringens infection on growth performance, including weight (ABW, g), average daily weight gain (ADG, g), average daily feed intake (ADFI, g) and feed conversion ratio (FCR, g diet: g BW), are shown in Table 3 . HS significantly reduced BW30 and BW36 and significantly increased the FCR ratio from 30 to 36 d (P < 0.05). The statistical results showed that the final body weight of the male chickens (BW36) and daily weight gain from 20 to 36 d were significantly higher in the L CP 0 group (p < 0.05) than in the HS groups (H CP 0 and H CP 2). No significant differences between the groups were observed in the ADFI from 20 to 36 d. However, the highest and lowest average feed conversions were recorded in the L CP 0 and H CP 0 groups, respectively (Table 3 ). Table 3 Effect of high temperature and Clostridium perfringens on the growth performance of Qingyuan Patridge chicken. Parameters BW (g) BWG (g/bird/day) FI (g/bird/day) FCR (g feed/g gain) 20d 30d 36d 20–30 d 30–36 d 20–36 d 20–36 d 20–36 d Ambient Temperature (T) L, 25°C 187 328.5 a 467 a 14.16 a 22.99 a 17.50 a 35.14 2.01 b H, 35°C 186 322.5 b 435 b 13.61 b 18.85 b 15.56 b 33.83 2.18 a Clostridium perfringens ( CP ) CP0 , 0 dose 186 324 450 13.77 20.95 16.47 34.28 2.10 CP2 , 2×10 8 cfu 187 324 452 14.00 20.90 16.59 34.69 2.10 Interaction T x CP L CP 0 187 328 469 a 14.11 23.31 17.60 a 35.07 2.00 L CP 2 187 329 465 a 14.21 22.68 17.41 a 35.21 2.03 H CP 0 186 320 431 b 13.43 18.58 15.35 b 33.49 2.20 H CP 2 187 325 439 b 13.80 19.12 14.77 b 34.18 2.17 SEM 0.08 0.57 2.68 0.05 0.35 0.16 0.10 0.01 Two ways probabilities* T 0.127 0.037 0.001 0.039 0.001 0.001 0.118 0.001 CP 0.472 0.621 0.531 0.674 0.621 0.118 0.208 0.623 T × CP 0.749 0.673 0.562 0.117 0.563 0.174 0.102 0.210 T = temperature; CP = Clostridium perfringens ; SEM = standard error of means; BW = body weight; DWG = daily weight gain; DFI = daily feed intake; FCR = feed conversion ratio; ns = non-significance; 1 n = 6 per treatment group with 12 birds per replicate. Blood biochemical variables in response to exposure of chickens to HS and C. perfringens infection The plasma concentrations of immune cytokines are shown in Table 4 . The plasma concentration of cortisol was significantly higher ( P < 0.05) in the H CP 2 group than in the L CP 0 group, whereas the plasma concentration of IL-6 was significantly higher ( P < 0.001) in the L CP 0 group than in the H CP 2 group. The highest concentrations ( P < 0.05) of IL-10 and TNF-α were observed in the L CP 2 and H CP 0 groups, respectively. For plasma immunoglobulin variables, the statistical results showed that the lowest ( P < 0.05) plasma concentration of immunoglobulin (Ig) M was in the L CP 0 group. In contrast, no significant differences were observed in the plasma concentrations of IgA and IgG between the groups (Table 4 ). Table 4 Effects of high temperature and Clostridium perfringens on the plasma immune parameters of Qingyuan Patridge chicken. Parameters Cortisol (µg/L) IL- 1β (ng/L) IL-6 (ng/L) IL-10 (ng/L) IL-22 (ng/L) TNF-α ( ng/L) IFN-γ (pg/mL) IgM ( ng/mL ) IgA ( ng/mL) IgG (µg /mL) Ambient Temperature (T) L, 25°C 231 b 110.5 31.29 55.60 a 19.89 55.18 b 88.72 270.15 122.03 65.75 H, 35°C 250 a 118.5 29.17 50.44 b 16.74 58.72 a 92.29 286.65 116.07 68.43 Clostridium perfringens ( CP ) CP0 , 0 dose 234 117 32.67 a 52.49 18.40 57.50 89.92 268.11 126.03 69.85 CP2 ,2×10 8 cfu 246 112 27.79 b 53.56 18.24 56.40 91.09 288.72 117.86 64.33 Interaction T x CP L CP 0 225 b 1 10 33.65 a 53.71 ab 20.80 53.70 b 89.72 247.43 b 129.79 68.00 L CP 2 237 ab 1 11 29.94 a 57.50 a 1 8.99 56.66 ab 87.73 292.91 a 125.86 63.50 H CP 0 244 ab 1 24 32.69 a 51.27 ab 1 6.00 61.30 a 90.12 288.83 a 122.28 71.71 H CP 2 256 a 113 25.65 b 49.62 b 17.49 56.15 ab 94.47 284.57 a 109.86 65.16 SEM 3.76 4.64 0.65 1.05 1.07 0.93 2.64 6.98 5.22 1.58 Two ways probabilities* T 0.015 0.420 0.109 0.025 0.149 0.033 0.502 0.175 0.267 0.395 CP 0.121 0.614 0.001 0.709 0.942 0.559 0.823 0.092 0.438 0.089 T × CP 0.013 0.525 0.001 0.049 0.445 0.035 0.551 0.013 0.686 0.739 T = temperature; CP = Clostridium perfringens ; SEM = standard error of means; IL-1β = interleukin-1β; IL-6 = interleukin-6; IL-10 = interleukin-10; IL-22 = interleukin-22; TNF-α = tumour necrosis factor- α; IFN- γ = interferon- γ; IgM = immunoglobulin M; IgA = immunoglobulin A; and IgG = immunoglobulin G. a, b Different letters in the same column indicate a significant difference ( P < 0.05); 1 n = 6 per treatment group with 12 birds per replicate Jejunum morphometry variables in response to exposure of chickens to HS and C. perfringens infection The effects of HS and C. perfringens infection on jejunum morphometry variables, including villus height, crypt depth, and villus/crypt ratio, as affected by HS and infection with C. perfringens are shown in Fig. 1 A. Villus height was significantly lower ( P < 0.05) in the H CP 2 group than in the other groups. Furthermore, jejunum SIgA concentration increased ( P < 0.10) in the L CP 2 group compared to that in the L CP 0 group (Fig. 1 A). Genes and protein expression of jejunal lamina propria in response to exposure of chickens to HS and C. perfringens infection The gene and protein expression results of the jejunal lamina propria, as affected by HS and C. perfringens infection, are shown in Tables 5 and 6 and Fig. 1 B. The relative mRNA expression of IL-6 was significantly ( P < 0.05) upregulated in the jejunal lamina propria tissue of the L CP 0 group compared to that in the other groups (Table 5 ). In addition, the relative mRNA expression of IL-4, IL-10, NF-κB , and Muc-2 was significantly ( P < 0.05) upregulated in the jejunal lamina propria tissue of the L CP 2 group compared with that of the other groups (Tables 5 and 6 ). The highest mRNA expression ( P < 0.05) of IL-8, IFN-γ, NLRP-3, Zo-1 , and Claudin-1 was detected in the jejunal lamina propria tissue of the H CP 0 group compared with that of the H CP 2 group (Tables 5 and 6 ). In contrast, the highest mRNA expression ( P < 0.05) of TNF-α was detected in the jejunal lamina propria tissue of the H CP 2 group compared with that of the other groups (Table 6 ). Western blot results also revealed a higher ( P < 0.05) protein expression of TNF-α in the jejunal lamina propria tissue of the H CP 0 group than in the other groups (Fig. 1 B). Table 5 Effects of high temperature and Clostridium perfringens on the mRNA expression of cytokine gene in the jejunum mucosa of Qingyuan Patridge chicken. Parameters IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-17 Ambient Temperature (T) L, 25°C 1.56 a 1.28 1.25 a 1.02 a 2.01 b 1.42 a 1.01 H, 35°C 0.62 b 1.36 0.72 b 0.75 b 3.87 a 0.41 b 0.87 Clostridium perfringens ( CP ) CP0 , 0 dose 1.06 1.27 0.98 0.98 2.58 0.86 0.90 CP2 , 2×10 8 cfu 1.12 1.37 0.98 0.79 3.21 0.97 0.98 Interaction T x CP L CP 0 1.40 ab 1.04 1.14 ab 1.07 a 1.17 b 1.05 b 1.10 L CP 2 1.73 a 1.52 1.36 a 0.98 a 2.85 a 1.80 a 0.93 H CP 0 0.72 b 1.51 0.83 ab 0.90 a 4.00 a 0.67 bc 0.71 H CP 2 0.52 b 1.22 0.61 b 0.61 b 3.57 a 0.15 c 1.03 SEM 0.15 0.11 0.12 0.05 0.23 0.07 0.12 Two ways probabilities* T 0.004 0.675 0.030 0.011 0.001 0.001 0.513 CP 0.824 0.653 0.998 0.073 0.181 0.398 0.758 T × CP 0.037 0.071 0.020 0.038 0.027 0.001 0.277 T = temperature; CP = Clostridium perfringens ; SEM = standard error of means; IL-1β = interleukin-1β; IL-2 = interleukin-2; IL-4 = interleukin-4; IL-6 = interleukin-6; IL-8 = interleukin-8; IL-10 = interleukin-10; IL-17 = interleukin-17. a, b, c Different letters in the same column indicate significant difference ( P < 0.05); 1 n = 6 per treatment group with 12 birds per replicate. Table 6 Effects of high temperature and Clostridium perfringens on the expression of immune-related genes in the jejunum mucosa of Qingyuan Patridge chicken. Parameters TLR-4 TNF-α NF-κB TGF-β IFN-γ NLRP-3 MYLK Occiudin-1 Muc-2 Zo-1 Claudin-1 Ambient Temperature (T) L, 25°C 1.23 1.14 b 1.20 2.08 1.05 1.09b 4.28 1.07 1.24 1.11 1.10 b H, 35°C 1.45 1.93 a 1.19 2.58 1.18 1.48a 4.88 1.12 1.05 1.47 1.62 a Clostridium perfringens ( CP ) CP0 , 0 dose 1.28 1.43 1.14 2.01 1.28 a 1.15 4.43 1.10 1.14 1.29 1.35 CP2 , 2×10 8 cfu 1.40 1.64 1.25 2.65 0.95 b 1.11 4.71 1.05 0.70 1.30 1.38 Interaction T x CP L CP 0 1.11 1.03 b 1.02 b 1.60 1.09 ab 1.11 b 3.90 1.05 1.08 ab 1.04 c 1.04 b L CP 2 1.36 1.25 b 1.39 a 2.56 1.02 b 1.07 b 4.67 1.10 1.40 a 1.19 bc 1.17 b H CP 0 1.46 1.83 a 1.27 ab 2.42 1.47 a 1.82 a 4.96 1.15 1.20 ab 1.54 a 1.66 a H CP 2 1.45 2.03 a 1.12 ab 2.74 0.89 b 1.15 b 4.76 1.10 0.90 b 1.41 ab 1.59 a SEM 0.11 0.07 0.06 0.33 0.06 0.07 0.72 0.07 0.08 0.06 0.06 Two ways probabilities* T 0.315 0.001 0.941 0.449 0.316 0.010 0.688 0.716 0.203 0.002 0.001 CP 0.605 0.144 0.336 0.335 0.012 0.120 0.842 0.984 0.953 0.923 0.772 T × CP 0.545 0.932 0.031 0.628 0.047 0.039 0.734 0.706 0.043 0.225 0.361 T = temperature; CP = Clostridium perfringens ; SEM = standard error of means; TLR-4 = toll-like receptor-4; TNF-α = tumour necrosis factor- α; NF-κB = nuclear factor kappa B; TFG-β = transforming growth factor –β; IFN- γ = interferon- γ; NLRP-3 = non-like receptor protein-3; MUC-2 = mucin-2; ZO = zonula occludens. a, b, c Different letters in the same column indicate significant differences ( P < 0.05); 1 n = 6 per treatment group with 12 birds per replicate. Dynamic shifts in T cells in the blood and jejunal lamina propria in response to exposure of chickens to HS and C. perfringens infection The dynamic shifts in T cells in the blood induced by exposure to HS and C. perfringens infection are shown in Figs. 2 and 3 . In the blood, the T lymphocyte subsets of peripheral blood, total CD3 + and CD4 + T cells, CD3 + CD4 + T cells, CD3 + CD8 + T cells, and CD3 + CD4 + /CD3 + CD8 + ratio significantly decreased ( P < 0.01) in the L CP 2 group compared to the other groups. However, in the chickens of the L CP 2 group, CD3 + CD4 + T cells, CD3 + CD4 + T cells, and the CD3 + CD4 + /CD3 + CD8 + ratio decreased compared to those of the H CP 0 group. Further analysis indicated that in the chickens of the H CP 2 group, the CD4 + T-cell, CD3 + CD4 + T cells, and CD3 + CD4 + /CD3 + CD8 + ratios decreased ( P < 0.05) compared to those in the other groups (Fig. 2 ). In addition, the T lymphocyte subsets of the jejunal lamina propria are shown in Fig. 3 . The positive rates of jejunal CD3 + , CD3 + CD4 + T cells, CD3 + CD8 + T cells, and CD4 + CD8 + T cells in the H CP 2 group were significantly higher ( P < 0.05) than those in the other groups. Jejunal CD4 + T cells tended to increase ( P = 0.08) in the H CP 0 group, and the CD3 + , CD3 + CD4 + T cells, and CD3 + CD4 + /CD3 + CD8 + ratios in the H CP 0 group were significantly higher ( P < 0.05) than those in the other groups. Additionally, the number of positive CD4 + CD8 + T cells in the H CP 2 group was significantly higher ( P < 0.05) than that in the L CP 0 group. Dynamic shifts of the jejunum cecal microbiota in response to exposure of chickens to HS and C. perfringens infection The effects of exposure of animals to HS and C. perfringens infection were not significant on alpha measures (Chao1, Shannon, Simpson, dominance, Pielou, and observed OTUs) among the chick groups, as shown in Fig. 4 A. Principal coordinate analysis (PCoA) revealed significant differences ( P < 0.01) among the bacterial community groups (Fig. 4 B). The highest and lowest numbers of unique OTUs units were observed in the L CP0 and L CP2 groups, respectively (Fig. 4 C). The most common OTUs were observed in the H CP0 and H CP2 groups (Fig. 4 C). The taxonomic composition analysis at each classification level for the phyla and genera is presented in Fig. 4 D. At the phylum level, Firmicutes, Proteobacteria, Bacteroidota, Actinobacteriota, and Campilobacteria were predominant as top five phyla in the cecal microbiota. The highest average relative abundances of Firmicutes and Bacteroidota were observed in the L CP2 group, whereas the highest average relative abundances of Proteobacteria, Actinobacteriota, and Campilobacteria were observed in the H CP0 group. At the genus level, Lactobacillus , Esherichia-Shigella , Enterococcus , Bacteroides , and Candidatous_Arthromitus were the predominant genera in the cecal microbiota. The highest relative abundances of Lactobacillus and Enterococcus were observed in the L CP0 group, whereas the highest relative abundances of Esherichia-Shigella, Staphylococcus, Clostridia , and Bacteroides were observed in the H CP2 group (Fig. 4 D). The results of LEfSe and LDA analyses of the cecum microbiota communities for L CP0 vs. L CP2 + H CP0 + H CP2 are presented in Fig. 6A and B. The LEfSe analysis results revealed that the relative abundance of Bacillaceae and Parabacteroides increased ( P < 0.05) in the L CP0 group compared to the other groups (L CP2 + H CP0 + H CP2 ), whereas Phascolarctobacterium, Megasphaera , and Flavonifractor increased ( P < 0.05) in the L CP2 + H CP0 + H CP2 groups in comparison to the L CP0 group. The LDA analysis results showed that the relative abundances of Bacillus , Ligilactobacillus , and Sporichthyacea were the highest ( P < 0.05) in the L CP0 group, whereas Cyanobacteria , Cyanobacteriia , and Listeriaceae were significantly high ( P < 0.05) in the H CP2 group. Discussion HS could be a potential predisposing factor for inducing NE and gastrointestinal infections by pathogens, especially C. perfringens . Only one, HS or C. perfringens , is sufficient for NE occurrence; however, the harmful effects of both are extremely dangerous to animal health and survival. These harmful effects include performance deficits (decreased FI, weight gain, and nutrient absorption), intestinal diseases (inflammation, necrosis, ischemia, and increased permeability), and physiological disorders (immune dysfunction, impaired antioxidant responses, and disturbances in respiratory and digestive functions). Therefore, in this study, we investigated the effects of HS and C. perfringens infection on growth performance, plasma immune profiles, intestinal cytokine and immune-related gene expression, gut integrity, T-cell dynamics in blood and the jejunal lamina propria, and structural changes in the cecal microbiota of male Qingyuan Patridge chickens. The acute form of NE disturbance associated with HS and C. perfringens leads to increased mortality in broiler flocks, which is 1–5% per day. In the subclinical form, damage to the intestinal mucosa caused by HS and C. perfringens leads to decreased digestion and absorption, reduced GR, and increased FCR [ 7 , 4 ]. Our results showed a significant decrease in the final body weight (BW36) and average body weight gain in the H CP0 and H CP2 groups compared to those in the L CP0 group, which was caused by the exposure of the birds to HS [ 26 , 27 ]. HS is an environmental stressor that negatively affects the global poultry industry, reducing poultry production performance, favoring bacterial infection, inducing changes in the cellular and adaptive immune systems, and decreasing animal welfare [ 28 , 29 ]. HS alleviates the immune system of birds and makes them vulnerable against infection disease suppress or dysregulate the immune system via various physiological mechanisms chronic or acute stress triggers the release of corticotropin-releasing hormone (CRH) from the hypothalamus, stimulating the adrenal glands to produce glucocorticoids. Elevated glucocorticoids suppress