Changes in Oxidation-antioxidation Function on the Thymus of Chickens Infected with Reticuloendotheliosis Virus

Changes in Oxidation-antioxidation Function on the Thymus of Chickens Infected with Reticuloendotheliosis Virus | 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 Oxidation-antioxidation Function on the Thymus of Chickens Infected with Reticuloendotheliosis Virus Dahan Yang, Chenhui Zhao, Meixi Zhang, Shujun Zhang, Jie Zhai, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-30062/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Dec, 2020 Read the published version in BMC Veterinary Research → Version 1 posted 3 You are reading this latest preprint version Abstract Background Reticuloendotheliosis virus (REV) is a retrovirus that causes severe immunosuppression in infected poultry. Under oxidative stress,the animals grow slowly. In addition, long-term oxidative stress can impair immune function, as well as accelerate the aging and death of the animal.This study aimed to elucidate the pathogenesis of REV from the perspective of changes in oxidative-antioxidative function following REV infection. Results In specific pathogen free chickens infected with REV, the levels of H 2 O 2 and MDA in the thymus increased, the levels of T-AOC, SOD, CAT, and GPx1 decreased, and there was a reduction in CAT and Gpx1 mRNA expression, compared with the control group. The thymus index was also significantly reduced. Morphological analysis showed that after REV infection, there was an increase in thymic reticular endothelial cells, inflammatory cell infiltration, mitochondrial swelling, and destruction of the nuclear structure. Conclusions These results indicate that an increase in oxidative substances within the thymus, enhanced lipid peroxidation, a markedly decrease in antioxidant function, atrophy of the thymus, and immunosuppression in REV-infected chickens. Large Animal Medicine Small Animal Medicine reticuloendotheliosis virus thymus oxidation antioxidant imbalance histopathological and ultrastructural changes immunosuppression Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Reticuloendotheliosis (RE) constitutes a group of pathological syndromes caused by reticuloendotheliosis hyperplasia virus, including runting syndrome, chronic tumors of lymphoid and other tissues, acute reticulocytoma, and severe immunosuppression. Reticuloendotheliosis virus (REV) is a C-type avian retrovirus[ 1 , 2 ]. The REV group includes defective REV-T, non-defective REV-A, chick syncytial virus, duck infectious anemia virus, and spleen necrosis virus (SNV) [ 3 ]. In addition, REV infects a wide-range of hosts, including chickens, turkey, duck, goose, Japanese quail, and wild birds, among which, turkey is the most susceptible. Thus, turkeys and chickens are commonly used as experimental animals [ 4 , 5 , 6 , 7 ]. REV can be mixed with Marek’s disease virus (MDV), avian leukosis virus of subgroup J (ALV-J), and chicken anemia virus (CAV), resulting in reducing or loss of immune function in REV infection chickens. Thus, RE represents a serious threat to the development of the poultry industry[ 2 , 8 ]. Under normal physiological conditions, the body's oxidation-antioxidant system is in a dynamic equilibrium, and oxidative stress occurs when the antioxidant system is overwhelmed by the oxidation system[ 9 , 10 ]. Oxidative stress is an initial reaction of the body to external stimuli and may subsequently induce various signaling pathways and inflammation in the body[ 11 ]. Under oxidative stress, animals grow slowly, the feed conversion rate decreases, and there is a decrease in production performance[ 12 ]. In addition, long-term oxidative stress can deplete antioxidant vitamins and trace elements, impair immune function, as well as accelerate the aging and death of the animal, resulting in substantial economic losses to the poultry industry[ 13 ]. The oxidation system primarily includes reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS are by-products of the body’s metabolism that contain an unpaired electron. Moreover, ROS and RNS represent an important component of the pathogen resistance mechanism and may act as intracellular and intercellular signaling molecules; however, excessively high levels may also lead to oxidative stress[ 14 ]. ROS mainly include superoxide anions, hydroxyl radicals, other oxygen radicals, and H 2 O 2 . RNS includes NO, NO 2 , and peroxynitrite[ 15 ]. Antioxidant systems include enzymes and non-enzymatic antioxidants[ 4 ]. Enzyme antioxidants include SOD, peroxidase, CAT, GPx, and glutathione reductase, whereas non-enzymatic antioxidants include fat-soluble vitamin E, carotenoids, ubiquinone, and water-soluble vitamin C, glutathione, uric acid, and tryptophan metabolites. When the oxidation-antioxidant system is imbalanced, excessive ROS and RNS accumulate and destroy various biomacromolecules (e.g., lipids, proteins, and nucleic acids), which leads to cellular damage and ultimately cell death. Several studies have shown that oxidative stress plays an important role in inflammatory reactions, tumors, and many other diseases[ 16 , 17 , 18 ]. Currently, there are few existing studies that have investigated the changes in the oxidation-antioxidant system of poultry after a REV infection. This study examined the dynamic changes in the level of T-AOC, SOD activity, H 2 O 2 , MDA, CAT, and GPx1, as well as CAT and GPx1 mRNA expression in the thymus of one-day-old SPF chickens after a REV infection. Pathological sections and ultrastructural changes of the thymus were observed to further clarify the pathogenesis of REV infection and morphological changes resulting from oxidative stress and pathological processes, to facilitate the prevention and treatment of RE. Results Determination of oxidative stress biomarkers We first sought to study the oxidative-antioxidant capacity of the thymus in chickens infected with REV. We used commercial kits to detect the level of T-AOC, SOD, H 2 O 2 activity, and MDA of the chickens in both the infected and control groups. After the chickens were infected with REV, the thymus T-AOC (Fig. A) of the chickens was lower than that of the control chickens. Significant differences were found at 21 ( P༜0.05 ), 28 ( P༜0.01 ), and 35 ( P༜0.01 ) days post-REV infection. Compared with the control group, the SOD activity (Fig. B) of the thymus of the chickens