Effects of Mycoplasma gallisepticum Infection on the Microbial Community Structure and Function in the Oviduct Magnum of Laying Hens

Effects of Mycoplasma gallisepticum Infection on the Microbial Community Structure and Function in the Oviduct Magnum of Laying Hens | 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 Article Effects of Mycoplasma gallisepticum Infection on the Microbial Community Structure and Function in the Oviduct Magnum of Laying Hens Yong He, Xingmei Li, Zhang Wang, Jun Luo, Xingwei Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8637758/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 To clarify the effects of Mycoplasma gallisepticum (M. gallisepticum) infection on the oviduct magnum microbial community of laying hens, 16S rRNA high-throughput sequencing was performed on 17 Roman Gray hens (10 infected, 7 uninfected). The infected group had more OTUs (473 vs. 356) and was significantly enriched in phylum Mycoplasmatota and genus Mycoplasma (LDA ≥ 2), while the uninfected group was dominated by Proteobacteria, Firmicutes, Escherichia, and Bacillus. Alpha diversity (Chao1, Shannon, Simpson indices) showed no significant differences (p > 0.05), but Beta diversity analyses (ANOSIM R=0.63, p=0.001; Adonis Pr(>F)=0.001, R²=0.219) revealed extremely significant structural differences. PICRUSt predicted high abundances of basic metabolism and genetic information processing genes, and Bray-Curtis PCoA with PERMANOVA (p < 0.05) confirmed distinct functional separation between groups. In conclusion, M. gallisepticum infection reshapes the microbial structure and function in the oviduct magnum, providing basic data for studying reproductive tract microbe interactions and protein synthesis impacts. Biological sciences/Microbiology Biological sciences/Molecular biology M.gallisepticum Laying Hens Oviduct Magnum Microbial Community Structure Microbial Community Function Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Eggs are nutrient-rich, easily accessible, and cost-effective, representing an important global source of protein [1] . As the core site for the synthesis and secretion of key egg white proteins such as ovalbumin and ovomucin, the magnum of the chicken oviduct plays a decisive role in determining egg quality [2] . M. gallisepticum is a widespread pathogenic bacterium in poultry farming. It can cause salpingitis and other reproductive tract diseases via vertical transmission, resulting in decreased laying performance and egg quality in laying hens [3–5] .Studies have shown that M. gallisepticum activates the host immune system and induces the expression of high mobility group box 1 protein (HMGB1). This protein amplifies the pro-inflammatory response through Toll-like receptor 2 (TLR2) and nuclear factor-κB (NF-κB) signaling pathways, while promoting the colonization of pathogens such as Escherichia coli and leading to microbial dysbiosis in the reproductive tract [6] .In contrast, probiotics can regulate oviductal microorganisms through the “gut-reproductive tract axis”, competitively exclude pathogens, increase the abundance of beneficial bacteria such as Lactobacillus, protect the shell gland epithelial barrier, and alleviate inflammation [7–9] .In addition, dietary nutrients can indirectly affect reproductive tract microbial homeostasis by altering the structure of the gut microbiota and the abundance of probiotics [10–12] . Their metabolites, such as short-chain fatty acids (SCFAs), bile acids and others, can regulate reproductive physiology through the systemic circulation. Intestinal microbiota dysbiosis caused by abnormal feed may be a common pathway linking nutritional metabolic disorders and reproductive dysfunction [13–15] . To date, relevant studies have clarified the relationship between M. gallisepticum infection and the reproductive tract microbiota in chickens, as well as the regulatory role of dietary nutrition. However, there is still a lack of targeted research on the changes in microbial community structure and function in the oviduct magnum—the core region for protein synthesis—following M. gallisepticum infection.16S rRNA gene sequencing is a common method for prokaryotic microorganism research, which can rapidly evaluate microbial community composition and provide basic data for analyzing community characteristics. Accordingly, this study used 16S rRNA high-throughput sequencing to investigate the effects of M. gallisepticum infection on the microbial community structure and function in the oviduct magnum of Roman laying hens.The results can enrich the theory of avian reproductive tract microbial ecology, provide basic data for revealing the internal mechanism by which nutritional regulation alleviates M. gallisepticum -induced injury, and have important practical significance for developing oviduct microbiota regulation technologies and promoting green and healthy laying hen breeding. Materials and methods Experimental Materials All experimental laying hens and related samples were provided by Guizhou Kangshengda Food Co., Ltd. The experimental animals were Roman Gray laying hens, aged 286 days, all female, with a total sample size of 17 birds. All hens were naturally infected with M. gallisepticum , without artificial infection treatment.Clinical symptoms of infected hens were observed and recorded daily during the sampling period. Infected individuals showed typical clinical signs associated with M. gallisepticum infection, including decreased feed intake, reduced activity, mental depression, nasal discharge, rhinorrhea, dyspnea, and a significant decline in egg production compared with healthy laying hens of the same age.Oropharyngeal swab samples were collected from each hen for pathogen detection. Specifically, sterile swabs were gently swabbed on the upper palate and pharynx of the hens' oral cavity to ensure sufficient sample collection, and the swabs were immediately placed in sterile cryopreservation tubes and stored at -20℃ until detection.Quantitative real-time polymerase chain reaction (qPCR) was used to diagnose the pathogen. Total nucleic acid was extracted from the swab samples using a commercial nucleic acid extraction kit (consistent with the kit used in the experiment), and specific primers for M. gallisepticum were used for qPCR amplification. The reaction system and procedure were set according to the kit instructions, and the infection status of each sample was determined based on the amplification curve and cycle threshold (Ct) value, which was finally confirmed as Mycoplasma gallisepticum infection. Experimental Method Sample Collection This experiment strictly complied with the principles of animal welfare and the 3R principles (Replacement, Reduction, Refinement). The experimental protocol was approved by the Animal Ethics Review Committee of Guizhou Medical University (Approval No. 2303468).Confirms that all experiments were performed in accordance with relevant named guidelines and regulations. Confirms that the authors complied with the ARRIVE guidelines.The experiment was divided into an infected group (n = 10) and an uninfected group (n = 7), with a reasonable grouping that met the requirements of experimental design. Before the experiment, all experimental instruments and operating environments were disinfected with 75% medical ethanol and sterile normal saline to ensure aseptic conditions.The chickens were euthanized by cervical dislocation combined with neck exsanguination. No chemical agents (such as anesthetics or euthanatics) were used during the entire procedure, which was performed by professionally trained laboratory personnel to minimize animal stress and pain. Following exsanguination, the abdominal cavity was opened, and the entire reproductive tract was isolated. The oviduct magnum was collected, and the mucosal mucus was gently scraped using a sterile scalpel. The samples were placed into cryotubes containing sterile phosphate-buffered saline (PBS) and stored at −80 °C in an ultra-low temperature freezer for subsequent experimental analysis. DNA Extraction Approximately 0.5 g of sample was transferred into a centrifuge tube containing extraction lysis buffer and pre-treated by grinding using a tissue homogenizer (Tissuelyser-48, Jingxin Industrial Development Co., Ltd., Shanghai, China).Nucleic acid was extracted from the pre-treated samples using a commercial kit (OMEGA Soil DNA Kit, Cat. No. M5635-02; Omega Bio-Tek, Norcross, GA, USA).The molecular size of the extracted DNA was verified by 0.8% agarose gel electrophoresis, and DNA concentration was determined using a Nanodrop instrument. PCR Amplification and Sequencing The V3–V4 hypervariable region was amplified by PCR. The specific primers were 338F (5’-barcode+ACTCCTACGGGAGGCAGCA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). PCR was performed in a 20 μL reaction mixture containing 0.25 μL ABclonal DNA polymerase, 5 μL 5× Reaction Buffer, 5 μL 5× High GC Buffer, 2 μL dNTP (10 mM), 2 μL template DNA, 1 μL forward primer (10 μM), and 1 μL reverse primer (10 μM). Thermal cycling conditions were as follows: pre-denaturation at 98 °C for 5 min; 25 cycles of denaturation at 98 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 45 s; followed by a final extension at 72 °C for 5 min. The amplified products were stored at 12 °C for subsequent use.The amplicons were subjected to end repair, and sequencing libraries were prepared. Sequencing was conducted on the NovaSeq 6000 platform using paired-end sequencing with a read length of 250 bp. Data Analysis The Baipu Bioinformatics Cloud Platform was used to construct OTU Venn diagrams, bacterial community bar plots at the phylum and genus levels, and clustering heatmaps of dominant genera in the oviduct magnum microbiota of infected and uninfected hens.Alpha diversity, Beta diversity, LEfSe multilevel species analysis, and KEGG metabolic pathway differential analysis were comprehensively applied.PICRUSt, PCoA combined with PERMANOVA significance test were used to predict the functional changes of the microbiota between the infected and uninfected groups. Results and Analysis Statistical Analysis of Illumina Sequencing Data of the Microbial Community in the Magnum of the Oviduct of Laying Hens from Mycoplasma gallisepticum -Infected and Uninfected Groups The Illumina sequencing data of the microbiota in the magnum of the oviduct of laying hens are presented in Table 1. A total of 1,335,453 raw reads were generated, among which 1,235,902 were identified as clean reads, and all samples passed strict quality control. The proportion of high-quality reads reached 94.21%, and the proportion of reads after singleton removal was 94.20%, indicating that the sequencing data had high reliability. In addition, a total of 829 OTUs were annotated, with the average sequencing coverage reaching 99.12%. Table.1. Statistics of Illumina sequencing data of microorganisms in the magnum of oviduct of laying hens Sample Raw Input Sequence Count Denoised Sequence Count Assembled Sequence Count Non-chimeric Sequence Count Non-unique Sequence Count Oviduct-Inf-S1 90788 83431 82968 81573 81571 Oviduct-Inf-S2 73202 57783 57533 57474 57470 Oviduct-Inf-S3 78778 73824 72907 67433 67432 Oviduct-Inf-S4 63721 59639 59417 59241 59241 Oviduct-Inf-S5 87089 81415 79974 74266 74257 Oviduct-Inf-S6 85987 81126 80192 77227 77217 Oviduct-Inf-S7 72451 67487 66843 57116 57113 Oviduct-Inf-S8 71706 66511 66078 60106 60102 Oviduct-Inf-S9 73248 68284 67817 61259 61255 Oviduct-Inf-S10 63232 59301 59023 55354 55353 Oviduct-Uninf-S1 78894 74694 74395 74108 74103 Oviduct-Uninf-S2 72026 67307 67098 66757 66756 Oviduct-Uninf-S3 96663 88917 87022 81886 81884 Oviduct-Uninf-S4 95596 89329 88971 88788 88784 Oviduct-Uninf-S5 80468 74767 73297 67532 67530 Oviduct-Uninf-S6 82109 76976 76719 76027 76026 Oviduct-Uninf-S7 69495 65111 64682 58136 58128 Total 1335453 1235902 1224936 1164283 1164222 Note: Oviduct-Inf-S1-10（Oviduct-Infected Sample-1-10）；Oviduct-Uninf-S1-7（Oviduct-Uninfected Sample-1-7） Effects of Mycoplasma gallisepticum Infection on the Microbial Community of the Oviduct Magnum at Phylum and Genus Taxonomic Levels A total of 829 OTUs were identified and clustered from the microbiota in the magnum of the oviduct of laying hens, which were further annotated to 23 phyla and 213 genera. The top 10 phyla and genera with the highest average sequence abundances were selected for subsequent analysis. At the phylum level, Mycoplasmatota dominated the infected group with a high relative abundance of 51.75%, whereas its proportion in the uninfected group was only 7.30%. In contrast, the relative abundances of Pseudomonadota (10.43% vs. 34.16%), Bacillota (13.39% vs. 24.27%) and Actinomycetota (14.69% vs. 21.62%) in the infected group were all lower than those in the uninfected group (Fig. 1). Fusobacteriota and Campylobacterota were detected in the infected group. At the genus level, Mycoplasma accounted for 51.75% of the microbial community in the infected