pro-inflammatory cytokine production, inhibit T-cell proliferation, and reduce natural killer (NK) cell activity, weakening the body's ability to combat infections and respond to inflammation, which may act as a predisposing factor for NE development in chickens. Thus, exposure to HS for 28 d in chicken broilers resulted in reduced body weight, decreased daily weight gain, and an increased feed-to-weight gain ratio. This negative effect of HS on growth performance is potentially attributable to reduced nutrient absorption due to intestinal damage, inflammation, and necrosis [ 30 , 31 ]. In addition, BW36 and BWG20–36 d were decreased in the H CP2 group compared to the L CP0 group due to the harmful effects of exposure to HS and C. perfringens infection. Infecting chickens with 2×10 8 CFU/mL of C. perfringens type A showed increased diarrhea, narcotic enteritis, and energy loss [ 32 ]. The present study showed a significant reduction in villus height and villus-to-crypt ratio in the H CP 2 group compared to those in the L CP0 group. HS has a negative effect on the lining of the small intestine that separate the gut from the external environment, resulting in a condition known as leaky gut, which is detrimental to bird wellbeing, growth, and health [ 33 ]. Exposure of broiler chickens to HS increased leaky gut by damaging tight junction proteins (TJs), allowing partially digested food, toxins, and microbes to pass into the bloodstream [ 34 ]. TJs form a semipermeable barrier which aids in the passage of ions and solutes while preventing the passage of toxins and foreign bodies [ 35 ]. Defects in gastrointestinal physiology and function, such as intestinal permeability, gut barrier dysfunction, inflammatory bowel disease, celiac disease, and irritable bowel syndrome, are mainly associated with structural disorders of the gastrointestinal tract [ 36 ]. Similarly, functional gastrointestinal disorders, such as motility disturbance, visceral hypersensitivity, altered mucosal secretion and absorption, and dynamic changes in the gut microbiota, are identified by morphological and structural abnormalities [ 37 ]. Our findings revealed that the plasma cortisol concentration was significantly higher in the H CP2 group than in the L CP0 group. Corticosterone is a glucocorticoid hormone secreted by the adrenal glands when the body experiences different stressors, and excessive cortisol levels notably affect the digestive system [ 38 ]. In addition, mucosal cells and adrenal glands release several inflammatory cortisol and cytokines, causing increased intestinal disturbance, especially in bacterial-mucosal interactions, leading to the aggravation of diseases, such as inflammatory and irritable bowel syndrome [ 39 ]. On comparing the H CP2 and L CP0 groups, a significant decrease in the plasma concentration of IL-6 was observed. The results of the present study are consistent with those of a previous study which reported that C. perfringens infection under HS conditions changed the expression of pro- and anti-inflammatory cytokines involved in IL-6 activation [ 40 , 41 ]. In addition, the plasma concentration of IgM increased in the H CP2 group compared with the L CP0 group. IgM is a major antibody for immunity that increases in plasma during stressor conditions and protects the animal’s body against pathogenic infection by blocking detrimental pathogens, reducing the bacterial response in the organism [ 42 ]. The plasma concentration of IgM promote intestinal function, and animals exposed to HS and C. perfringens infection experience intestinal inflammation, increase pathogenic challenges, and induce the secretion of IgM for the immune response. The relative mRNA expression of TNF-α , NF-κB , and Zo-1 was upregulated in the jejunal mucosa of the H CP2 group compared to the L CP0 group owing to the stimulation of the expression of immune-related genes caused by exposure of the chickens to HS and C. perfringens infection. Cytokine mRNA expression in the intestine is a small protein secreted by gut cells to regulate host-microbe interactions via cellular immune signaling, promoting innate and adaptive immune responses [ 43 ]. Ohtsu et al. [ 44 ] found that HS induces spleen involution and affects the expression of splenic cytokines such as IL-12 and IFN-γ in broiler chickens. The expression of IFN-γ, IL-8, IL-17, and IL-18 was significantly increased during exposure of broiler chickens to HS [ 45 ]. Interleukins (ILs) are a group of cytokines that mainly secrete immune proteins and signaling molecules by leukocytes and gut mucosa. ILs (IL-8, IL-17, and IL-18) were observed to promote T-cell proliferation; however, interferons (IFN-γ and IFN-α) enhanced cell-mediated immunity, thereby increasing antibody-mediated immunity and antimicrobial activity [ 46 ]. These results are consistent with previous studies showing that HS increases IL-8 expression in chickens and mammals but decreases IL-8 plasma levels in chickens [ 47 , 48 ]. The downregulation of TGF-β, including a concurrent upregulation in IL-1β, IL-13, and IL-17, indicates that C. perfringens -induced subclinical NE triggers an inflammatory immune response in the intestine of broiler chicks [ 49 ]. The present study found that HS significantly decreased the positivity rate of CD4 + T cells and the CD3 + 4 + /CD3 + 8 + ratio in the blood of Qingyuan chickens. Tissue-resident immune cells in the chicken intestine are critical in protecting against pathogens. During pathogenic infection, adaptive immune T cells, CD4 + and CD8 + are the main sources of ILs production, regulating immune cell and cytokine production [ 50 ]. The synthesis of a lower range of pro- and anti-inflammatory cytokines and their deregulation can trigger the pathogenesis of toxigenic C. perfringens type A bacteria, destroying the signaling process of certain cytokines to lymphocytes and causing lymphoid tissue damage that causes abnormal T-cell responses, as described in growing pigs [ 51 ] and broilers [ 52 ]. Similarly, our results revealed that the H CP 2 group had significantly increased T lymphocyte subsets in peripheral blood, total CD3 + and CD4 + T cells, positive CD3 + 4 + cells, double positive CD4 + 8 + T cells, and the ratio of CD3 + 4 + /CD3 + 8 + in the chicken blood. These results indicate that infection with C. perfringens in the chickens rapidly expanded the T cells that activated the chicken adaptive immunity system. The findings support by the previous study on broilers for a 15 d trial, which reported that T-cell responses are enhanced after C. perfringens infection and secreted protein-stimulated cecal tonsil cells [ 11 ]. In the present study, significant changes were observed in the structure of cecal microbiota induced by exposure of Qingyuan chickens to HS and C. perfringens infection, with a decrease in the dominant Lactobacillus and Rikenellaceae_RC9 and an increase in Bacteroides and Escherichia-Shigella . A healthy microbial community colonized the gut of the L CP 0 group, which improved intestinal function and antimicrobial pathogenicity compared with the H CP 2 group. The relative abundance of Lactobacillus, Parabacteroides , and Enterococcus increased in the L CP 0 group; these genera are often used as beneficial probiotic products to ameliorate HS. The relative abundance of pathogenic bacteria, including Clostridia, Esherichia-Shigella, Staphylococcus , and Bacteroides , increased in the H CP0 group. Indeed, HS can disrupt the balance of beneficial gut bacteria, promoting the growth of pathogenic microbes. Dysbiosis can impair the fermentation of complex carbohydrates and the production of short-chain fatty acids (SCFAs), which are essential for gut health and energy supply to intestinal cells. These pathogenic genera increase chronically during HS and may cause the bile acid pool to promote intestinal inflammatory bowel diseases [ 53 , 54 ]. In addition, these pathogenic bacteria promote the harmful effects of LPS, which substantially affects animal health, primarily through interactions with the immune system and organ function failure, leading to abdominal congestion, intestinal sepsis, abnormal autoimmunity, and cellular senescence [ 5 ]. HS disrupts the intestinal microbiota balance and promotes the intestinal inflammatory response and colonization of pathogens [ 55 – 57 ]. Lactobacillus as probiotic strains can improve intestinal integrity, morphology, and microbial environment to enhance broiler growth performance under HS [ 58 , 59 ]. In contrast, at the genus level, the beneficial bacteria Rikenellaceae_RC9_gut_group was significantly reduced, and pathogenic bacteria Escherichia-Shigella and Coprococcus were increased. The results for microbial organisms are consistent with previous results [ 60 ]. Rikenellaceae_RC9 group beneficial bacteria have a potential protective function against metabolic and gastrointestinal diseases and are potential biomarkers of healthy animal guts [ 61 , 62 ]. In addition, the general levels of Ralstonia, Halomonas, and Roseomonas were lower, and those of Gca-900066575 and Flavonifractor were higher in the L CP 2 group in compared to the L CP 0 group. At the genus level, Parabacteroides and Roseomonas were reduced, whereas Ralstonia, Eubacterium hallii, and Flavonifactor were increased in the H CP 2 group in the jejunum mucosa. [ 63 ] demonstrated that the abundance of Parabacteroides and Roseomonas decreased and that of Flavonifactor increased in the small intestine of broilers under HS conditions with pathogenic infection. Conclusion In conclusion, our findings demonstrated that HS may accelerate the pathogenesis of C. perfringens infection in chickens. The common co-occurrence of Qingyuan Patridge chickens exposed to HS and C. perfringens infection will cause more harmful effects on growth performance, jejunum morphometry, immune-modulatory responses of T cells, expression of cytokines and immune-related genes, and gut microbiota dynamics, and exacerbates NE-related disturbances compared to HS or C. perfringens infection treatment. The common co-occurrence of HS and C. perfringens infection could be used to induce NE model. Abbreviations HS Heat stress NF-α Tumor necrosis factor-α NF-κB Nuclear factor kappa B Zo-1 Zonula occludens-1 NE Necrotic enteritis LPS Lipopolysaccharide bacteria LP Lipid peroxidation GIT Gastrointestinal tract PBS Phosphate buffered saline RIPA Radioimmunoprecipitation assay NC Nitrocellulose OTUs Operational taxonomic unit PCA Principal component analysis PCoA Principal coordinate analysis FLASH Fast length adjustment of short reads, v1.2.11 RDP Ribosomal database project LEfSe Linear discriminant analysis Effect size Declarations Acknowledgements The authors thank the staff of the Animal Research of Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Huazhong Agricultural University, and Zhongkai University of Agriculture and Engineering for their assistance in animal husbandry and sample collection. Author contributions L. Jingdu, W. Chen, and C.T. Zheng designed the project. C.T. Zheng and W. Chen supervised the project. L. Jingdu, Z. Qi, Y. Lv, S. Wang, W. Xia, Y. Zhang, C. Jin, S. Wang X. Huang, K. Li, C.T Zheng and W. Chen collected the samples and performed the laboratory work. L. Jingdu, A.A. Elokil, M. T. Sarker, K. Abuelezz, and W. Chen carried out the analyses and interpreted the data. A.A. Elokil, M. T. Sarker, K. Abuelezz, and w. Chen drafted the paper. A.A. Elokil, K. Abuelezz, C.T. Zheng and W. Chen edited the manuscript. All authors reviewed the manuscript. Funding This work was supported by the National Key R&D Program of China (Grant No.2018YFE0128200), State Key Laboratory of Swine and Poultry Breeding Industry (GDNKY-ZQQZ-K3, GDNKY-ZQQZ-K22), the Project of Science and Technology innovation strategy (ZX202401-06), the Foreign Expert Project (Grant No. H20240494). Talented Young Scientists Program TYSP (No. P19U42006), Ministry of Science and Technology (MOST), China, the Opening Project of Guangdong Provincial Key Laboratory of Animal Breeding and Nutrition (2022SZ01). The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Data availability The bacterial sequences supporting the findings of this study are available at the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1285695) under the BioProject ID PRJNA1285695. Ethics approval and consent to participate The Institutional Animal Care and Use Committee of Guangdong Academy of Agricultural Sciences (ACUCGAAC 1.2019) approved all the animal procedures, and all methods were performed in accordance with the relevant guidelines and regulations, following ARRIVE guidelines to minimize the animal suffering during the experiment. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Xu W, Wang H, Liu L, Miao Z, Huo Y, Zhong Z. 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Tavella T, Rampelli S, Guidarelli G, Bazzocchi A, Gasperini C, Pujos-Guillot E, Comte B, Barone M, Biagi E, Candela M. Elevated gut microbiome abundance of Christensenellaceae, Porphyromonadaceae and Rikenellaceae is associated with reduced visceral adipose tissue and healthier metabolic profile in Italian elderly. Gut Microbes. 2021;13(1):1880221. Wang X, Feng J, Zhang M, Li X, Ma D, Chang S. Effects of high ambient temperature on the community structure and composition of ileal microbiome of broilers. Poult Sci. 2018;97(6):2153–8. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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12:39:20","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":254341,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-6855798/v1/42e665e41858589e1d0b0adf.html"},{"id":95735611,"identity":"f7c0fc11-e783-4c30-a3aa-4ad06933fec4","added_by":"auto","created_at":"2025-11-12 12:39:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":876449,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of high temperature and \u003cem\u003eClostridium perfringens\u003c/em\u003e on the intestinal morphology of Qingyuan Patridge chicken. (A) H \u0026amp; E staining of the jejunum segment, magnification 10X; scale bar was 200 µm. Data are expressed as mean with standard error of the mean. \"*\" means \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \"**\" means \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \"***\" means \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, “# \" means \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.1.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6855798/v1/86bea76d6f76fcfa096b9ec4.png"},{"id":95735613,"identity":"d79dcadd-684a-45f5-9334-8c980b21b7a7","added_by":"auto","created_at":"2025-11-12 12:39:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":621908,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic shifts in T cells in blood induced by exposure animal to HS and C. perfringens infection of Qingyuan Patridge chicken. Data are expressed as mean with standard error of the mean. \"*\" means \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \"**\" means \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \"***\" means \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001,\" #” means \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.1.