decreased to varying degrees after REV infection, and exhibited significant differences on days 14 ( P༜0.01 ), 21 ( P༜0.05 ), and 28 ( P༜0.05 ) post REV-infection. Moreover, the level of MDA (Fig. C) in the thymus of REV-infected chickens increased from day 7 to 35 compared with the control group, and exhibited significant changes on day 28 ( P༜0.05 ). Compared with the control group, the H 2 O 2 content (Fig. D) in the thymus of the chickens increased between 14–35 days after REV infection, and showed significant changes on day 35 ( P༜0.05 ). Compared with the control group, the level of CAT (Fig. E) in the thymus showed a downward trend from day 14 to 35 in REV-infected SPF chickens, and showed significant changes on days 14 ( P < 0.01 ), 21 ( P < 0.01 ), and 28 ( P < 0.05 ). The (Fig. F) level of GPx1 in the thymus was lower than that of the control SPF chickens after REV infection, and showed significant changes on day 28 ( P < 0.05 ) post-infection. CAT And GPx1 mRNA Expression In The Thymus In REV-infected SPF chickens, the level of CAT (Fig. 2 a) expression in the thymus decreased from days 14 to 35 compared to the control group, and was significantly decreased on days 21 ( P < 0.05 ) and 28 ( P < 0.05 ). From day 7 to 35 post-REV infection, the level of GPx1 expression (Fig. 2 b) in the thymus of SPF chickens in the infected group was lower than that in the control chickens; significant changes were observed on days 28 ( P < 0.05 ) and 35 ( P < 0.05 ). Determination Of The Thymus Organ Index In SPF chickens infected with REV, the thymus organ index (Fig. 3 ) was lower than that of the control chickens, and was significantly lower than the control chickens on days 28 ( P < 0.05 ) and 35 ( P < 0.05 ). Histopathological Changes In The Thymus Of REV-infected SPF Chickens The histomorphology and ultrastructural changes of the thymus are presented in Fig. 4 . Compared with the control group, the thymic lobule was atrophied, the cortex and medulla exhibited hemorrhaging, and there was narrowing of the cortical area. In addition, there was a reduced number of lymphocytes in the medulla, an increased number of reticuloendothelial cells, and enhanced inflammatory cell infiltration. Under an electron microscope, the ultrastructure of the control group appeared normal. However, in the infected group, there was obvious cell damage, which primarily manifested as mitochondrial swelling, sputum rupture, Golgi vesicle expansion, and decreased cytoplasm. In addition, the nuclear membrane displayed breaks and had dissolved, and the nucleolus was enlarged. Discussion After the body is infected with a virus, ROS activates the immune system to eliminate the infection and provides a positive regulatory role[ 19 , 20 ]. Simultaneously, the virus breaks the oxidation-antioxidant balance of the host cells, causing the accumulation of a large amount of ROS, which results in cellular damage and apoptosis[ 21 ]. RE is an immunosuppressive tumor disease caused by REV. This test detects changes in the oxidative-antioxidative function of the thymus in chickens after REV infection. It was found that REV can cause oxidative stress in the thymus and reduce antioxidant function. Under conditions of oxidative stress, ROS and RNS promote the accumulation of lipid peroxidation in polyunsaturated fatty acids in oxidized biofilms, and produce lipid peroxides (e.g., MDA, ketone, hydroxyl, and carboxyl)[ 22 ]. Moreover, MDA can reflect the degree of lipid peroxidation in the body and can be used as an indicator to determine whether a cell is oxidatively damaged[ 23 ]. H 2 O 2 is an important ROS that can be scavenged by catalase or peroxidase, and can also react with free Fe 2+ to produce ·OH to initiate lipid peroxidation, destroy the cell membrane structure, and accelerate protein decomposition[ 24 ]. These findings reveal that the MDA and H 2 O 2 content in the thymus of REV-infected SPF chickens was higher than that of the control chickens. The level of H 2 O 2 in the thymus on day 35 in REV-infected chickens was significantly higher than that of the control chickens. Moreover, the MDA content in the thymus was significantly higher on day 28 in REV-infected chickens than that of the control chickens. After REV invades the immune system of the chicken host, both non-specific immunity and specific immunity play a role in the generation of a large amount of free radicals, oxidative stress within the body, and enhanced lipid peroxidation[ 25 ]. After REV enters a host, it induces NF-κB inhibitory protein I-κB phosphorylation, NF-κB activation, NF-κB translocation into the nucleus to bind to a specific nucleotide sequence of the iNOS gene promoter region, and the initiation of iNOS expression[ 26 ]. Studies have found that in the context of sepsis, inflammatory factors can mediate a decrease in nNOS expression in cardiomyocytes, up-regulate iNOS expression, and induce NO synthesis[ 27 , 28 ]. Moreover, iNOS expression can stimulate macrophages to produce NO, which in turn stimulates the cells to produce more ROS, triggering oxidative stress. Under normal conditions, SOD can catalyze the disproportionation of O 2 ____˙ in the body to produce both H 2 O and H 2 O 2 [14]. H 2 O 2 can break the structure and function of the membrane through the cell membrane, react with purines and pyrimidines, and result in nucleic acid damage[29]. CAT and GPx1 work together to clear H 2 O 2 from the cell[11]. This study found that REV-infected SPF chickens exhibited lower levels of T-AOC, SOD activity, CAT, GPx1 content, as well as CAT and GPx1 mRNA expression, compared to the control group. We consider one potential reason for this phenomenon is that MDA can disrupt the synthetic SOD pathway, thereby reducing SOD activity. Moreover, the MDA content is negatively correlated with SOD activity. The peroxide deposition causes a large amount of antioxidant enzymes to be consumed in the body, and the function of the antioxidant system is suppressed. Decreased antioxidant enzyme activity leads to an accumulation of oxidizing substances, which can cause inflammatory reactions, cellular damage, and apoptosis. In addition, ROS can activate NF-κB, which can induce the expression of various inflammatory factors (e.g., IL-1, IL-6, and TNF-α) and aggravate the inflammatory response [30,31,32]. Chickens infected with REV exhibit immunosuppression. Combined with the results