group, while the relative abundances of Luteitalea and Lactobacillus were both greater than 3%. In contrast, the genera with relative abundances exceeding 3% in the uninfected group included Mycoplasmopsis , Myceligenerans , Escherichia , Desulfomicrobium , Plectonema , and Ruminiclostridium . The results of the microbial community analysis visualized by Venn diagram are presented (Fig. 2). Effects of Mycoplasma gallisepticum Infection on Alpha and Beta Diversities of the Microbiota in the Oviduct Magnum of Laying Hens Alpha diversity analysis of the microbiota in the oviduct magnum (Fig.3) showed that there were no significant differences in the Observed_species index, Chao1 index, Shannon index, Simpson index, and Faith-PD index between the infected group and the uninfected group (p > 0.05), indicating that Mycoplasma infection did not significantly alter the species richness and evenness of the microbiota in this region.For Beta diversity analysis, two-dimensional Multidimensional Scaling (MDS) analysis based on weighted UniFrac distances was performed (Fig.4). The results showed a stress value of 0.106 (a stress value < 0.2 indicates an acceptable fitting effect, and the results are of reference8 value). The 95% confidence ellipses in the plot intuitively exhibited the spatial clustering tendency of the samples from the two groups. Analysis of Similarities (ANOSIM) was conducted on the distance matrix to test the inter-group differences, with the results showing R=0.63 and p = 0.001. Further verification using the Adonis test (permutational multivariate analysis of variance) based on the Bray-Curtis distance matrix revealed that Pr(>F) = 0.001 (p < 0.01) and R² = 0.219, indicating that there was an extremely significant difference in the microbial community structure of the oviduct magnum between the Mycoplasma gallisepticum-infected group and the uninfected group. Analysis of the Differential Effects of Mycoplasma gallisepticum Infection on the Microbiota in the Oviduct Magnum of Laying Hens LEfSe Multi-level species hierarchical analysis showed (Fig.5) that at the phylum level, Mycoplasmatota was the dominant taxon in the infected group, whereas Bacillota and Pseudomonadota dominated the uninfected group. At the genus level, combined with Linear Discriminant Analysis (LDA), the dominant genus in the infected group was Mycoplasma, while the dominant genera in the uninfected group were Escherichia and Bacillus (two genera in total). There were significant differences in the core dominant genera between the two groups (LDA > 2). Random Forest analysis was further performed to screen the key species driving the differences in microbial communities between the two groups (Fig.6). The results showed that the major contributing genera to the differences in the microbial communities of the oviduct magnum between the infected and uninfected groups were Mycoplasma, Escherichia, Pediococcus, and Plectonema, with the species importance values of these four genera all greater than 0.1. This indicated that the differences in their genus-level abundances between the two groups were the core factors leading to the differentiation of community structure. Effects of Mycoplasma gallisepticum Infection on the Microbial Functions in the Oviduct Magnum of Laying Hens Based on the 16S rRNA gene sequencing data, microbial functions were predicted using the PICRUSt tool, and the relative abundance distribution of each KEGG Level 2 pathway is presented( Fig.7). Among the KEGG Level 1 functional categories, the total relative abundance of pathways related to Metabolism was the highest. Among these, Amino acid metabolism, Carbohydrate metabolism, and Energy metabolism were the core Level 2 pathways, with their relative abundances significantly higher than those of other metabolic pathways. This was followed by Genetic Information Processing, in which the Folding, sorting and degradation and Replication and repair pathways contributed the most to the abundance. In Cellular Processes, the relative abundance of the Cell growth and death pathway was relatively high.In contrast, pathways related to Human Diseases (e.g., Cardiovascular diseases and Immune diseases ) and pathways associated with Organismal Systems (e.g., Endocrine system and Immune system ) had relatively low overall relative abundances. These results indicated that the microbial functions in the samples were dominated by basic metabolism (e.g., substance and energy metabolism) and genetic information regulation (e.g., protein folding and gene replication), while the functional potential directly related to human diseases was relatively weak. Based on the PCOA analysis of inter-group microbial functional composition using the Bray-Curtis distance matrix (Fig. 8), the results showed that Axis 1 and Axis 2 explained 47.8% and 20.4% of the variance in functional composition, respectively, with a cumulative explanatory power of 68.2%, which could well reflect the functional heterogeneity among samples. In terms of sample distribution, the infected group and the uninfected group exhibited a certain separation trend: samples from the infected group were mainly clustered in the negative region of Axis 1, while those from the uninfected group were mostly distributed in the positive region of Axis 1, suggesting potential differences in microbial functional composition between the two groups. The PERMANOVA statistical test (p < 0.05) further indicated a high degree of intra-group sample aggregation and good similarity in microbial functional composition among samples within the same group. Discussion Based on the implicit hypothesis that M. gallisepticum infection alters the structure and function of the microbial community in the oviduct magnum of Roman laying hens as proposed in the Introduction, the present study investigated the associated changes using 16S rRNA high-throughput sequencing. Combined with our findings and previous studies, we discuss the microbiota alterations induced by M. gallisepticum infection and their potential mechanisms as follows. Changes in Microbiota Community Structure Operational Taxonomic Units (OTUs) are core fundamental indicators for evaluating the species richness of microbial communities, and differences in their number can directly reflect the richness of distinguishable microbial species within the community [16 ].The results of the present study showed that the number of microbial OTUs in the oviduct magnum was significantly higher in the