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6855798/v1/643dd3eb86461f21342f76b3.png"},{"id":95735612,"identity":"856bbb08-368d-4092-bbbe-80474aa8e5be","added_by":"auto","created_at":"2025-11-12 12:39:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":609746,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic shifts in T cells in the jejunal lamina propria induced by exposure animal to HS and C. perfringens infection of Qingyuan Patridge chicken. Data are expressed as mean with standard error of the mean. \"*\" means \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \"**\" means \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \"***\" means \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001,\" #” means \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.1.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6855798/v1/6d8684af1e82b405c40739c6.png"},{"id":95735623,"identity":"c2a8826e-4c9d-419c-a62c-39945a54ce66","added_by":"auto","created_at":"2025-11-12 12:39:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":443321,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in structural cecal microbiota induced by exposure animal to HS and C. perfringens infection of Qingyuan Patridge chicken. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(A) α-diversity measures of bacterial communities among groups, (B) Principal coordinate analysis (PCoA) among groups of bacterial communities based Bray Curtis distance and matrix of P-values generated by permutation multiple variance analysis (PERMANOVA) of the gut communities of bacteria (C) Venn diagram of OUT number among groups of common and unique units, and (D) Taxonomic composition analysis at each classification level of phylum and genus of bacterial communities.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6855798/v1/245bf9eb3188677625a00bbe.png"},{"id":95735618,"identity":"d804890f-b6ed-467b-a6ce-d219bfe10639","added_by":"auto","created_at":"2025-11-12 12:39:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":392882,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eLEfSe analysis of cecum microbiota communities LCP0 vs. LCP2 + HCP0 + HCP2. (B) LDA analysis of bacterial communities among groups of Qingyuan Patridge chicken.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6855798/v1/f04c2a32186074f03952c1de.png"},{"id":96708045,"identity":"d8ead259-ad13-4411-994f-fb79cf7820a6","added_by":"auto","created_at":"2025-11-25 09:53:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4449778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6855798/v1/1acbec66-c42a-44ae-863f-afd4f3a810f0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eChanges in intestinal immunity and gut microbiota associated necrotic enteritis induced by \u003cem\u003eClostridium perfringens\u003c/em\u003e infection and exposure to heat stress in chicken\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eClostridium perfringens\u003c/em\u003e (\u003cem\u003eC. perfringens\u003c/em\u003e) is a common pathogenic that can cause both of animal and human diseases, such as necrotic enteritis (NE) in poultry [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Heat stress (HS) is one of the most important stressors that compromise the immune system of birds and increases their susceptibility to enteric pathogens such as \u003cem\u003eC. perfringens\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. NE is caused by \u003cem\u003eC. perfringens\u003c/em\u003e type A, which produces alpha toxin and has become increasingly prevalent in countries that have ceased using growth-promoting antibiotics [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, abdominal hyperthermia by HS and infection with \u003cem\u003eC. perfringens\u003c/em\u003e cause NE-associated disturbances, such as intestinal congestion, mucosal perturbation, barrier dysfunction, ischemia, hyperpermeability, and cholangiohepatitis, via the invasion of enteric pathogens from bacterial lipopolysaccharide (LPS) and opportunistic gut microbiota communities [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Under the predominance of NE, excessive production of both reactive oxygen species and reactive nitrogen species negatively affects enteric antioxidant defense and increases cellular lipid peroxidation (LP) and intestinal tissue congestion, thereby reducing feed intake (FI) and growth rate (GR) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eC. perfringens\u003c/em\u003e is a gram-positive, spore-forming, anaerobic bacterium classified based on its toxins into over 20 types, including seven toxin-producing strains predominant in poultry, known for alpha, beta, epsilon, and iota toxins [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Similarly, \u003cem\u003eC. perfringens\u003c/em\u003e infection is a persistent threat to animal and public health and is associated with NE via the production of alpha- and beta-toxins that enter the bloodstream. \u003cem\u003eC. perfringens\u003c/em\u003e infection and HS are key factors that induce NE in poultry, leading to huge economic losses in the broiler industry through performance degradation, inefficient feed use, and increased mortality.\u003c/p\u003e\u003cp\u003eReduced FI during the exposure of animals to HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection decreases systemic energy homeostasis. This reduces the panting rate to dissipate excess body heat, and the immune response against pathogenic infections [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Notably, HS reduces intestinal absorption owing to epithelial cell injury and hyperpermeability, negatively affecting cellular functions [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Host health efficiency regulates the pattern of microbiota interactions by determining the motility, proliferation, and lifestyle of the gut microbial communities. The resulting bacteria can produce essential amino acids and vitamins and regulate digestion to enhance host health and function [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, the composition and structure of the intestinal microbiota have been identified as novel molecular markers to investigate community diversity-regulated candidate performance, including GR [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], egg performance (Elokil, et al., 2020a), immune responses [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and fertility [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLymphoid tissues in the gastrointestinal tract (GIT) are well developed and form the primary defense against enteric pathogens. The T-cell-mediated immune response is important in the early process of cellular defense, including CD4\u003csup\u003e+\u003c/sup\u003e as inducer T cells that stimulate the phagocytosis of macrophages and antibody production by B cells, whereas CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells kill infected cells. Jiang et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] reported that the percentage of T-cell subsets and the expression level of cytokine mRNA in the small intestine was reduced with contaminated chicks diet with 0.6 mg/kg aflatoxin B1, implying a negative effect on the immunity and function of the intestinal mucosa. In contrast, probiotic strains, such as \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eLactobacilli\u003c/em\u003e, have improved the activation and proliferation of T-helper populations of CD4\u0026thinsp;+\u0026thinsp;T cells and stimulated cytotoxic T cells (CD8\u0026thinsp;+\u0026thinsp;T cells), presumably through surface contact [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Collectively, avian NE induced by \u003cem\u003eC. perfringens\u003c/em\u003e infection and exposure to HS is a serious disease causing huge losses in chicken farms. Therefore, the present study was performed to explore the impacts of \u003cem\u003eC. perfringens\u003c/em\u003e and exposure to HS on performance and stability of gut health (i.e., jejunum morphometry, immune-modulatory responses of T cells, expression of cytokines and immune-related genes), together with the dynamic changes (composition and structure) of endogenous key microbial genera and functional groups in gastrointestinal tract of Qingyuan Patridge chickens. To the best of our knowledge, this is the first study to explore changes in intestinal immunity and gut microbiota associated NE induced by \u003cem\u003eC. perfringens\u003c/em\u003e infection and exposure to HS in chicken.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cem\u003eAnimals and experimental design\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of 192 male Qingyuan Patridge chicken at 3 weeks old were obtained from Guangdong Aijiankang Biotechnology Co., Ltd., Qingyuan City, China, and randomly assigned to a 2\u0026times;2 factorial arrangement experiment, including four treatments, each comprising 12 replicates (n\u0026thinsp;=\u0026thinsp;48 chicks/treatment; n\u0026thinsp;=\u0026thinsp;4 chicks/replicate). A 2 \u0026times; 2 factorial arrangement experiment was performed, including two levels of oral \u003cem\u003eC. perfringens\u003c/em\u003e dosages without (\u003cem\u003eCP0\u003c/em\u003e) and with 2\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL (\u003cem\u003eCP2\u003c/em\u003e) under two ambient temperatures of 25\u0026deg;C (L) and 35\u0026deg;C (H) for 30 d. Four experimental groups were assigned as follows: L\u003cem\u003eCP0\u003c/em\u003e group, chicks reared under L of ambient temperature and without an oral dosage of \u003cem\u003eC. perfringens\u003c/em\u003e (\u003cem\u003eCP\u003c/em\u003e0); L\u003cem\u003eCP2\u003c/em\u003e group, chicks reared under L of ambient temperature and with an oral dosage of \u003cem\u003eC. perfringens\u003c/em\u003e (\u003cem\u003eCP\u003c/em\u003e2); H\u003cem\u003eCP0\u003c/em\u003e group, chicks reared under H of ambient temperature and without an oral dosage of \u003cem\u003eC. perfringens\u003c/em\u003e (\u003cem\u003eCP\u003c/em\u003e0); and H\u003cem\u003eCP2\u003c/em\u003e group, chicks reared under H of ambient temperature and with an oral dosage of \u003cem\u003eC. perfringens\u003c/em\u003e (\u003cem\u003eCP\u003c/em\u003e2). The HS application involved exposing birds to an ambient temperature of 35\u0026deg;C for 10 h (from 9:00 am to 7:00 pm), after which the ambient temperature returned to 25\u0026deg;C. All birds were housed in cages of the same size (40 \u0026times; 50 \u0026times; 70 cm), and the experimental period lasted for 15 days. The diet was formulated to meet or exceed the nutritional requirements of yellow chickens (Ministry of Agriculture of China), according to the national research council (NRC) recommendations [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. The composition of the feed ingredients and the estimated nutrient levels are presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eComposition and nutrition levels of the basal diet (%, as fed-basis).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIngredients\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePercentage (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNutrient content\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLevel (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCorn\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e53.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMetabolizable energy (MJ/kg)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12.13\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWheat bran\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.70\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCrude protein\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20.59\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSoybean meal (CP 43%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e28.30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCalcium\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.96\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCorn protein flour\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePhosphorus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.77\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSoybean oil\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAvailable phosphorus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eL-lysine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLysine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.33\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDL-Methionine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMethionine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThreonine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStone powder\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCalcium hydrogen phosphate\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSalt\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePremix\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTotal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003csup\u003e1\u003c/sup\u003eEach kilogram of diet (including feed ingredients) provides VA 12,000 IU, VD3 600 IU, VE 45 IU, VK 2.5 mg, VB1 2.4 mg, VB2 5.0 mg, VB6 2.8 mg, niacin 42 mg, pantothenic acid 12 mg, Folic acid 1.0 mg, choline 1,300 mg, Fe 80 mg, Cu 7 mg, Mn 80 mg, Zn 85 mg, I 0.70 mg, Se 0.15 mg.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003e\u003csup\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sup\u003eThe crude protein, calcium, total phosphorus are analyzed values and metabolizable energy (MJ/kg) and others were calculated values.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eInoculation protocol of C. perfringens\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe pathogenic strain of \u003cem\u003eC. perfringens\u003c/em\u003e type A (No. ATCC13124) was provided from the China Veterinary Culture Collection Center (Beijing, China). The bacterial strain was kept in our laboratory Institute of Animal Health, Guangdong Academy of Agricultural Sciences in glycerol at -80\u0026deg;C and the bacterial culture protocol was aseptically inoculated into fluid thioglycollate medium overnight at 37\u0026deg;C in an anaerobic environment before being used for the inoculation [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, chicken in the L\u003cem\u003eCP\u003c/em\u003e2 and H\u003cem\u003eCP\u003c/em\u003e2 groups orally gavage 1 mL actively growing culture of \u003cem\u003eC. perfringens\u003c/em\u003e type A with a concentration of 2\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL (Colony-forming units per milliliter) from 31d to 34d. Whereas at the same time, two no-inoculated groups (L\u003cem\u003eCP\u003c/em\u003e0 and H\u003cem\u003eCP\u003c/em\u003e0) of chickens received 1 dose of 1 mL PBS (phosphate buffered saline).