of this study, the immunosuppression caused by REV may be related to an accumulation of oxidizing substances and a decline in antioxidant system functionality. Conclusion Following REV infection in one day old SPF chickens, the content of MDA and H 2 O 2 and superoxide in the thymus was increased, the T-AOC and SOD activity decreased, and the thymus organ index was decreased. These changes exceeded the body's ability to scavenge antioxidant substances, resulting in oxidative stress in the body. Materials And Methods Experimental animals and infectious virus strain All of the chickens used for experiments were one-day-old SPF chickens obtained from Harbin Veterinary Research Institute (HVRI), Chinese Academy of Agricultural Sciences (CAAS), China. The REV-T strain (CVCC No. CACCAV107) was purchased from China Veterinary Culture Collection Center (CVCC). The virus TCID 50 was 10 4.62 /0.1 mL as determined by cell breeding. Experimental Design And Sample Collection A total of 80 one-day-old SPF chickens were randomly divided into a control group (Group C) and a REV-infected group(Group I). Group I were intraperitoneally administered with 100 µL of viral suspension at one-day-old. Group C were intraperitoneally administered with 100 µL of sterile physiological saline. The two groups of chickens were housed in isolators with similar environments,and chickens were given free access to feed and water. During housing, animals were monitored twice daily for health status. No adverse events were observed. On days 3, 7, 14, 21, 28, 35, and 49 post-REV infection, five chickens were randomly selected from each group and euthanasia was performed by sedation using Rompun/Ketamine mixture as an intramuscular injection followed by an intravenous injection of Pentobarbitone. Thymus were dissected and homogenized in chilled sterile physiological saline using a glass homogenizer with a teflon pestle under cold condition. The homogenate was centrifuged at 4000 g for 10 min at 4 °C. The supernatant was used for the assay of oxidative stress biomarkers. Determination Of Thymic Oxidative Stress Biomarkers The prepared 10% tissue homogenate was removed from − 80 °C. The T-AOC, SOD, H 2 O 2 activity, and MDA level in the thymus was determined using commercially available detection kits (Nanjing Jiancheng Bioengineering Research Institute, China). The level of CAT and GPx1 was measured by an ELISA, according to the manufacturer's instructions (Qiyi Biological Technology Co., Ltd. China). Total RNA Extraction And Detection The total RNA of thymus was extracted using Trizol reagent(Invitrogen ,Shanghai, China) The quality of RNA was assessed using a NanoDrop2 000 spectrophotometer (Thermo Fisher Scientific) through the ratio of absorbance at 260 nm and 280 nm. cDNA was obtained by reverse transcription of the RNA using the M-MLV Reverse Transcriptase kit (Invitrogen) according to the manufacturer's instructions. Real-time PCR Determination of CAT and GPx1 mRNA in the thymus by real time PCR. The CAT, Gpx1, and β-actin primers were designed using Primer5.0 software. CAT: forward primer 5'- ATGTCCGTTTCAGGAGATGTGCAGC-3', reverse primer 5'- CCAGCAGTGCCTGAATACG − 3'; GPx1: forward primer 5'- ATGACCAACCCGCAGTACATCATCT-3', reverse primer 5'- GCAGTTTGATGGTCTCGAAGTGGC-3'; β-actin forward primer 5'- CGGGACGGATGAGAAGAA − 3', reverse primer 5'- TCGGCGCTCCAGATGTAC − 3'. Amplification of the target gene using the LightCycler2.0 real-time PCR System (Roche480, USA). The real-time PCR reaction procedure was 95 °C for 2 min, 45 cycles of 95℃ for 20 s, 59℃ for 20 s, and 72℃ for 10 s. To ensure the repeatability of the amplified samples, all samples were analyzed in triplicate. β-actin was used as a reliable normalization gene, and the expression of the samples were evaluated in relation to this housekeeping gene. The level of CAT and GPx1 mRNA expression were analyzed in accordance with the 2 −ΔΔCt method. Determination Of The Thymus Index Weight of the chickens and thymus at the time of sampling for calculating the thymus index. The thymus index was calculated by the weight of the thymus (g)/chicken weight(kg). Pathological Changes Of The Thymus After A REV Infection Thymus tissues were fixed in 4% formalin. The samples were dehydrated with ethanol and embedded in paraffin. Each sample was cut into 5-µm sections with a microtome and placed onto a glass slide, and stained with hematoxylin and eosin (H&E). The pathological changes of the thymus were observed under a both a light microscope (Nikon, H600, Japan) and an electron microscope (Hitachi 7650, Tokyo, Japan). Statistical Analyses The data were analyzed with SPSS 17.0 software, and the differences were compared between the groups were analyzed using one-way ANOVA followed by Tukey’s post-hoc test. All data were expressed as the mean ± SD. P < 0.05 was considered to be statistically significant. Abbreviations REV: Reticuloendotheliosis virus; SPF: Specific pathogen free; MDA: malondialdehyde; T-AOC: Total-antioxidante capacity; SOD: superoxide dismutase; ELISA: Enzyme linked immunosorbent assay; CAT: catalase; GPx1: Glutathione peroxidase 1; ROS: Reactive oxygen species; RNS: Reactive nitrogen species Declarations Ethics approval and consent to participate According to the Australian National Health and Medical Research Council's Australian Nursing and Use Regulations, the Animal Ethics Committee of the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences approved all experimental procedures with animals (SYXK [Hei] 2011022) for scientific purposes. All procedures were performed in accordance with the institutional guidelines. Authors’ contributions Z.SM designed the experiments and revised manuscript. Z.MX performed the experiments of REV infection. Z.CH and Z.SJ performed the experiments of Morphological analysis. Y.DH analyzed the data and drafted manuscript. G.XL and Z.J oversaw the experiments. L.XP and L.CN aided with analysis of results. All authors have read and approved the manuscript and ensure that this is the case. Consent for publication Not applicable Availability of data and materials All data generated or analysed during this study are included in this published article Competing interests The authors declare that they have no competing interests Funding This work was supported by the National Natural Science Foundation of China (31472169). The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Acknowledgments Not Applicable. Author details 1 College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, P.R. China. 