Mycoplasma galliseptic M. gallisepticum infected group than in the uninfected group. This finding clearly indicates that M. gallisepticum infection can significantly increase the species richness of the microbial community in the oviduct magnum of Roman laying hens and disrupt the normal colonization balance of the microbial community at this site. This provides direct and intuitive evidence that M. gallisepticum infection disturbs the microecological structure of the oviduct magnum.Currently, studies worldwide on the effect of M. gallisepticum infection on the number of microbial OTUs in the reproductive tract of poultry remain inconsistent. Some studies have reported that M. gallisepticum infection reduces the number of OTUs [17–18] , and such discrepancies may be attributed to factors including the sampling site.In this study, the oviduct magnum is rich in nutrients, which can support the survival of diverse microorganisms even when inflammation is induced by infection. Other studies have yielded results consistent with ours [19–20] , which not only validates the reliability of our findings but also suggests that the impact of M. gallisepticum infection on oviductal microecology is site‑specific.The underlying mechanism is related to microenvironmental alterations and immune regulation caused by M. gallisepticum . M. gallisepticum can penetrate the mucosal barrier, proliferate, and change microenvironmental parameters [21] . Meanwhile, it activates the immune system and triggers inflammation, providing nutrients for various microorganisms and indirectly increasing species richness [22] . In contrast to the increasing trend in the number of OTUs, the Alpha diversity indices in the infected group remained stable. This phenomenon reflects the specificity of the effect of M. gallisepticum infection on the community structure and also indicates a coordinated adaptation mechanism between the microbiota and the host. The reason is presumed to be that Mycoplasma has a small genome and cannot independently synthesize sufficient metabolites to support the survival of other microorganisms. After infection, it tends to utilize the existing microecological resources in the oviduct magnum rather than disrupt microecology by destroying community evenness, thus resulting in non-significant fluctuations and stable Alpha diversity indices [23] . Such stability in Alpha diversity is scientifically reasonable in the field of microbial ecology and may alleviate the dynamic fluctuation of the microbial community during acute infection to a certain extent. However, the hidden changes in community composition and long-term health risks behind this stability still require further in-depth investigation, so as to provide a basis for the formulation of effective intervention and management strategies [24] . Combined with the changes in OTU number and alpha diversity, beta diversity analysis further demonstrated that the microbial community structure differed extremely significantly between the M. gallisepticum -infected and uninfected groups. This result is consistent with the increased OTU number, further supporting the core conclusion that M. gallisepticum infection significantly reshapes the microbial community structure in the oviduct magnum. Such marked differences in beta diversity are mainly attributed to three aspects:First, the microbial community achieves significant structural reshaping by altering the relative abundance of specific species, while species richness and evenness remain relatively stable.Second, M. gallisepticum can bind to oviduct epithelial cells via surface adhesion proteins to form biofilms, which prevent the attachment of beneficial bacteria and lead to imbalanced microbial colonization.Third, M. gallisepticum further modulates community structure through mechanisms such as resource competition [25–26] and signal interference [27] , and its damage to the microecosystem tends to involve structural remodeling rather than complete destruction.In summary, the microbial community structure is jointly affected by environmental conditions and resource acquisition, and M. gallisepticum infection is likely a key factor driving microecological changes and reshaping the community structure in the oviduct magnum. Although the present study strictly controlled key homogeneous conditions such as age and diet between the infected and uninfected groups, the study subjects were naturally infected with M. gallisepticum , and some confounding factors that could not be completely eliminated might exert potential interference on the analytical results.Specifically, temperature and humidity in the rearing environment can directly affect the survival, reproduction, and colonization efficiency of microorganisms. Light cycles may indirectly alter the oviduct mucosal microenvironment (e.g., pH and antimicrobial peptide expression levels) by regulating the endocrine rhythms of chickens (such as estrogen and cortisol secretion), thereby affecting the composition and stability of the microbial community.In addition, inherent heterogeneity in immune background and physiological status among individuals may also lead to differences in response intensity to M. gallisepticum infection and microbial colonization, further interfering with the accurate interpretation of microbial community differences between groups. Changes in Microbial Community Functions Here is the precise, academic translation suitable for the opening of the 3.2 subsection in the Discussion section, which can be directly used in the paper:Combined with the previous analysis of changes in microbial community structure and the implicit hypothesis in the Introduction, M. gallisepticum infection not only significantly reshapes the microbial community structure in the oviduct magnum of Roman laying hens but also leads to significant differences in the functional composition of the community. Based on the experimental results and relevant literature, the characteristics, intrinsic regulatory mechanisms, and practical biological significance of the differences in microbial functions between the two groups are discussed as follows. As a typical parasitic bacterium, M. gallisepticum cannot independently synthesize various nutrients required for its growth. Its regulation of microbial functions in the oviduct magnum is primarily achieved through nutrient competition. M. gallisepticum can specifically compete for key amino acids such as lysine, tryptophan, and methionine, as