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGrowth performance assessment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe supplied and residual feed amounts were recorded daily for each replicate to calculate the average daily feed intake (ADFI). The number of dead birds was recorded daily to calculate the percentage of the surviving birds. Chicks in each replicate were weighed on days 20, 30, and 36 in the morning after 12 h of fasting without feeding to measure body weight and calculate the average daily gain (ADG) and feed-to-gain ratio (F/G). FI, daily gain (DG), and feed conversion rate (FCR) were calculated based on the following formulas: FI (g/d)\u0026thinsp;=\u0026thinsp;total food intake (g)/feed days (d); DG (g/d) = (final weight - initial weight) (g)/feed days (d); FCR (g/g)\u0026thinsp;=\u0026thinsp;ADFI (g/d)/ADG (g/d). On day 36, 12 birds, approximately the average body weight for each group, were selected to collect samples, including plasma, organs, and cecal digesta [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHematoxylin\u0026thinsp;\u0026minus;\u0026thinsp;Eosin staining and intestinal morphometric analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze the jejunal morphometric traits, tissue sections were stained using the hematoxylin and eosin staining. Briefly, the jejunum segments were fixed using a 4% paraformaldehyde solution, dehydrated using xylene, and embedded in paraffin. The paraffin-embedded jejunal tissues were sliced and placed on glass slides. Two sections (100 \u0026micro;m) were obtained from each sample and stained with hematoxylin and eosin for 1 min and 10 s, respectively. All morphometric variables were measured using a camera (Olympus; TH4-200; Tokyo, Japan) coupled with computer-assisted digital Image-Pro Plus analysis software (Image-Pro Plus 4.5, Media Cybernetics, Silver Spring, MD, United States) to assess the maximum villus length, crypt depth, and submucosa/muscularis/serosa.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePlasma biochemical variables\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe plasma concentration (n\u0026thinsp;=\u0026thinsp;6) of different biochemical parameters, including interleukins (IL-1\u0026beta;, IL-6, IL-10, IL-22), tumour necrosis factor-\u0026alpha; (TNF-\u0026alpha;), interferon-\u0026gamma; (IFN- \u0026gamma;), immunoglobulins (IgG, IgA, and IgM) were measured by ELISA kits. The experimental kits were acquired from Jiangsu Enzyme Immunization Industrial Co., Ltd. (Yancheng, China), and the procedure was carried out according to the manufacturer's instructions. Preparation of target cells for detection of immune-related cytokines add 10 \u0026micro;L of sample with 40 \u0026micro;L of sample diluent placed in each well then seal and place in incubator 37\u0026deg;C for 30 min. The well with diluted was washed solution5 times, then added 60 \u0026micro;L of enzymes to each well, seal with film and place in an incubator 37\u0026deg;C for 30 min then wash it 5 times and let stand 30 s. Add chromogen A50 and B50 to each well, mix and place in the dark at 37\u0026deg;C for 10 min for colour development. To terminate the reaction, we added 50 \u0026micro;L stop solution to each and every well. The microplate reader was used to measure the absorbance value of each and every well at different wavelengths.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDetection of blood and jejunal lamina propria T lymphocytes by flow cytometer\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples (2.5 mL) were collected for T lymphocyte analysis, and then phosphate-buffered saline (PBS) and 4 mL Ficoll (Paque TM Plus, Amersham) were added to separate peripheral blood mononuclear cells (PBMC). The samples were centrifuged at 2000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 min at 20\u0026deg;C to separate the uppermost layer and transferred to a 5 mL tube for centrifuge again at 1500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min at room temperature. The procedure was repeated twice and washed 2\u0026ndash;3 times with 2 mL of PBS. To determine the T lymphocyte of intestinal lamina propria, a 1 cm jejunum sample was taken, then digestion at 37\u0026deg;C in a water bath for 10 min by the addition of 5 mL of predigestion solution (5 mL PBS\u0026thinsp;+\u0026thinsp;1 \u0026micro;M DTT) and 10 mL of predigestion solution (10 mL PBS\u0026thinsp;+\u0026thinsp;2% FCS\u0026thinsp;+\u0026thinsp;1 mM EDTA). The sample was washed 2\u0026ndash;3 times with PBS, then 1 mL of digestion solution (5 mL RPMI 1640 cell culture medium\u0026thinsp;+\u0026thinsp;2% FCS\u0026thinsp;+\u0026thinsp;200 \u0026micro;g/mL Dnase 1\u0026thinsp;+\u0026thinsp;1 mg/mL Collagenase A) was added for digestion at 37\u0026deg;C in a water bath for 60 min. The samples were centrifuged at 1500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min at room temperature, and the supernatant was washed with 1 mL of PBS. Using the cell counting machine to measure the viable cell concentration, 10 \u0026micro;L of the sample was mixed with 0.4% trypan blue stain and dropped into the counting board for assessment. For the concentration calculation, 50 mL of suspended viable PBMC (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells) samples were transferred to a 96-well plate, followed by the addition of 50 mL of the appropriate antibody (Southern Biotech, Birmingham, USA). The PBMC cell suspensions were multi-stained with a cocktail of T lymphocyte CD marker antibodies (CD3\u0026ndash;FITC, CD4-APC, and CD8-PE, Thermo Fisher Scientific). The well plate was shaken manually for 15 s and incubated at 4\u0026deg;C in the dark for 30 min for each step of antibody labeling. Subsequently, the plate was pipetted and washed three times with the gentle addition of 1 mL of PBS. The cells were resuspended with 500 \u0026micro;L of PBS solution. A total of 50,000 cells were acquired and analyzed using a Becton Dickinson flow cytometer (BD Accuri C6 plus, USA) and Cell Quest software (Becton Dickinson Immunocytometer System, San Jose, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRNA extraction and real-time qPCR analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRNA extraction, cDNA synthesis, and qPCR analyses were performed [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e], approximately 0.05 g of a jejunal mucosal sample was obtained for total RNA extraction using TRIzol reagent (Tiangen Biochemical Technology, Beijing, China). The RNA samples were purified by incubation with RNase-free DNase (Dalian Takara Co. Ltd., Dalian, China) to clean the genomic DNA according to the manufacturer\u0026rsquo;s protocol. The integrity and purity were evaluated using 1.2% agarose gel electrophoresis, and a NanoDrop ND-1000 UV (NanoDrop Technologies, Wilmington, DE, USA), and isolated RNA was chosen for further reactions. After dilution, 1 \u0026micro;L was taken for reverse-transcribed into cDNA using a Primer Script RT reagent kit (Takara, Japan). Real-time fluorescence quantitative PCR was performed using an ABI 7500 PCR system (Applied Biosystems, USA). The amplification was performed in a total volume of 20 \u0026micro;L which comprised 2 \u0026micro;L of cDNA product, 10 \u0026micro;L SYBR primer mix (Takara, Biotechnology Co. Ltd. Dalian, China), 0.4 \u0026micro;L each of the forward and reverse primers, and 7.6 \u0026micro;L of dH2O. The primer sequence was based on the chicken gene sequence uploaded from GenBank, designed using Primer 3.0 software, and synthesized by Shanghai Sangon Biotechnology Company, China. The primer sequences are listed in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. For housekeeping gene, GAPDH was used to normalize target gene expression levels. The 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e technique [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e] was used to calculate the expression results.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003ePrimer sequences used for real-time PCR\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eGene\u003c/em\u003e\u003csup\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePrimer sequences( 5'-3')\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAccession ID\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-GGTGAAAGTCGGAGTCAACGG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_204305.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-TCGATGAAGGGATCATTGATGGC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eIL-1\u0026beta;\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-CTTCACCCTCAGCTTTCACG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_204524.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-TGTGGTGTGCTCAGAATCCA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eIL-2\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-ACCGGAAGTGAATGCAAGAT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_204153.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-AGTGGTCCCAGAATGGACAG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eIL-4\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-GCCAGCACTGCCACAAGAAC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_001398460.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-CGTGGGACATGGTGCCTTGAG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eIL-6\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-CTCCTCGCCAATCTGAAGTC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_204628.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-GGATTGTGCCCGAACTAAAA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eIL-8\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-GCTCTGTCGCAAGGTAGGAC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_205498.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-GGCCATAAGTGCCTTTACGA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e1 L-10\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-GATGCTGCGCTTCTACACAG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_001004414.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-TCCCGTTCTCATCCCATCTTC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eI L-17\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-ATGTTGTCAGCCAGCATTTC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_204460.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-GGAGCCAGTGAGCGTTTG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-CTGTTCTATGACCGCCCAGT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_204267.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-TCAGAGCATCAACGCAAAAG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eNLRP-3\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-CTGGTGAACCCCAAGTCAGT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_001348947.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-GGAAGGTGTCCAAATCCTCA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eIFN-\u0026gamma;\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-TCAAAGCCGCACATCAAACA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_205149.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-GAAGAGTTCATTCGCGGCTT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eTLR-4\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-CACAGCTCTGGATTTCAGCA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_001030693.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-TTCCGCAGTAGATCCTGCTT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eTGF-\u0026beta;\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-GGAGGAGGAGAAGGAGGAGA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_205454.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-GGAACTCTGCTCGAAACAGG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eNF-\u0026kappa;B\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-AGAGGATGCTTCGTTGTGCT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_001396396.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-TCCTGGACAGCAGTGAGATG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eMYLK-1\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-CATCATGCAGTGCTCAGGTC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_001322361.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-TTCTTTCCCATTGTGCAGCC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eMUC-2\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-GCTACAGGATCTGCCTTTGC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eXM_040673077.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-AATGGGCCCTCTGAGTTTTT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOccludin\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-GTCTGTGGGTTCCTCATCGT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_205128.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-TTCTTCACCCACTCCTCCAC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eZO-1\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-ACCAGAGGTCAGAGCCTTCA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eXM_046925214.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-AGCGGTGGTGTTTGTTTTTC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eClaudin-1\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF-GGTTGGTGTGTTTGTTGCTG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNM_001013611.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-TCTGGTGTTAACGGGTGTGA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eIL-1\u0026beta;\u0026thinsp;=\u0026thinsp;interleukin-1\u0026beta;; IL-2\u0026thinsp;=\u0026thinsp;interleukin-2; IL-4\u0026thinsp;=\u0026thinsp;interleukin-4; IL-6\u0026thinsp;=\u0026thinsp;interleukin-6; IL-8\u0026thinsp;=\u0026thinsp;interleukin-8; IL-10\u0026thinsp;=\u0026thinsp;interleukin-10; IL-17\u0026thinsp;=\u0026thinsp;interleukin-17; TNF-\u0026alpha;\u0026thinsp;=\u0026thinsp;tumour necrosis factor- \u0026alpha;; NLRP-3\u0026thinsp;=\u0026thinsp;non-like receptor protein; IFN- \u0026gamma;\u0026thinsp;=\u0026thinsp;interferon- \u0026gamma;; TLR-4\u0026thinsp;=\u0026thinsp;toll-like receptor-4; TFG-\u0026beta;\u0026thinsp;=\u0026thinsp;transforming growth factor \u0026ndash;\u0026beta;; NF-\u0026kappa;B\u0026thinsp;=\u0026thinsp;nuclear factor kappa B; MUC-2\u0026thinsp;=\u0026thinsp;mucin-2; ZO\u0026thinsp;=\u0026thinsp;zonula occludens.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eWestern blot analysis for jejunal lamina propria protein expressions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe frozen specimens were powdered in liquid nitrogen and homogenized in a radioimmunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor. The supernatants samples was incubated on ice for 10 min and centrifuged at 12,500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 6 min. BCA protein quantification kit (Beyotime, Jiangsu, China) was used to determine the total protein concentration. The denatured protein was separated on an 8\u0026ndash;12% SDS-PAGE gel and transferred onto the nitrocellulose (NC) membrane in equal amounts. Then, the separated proteins were incubated for 1 h at room temperature in a blocking buffer (5% skimmed milk in Tris-buffered saline containing 1% Tween-20) and incubated overnight at 4 ℃ with target primary antibodies. All antibodies were obtained from Proteintech Group, Inc., Wuhan, China, with the catalog numbers for ZO-1:21773-1-AP, IL8:27095-1-AP, IL10:60269-1-AP, TNF-\u0026alpha;: 17590-1-AP, and Claudin-1:28674-1-AP. After washing, the cells were incubated with a horseradish peroxidase-conjugated secondary antibody (1:10,000, Biosharp, BL003A) for 1 h at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicrobial genomic DNA extraction, sequencing quality assessment, and 16S rRNA gene sequencing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo extract microbial genomic, total DNA of jejunum digesta samples (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6) were extracted using the MagBind Soil DNA Kit (OmegaBiotec, Norcross, GA, USA) according to the manufacturer\u0026rsquo;s instructions. The cecal microbiota composition was analyzed by sequencing the V3\u0026ndash;V4 hypervariable region of \u003cem\u003e16S rRNA\u003c/em\u003e genes, as described by [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. PCR amplification of the bacterial hypervariable regions V3\u0026ndash;V4 was performed using the forward primer 338F of \u003cem\u003e16S rRNA\u003c/em\u003e gene (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). High-quality and pure clean reads were quantified using Qubit and qPCR (CFX 96Touch, Bio-Rad, Singapore) and merged using FLASH (Fast Length Adjustment of Short reads, v1.2.11) software. The sequencing data were analyzed and classified using RDP, and QIIME 2 software with operational taxonomic units with a 97% similarity level was set. Additionally, alpha diversity (observed species, coverage, Chao, OTUs, Simpson, and dominance) and beta diversity principal coordinate analysis (PCoA) were calculated in different groups. LEfSe analysis was performed by using LEfSe software.