2 Heilongjiang Key Laboratory of Laboratory Animals and Comparative Medicine Harbin 150030, P.R. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-30062","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":681897,"identity":"d5235e78-d803-4380-9159-26df7ecb2438","order_by":0,"name":"Dahan Yang","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dahan","middleName":"","lastName":"Yang","suffix":""},{"id":681898,"identity":"4d6289d2-2d12-4960-9b49-4c82a61a5321","order_by":1,"name":"Chenhui Zhao","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chenhui","middleName":"","lastName":"Zhao","suffix":""},{"id":681899,"identity":"df6e020f-5ae2-4b5d-bfc3-0081384cc80b","order_by":2,"name":"Meixi Zhang","email":"","orcid":"","institution":"WuXi AppTec","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meixi","middleName":"","lastName":"Zhang","suffix":""},{"id":681900,"identity":"27981742-6bfa-4b53-aecb-c574543dd651","order_by":3,"name":"Shujun Zhang","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shujun","middleName":"","lastName":"Zhang","suffix":""},{"id":681901,"identity":"5b7e2141-42c9-4ea1-91dd-6ef030c0a7ab","order_by":4,"name":"Jie Zhai","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhai","suffix":""},{"id":681902,"identity":"686269ad-7d9a-46aa-a2df-1219c1ab713c","order_by":5,"name":"XueLi Gao","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"XueLi","middleName":"","lastName":"Gao","suffix":""},{"id":681903,"identity":"b53e1a25-422a-4529-9fa4-8f85435d145d","order_by":6,"name":"Chaonan Liu","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chaonan","middleName":"","lastName":"Liu","suffix":""},{"id":681904,"identity":"a60911be-5962-4f94-89b7-34709a4b27d3","order_by":7,"name":"Xiaoping Lv","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoping","middleName":"","lastName":"Lv","suffix":""},{"id":681905,"identity":"6230b8f0-9b23-4969-9e15-e7764832b69f","order_by":8,"name":"Shimin Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYDACCTB5gIeBmYGN4UEFA4MBaVoSzpCgBUSwMSS2EaFFfnbzsYdf/tyR0W3nPfYgcV6dnLlEAuOHjzm4tTDOOZZuLNv2jMfsMF+6QeK2w8aWMxKYJWduw62FWSLHTFqy4TBQC4+ZROK2A4kbbiSwMfPi0cImkf9NWuIPTMucOsJaeCRy2CQ/sMG0NDAT1iIhkWYmzdgG1mJukHDssLHBmYfNeP0iPyP5meSPP4ftzc6fMXvwoaZOzuB48sEPH/FoAQcBDyqfsQG/epCSHwSVjIJRMApGwYgGAMRNUMGnYvp7AAAAAElFTkSuQmCC","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shimin","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2020-05-19 05:37:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-30062/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-30062/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-020-02708-6","type":"published","date":"2020-12-11T15:01:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":1359934,"identity":"af05bc96-76a1-4005-89ca-101ae59b2592","added_by":"auto","created_at":"2020-06-18 16:22:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":73802,"visible":true,"origin":"","legend":"Changes in the oxidative stress biomarkers in chickens infected with REV. (A) The total antioxidant capacity (T-AOC) of the thymus. (B) The superoxide dismutase (SOD) activity in the thymus. (C) The level of malondialdehyde (MDA) in the thymus. (D) The level of H2O2 in the thymus. (E) The level of catalase (CAT) in the thymus. (F) The level of glutathione peroxidase 1 (GPx1) in the thymus. Data are presented as the means ± SD (n = 5).*(P\u003c 0.05) and **(P\u003c 0.01) indicates a significant difference when compared to the control.","description":"","filename":"OnlineFig.1.Png","url":"https://assets-eu.researchsquare.com/files/rs-30062/v1/OnlineFig.1.Png"},{"id":1359935,"identity":"0271bb29-a14c-4c94-b32e-bbd1f63e8b8e","added_by":"auto","created_at":"2020-06-18 16:22:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":29459,"visible":true,"origin":"","legend":"Changes in CAT and GPx1 mRNA expression. (A) CAT mRNA expression in the thymus. (B) Level of GPx1 mRNA expression in the thymus. Data are expressed as the means ± SD (n = 5).*(P\u003c 0.05) and **(P \u003c 0.01) indicates a significant difference compared to the control group.","description":"","filename":"OnlineFig.2.Png","url":"https://assets-eu.researchsquare.com/files/rs-30062/v1/OnlineFig.2.Png"},{"id":1359936,"identity":"267d9232-145c-4569-8be0-e93c53f749f0","added_by":"auto","created_at":"2020-06-18 16:22:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15884,"visible":true,"origin":"","legend":"Changes in the thymus organ index. Data are expressed as the means ± SD (n = 5).*(P\u003c 0.05) and **(P\u003c 0.01) indicate a significant difference compared to the control group.","description":"","filename":"OnlineFig.3.Png","url":"https://assets-eu.researchsquare.com/files/rs-30062/v1/OnlineFig.3.Png"},{"id":1359937,"identity":"c00f34e4-782a-4f5e-90af-80f78ca49c37","added_by":"auto","created_at":"2020-06-18 16:22:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":136783,"visible":true,"origin":"","legend":"Histological and ultrastructural changes in the chicken thymus after REV infection. Histological examination of the thymus in the control group (A) and REV infection group (B) of chickens by HE staining (200x). Histological results showed significant necrosis (red ↑) and haemorrhage (black ↑) in the REV infection group. Transmission electron microscopy examination of the thymus in the control group (C) and REV infection group (D) of chickens (15 000x).Ultrastructural results showed that the REV infection group showed nuclear membrane rupture (red ↑), mitochondrial vacuolization (black ↑) and enlarged intercellular space (blue ↑).","description":"","filename":"OnlineFig.4.Png","url":"https://assets-eu.researchsquare.com/files/rs-30062/v1/OnlineFig.4.Png"},{"id":13542518,"identity":"7add5def-2d12-4183-9ae0-7cf8679a7b45","added_by":"auto","created_at":"2021-09-17 01:54:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":719862,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-30062/v1/43153881-d2ec-47b4-b80d-41a05ecacd8f.pdf"},{"id":2626415,"identity":"1f98379d-ea7f-43c0-927b-dec868602ac0","added_by":"acdc","created_at":"2020-09-25 