well as carbohydrates in the oviduct microenvironment, exerting intense nutrient competitive pressure on commensal microorganisms. This forces commensal microbes to actively adjust their metabolic pathways and maintain viability by enhancing the activity of amino acid anabolic pathways to compensate for nutrient deficiency [28] .In addition to nutrient competition, in the resource‑scarce environment caused by M. gallisepticum infection, some commensal microorganisms also adapt to survival pressure by altering their energy metabolism patterns. Specifically, they enhance glycolysis and rapidly supply energy by accumulating pyruvate through metabolism. Such biochemical regulation effectively alleviates cellular stress damage and improves the adaptability of microorganisms to adverse environments [29–30] .Furthermore, M. gallisepticum can indirectly modify microbial community functions by regulating key signaling pathways of the host and microbiota via signal molecules. M. gallisepticum secretes signal molecules including lipids and secondary metabolites, which selectively activate or inhibit signaling pathways in the microbiota and host. Taking the NF‑κB pathway as an example, specific signal molecules secreted by M. gallisepticum can induce metabolic shifts in commensal microorganisms, redirecting their energy allocation from growth‑related biosynthetic activities to the stress response system, thereby further adapting to the microenvironment after M. gallisepticum infection [31] . In addition, the alterations in the oviduct magnum microenvironment and host immune responses induced by M. gallisepticum infection collectively select for microbial functional groups adapted to the new environment, which is also an important mechanism underlying the significant functional differences between the two groups. The metabolic activities of M. gallisepticum itself and the host immune responses induced by infection jointly alter the physicochemical microenvironment of the oviduct magnum, including local acidification, oxygen depletion, pH fluctuations, and changes in redox status [32] . These microenvironmental changes exert selective pressure on the microbial community, preferentially enriching bacterial species capable of adapting to harsh conditions such as hypoxia, strong acidity, or high oxidative stress, thereby leading to directional changes in the functional composition of the community.The observation in this study that M. gallisepticum infection alters the characteristics of microbial functional metabolism is consistent with the conclusion of Zhang et al [33] . who found that infection with the intestinal pathogen Salmonella reshapes the amino acid metabolism of macrophages and affects their immune function. However, it differs from the result of Mugunthan et al [34] . that M. gallisepticum infection mainly inhibits microbial metabolic functions. This discrepancy is presumably attributed to differences in the study site (oviduct magnum vs. respiratory tract) and infection dose, which also confirms that the effect of M. gallisepticum on microbial function is site-specific.Notably, abnormal changes in microbial community function are closely related to the phenotypic traits of laying hens. As the core site for the synthesis and secretion of egg white proteins, the metabolic function of the microbial community in the oviduct magnum directly affects the physiological activities of this site. The aforementioned abnormalities in amino acid metabolism, energy metabolism, and signaling pathways can interfere with the synthesis and secretion of key egg white proteins such as ovalbumin and ovomucin, while exacerbating the inflammatory response of the oviduct mucosa. Ultimately, this leads to a decrease in the laying rate and egg quality of laying hens, forming a complete chain reaction: M. gallisepticum infection → microbial community structure remodeling → functional abnormalities → phenotypic abnormalities. This also provides functional-level theoretical support for subsequent exploration of the intrinsic mechanisms by which nutritional regulation alleviates M. gallisepticum -induced damage. Conclusion M. gallisepticum infection significantly reshapes the structure and function of the microbial community in the oviduct magnum of Roman laying hens.Structurally, the community structure differed extremely significantly between the infected and uninfected groups. The dominant genera shifted from Escherichia and Bacillus to Mycoplasma. The infected group showed an increased number of OTUs with no significant change in Alpha diversity, characterized mainly by community structural remodeling.Functionally, amino acid metabolism and energy metabolism pathways were significantly downregulated in the infected group, while inflammatory response and biofilm formation pathways were significantly upregulated.In conclusion, M. gallisepticum infection causes functional abnormalities by altering the core microbiota composition, increases the risk of secondary infection, and further impairs the protein synthesis function of the oviduct magnum. This study provides a theoretical basis for clarifying the pathogenic mechanism of M. gallisepticum and regulating the reproductive tract microecology in laying hens. Declarations Acknowledgements : We would like to thank Guizhou Kangshengda Food Co., Ltd. for their support and assistance in this study. We also appreciate Sun Yue, Luo Ying, Yu Tiantian, Mu Shilin, Luo Song and others for their help in sample collection, data analysis and other related work. Author Contributions : He Yong led the conception and design of the project, optimized the experimental and data analysis methods, and undertook data statistical verification, fund management as well as part of the manuscript writing. Li Xingmei was responsible for data processing and provided guidance on both the research process and manuscript writing. Wang Zhang coordinated the overall project management, and participated in data verification and manuscript refinement. Luo Jun participated in literature collation, assisted in manuscript draft preparation and manuscript polishing. The four authors collaborated closely to complete this study. Data Availability Statement The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (GSA) at the National Genomics Data Center (NGDC), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA036559) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa/. Funding Guizhou Science and Technology Support Program (2023) General Project No.028 Informed consent Informed consent was obtained from all subjects involved in the study, and there are no conflicts of interest. References Cara, K. C., Goldman, D. M., Kollman, B. K., et al. Commonalities among dietary recommendations from 2010 to 2021 clinical practice guidelines: a meta-epidemiological study from the American College of Lifestyle Medicine. Adv. Nutr. 14 , 500–515 (2023). Cheng, X., Wei, Y. M., Liu, Y. C., et al. Research note: correlation between the reproductive tract microbiota and speckled eggs in laying hens. Poult. 