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eData from this study were statistically analyzed based on a 2 \u0026times; 2 factorial design using the following model: Y\u003csub\u003eijk\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026micro;\u0026thinsp;+\u0026thinsp;S\u003csub\u003ei\u003c/sub\u003e + D\u003csub\u003ej\u003c/sub\u003e + SD\u003csub\u003eij\u003c/sub\u003e + e\u003csub\u003eijk\u003c/sub\u003e, where Y\u003csub\u003eijk\u003c/sub\u003e = an observation, \u0026micro;\u0026thinsp;=\u0026thinsp;the overall mean, Si\u0026thinsp;=\u0026thinsp;effect of ambient temperature (\u003csub\u003ei\u003c/sub\u003e = L, 25\u0026deg;C and H, 35\u0026deg;C), Dj\u0026thinsp;=\u0026thinsp;effect of inclusion of \u003cem\u003eC. perfringens\u003c/em\u003e (\u003csub\u003ej\u003c/sub\u003e = \u003cem\u003eCP0\u003c/em\u003e, 0 CFU/g feed and \u003cem\u003eCP2\u003c/em\u003e, 2 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/g feed), SD\u003csub\u003eij\u003c/sub\u003e = the interaction between levels of Si and Dj, and e\u003csub\u003eijk\u003c/sub\u003e = random error. Data were analyzed using SPSS software (IBM Statistics 26.0, Armonk, NY, USA). Replicates were used as experimental units. Duncan\u0026rsquo;s new multiple range test was used to calculate the differences among means within the same factor and significance are based on P\u0026thinsp;\u0026le;\u0026thinsp;0.05. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean, with different superscript letters indicating significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Gut microbial analysis was performed using the R program version 3.4.1 (R Foundation for Statistical Computing, Vienna, Austria).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eGrowth performance decreases in response to exposure of chickens to HS and C. perfringens infection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection on growth performance, including weight (ABW, g), average daily weight gain (ADG, g), average daily feed intake (ADFI, g) and feed conversion ratio (FCR, g diet: g BW), are shown in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. HS significantly reduced BW30 and BW36 and significantly increased the FCR ratio from 30 to 36 d (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The statistical results showed that the final body weight of the male chickens (BW36) and daily weight gain from 20 to 36 d were significantly higher in the L\u003cem\u003eCP\u003c/em\u003e0 group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than in the HS groups (H\u003cem\u003eCP\u003c/em\u003e0 and H\u003cem\u003eCP\u003c/em\u003e2). No significant differences between the groups were observed in the ADFI from 20 to 36 d. However, the highest and lowest average feed conversions were recorded in the L\u003cem\u003eCP\u003c/em\u003e0 and H\u003cem\u003eCP\u003c/em\u003e0 groups, respectively (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of high temperature and \u003cem\u003eClostridium perfringens\u003c/em\u003e on the growth performance of Qingyuan Patridge chicken.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eBW (g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eBWG (g/bird/day)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFI (g/bird/day)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFCR\u003c/p\u003e\n \u003cp\u003e(g feed/g gain)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e20d\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e30d\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e36d\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e20\u0026ndash;30 d\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e30\u0026ndash;36 d\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e20\u0026ndash;36 d\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e20\u0026ndash;36 d\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e20\u0026ndash;36 d\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eAmbient Temperature (T)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL, 25\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e328.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e467\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.99\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH, 35\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e186\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e322.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e435\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.56\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eClostridium perfringens\u003c/em\u003e (\u003cem\u003eCP\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCP0\u003c/em\u003e, 0 dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e186\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e324\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCP2\u003c/em\u003e, 2\u0026times;10\u003csup\u003e8\u003c/sup\u003e cfu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e324\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e452\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eInteraction T x \u003cem\u003eCP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL\u003cem\u003eCP\u003c/em\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e328\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e469\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.60\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL\u003cem\u003eCP\u003c/em\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e329\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e465\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003cem\u003eCP\u003c/em\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e186\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e431\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003cem\u003eCP\u003c/em\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e325\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e439\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.77\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eTwo ways probabilities*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.127\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.472\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.621\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.531\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.674\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.621\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.623\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT \u0026times; \u003cem\u003eCP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.749\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.673\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.562\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.210\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eT\u0026thinsp;=\u0026thinsp;temperature; CP\u0026thinsp;=\u0026thinsp;\u003cem\u003eClostridium perfringens\u003c/em\u003e; SEM\u0026thinsp;=\u0026thinsp;standard error of means; BW\u0026thinsp;=\u0026thinsp;body weight; DWG\u0026thinsp;=\u0026thinsp;daily weight gain; DFI\u0026thinsp;=\u0026thinsp;daily feed intake; FCR\u0026thinsp;=\u0026thinsp;feed conversion ratio; ns\u0026thinsp;=\u0026thinsp;non-significance; \u003csup\u003e1\u003c/sup\u003e\u003cem\u003en\u003c/em\u003e = 6 per treatment group with 12 birds per replicate.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cem\u003eBlood biochemical variables in response to exposure of chickens to HS and C. perfringens infection\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eThe plasma concentrations of immune cytokines are shown in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The plasma concentration of cortisol was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the H\u003cem\u003eCP\u003c/em\u003e2 group than in the L\u003cem\u003eCP\u003c/em\u003e0 group, whereas the plasma concentration of IL-6 was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the L\u003cem\u003eCP\u003c/em\u003e0 group than in the H\u003cem\u003eCP\u003c/em\u003e2 group. The highest concentrations (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of IL-10 and TNF-\u0026alpha; were observed in the L\u003cem\u003eCP\u003c/em\u003e2 and H\u003cem\u003eCP\u003c/em\u003e0 groups, respectively. For plasma immunoglobulin variables, the statistical results showed that the lowest (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) plasma concentration of immunoglobulin (Ig) M was in the L\u003cem\u003eCP\u003c/em\u003e0 group. In contrast, no significant differences were observed in the plasma concentrations of IgA and IgG between the groups (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\" style=\"margin-right: calc(0%); width: 100%;\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of high temperature and \u003cem\u003eClostridium perfringens\u003c/em\u003e on the plasma immune parameters of Qingyuan Patridge chicken.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCortisol (\u0026micro;g/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIL- 1\u0026beta;\u003c/p\u003e\n \u003cp\u003e(ng/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIL-6\u003c/p\u003e\n \u003cp\u003e(ng/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIL-10\u003c/p\u003e\n \u003cp\u003e(ng/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIL-22\u003c/p\u003e\n \u003cp\u003e(ng/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTNF-\u0026alpha;\u003c/p\u003e\n \u003cp\u003e( ng/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIFN-\u0026gamma;\u003c/p\u003e\n \u003cp\u003e(pg/mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIgM\u003c/p\u003e\n \u003cp\u003e( ng/mL )\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIgA\u003c/p\u003e\n \u003cp\u003e( ng/mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIgG\u003c/p\u003e\n \u003cp\u003e(\u0026micro;g /mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"12\" style=\"width: 98.6305%;\"\u003e\n \u003cp\u003eAmbient Temperature (T)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\" style=\"width: 1.2717%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL, 25\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e231\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e110.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.60\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e270.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e122.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH, 35\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e118.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.44\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.72\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e286.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e116.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"13\"\u003e\n \u003cp\u003e\u003cem\u003eClostridium perfringens\u003c/em\u003e (\u003cem\u003eCP\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCP0\u003c/em\u003e, 0 dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e268.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e126.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCP2\u003c/em\u003e,2\u0026times;10\u003csup\u003e8\u003c/sup\u003e cfu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e246\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.79\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e288.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e117.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"13\"\u003e\n \u003cp\u003eInteraction T x \u003cem\u003eCP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL\u003cem\u003eCP\u003c/em\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e225\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.71\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.70\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e247.43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e129.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL\u003cem\u003eCP\u003c/em\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e237\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.94\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 8.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.66\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e292.91\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003cem\u003eCP\u003c/em\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e244\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.69\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.27\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 6.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e288.83\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e122.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003cem\u003eCP\u003c/em\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e256\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.65\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.62\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e284.57\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e109.