21:02:49","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":676234,"visible":true,"origin":"acdc-supplements-supplement","legend":"","description":"{\"primaryId\":\"undefined\",\"secondaryId\":\"BVET-D-20-00449\",\"acdcId\":\"undefined\",\"revision\":\"1\",\"timestamp\":\"2020-06-17T14:58:46\",\"document\":\"supplements\",\"linkRel\":\"supplement\"}","filename":"supplement8.docx","url":"https://assets-eu.researchsquare.com/files/rs-30062/v1/supplement_8.docx"}],"financialInterests":"","formattedTitle":"Changes in Oxidation-antioxidation Function on the Thymus of Chickens Infected with Reticuloendotheliosis Virus","fulltext":[{"header":"Background","content":" \u003cp\u003eReticuloendotheliosis (RE) constitutes a group of pathological syndromes caused by reticuloendotheliosis hyperplasia virus, including runting syndrome, chronic tumors of lymphoid and other tissues, acute reticulocytoma, and severe immunosuppression. Reticuloendotheliosis virus (REV) is a C-type avian retrovirus[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The REV group includes defective REV-T, non-defective REV-A, chick syncytial virus, duck infectious anemia virus, and spleen necrosis virus (SNV) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In addition, REV infects a wide-range of hosts, including chickens, turkey, duck, goose, Japanese quail, and wild birds, among which, turkey is the most susceptible. Thus, turkeys and chickens are commonly used as experimental animals [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. REV can be mixed with Marek\u0026rsquo;s disease virus (MDV), avian leukosis virus of subgroup J (ALV-J), and chicken anemia virus (CAV), resulting in reducing or loss of immune function in REV infection chickens. Thus, RE represents a serious threat to the development of the poultry industry[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnder normal physiological conditions, the body's oxidation-antioxidant system is in a dynamic equilibrium, and oxidative stress occurs when the antioxidant system is overwhelmed by the oxidation system[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Oxidative stress is an initial reaction of the body to external stimuli and may subsequently induce various signaling pathways and inflammation in the body[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Under oxidative stress, animals grow slowly, the feed conversion rate decreases, and there is a decrease in production performance[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, long-term oxidative stress can deplete antioxidant vitamins and trace elements, impair immune function, as well as accelerate the aging and death of the animal, resulting in substantial economic losses to the poultry industry[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe oxidation system primarily includes reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS are by-products of the body\u0026rsquo;s metabolism that contain an unpaired electron. Moreover, ROS and RNS represent an important component of the pathogen resistance mechanism and may act as intracellular and intercellular signaling molecules; however, excessively high levels may also lead to oxidative stress[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. ROS mainly include superoxide anions, hydroxyl radicals, other oxygen radicals, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. RNS includes NO, NO\u003csub\u003e2\u003c/sub\u003e, and peroxynitrite[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntioxidant systems include enzymes and non-enzymatic antioxidants[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Enzyme antioxidants include SOD, peroxidase, CAT, GPx, and glutathione reductase, whereas non-enzymatic antioxidants include fat-soluble vitamin E, carotenoids, ubiquinone, and water-soluble vitamin C, glutathione, uric acid, and tryptophan metabolites. When the oxidation-antioxidant system is imbalanced, excessive ROS and RNS accumulate and destroy various biomacromolecules (e.g., lipids, proteins, and nucleic acids), which leads to cellular damage and ultimately cell death. Several studies have shown that oxidative stress plays an important role in inflammatory reactions, tumors, and many other diseases[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, there are few existing studies that have investigated the changes in the oxidation-antioxidant system of poultry after a REV infection. This study examined the dynamic changes in the level of T-AOC, SOD activity, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, MDA, CAT, and GPx1, as well as CAT and GPx1 mRNA expression in the thymus of one-day-old SPF chickens after a REV infection. Pathological sections and ultrastructural changes of the thymus were observed to further clarify the pathogenesis of REV infection and morphological changes resulting from oxidative stress and pathological processes, to facilitate the prevention and treatment of RE.\u003c/p\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of oxidative stress biomarkers\u003c/h2\u003e \u003cp\u003eWe first sought to study the oxidative-antioxidant capacity of the thymus in chickens infected with REV. We used commercial kits to detect the level of T-AOC, SOD, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e activity, and MDA of the chickens in both the infected and control groups. After the chickens were infected with REV, the thymus T-AOC (Fig. A) of the chickens was lower than that of the control chickens. Significant differences were found at 21 (\u003cem\u003eP༜0.05\u003c/em\u003e), 28 (\u003cem\u003eP༜0.01\u003c/em\u003e), and 35 (\u003cem\u003eP༜0.01\u003c/em\u003e) days post-REV infection. Compared with the control group, the SOD activity (Fig. B) of the thymus of the chickens decreased to varying degrees after REV infection, and exhibited significant differences on days 14 (\u003cem\u003eP༜0.01\u003c/em\u003e), 21 (\u003cem\u003eP༜0.05\u003c/em\u003e), and 28 (\u003cem\u003eP༜0.05\u003c/em\u003e) post REV-infection. Moreover, the level of MDA (Fig. C) in the thymus of REV-infected chickens increased from day 7 to 35 compared with the control group, and exhibited significant changes on day 28 (\u003cem\u003eP༜0.05\u003c/em\u003e). Compared with the control group, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content (Fig. D) in the thymus of the chickens increased between 14\u0026ndash;35 days after REV infection, and showed significant changes on day 35 (\u003cem\u003eP༜0.05\u003c/em\u003e). Compared with the control group, the level of CAT (Fig. E) in the thymus showed a downward trend from day 14 to 35 in REV-infected SPF chickens, and showed significant changes on days 14 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), 21 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), and 28 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The (Fig. F) level of GPx1 in the thymus was lower than that of the control SPF chickens after REV infection, and showed significant changes on day 28 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) post-infection.