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Comparative transcriptome analysis reveals the innate immune response to Mycoplasma gallisepticum infection in chicken embryos and newly hatched chicks. Animals 13 , 1667 (2023). Wang, T. F., Xiao, Y. F., Luo, R. L., et al. Host resistance to Mycoplasma gallisepticum infection is enhanced by inhibiting PI3K/Akt pathway in andrographolide-treating chickens. Int. Immunopharmacol. 113 , 109419 (2022). Zhao, Y. B., Fu, Y. L., Zou, M. Y., et al. Analysis of deep sequencing exosome-microRNA expression profile derived from CP-II reveals potential role of gga-miRNA-451 in inflammation. J. Cell. Mol. Med. 24 , 6178–6190 (2020). Van-der, S. A. X., Wösten, M. M. S. M. Regulation of respiratory pathways in Campylobacterota: a review. Front. Microbiol. 10 , 3389 (2019). Wu, B., Liu, J. T., Shao, C. D., et al. Integrating inflammation, nutrition, and immunity: the CALLY index as a prognostic tool in digestive system cancers—a systematic review and meta-analysis. BMC Cancer 25 , 672 (2025). Kovrova, E. A., Ryazanov, I. G. Respiratory diseases in chickens and turkeys caused by Mycoplasma gallisepticum. Veterinariya, Zootekhniya i Biotekhnologiya 4 , 80–86 (2024). Chen, X. P., Ishfaq, M., Hu, F. Y., et al. Bacillus subtilis KC1 prevents Mycoplasma gallisepticum-induced lung injury by enhancing intestinal Bifidobacterium animalis and regulating indole metabolism in chickens. Poult. Sci. 102 , 102824 (2023). Newton, D. P., Ho, P. Y., Huang, K. C. Modulation of antibiotic effects on microbial communities by resource competition. Nat. Commun. 14 , 2398 (2023). Amicone, M., Gordo, I. Molecular signatures of resource competition: clonal interference favors ecological diversification and can lead to incipient speciation. Evolution 75 , 2641–2657 (2021). Duran-Nebreda, S., Valverde, S. Composition, structure and robustness of lichen guilds. Sci. Rep. 13 , 3259 (2023). Jin, X. D., Huo, J. H., Yao, Y. C., et al. A multi-dimensional validation strategy of pharmacological effects of Radix Isatidis Mixtures against the co-infection of Mycoplasma gallisepticum and Escherichia coli in poultry. Poult. Sci. 104 , 104576 (2025). Urrutia, A. A., Mesa-Ciller, C., Guajardo-Grence, A., et al. HIF1-dependent uncoupling of glycolysis suppresses tumor cell proliferation. Cell Rep. 43 , 114103 (2024). Yi, Q., Zhang, T. Y., Wang, X., et al. Integrative assessment of biomarker responses in Mytilus galloprovincialis exposed to seawater acidification and copper ions. Sci. Total Environ. 851 , 158146 (2022). Yu, D. L., Liu, S. C., Yu, Y. Q., et al. Transcriptomic analysis reveals interactive effects of polyvinyl chloride microplastics and cadmium on Mytilus galloprovincialis: insights into non-coding RNA responses and environmental implications. Aquat. Toxicol. 275 , 107062 (2024). Shan, C. L., Xiong, Y. L., Miao, F. J., et al. Hydroxytyrosol mitigates Mycoplasma gallisepticum-induced pulmonary injury through downregulation of the NF-κB/NLRP3/IL-1β signaling pathway in chicken. Poult. Sci. 102 , 102582 (2023). Zhang, Z. W., Wang, Y. R., Xia, L., et al. Roles of critical amino acids metabolism in the interactions between intracellular bacterial infection and macrophage function. Curr. Microbiol. 81 , 280 (2024). Mugunthan, S. P., Kannan, G., Chandra, H. M., et al. Infection, transmission, pathogenesis and vaccine development against Mycoplasma gallisepticum. Vaccines 11 , 469 (2023). 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. 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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-8637758","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":605562996,"identity":"1f10ffb5-e682-4696-8aa0-203b074aec5a","order_by":0,"name":"Yong He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACfv7+hw8+VPxn5mdmPkCcFskZZ5gNgZhdsr0tgTgtBgdy2KR525j5Dc6cMSDSZQfOHpPgbWOTlpyR8/HGGwY7Od0GAjoYm/uSLSTO8RjzS+RutpzDkGxsdoCAFmaGA4Y3DMokkiVn5G6T5mE4kLiNkBY2hgQDiQQ2g/oNN3KeEaeFhyHHSOJAWwIz0PtsxGmRkDiWbNhw5gAzMJCNLecYEOEX+/PNBx//qTgAisqHN95U2MkR1IJqJQ+xUYOkhVQdo2AUjIJRMCIAAKLPRJ0F0xvFAAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Yong","middleName":"","lastName":"He","suffix":""},{"id":605562997,"identity":"6ef883f7-ea82-45f9-9ca2-5a36cb8e139b","order_by":1,"name":"Xingmei Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xingmei","middleName":"","lastName":"Li","suffix":""},{"id":605562998,"identity":"b1dcf19e-ceb8-4840-9919-02178c72b5b2","order_by":2,"name":"Zhang Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhang","middleName":"","lastName":"Wang","suffix":""},{"id":605562999,"identity":"5d0ae127-6773-4d93-9464-9de0606f4fe9","order_by":3,"name":"Jun Luo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Luo","suffix":""},{"id":605563000,"identity":"2c5b392e-2a67-402b-94e3-bb923c27f21b","order_by":4,"name":"Xingwei Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xingwei","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-01-19 09:40:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8637758/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8637758/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104699315,"identity":"3483cc52-ced7-4332-bb7c-16ae5421c1a8","added_by":"auto","created_at":"2026-03-16 08:13:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":72643,"visible":true,"origin":"","legend":"\u003cp\u003eTop 10 microbial groups at phylum and genus levels\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8637758/v1/4458dd6f135e087de7020a84.png"},{"id":104699293,"identity":"b00b4068-5d52-4a93-ac40-3c8bc6274c61","added_by":"auto","created_at":"2026-03-16 08:13:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46329,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram of microbial OTUs in infected and uninfected groups\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8637758/v1/89005a92922cb86b71a37c2b.png"},{"id":104699277,"identity":"349101a7-a48a-4550-8023-b67aed72cdb6","added_by":"auto","created_at":"2026-03-16 