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"13\"\u003e\n \u003cp\u003eTwo ways probabilities*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.420\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.502\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.395\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.614\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.709\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.942\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.559\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.823\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.092\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.438\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.089\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT \u0026times; \u003cem\u003eCP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.525\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.445\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.551\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.686\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.739\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"13\"\u003eT\u0026thinsp;=\u0026thinsp;temperature; CP\u0026thinsp;=\u0026thinsp;\u003cem\u003eClostridium perfringens\u003c/em\u003e; SEM\u0026thinsp;=\u0026thinsp;standard error of means; IL-1\u0026beta;\u0026thinsp;=\u0026thinsp;interleukin-1\u0026beta;; IL-6\u0026thinsp;=\u0026thinsp;interleukin-6; IL-10\u0026thinsp;=\u0026thinsp;interleukin-10; IL-22\u0026thinsp;=\u0026thinsp;interleukin-22; TNF-\u0026alpha;\u0026thinsp;=\u0026thinsp;tumour necrosis factor- \u0026alpha;; IFN- \u0026gamma;\u0026thinsp;=\u0026thinsp;interferon- \u0026gamma;; IgM\u0026thinsp;=\u0026thinsp;immunoglobulin M; IgA\u0026thinsp;=\u0026thinsp;immunoglobulin A; and IgG\u0026thinsp;=\u0026thinsp;immunoglobulin G. \u003csup\u003ea, b\u003c/sup\u003e Different letters in the same column indicate a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); \u003csup\u003e1\u003c/sup\u003e\u003cem\u003en\u003c/em\u003e = 6 per treatment group with 12 birds per replicate\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cem\u003eJejunum morphometry variables in response to exposure of chickens to HS and C. perfringens infection\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of HS and C. perfringens infection on jejunum morphometry variables, including villus height, crypt depth, and villus/crypt ratio, as affected by HS and infection with \u003cem\u003eC. perfringens\u003c/em\u003e are shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA. Villus height was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the H\u003cem\u003eCP\u003c/em\u003e2 group than in the other groups. Furthermore, jejunum SIgA concentration increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.10) in the L\u003cem\u003eCP\u003c/em\u003e2 group compared to that in the L\u003cem\u003eCP\u003c/em\u003e0 group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGenes and protein expression of jejunal lamina propria in response to exposure of chickens to HS and C. perfringens infection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe gene and protein expression results of the jejunal lamina propria, as affected by HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection, are shown in Tables \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB. The relative mRNA expression of \u003cem\u003eIL-6\u003c/em\u003e was significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) upregulated in the jejunal lamina propria tissue of the L\u003cem\u003eCP\u003c/em\u003e0 group compared to that in the other groups (Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). In addition, the relative mRNA expression of \u003cem\u003eIL-4, IL-10, NF-\u0026kappa;B\u003c/em\u003e, and \u003cem\u003eMuc-2\u003c/em\u003e was significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) upregulated in the jejunal lamina propria tissue of the L\u003cem\u003eCP\u003c/em\u003e2 group compared with that of the other groups (Tables \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). The highest mRNA expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of \u003cem\u003eIL-8, IFN-\u0026gamma;, NLRP-3, Zo-1\u003c/em\u003e, and \u003cem\u003eClaudin-1\u003c/em\u003e was detected in the jejunal lamina propria tissue of the H\u003cem\u003eCP\u003c/em\u003e0 group compared with that of the H\u003cem\u003eCP\u003c/em\u003e2 group (Tables \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). In contrast, the highest mRNA expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of \u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e was detected in the jejunal lamina propria tissue of the H\u003cem\u003eCP\u003c/em\u003e2 group compared with that of the other groups (Table \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Western blot results also revealed a higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) protein expression of \u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e in the jejunal lamina propria tissue of the H\u003cem\u003eCP\u003c/em\u003e0 group than in the other groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of high temperature and \u003cem\u003eClostridium perfringens\u003c/em\u003e on the mRNA expression of cytokine gene in the jejunum mucosa of Qingyuan Patridge chicken.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIL-1\u0026beta;\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIL-2\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIL-4\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIL-6\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIL-8\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIL-10\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIL-17\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"8\"\u003e\n \u003cp\u003eAmbient Temperature (T)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL, 25\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH, 35\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.62\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.72\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.75\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.87\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"8\"\u003e\n \u003cp\u003e\u003cem\u003eClostridium perfringens\u003c/em\u003e (\u003cem\u003eCP\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCP0\u003c/em\u003e, 0 dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCP2\u003c/em\u003e, 2\u0026times;10\u003csup\u003e8\u003c/sup\u003e cfu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"8\"\u003e\n \u003cp\u003eInteraction T x \u003cem\u003eCP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL\u003cem\u003eCP\u003c/em\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.40\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.14\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL\u003cem\u003eCP\u003c/em\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.85\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.80\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003cem\u003eCP\u003c/em\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.72\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.83\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.90\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.67\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003cem\u003eCP\u003c/em\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.52\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.57\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.15\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"8\"\u003e\n \u003cp\u003eTwo ways probabilities*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.675\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.513\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.824\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.653\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.073\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.181\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.398\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.758\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT \u0026times; \u003cem\u003eCP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.071\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.038\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.027\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.277\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eT\u0026thinsp;=\u0026thinsp;temperature; CP\u0026thinsp;=\u0026thinsp;\u003cem\u003eClostridium perfringens\u003c/em\u003e; SEM\u0026thinsp;=\u0026thinsp;standard error of means; IL-1\u0026beta;\u0026thinsp;=\u0026thinsp;interleukin-1\u0026beta;; IL-2\u0026thinsp;=\u0026thinsp;interleukin-2; IL-4\u0026thinsp;=\u0026thinsp;interleukin-4; IL-6\u0026thinsp;=\u0026thinsp;interleukin-6; IL-8\u0026thinsp;=\u0026thinsp;interleukin-8; IL-10\u0026thinsp;=\u0026thinsp;interleukin-10; IL-17\u0026thinsp;=\u0026thinsp;interleukin-17. \u003csup\u003ea, b, c\u003c/sup\u003e Different letters in the same column indicate significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); \u003csup\u003e1\u003c/sup\u003e\u003cem\u003en\u003c/em\u003e = 6 per treatment group with 12 birds per replicate.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of high temperature and \u003cem\u003eClostridium perfringens\u003c/em\u003e on the expression of immune-related genes in the jejunum mucosa of Qingyuan Patridge chicken.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTLR-4\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNF-\u0026kappa;B\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTGF-\u0026beta;\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIFN-\u0026gamma;\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNLRP-3\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMYLK\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eOcciudin-1\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMuc-2\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eZo-1\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eClaudin-1\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"12\"\u003e\n \u003cp\u003eAmbient Temperature (T)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL, 25\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.09b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH, 35\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.48a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.62\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"13\"\u003e\n \u003cp\u003e\u003cem\u003eClostridium perfringens\u003c/em\u003e (\u003cem\u003eCP\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCP0\u003c/em\u003e, 0 dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCP2\u003c/em\u003e, 2\u0026times;10\u003csup\u003e8\u003c/sup\u003e cfu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.95\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"13\"\u003e\n \u003cp\u003eInteraction T x \u003cem\u003eCP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL\u003cem\u003eCP\u003c/em\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.09\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.08\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL\u003cem\u003eCP\u003c/em\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.19\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003cem\u003eCP\u003c/em\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.83\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.27\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.82\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.20\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003cem\u003eCP\u003c/em\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.12\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.89\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.90\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.41\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.59\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"13\"\u003e\n \u003cp\u003eTwo ways probabilities*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.315\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.941\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.449\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.688\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.716\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.336\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.335\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.842\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.984\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.953\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.923\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.772\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT \u0026times; \u003cem\u003eCP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.545\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.628\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.047\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.734\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.706\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.043\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.225\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"13\"\u003eT\u0026thinsp;=\u0026thinsp;temperature; CP\u0026thinsp;=\u0026thinsp;\u003cem\u003eClostridium perfringens\u003c/em\u003e; SEM\u0026thinsp;=\u0026thinsp;standard error of means; TLR-4\u0026thinsp;=\u0026thinsp;toll-like receptor-4; TNF-\u0026alpha;\u0026thinsp;=\u0026thinsp;tumour necrosis factor- \u0026alpha;; NF-\u0026kappa;B\u0026thinsp;=\u0026thinsp;nuclear factor kappa B; TFG-\u0026beta;\u0026thinsp;=\u0026thinsp;transforming growth factor \u0026ndash;\u0026beta;; IFN- \u0026gamma;\u0026thinsp;=\u0026thinsp;interferon- \u0026gamma;; NLRP-3\u0026thinsp;=\u0026thinsp;non-like receptor protein-3; MUC-2\u0026thinsp;=\u0026thinsp;mucin-2; ZO\u0026thinsp;=\u0026thinsp;zonula occludens. \u003csup\u003ea, b, c\u003c/sup\u003e Different letters in the same column indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); \u003csup\u003e1\u003c/sup\u003e\u003cem\u003en\u003c/em\u003e = 6 per treatment group with 12 birds per replicate.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cem\u003eDynamic shifts in T cells in the blood and jejunal lamina propria in response to exposure of chickens to HS and C. perfringens infection\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eThe dynamic shifts in T cells in the blood induced by exposure to HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection are shown in Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. In the blood, the T lymphocyte subsets of peripheral blood, total CD3\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells, CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells, and CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e/CD3\u003csup\u003e+\u003c/sup\u003e CD8\u0026thinsp;+\u0026thinsp;ratio significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in the L\u003cem\u003eCP\u003c/em\u003e2 group compared to the other groups. However, in the chickens of the L\u003cem\u003eCP\u003c/em\u003e2 group, CD3\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells, and the CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e/CD3\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e ratio decreased compared to those of the H\u003cem\u003eCP\u003c/em\u003e0 group. Further analysis indicated that in the chickens of the H\u003cem\u003eCP\u003c/em\u003e2 group, the CD4\u003csup\u003e+\u003c/sup\u003e T-cell, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells, and CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e/CD3\u003csup\u003e+\u003c/sup\u003e CD8\u0026thinsp;+\u0026thinsp;ratios decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to those in the other groups (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, the T lymphocyte subsets of the jejunal lamina propria are shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The positive rates of jejunal CD3\u003csup\u003e+\u003c/sup\u003e, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells, CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells, and CD4\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells in the H\u003cem\u003eCP\u003c/em\u003e2 group were significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than those in the other groups. Jejunal CD4\u003csup\u003e+\u003c/sup\u003e T cells tended to increase (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.08) in the H\u003cem\u003eCP\u003c/em\u003e0 group, and the CD3\u003csup\u003e+\u003c/sup\u003e, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells, and CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e/CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e ratios in the H\u003cem\u003eCP\u003c/em\u003e0 group were significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than those in the other groups. Additionally, the number of positive CD4\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells in the H\u003cem\u003eCP\u003c/em\u003e2 group was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than that in the L\u003cem\u003eCP\u003c/em\u003e0 group.