\u003c/p\u003e \u003c/div\u003e \n\u003ch2\u003eCAT And GPx1 mRNA Expression In The Thymus\u003c/h2\u003e\n \u003cp\u003eIn REV-infected SPF chickens, the level of CAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) expression in the thymus decreased from days 14 to 35 compared to the control group, and was significantly decreased on days 21 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and 28 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). From day 7 to 35 post-REV infection, the level of GPx1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) in the thymus of SPF chickens in the infected group was lower than that in the control chickens; significant changes were observed on days 28 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and 35 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \n\u003ch2\u003eDetermination Of The Thymus Organ Index\u003c/h2\u003e\n \u003cp\u003eIn SPF chickens infected with REV, the thymus organ index (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) was lower than that of the control chickens, and was significantly lower than the control chickens on days 28 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and 35 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \n\u003ch2\u003eHistopathological Changes In The Thymus Of REV-infected SPF Chickens\u003c/h2\u003e\n \u003cp\u003eThe histomorphology and ultrastructural changes of the thymus are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Compared with the control group, the thymic lobule was atrophied, the cortex and medulla exhibited hemorrhaging, and there was narrowing of the cortical area. In addition, there was a reduced number of lymphocytes in the medulla, an increased number of reticuloendothelial cells, and enhanced inflammatory cell infiltration. Under an electron microscope, the ultrastructure of the control group appeared normal. However, in the infected group, there was obvious cell damage, which primarily manifested as mitochondrial swelling, sputum rupture, Golgi vesicle expansion, and decreased cytoplasm. In addition, the nuclear membrane displayed breaks and had dissolved, and the nucleolus was enlarged.\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eAfter the body is infected with a virus, ROS activates the immune system to eliminate the infection and provides a positive regulatory role[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Simultaneously, the virus breaks the oxidation-antioxidant balance of the host cells, causing the accumulation of a large amount of ROS, which results in cellular damage and apoptosis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. RE is an immunosuppressive tumor disease caused by REV. This test detects changes in the oxidative-antioxidative function of the thymus in chickens after REV infection. It was found that REV can cause oxidative stress in the thymus and reduce antioxidant function.\u003c/p\u003e \u003cp\u003eUnder conditions of oxidative stress, ROS and RNS promote the accumulation of lipid peroxidation in polyunsaturated fatty acids in oxidized biofilms, and produce lipid peroxides (e.g., MDA, ketone, hydroxyl, and carboxyl)[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Moreover, MDA can reflect the degree of lipid peroxidation in the body and can be used as an indicator to determine whether a cell is oxidatively damaged[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is an important ROS that can be scavenged by catalase or peroxidase, and can also react with free Fe\u003csup\u003e2+\u003c/sup\u003e to produce \u0026middot;OH to initiate lipid peroxidation, destroy the cell membrane structure, and accelerate protein decomposition[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These findings reveal that the MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in the thymus of REV-infected SPF chickens was higher than that of the control chickens. The level of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the thymus on day 35 in REV-infected chickens was significantly higher than that of the control chickens. Moreover, the MDA content in the thymus was significantly higher on day 28 in REV-infected chickens than that of the control chickens. After REV invades the immune system of the chicken host, both non-specific immunity and specific immunity play a role in the generation of a large amount of free radicals, oxidative stress within the body, and enhanced lipid peroxidation[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. After REV enters a host, it induces NF-κB inhibitory protein I-κB phosphorylation, NF-κB activation, NF-κB translocation into the nucleus to bind to a specific nucleotide sequence of the iNOS gene promoter region, and the initiation of iNOS expression[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Studies have found that in the context of sepsis, inflammatory factors can mediate a decrease in nNOS expression in cardiomyocytes, up-regulate iNOS expression, and induce NO synthesis[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Moreover, iNOS expression can stimulate macrophages to produce NO, which in turn stimulates the cells to produce more ROS, triggering oxidative stress.\u003c/p\u003e \u003cp\u003eUnder normal conditions, SOD can catalyze the disproportionation of O\u003csub\u003e2\u003c/sub\u003e____˙ in the body to produce both H\u003csub\u003e2\u003c/sub\u003eO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e[14]. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e can break the structure and function of the membrane through the cell membrane, react with purines and pyrimidines, and result in nucleic acid damage[29]. CAT and GPx1 work together to clear H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e from the cell[11]. This study found that REV-infected SPF chickens exhibited lower levels of T-AOC, SOD activity, CAT, GPx1 content, as well as CAT and GPx1 mRNA expression, compared to the control group. We consider one potential reason for this phenomenon is that MDA can disrupt the synthetic SOD pathway, thereby reducing SOD activity. Moreover, the MDA content is negatively correlated with SOD activity. The peroxide deposition causes a large amount of antioxidant enzymes to be consumed in the body, and the function of the antioxidant system is suppressed. Decreased antioxidant enzyme activity leads to an accumulation of oxidizing substances, which can cause inflammatory reactions, cellular damage, and apoptosis. In addition, ROS can activate NF-κB, which can induce the expression of various inflammatory factors (e.g., IL-1, IL-6, and TNF-α) and aggravate the inflammatory response [30,31,32]. Chickens infected with REV exhibit immunosuppression. Combined with the results of this study, the immunosuppression caused by REV may be related to an accumulation of oxidizing substances and a decline in antioxidant system functionality.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eFollowing REV infection in one day old SPF chickens, the content of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and superoxide in the thymus was increased, the T-AOC and SOD activity decreased, and the thymus organ index was decreased. These changes exceeded the body's ability to scavenge antioxidant substances, resulting in oxidative stress in the body.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals and infectious virus strain\u003c/h2\u003e \u003cp\u003eAll of the chickens used for experiments were one-day-old SPF chickens obtained from Harbin Veterinary Research Institute (HVRI), Chinese Academy of Agricultural Sciences (CAAS), China. The REV-T strain (CVCC No. CACCAV107) was purchased from China Veterinary Culture Collection Center (CVCC). The virus TCID\u003csub\u003e50\u003c/sub\u003e was 10\u003csup\u003e4.62\u003c/sup\u003e/0.1\u0026nbsp;mL as determined by cell breeding.\u003c/p\u003e \u003c/div\u003e \n\u003ch2\u003eExperimental Design And Sample Collection\u003c/h2\u003e\n \u003cp\u003eA total of 80 one-day-old SPF chickens were randomly divided into a control group (Group C) and a REV-infected group(Group I). Group I were intraperitoneally administered with 100 \u0026micro;L of viral suspension at one-day-old. Group C were intraperitoneally administered with 100 \u0026micro;L of sterile physiological saline. The two groups of chickens were housed in isolators with similar environments,and chickens were given free access to feed and water. During housing, animals were monitored twice daily for health status. No adverse events were observed. On days 3, 7, 14, 21, 28, 35, and 49 post-REV infection, five chickens were randomly selected from each group and euthanasia was performed by sedation using Rompun/Ketamine mixture as an intramuscular injection followed by an intravenous injection of Pentobarbitone. Thymus were dissected and homogenized in chilled sterile physiological saline using a glass homogenizer with a teflon pestle under cold condition. The homogenate was centrifuged at 4000\u0026nbsp;g for 10\u0026nbsp;min at 4\u0026nbsp;\u0026deg;C. The supernatant was used for the assay of oxidative stress biomarkers.\u003c/p\u003e \n\u003ch2\u003eDetermination Of Thymic Oxidative Stress Biomarkers\u003c/h2\u003e\n \u003cp\u003eThe prepared 10% tissue homogenate was removed from \u0026minus;\u0026thinsp;80\u0026nbsp;\u0026deg;C. The T-AOC, SOD, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e activity, and MDA level in the thymus was determined using commercially available detection kits (Nanjing Jiancheng Bioengineering Research Institute, China). The level of CAT and GPx1 was measured by an ELISA, according to the manufacturer's instructions (Qiyi Biological Technology Co., Ltd. China).\u003c/p\u003e \n\u003ch2\u003eTotal RNA Extraction And Detection\u003c/h2\u003e\n \u003cp\u003eThe total RNA of thymus was extracted using Trizol reagent(Invitrogen ,Shanghai, China) The quality of RNA was assessed using a NanoDrop2 000 spectrophotometer (Thermo Fisher Scientific) through the ratio of absorbance at 260\u0026nbsp;nm and 280\u0026nbsp;nm. cDNA was obtained by reverse transcription of the RNA using the M-MLV Reverse Transcriptase kit (Invitrogen) according to the manufacturer's instructions.\u003c/p\u003e \n\u003ch2\u003eReal-time PCR\u003c/h2\u003e\n \u003cp\u003eDetermination of CAT and GPx1 mRNA in the thymus by real time PCR. The CAT, Gpx1, and β-actin primers were designed using Primer5.0 software. CAT: forward primer 5'- ATGTCCGTTTCAGGAGATGTGCAGC-3', reverse primer 5'- CCAGCAGTGCCTGAATACG \u0026minus;\u0026thinsp;3'; GPx1: forward primer 5'- ATGACCAACCCGCAGTACATCATCT-3', reverse primer 5'- GCAGTTTGATGGTCTCGAAGTGGC-3'; β-actin forward primer 5'- CGGGACGGATGAGAAGAA \u0026minus;\u0026thinsp;3', reverse primer 5'- TCGGCGCTCCAGATGTAC \u0026minus;\u0026thinsp;3'. Amplification of the target gene using the LightCycler2.0 real-time PCR System (Roche480, USA). The real-time PCR reaction procedure was 95\u0026nbsp;\u0026deg;C for 2\u0026nbsp;min, 45 cycles of 95℃ for 20\u0026nbsp;s, 59℃ for 20\u0026nbsp;s, and 72℃ for 10\u0026nbsp;s. To ensure the repeatability of the amplified samples, all samples were analyzed in triplicate. β-actin was used as a reliable normalization gene, and the expression of the samples were evaluated in relation to this housekeeping gene. The level of CAT and GPx1 mRNA expression were analyzed in accordance with the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \n\u003ch2\u003eDetermination Of The Thymus Index\u003c/h2\u003e\n \u003cp\u003eWeight of the chickens and thymus at the time of sampling for calculating the thymus index. The thymus index was calculated by the weight of the thymus (g)/chicken weight(kg).\u003c/p\u003e \n\u003ch2\u003ePathological Changes Of The Thymus After A REV Infection\u003c/h2\u003e\n \u003cp\u003eThymus tissues were fixed in 4% formalin. The samples were dehydrated with ethanol and embedded in paraffin. Each sample was cut into 5-\u0026micro;m sections with a microtome and placed onto a glass slide, and stained with hematoxylin and eosin (H\u0026amp;E). The pathological changes of the thymus were observed under a both a light microscope (Nikon, H600, Japan) and an electron microscope (Hitachi 7650, Tokyo, Japan).