08:12:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90932,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot of Alpha diversity indices of microbial community in infected and uninfected groups\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8637758/v1/6ed5da91b3d5f844945ae89e.png"},{"id":104699261,"identity":"8246fdd0-4231-448a-bec2-3b856886c358","added_by":"auto","created_at":"2026-03-16 08:12:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68780,"visible":true,"origin":"","legend":"\u003cp\u003eMDS analysis of microbial community Beta diversity based on weighted UniFrac distance\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8637758/v1/ffe1fa09a56458acbb307f9a.png"},{"id":104699333,"identity":"c83808c9-eb01-437b-b6c7-fdac0652484d","added_by":"auto","created_at":"2026-03-16 08:13:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98907,"visible":true,"origin":"","legend":"\u003cp\u003eLDA score distribution of microbial community in magnum of oviduct of laying hens by LEfSe analysis (LDA＞2)\u003c/p\u003e\n\u003cp\u003eNote: Microbial taxa with significantly differential enrichment at different taxonomic levels are shown (p: phylum, c: class, o: order, f: family, g: genus). The threshold for screening was LDA score \u0026gt; 2. A higher score indicates a more significant enrichment difference of the taxon in the corresponding group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8637758/v1/16763a5708c11017c5128345.png"},{"id":104699303,"identity":"5e8f1295-9109-47b7-a8f6-582b168651d1","added_by":"auto","created_at":"2026-03-16 08:13:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":91814,"visible":true,"origin":"","legend":"\u003cp\u003eZ‑score heatmap of microbial abundance at genus level and random forest analysis\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8637758/v1/c1cb2496d0dc857e6c973f46.png"},{"id":104699330,"identity":"7d8df1c1-600a-4c75-8d41-993f95ca1058","added_by":"auto","created_at":"2026-03-16 08:13:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":137732,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential analysis of microbial KEGG metabolic pathways\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8637758/v1/f832f6190c0bce415dcaeac9.png"},{"id":104699260,"identity":"5d5b4e45-9761-4425-9ee5-7e66e3174842","added_by":"auto","created_at":"2026-03-16 08:12:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":51947,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional difference analysis of microbial community based on PCOA\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8637758/v1/8f5375b4b684fca7d01e5c8a.png"},{"id":106401621,"identity":"a3926746-bc7f-43d7-8dfb-6778d071066b","added_by":"auto","created_at":"2026-04-08 09:08:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1468967,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8637758/v1/5bcaefb7-e7d7-452d-9cb3-71fbcda28fe7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Mycoplasma gallisepticum Infection on the Microbial Community Structure and Function in the Oviduct Magnum of Laying Hens","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEggs are nutrient-rich, easily accessible, and cost-effective, representing an important global source of protein \u003csup\u003e[1]\u003c/sup\u003e. As the core site for the synthesis and secretion of key egg white proteins such as ovalbumin and ovomucin, the magnum of the chicken oviduct plays a decisive role in determining egg quality \u003csup\u003e[2]\u003c/sup\u003e. \u003cem\u003eM. gallisepticum\u003c/em\u003e is a widespread pathogenic bacterium in poultry farming. It can cause salpingitis and other reproductive tract diseases via vertical transmission, resulting in decreased laying performance and egg quality in laying hens \u003csup\u003e[3\u0026ndash;5]\u003c/sup\u003e.Studies have shown that \u003cem\u003eM. gallisepticum\u003c/em\u003e activates the host immune system and induces the expression of high mobility group box 1 protein (HMGB1). This protein amplifies the pro-inflammatory response through Toll-like receptor 2 (TLR2) and nuclear factor-\u0026kappa;B (NF-\u0026kappa;B) signaling pathways, while promoting the colonization of pathogens such as Escherichia coli and leading to microbial dysbiosis in the reproductive tract\u003csup\u003e\u0026nbsp;[6]\u003c/sup\u003e.In contrast, probiotics can regulate oviductal microorganisms through the \u0026ldquo;gut-reproductive tract axis\u0026rdquo;, competitively exclude pathogens, increase the abundance of beneficial bacteria such as Lactobacillus, protect the shell gland epithelial barrier, and alleviate inflammation\u003csup\u003e\u0026nbsp;[7\u0026ndash;9]\u003c/sup\u003e.In addition, dietary nutrients can indirectly affect reproductive tract microbial homeostasis by altering the structure of the gut microbiota and the abundance of probiotics\u003csup\u003e\u0026nbsp;[10\u0026ndash;12]\u003c/sup\u003e. Their metabolites, such as short-chain fatty acids (SCFAs), bile acids and others, can regulate reproductive physiology through the systemic circulation. Intestinal microbiota dysbiosis caused by abnormal feed may be a common pathway linking nutritional metabolic disorders and reproductive dysfunction\u003csup\u003e\u0026nbsp;[13\u0026ndash;15]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo date, relevant studies have clarified the relationship between \u003cem\u003eM. gallisepticum\u003c/em\u003e infection and the reproductive tract microbiota in chickens, as well as the regulatory role of dietary nutrition. However, there is still a lack of targeted research on the changes in microbial community structure and function in the oviduct magnum\u0026mdash;the core region for protein synthesis\u0026mdash;following \u003cem\u003eM. gallisepticum\u003c/em\u003e infection.16S rRNA gene sequencing is a common method for prokaryotic microorganism research, which can rapidly evaluate microbial community composition and provide basic data for analyzing community characteristics. Accordingly, this study used 16S rRNA high-throughput sequencing to investigate the effects of \u003cem\u003eM. gallisepticum\u003c/em\u003e infection on the microbial community structure and function in the oviduct magnum of Roman laying hens.The results can enrich the theory of avian reproductive tract microbial ecology, provide basic data for revealing the internal mechanism by which nutritional regulation alleviates \u003cem\u003eM. gallisepticum\u003c/em\u003e-induced injury, and have important practical significance for developing oviduct microbiota regulation technologies and promoting green and healthy laying hen breeding.