\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cem\u003eDynamic shifts of the jejunum cecal microbiota in response to exposure of chickens to HS and C. perfringens infection\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eThe effects of exposure of animals to HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection were not significant on alpha measures (Chao1, Shannon, Simpson, dominance, Pielou, and observed OTUs) among the chick groups, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA. Principal coordinate analysis (PCoA) revealed significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) among the bacterial community groups (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). The highest and lowest numbers of unique OTUs units were observed in the L\u003cem\u003eCP0\u003c/em\u003e and L\u003cem\u003eCP2\u003c/em\u003e groups, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). The most common OTUs were observed in the H\u003cem\u003eCP0\u003c/em\u003e and H\u003cem\u003eCP2\u003c/em\u003e groups (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). The taxonomic composition analysis at each classification level for the phyla and genera is presented in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD. At the phylum level, Firmicutes, Proteobacteria, Bacteroidota, Actinobacteriota, and Campilobacteria were predominant as top five phyla in the cecal microbiota. The highest average relative abundances of Firmicutes and Bacteroidota were observed in the L\u003cem\u003eCP2\u003c/em\u003e group, whereas the highest average relative abundances of Proteobacteria, Actinobacteriota, and Campilobacteria were observed in the H\u003cem\u003eCP0\u003c/em\u003e group. At the genus level, \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eEsherichia-Shigella\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, \u003cem\u003eBacteroides\u003c/em\u003e, and \u003cem\u003eCandidatous_Arthromitus\u003c/em\u003e were the predominant genera in the cecal microbiota. The highest relative abundances of \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eEnterococcus\u003c/em\u003e were observed in the L\u003cem\u003eCP0\u003c/em\u003e group, whereas the highest relative abundances of \u003cem\u003eEsherichia-Shigella, Staphylococcus, Clostridia\u003c/em\u003e, and \u003cem\u003eBacteroides\u003c/em\u003e were observed in the H\u003cem\u003eCP2\u003c/em\u003e group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). The results of LEfSe and LDA analyses of the cecum microbiota communities for L\u003cem\u003eCP0\u003c/em\u003e vs. L\u003cem\u003eCP2\u003c/em\u003e\u0026thinsp;+\u0026thinsp;H\u003cem\u003eCP0\u003c/em\u003e\u0026thinsp;+\u0026thinsp;H\u003cem\u003eCP2\u003c/em\u003e are presented in Fig. 6A and B. The LEfSe analysis results revealed that the relative abundance of \u003cem\u003eBacillaceae\u003c/em\u003e and \u003cem\u003eParabacteroides\u003c/em\u003e increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the L\u003cem\u003eCP0\u003c/em\u003e group compared to the other groups (L\u003cem\u003eCP2\u003c/em\u003e\u0026thinsp;+\u0026thinsp;H\u003cem\u003eCP0\u003c/em\u003e\u0026thinsp;+\u0026thinsp;H\u003cem\u003eCP2\u003c/em\u003e), whereas \u003cem\u003ePhascolarctobacterium, Megasphaera\u003c/em\u003e, and \u003cem\u003eFlavonifractor\u003c/em\u003e increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the L\u003cem\u003eCP2\u003c/em\u003e\u0026thinsp;+\u0026thinsp;H\u003cem\u003eCP0\u003c/em\u003e\u0026thinsp;+\u0026thinsp;H\u003cem\u003eCP2\u003c/em\u003e groups in comparison to the L\u003cem\u003eCP0\u003c/em\u003e group. The LDA analysis results showed that the relative abundances of \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eLigilactobacillus\u003c/em\u003e, and \u003cem\u003eSporichthyacea\u003c/em\u003e were the highest (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the L\u003cem\u003eCP0\u003c/em\u003e group, whereas \u003cem\u003eCyanobacteria\u003c/em\u003e, \u003cem\u003eCyanobacteriia\u003c/em\u003e, and \u003cem\u003eListeriaceae\u003c/em\u003e were significantly high (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the H\u003cem\u003eCP2\u003c/em\u003e group.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHS could be a potential predisposing factor for inducing NE and gastrointestinal infections by pathogens, especially \u003cem\u003eC. perfringens\u003c/em\u003e. Only one, HS or \u003cem\u003eC. perfringens\u003c/em\u003e, is sufficient for NE occurrence; however, the harmful effects of both are extremely dangerous to animal health and survival. These harmful effects include performance deficits (decreased FI, weight gain, and nutrient absorption), intestinal diseases (inflammation, necrosis, ischemia, and increased permeability), and physiological disorders (immune dysfunction, impaired antioxidant responses, and disturbances in respiratory and digestive functions). Therefore, in this study, we investigated the effects of HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection on growth performance, plasma immune profiles, intestinal cytokine and immune-related gene expression, gut integrity, T-cell dynamics in blood and the jejunal lamina propria, and structural changes in the cecal microbiota of male Qingyuan Patridge chickens. The acute form of NE disturbance associated with HS and \u003cem\u003eC. perfringens\u003c/em\u003e leads to increased mortality in broiler flocks, which is 1\u0026ndash;5% per day. In the subclinical form, damage to the intestinal mucosa caused by HS and \u003cem\u003eC. perfringens\u003c/em\u003e leads to decreased digestion and absorption, reduced GR, and increased FCR [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur results showed a significant decrease in the final body weight (BW36) and average body weight gain in the H\u003cem\u003eCP0\u003c/em\u003e and H\u003cem\u003eCP2\u003c/em\u003e groups compared to those in the L\u003cem\u003eCP0\u003c/em\u003e group, which was caused by the exposure of the birds to HS [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. HS is an environmental stressor that negatively affects the global poultry industry, reducing poultry production performance, favoring bacterial infection, inducing changes in the cellular and adaptive immune systems, and decreasing animal welfare [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. HS alleviates the immune system of birds and makes them vulnerable against infection disease suppress or dysregulate the immune system via various physiological mechanisms chronic or acute stress triggers the release of corticotropin-releasing hormone (CRH) from the hypothalamus, stimulating the adrenal glands to produce glucocorticoids. Elevated glucocorticoids suppress pro-inflammatory cytokine production, inhibit T-cell proliferation, and reduce natural killer (NK) cell activity, weakening the body's ability to combat infections and respond to inflammation, which may act as a predisposing factor for NE development in chickens. Thus, exposure to HS for 28 d in chicken broilers resulted in reduced body weight, decreased daily weight gain, and an increased feed-to-weight gain ratio. This negative effect of HS on growth performance is potentially attributable to reduced nutrient absorption due to intestinal damage, inflammation, and necrosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In addition, BW36 and BWG20\u0026ndash;36 d were decreased in the H\u003cem\u003eCP2\u003c/em\u003e group compared to the L\u003cem\u003eCP0\u003c/em\u003e group due to the harmful effects of exposure to HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection. Infecting chickens with 2\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL of \u003cem\u003eC. perfringens\u003c/em\u003e type A showed increased diarrhea, narcotic enteritis, and energy loss [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe present study showed a significant reduction in villus height and villus-to-crypt ratio in the H\u003cem\u003eCP\u003c/em\u003e2 group compared to those in the L\u003cem\u003eCP0\u003c/em\u003e group. HS has a negative effect on the lining of the small intestine that separate the gut from the external environment, resulting in a condition known as leaky gut, which is detrimental to bird wellbeing, growth, and health [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Exposure of broiler chickens to HS increased leaky gut by damaging tight junction proteins (TJs), allowing partially digested food, toxins, and microbes to pass into the bloodstream [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. TJs form a semipermeable barrier which aids in the passage of ions and solutes while preventing the passage of toxins and foreign bodies [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Defects in gastrointestinal physiology and function, such as intestinal permeability, gut barrier dysfunction, inflammatory bowel disease, celiac disease, and irritable bowel syndrome, are mainly associated with structural disorders of the gastrointestinal tract [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Similarly, functional gastrointestinal disorders, such as motility disturbance, visceral hypersensitivity, altered mucosal secretion and absorption, and dynamic changes in the gut microbiota, are identified by morphological and structural abnormalities [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur findings revealed that the plasma cortisol concentration was significantly higher in the H\u003cem\u003eCP2\u003c/em\u003e group than in the L\u003cem\u003eCP0\u003c/em\u003e group. Corticosterone is a glucocorticoid hormone secreted by the adrenal glands when the body experiences different stressors, and excessive cortisol levels notably affect the digestive system [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In addition, mucosal cells and adrenal glands release several inflammatory cortisol and cytokines, causing increased intestinal disturbance, especially in bacterial-mucosal interactions, leading to the aggravation of diseases, such as inflammatory and irritable bowel syndrome [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. On comparing the H\u003cem\u003eCP2\u003c/em\u003e and L\u003cem\u003eCP0\u003c/em\u003e groups, a significant decrease in the plasma concentration of IL-6 was observed. The results of the present study are consistent with those of a previous study which reported that \u003cem\u003eC. perfringens\u003c/em\u003e infection under HS conditions changed the expression of pro- and anti-inflammatory cytokines involved in IL-6 activation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In addition, the plasma concentration of IgM increased in the H\u003cem\u003eCP2\u003c/em\u003e group compared with the L\u003cem\u003eCP0\u003c/em\u003e group. IgM is a major antibody for immunity that increases in plasma during stressor conditions and protects the animal\u0026rsquo;s body against pathogenic infection by blocking detrimental pathogens, reducing the bacterial response in the organism [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The plasma concentration of IgM promote intestinal function, and animals exposed to HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection experience intestinal inflammation, increase pathogenic challenges, and induce the secretion of IgM for the immune response.\u003c/p\u003e\u003cp\u003eThe relative mRNA expression of \u003cem\u003eTNF-α\u003c/em\u003e, \u003cem\u003eNF-κB\u003c/em\u003e, and \u003cem\u003eZo-1\u003c/em\u003e was upregulated in the jejunal mucosa of the H\u003cem\u003eCP2\u003c/em\u003e group compared to the L\u003cem\u003eCP0\u003c/em\u003e group owing to the stimulation of the expression of immune-related genes caused by exposure of the chickens to HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection. Cytokine mRNA expression in the intestine is a small protein secreted by gut cells to regulate host-microbe interactions via cellular immune signaling, promoting innate and adaptive immune responses [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Ohtsu et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] found that HS induces spleen involution and affects the expression of splenic cytokines such as IL-12 and IFN-γ in broiler chickens. The expression of IFN-γ, IL-8, IL-17, and IL-18 was significantly increased during exposure of broiler chickens to HS [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Interleukins (ILs) are a group of cytokines that mainly secrete immune proteins and signaling molecules by leukocytes and gut mucosa. ILs (IL-8, IL-17, and IL-18) were observed to promote T-cell proliferation; however, interferons (IFN-γ and IFN-α) enhanced cell-mediated immunity, thereby increasing antibody-mediated immunity and antimicrobial activity [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. These results are consistent with previous studies showing that HS increases IL-8 expression in chickens and mammals but decreases IL-8 plasma levels in chickens [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The downregulation of TGF-β, including a concurrent upregulation in IL-1β, IL-13, and IL-17, indicates that \u003cem\u003eC. perfringens\u003c/em\u003e-induced subclinical NE triggers an inflammatory immune response in the intestine of broiler chicks [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe present study found that HS significantly decreased the positivity rate of CD4\u003csup\u003e+\u003c/sup\u003e T cells and the CD3\u003csup\u003e+\u003c/sup\u003e4\u003csup\u003e+\u003c/sup\u003e/CD3\u003csup\u003e+\u003c/sup\u003e8\u003csup\u003e+\u003c/sup\u003e ratio in the blood of Qingyuan chickens. Tissue-resident immune cells in the chicken intestine are critical in protecting against pathogens. During pathogenic infection, adaptive immune T cells, CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e are the main sources of ILs production, regulating immune cell and cytokine production [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The synthesis of a lower range of pro- and anti-inflammatory cytokines and their deregulation can trigger the pathogenesis of toxigenic \u003cem\u003eC. perfringens\u003c/em\u003e type A bacteria, destroying the signaling process of certain cytokines to lymphocytes and causing lymphoid tissue damage that causes abnormal T-cell responses, as described