\u003c/p\u003e \n\u003ch2\u003eStatistical Analyses\u003c/h2\u003e\n \u003cp\u003eThe data were analyzed with SPSS 17.0 software, and the differences were compared between the groups were analyzed using one-way ANOVA followed by Tukey\u0026rsquo;s post-hoc test. All data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e was considered to be statistically significant.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eREV: Reticuloendotheliosis virus; SPF: Specific pathogen free; MDA: malondialdehyde; T-AOC: Total-antioxidante capacity; SOD: superoxide dismutase; ELISA: Enzyme linked immunosorbent assay; CAT: catalase; GPx1: Glutathione peroxidase 1; ROS: Reactive oxygen species; RNS: Reactive nitrogen species\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the Australian National Health and Medical Research Council's Australian Nursing and Use Regulations, the Animal Ethics Committee of the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences approved all experimental procedures with animals (SYXK [Hei] 2011022) for scientific purposes. All procedures were performed in accordance with the institutional guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.SM designed the experiments and revised manuscript. Z.MX performed the experiments of REV infection. Z.CH and Z.SJ performed the experiments of\u0026nbsp; Morphological analysis. Y.DH analyzed the data and drafted manuscript. G.XL and Z.J oversaw the experiments. L.XP and L.CN aided with analysis of results. All authors have read and approved the manuscript and ensure that this is the case.\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\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (31472169). The funding bodies 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\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1 \u003c/sup\u003eCollege of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, P.R. China.\u003csup\u003e2 \u003c/sup\u003eHeilongjiang Key Laboratory of Laboratory Animals and Comparative Medicine Harbin 150030, P.R. China.\u003csup\u003e3\u003c/sup\u003eWuXi AppTec (Suzhou)Co.,Ltd,Suzhou 215000 P.R .China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e \u003cspan\u003eGao C, Zhai J, Dang SY, Zheng SM. Analysis of alternative splicing in chicken embryo fibroblasts in response to reticuloendotheliosis virus infection. 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EBV and genomic instability\u0026ndash;a new look at the role of the virus in the pathogenesis of Burkitt's lymphoma. Semin Cancer Biol. 2009;19(6):394\u0026ndash;400.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eLiu B, Li A, Qin Y, Tian X, Gao M, Jiang W, Gong G. Comparative study on isolation and mitochondrial function of adult mouse and rat cardiomyocytes. J Mol Cell Cardiol. 2019;136:64\u0026ndash;71.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eZhang Z, Rong L, Li YP. Flaviviridae Viruses and Oxidative Stress: Implications for Viral Pathogenesis. Oxid Med Cell Longev 2019; 2019:1409582.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eMalik UU, Siddiqui IA, Hashim Z, Zarina S. Measurement of serum paraoxonase activity and MDA concentrations in patients suffering with oral squamous cell carcinoma. Clin Chim Acta. 2014;430:38\u0026ndash;42.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eVeal E, Day A. 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Mol Biol Rep. 2016;43(7):607\u0026ndash;28.\u003c/span\u003e \u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"reticuloendotheliosis virus, thymus, oxidation antioxidant imbalance, histopathological and ultrastructural changes, immunosuppression","lastPublishedDoi":"10.21203/rs.3.rs-30062/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-30062/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eReticuloendotheliosis virus (REV) is a retrovirus that causes severe immunosuppression in infected poultry. Under oxidative stress,the animals grow slowly. In addition, long-term oxidative stress can impair immune function, as well as accelerate the aging and death of the animal.This study aimed to elucidate the pathogenesis of REV from the perspective of changes in oxidative-antioxidative function following REV infection.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn specific pathogen free chickens infected with REV, the levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA in the thymus increased, the levels of T-AOC, SOD, CAT, and GPx1 decreased, and there was a reduction in CAT and Gpx1 mRNA expression, compared with the control group. The thymus index was also significantly reduced. Morphological analysis showed that after REV infection, there was an increase in thymic reticular endothelial cells, inflammatory cell infiltration, mitochondrial swelling, and destruction of the nuclear structure.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese results indicate that an increase in oxidative substances within the thymus, enhanced lipid peroxidation, a markedly decrease in antioxidant function, atrophy of the thymus, and immunosuppression in REV-infected chickens.\u003c/p\u003e","manuscriptTitle":"Changes in Oxidation-antioxidation Function on the Thymus of Chickens Infected with Reticuloendotheliosis Virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-06-18 16:22:14","doi":"10.21203/rs.3.rs-30062/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2020-06-09T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-06-08T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-06-08T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"93d5581b-8266-4ab6-a104-ae2f9ea2fdd6","owner":[],"postedDate":"June 18th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":123850,"name":"Large Animal Medicine"},{"id":123851,"name":"Small Animal Medicine"}],"tags":[],"updatedAt":"2020-12-13T15:02:48+00:00","versionOfRecord":{"articleIdentity":"rs-30062","link":"https://doi.org/10.1186/s12917-020-02708-6","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2020-12-11 15:01:22","publishedOnDateReadable":"December 11th, 2020"},"versionCreatedAt":"2020-06-18 16:22:14","video":"","vorDoi":"10.1186/s12917-020-02708-6","vorDoiUrl":"https://doi.org/10.1186/s12917-020-02708-6","workflowStages":[]},"version":"v1","identity":"rs-30062","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-30062","identity":"rs-30062","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}