in growing pigs [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and broilers [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Similarly, our results revealed that the H\u003cem\u003eCP\u003c/em\u003e2 group had significantly increased T lymphocyte subsets in peripheral blood, total CD3\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cells, positive CD3\u003csup\u003e+\u003c/sup\u003e4\u003csup\u003e+\u003c/sup\u003e cells, double positive CD4\u003csup\u003e+\u003c/sup\u003e8\u003csup\u003e+\u003c/sup\u003e T cells, and the ratio of CD3\u003csup\u003e+\u003c/sup\u003e4\u003csup\u003e+\u003c/sup\u003e /CD3\u003csup\u003e+\u003c/sup\u003e 8\u003csup\u003e+\u003c/sup\u003e in the chicken blood. These results indicate that infection with \u003cem\u003eC. perfringens\u003c/em\u003e in the chickens rapidly expanded the T cells that activated the chicken adaptive immunity system. The findings support by the previous study on broilers for a 15 d trial, which reported that T-cell responses are enhanced after \u003cem\u003eC. perfringens\u003c/em\u003e infection and secreted protein-stimulated cecal tonsil cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the present study, significant changes were observed in the structure of cecal microbiota induced by exposure of Qingyuan chickens to HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection, with a decrease in the dominant \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eRikenellaceae_RC9\u003c/em\u003e and an increase in \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003eEscherichia-Shigella\u003c/em\u003e. A healthy microbial community colonized the gut of the L\u003cem\u003eCP\u003c/em\u003e0 group, which improved intestinal function and antimicrobial pathogenicity compared with the H\u003cem\u003eCP\u003c/em\u003e2 group. The relative abundance of \u003cem\u003eLactobacillus, Parabacteroides\u003c/em\u003e, and \u003cem\u003eEnterococcus\u003c/em\u003e increased in the L\u003cem\u003eCP\u003c/em\u003e0 group; these genera are often used as beneficial probiotic products to ameliorate HS. The relative abundance of pathogenic bacteria, including Clostridia, \u003cem\u003eEsherichia-Shigella, Staphylococcus\u003c/em\u003e, and \u003cem\u003eBacteroides\u003c/em\u003e, increased in the H\u003cem\u003eCP0\u003c/em\u003e group. Indeed, HS can disrupt the balance of beneficial gut bacteria, promoting the growth of pathogenic microbes. Dysbiosis can impair the fermentation of complex carbohydrates and the production of short-chain fatty acids (SCFAs), which are essential for gut health and energy supply to intestinal cells. These pathogenic genera increase chronically during HS and may cause the bile acid pool to promote intestinal inflammatory bowel diseases [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In addition, these pathogenic bacteria promote the harmful effects of LPS, which substantially affects animal health, primarily through interactions with the immune system and organ function failure, leading to abdominal congestion, intestinal sepsis, abnormal autoimmunity, and cellular senescence [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHS disrupts the intestinal microbiota balance and promotes the intestinal inflammatory response and colonization of pathogens [\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. \u003cem\u003eLactobacillus\u003c/em\u003e as probiotic strains can improve intestinal integrity, morphology, and microbial environment to enhance broiler growth performance under HS [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In contrast, at the genus level, the beneficial bacteria Rikenellaceae_RC9_gut_group was significantly reduced, and pathogenic bacteria \u003cem\u003eEscherichia-Shigella\u003c/em\u003e and Coprococcus were increased. The results for microbial organisms are consistent with previous results [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. \u003cem\u003eRikenellaceae_RC9\u003c/em\u003e group beneficial bacteria have a potential protective function against metabolic and gastrointestinal diseases and are potential biomarkers of healthy animal guts [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In addition, the general levels of Ralstonia, Halomonas, and Roseomonas were lower, and those of Gca-900066575 and Flavonifractor were higher in the L\u003cem\u003eCP\u003c/em\u003e2 group in compared to the L\u003cem\u003eCP\u003c/em\u003e0 group. At the genus level, Parabacteroides and Roseomonas were reduced, whereas Ralstonia, Eubacterium hallii, and Flavonifactor were increased in the H\u003cem\u003eCP\u003c/em\u003e2 group in the jejunum mucosa. [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] demonstrated that the abundance of Parabacteroides and Roseomonas decreased and that of Flavonifactor increased in the small intestine of broilers under HS conditions with pathogenic infection.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our findings demonstrated that HS may accelerate the pathogenesis of \u003cem\u003eC. perfringens\u003c/em\u003e infection in chickens. The common co-occurrence of Qingyuan Patridge chickens exposed to HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection will cause more harmful effects on growth performance, jejunum morphometry, immune-modulatory responses of T cells, expression of cytokines and immune-related genes, and gut microbiota dynamics, and exacerbates NE-related disturbances compared to HS or \u003cem\u003eC. perfringens\u003c/em\u003e infection treatment. The common co-occurrence of HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection could be used to induce NE model.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHeat stress\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNF-α\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTumor necrosis factor-α\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eNF-κB\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNuclear factor kappa B\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eZo-1\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eZonula occludens-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNecrotic enteritis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLipopolysaccharide bacteria\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLipid peroxidation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGIT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGastrointestinal tract\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhosphate buffered saline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRIPA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRadioimmunoprecipitation assay\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNitrocellulose\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOTUs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOperational taxonomic unit\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePrincipal component analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCoA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePrincipal coordinate analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFLASH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFast length adjustment of short reads, v1.2.11\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRDP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRibosomal database project\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLEfSe\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLinear discriminant analysis Effect size\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the staff of the Animal Research of Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Huazhong Agricultural University, and Zhongkai University of Agriculture and Engineering for their assistance in animal husbandry and sample collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL. Jingdu, W. Chen, and C.T. Zheng designed the project. \u0026nbsp;C.T. Zheng and W. Chen supervised the project. L. Jingdu, Z. Qi, Y. Lv, S. Wang, W. Xia, Y. Zhang, C. Jin, S. Wang X. Huang, K. Li, C.T Zheng and W. Chen collected the samples and performed the laboratory work. L. Jingdu, A.A. Elokil, M. T. Sarker, K. Abuelezz, and W. Chen carried out the analyses and interpreted the data. \u0026nbsp;A.A. Elokil, M. T. Sarker, K. Abuelezz, and w. Chen drafted the paper. A.A. Elokil, K. Abuelezz, C.T. Zheng and W. Chen edited the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key R\u0026amp;D Program of China (Grant No.2018YFE0128200), State Key Laboratory of Swine and Poultry Breeding Industry (GDNKY-ZQQZ-K3, GDNKY-ZQQZ-K22), the Project of Science and Technology innovation strategy (ZX202401-06), the Foreign Expert Project (Grant No. H20240494). Talented Young Scientists Program TYSP (No. P19U42006), Ministry of Science and Technology (MOST), China, the Opening Project of Guangdong Provincial Key Laboratory of Animal Breeding and Nutrition (2022SZ01). The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bacterial sequences supporting the findings of this study are available at the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1285695) under the BioProject ID PRJNA1285695.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Institutional Animal Care and Use Committee of Guangdong Academy of Agricultural Sciences (ACUCGAAC 1.2019) approved all the animal procedures, and all methods were performed in accordance with the relevant guidelines and regulations, following ARRIVE guidelines to minimize the animal suffering during the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXu W, Wang H, Liu L, Miao Z, Huo Y, Zhong Z. 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Poult Sci. 2018;97(11):4073\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Chen H, Cong W, Zhang K, Jia Y, Wu L. Chronic Heat Stress Affects Bile Acid Profile and Gut Microbiota in Broilers. Int J Mol Sci 2023, 24(12).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDaniel N, L\u0026eacute;cuyer E, Chassaing B. Host/microbiota interactions in health and diseases-Time for mucosal microbiology! Mucosal Immun. 2021;14(5):1006\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong B, Li H, Wu Y, Zhen W, Wang Z, Xia Z, Guo Y. Effect of microencapsulated sodium butyrate dietary supplementation on growth performance and intestinal barrier function of broiler chickens infected with necrotic enteritis. Anim Feed Sci Technol. 2017;232:6\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFarag MR, Alagawany M. Physiological alterations of poultry to the high environmental temperature. 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Elevated gut microbiome abundance of Christensenellaceae, Porphyromonadaceae and Rikenellaceae is associated with reduced visceral adipose tissue and healthier metabolic profile in Italian elderly. Gut Microbes. 2021;13(1):1880221.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang X, Feng J, Zhang M, Li X, Ma D, Chang S. Effects of high ambient temperature on the community structure and composition of ileal microbiome of broilers. Poult Sci. 2018;97(6):2153\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Heat-stressed chickens, Clostridium perfringens infection, Necrotic enteritis, Intestinal health, Host microbiota composition, Metagenomics","lastPublishedDoi":"10.21203/rs.3.rs-6855798/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6855798/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground The present study investigated the effects of heat stress (HS) and Clostridium perfringens (C. perfringens) infection on the performance, jejunal morphometry, immunomodulatory responses of T cells, expression of cytokines and immune-related genes, and gut microbiota dynamics of male Qingyuan Patridge chickens. Healthy 20-day-old of male Qingyuan chicks (192) were randomly assigned to four groups of 12 replicates (n\u0026thinsp;=\u0026thinsp;4 chicks/replicate). A 2 \u0026times; 2 factorial arrangement experiment was performed, including two levels of oral C. perfringens dosages, without (CP0) and with 2\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL (CP2) under two ambient temperatures of 25\u0026deg;C (L) and 35\u0026deg;C (H) for 30 d.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e The results showed that exposure of chicks to HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection in the H\u003cem\u003eCP\u003c/em\u003e2 (chicks reared under H ambient temperature with an oral dosage of \u003cem\u003eCP\u003c/em\u003e) group decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) feed intake, body weight, daily weight gain, feed conversion ratio, villus height, and villus-to-crypt depth ratio in the jejunum compared to the LCP0 group. The plasma concentrations of interleukin (IL)-6 and IL-10 were lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the H\u003cem\u003eCP\u003c/em\u003e2 group than in the L\u003cem\u003eCP\u003c/em\u003e0 group (chicks reared under L ambient temperature without an oral dosage of CP). The T lymphocyte subsets of peripheral blood CD4\u003csup\u003e+\u003c/sup\u003eT cells, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells, and the CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e/CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e ratio decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the H\u003cem\u003eCP\u003c/em\u003e2 group compared with the other groups. Intestinal mRNA expression of \u003cem\u003eIL-4\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eIL-10\u003c/em\u003e, IFN-γ was downregulated; however, IL-8, tumor necrosis factor-α (\u003cb\u003eTNF-α\u003c/b\u003e), nuclear factor kappa B (\u003cb\u003eNF-κB\u003c/b\u003e), zonula occludens-1 (\u003cb\u003eZo-1\u003c/b\u003e), and Claudin-1 were upregulated in the H\u003cem\u003eCP\u003c/em\u003e2 group compared to the L\u003cem\u003eCP0\u003c/em\u003e group. The gut microbiota in the H\u003cem\u003eCP\u003c/em\u003e2 group had a higher relative abundance of \u003cem\u003eEsherichia-Shigella\u003c/em\u003e, \u003cem\u003eStaphylococcus, Clostridia, Cyanobacteria\u003c/em\u003e, and \u003cem\u003eListeriaceae\u003c/em\u003e compared to the other groups.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e These results demonstrate the potentially harmful effects of exposure to HS and \u003cem\u003eC. perfringens\u003c/em\u003e infection on necrotic enteritis-related disturbances, growth performance, jejunum morphometry, immune-modulatory responses of T cells, expression of cytokines and immune-related genes, and gut microbiota dynamics in Qingyuan Patridge chickens exposed to HS.\u003c/p\u003e","manuscriptTitle":"Changes in intestinal immunity and gut microbiota associated necrotic enteritis induced by Clostridium perfringens infection and exposure to heat stress in chicken","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 12:39:15","doi":"10.21203/rs.3.rs-6855798/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1fa67075-d3c0-40d6-a285-70d2b9f8d40c","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-20T11:08:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-12 12:39:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6855798","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6855798","identity":"rs-6855798","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}