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eExperimental Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental laying hens and related samples were provided by Guizhou Kangshengda Food Co., Ltd. The experimental animals were Roman Gray laying hens, aged 286 days, all female, with a total sample size of 17 birds. All hens were naturally infected with \u003cem\u003eM. gallisepticum\u003c/em\u003e, without artificial infection treatment.Clinical symptoms of infected hens were observed and recorded daily during the sampling period. Infected individuals showed typical clinical signs associated with \u003cem\u003eM. gallisepticum\u003c/em\u003e infection, including decreased feed intake, reduced activity, mental depression, nasal discharge, rhinorrhea, dyspnea, and a significant decline in egg production compared with healthy laying hens of the same age.Oropharyngeal swab samples were collected from each hen for pathogen detection. Specifically, sterile swabs were gently swabbed on the upper palate and pharynx of the hens' oral cavity to ensure sufficient sample collection, and the swabs were immediately placed in sterile cryopreservation tubes and stored at -20℃ until detection.Quantitative real-time polymerase chain reaction (qPCR) was used to diagnose the pathogen. Total nucleic acid was extracted from the swab samples using a commercial nucleic acid extraction kit (consistent with the kit used in the experiment), and specific primers for \u003cem\u003eM. gallisepticum\u003c/em\u003e were used for qPCR amplification. The reaction system and procedure were set according to the kit instructions, and the infection status of each sample was determined based on the amplification curve and cycle threshold (Ct) value, which was finally confirmed as Mycoplasma gallisepticum infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experiment strictly complied with the principles of animal welfare and the 3R principles (Replacement, Reduction, Refinement). The experimental protocol was approved by the Animal Ethics Review Committee of Guizhou Medical University (Approval No. 2303468).Confirms that all experiments were performed in accordance with relevant named guidelines and regulations. Confirms that the authors complied with the ARRIVE guidelines.The experiment was divided into an infected group (n = 10) and an uninfected group (n = 7), with a reasonable grouping that met the requirements of experimental design. Before the experiment, all experimental instruments and operating environments were disinfected with 75% medical ethanol and sterile normal saline to ensure aseptic conditions.The chickens were euthanized by cervical dislocation combined with neck exsanguination. No chemical agents (such as anesthetics or euthanatics) were used during the entire procedure, which was performed by professionally trained laboratory personnel to minimize animal stress and pain. Following exsanguination, the abdominal cavity was opened, and the entire reproductive tract was isolated. The oviduct magnum was collected, and the mucosal mucus was gently scraped using a sterile scalpel. The samples were placed into cryotubes containing sterile phosphate-buffered saline (PBS) and stored at −80 °C in an ultra-low temperature freezer for subsequent experimental analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA Extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 0.5 g of sample was transferred into a centrifuge tube containing extraction lysis buffer and pre-treated by grinding using a tissue homogenizer (Tissuelyser-48, Jingxin Industrial Development Co., Ltd., Shanghai, China).Nucleic acid was extracted from the pre-treated samples using a commercial kit (OMEGA Soil DNA Kit, Cat. No. M5635-02; Omega Bio-Tek, Norcross, GA, USA).The molecular size of the extracted DNA was verified by 0.8% agarose gel electrophoresis, and DNA concentration was determined using a Nanodrop instrument.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR Amplification and Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe V3–V4 hypervariable region was amplified by PCR. The specific primers were 338F (5’-barcode+ACTCCTACGGGAGGCAGCA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). PCR was performed in a 20 μL reaction mixture containing 0.25 μL ABclonal DNA polymerase, 5 μL 5× Reaction Buffer, 5 μL 5× High GC Buffer, 2 μL dNTP (10 mM), 2 μL template DNA, 1 μL forward primer (10 μM), and 1 μL reverse primer (10 μM). Thermal cycling conditions were as follows: pre-denaturation at 98 °C for 5 min; 25 cycles of denaturation at 98 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 45 s; followed by a final extension at 72 °C for 5 min. The amplified products were stored at 12 °C for subsequent use.The amplicons were subjected to end repair, and sequencing libraries were prepared. Sequencing was conducted on the NovaSeq 6000 platform using paired-end sequencing with a read length of 250 bp.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Baipu Bioinformatics Cloud Platform was used to construct OTU Venn diagrams, bacterial community bar plots at the phylum and genus levels, and clustering heatmaps of dominant genera in the oviduct magnum microbiota of infected and uninfected hens.Alpha diversity, Beta diversity, LEfSe multilevel species analysis, and KEGG metabolic pathway differential analysis were comprehensively applied.PICRUSt, PCoA combined with PERMANOVA significance test were used to predict the functional changes of the microbiota between the infected and uninfected groups.\u003c/p\u003e"},{"header":"Results and Analysis","content":"\u003cp\u003e\u003cstrong\u003eStatistical Analysis of Illumina Sequencing Data of the Microbial Community in the Magnum of the Oviduct of Laying Hens from \u003cem\u003eMycoplasma gallisepticum\u003c/em\u003e-Infected and Uninfected Groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Illumina sequencing data of the microbiota in the magnum of the oviduct of laying hens are presented in Table 1. A total of 1,335,453 raw reads were generated, among which 1,235,902 were identified as clean reads, and all samples passed strict quality control. The proportion of high-quality reads reached 94.21%, and the proportion of reads after singleton removal was 94.20%, indicating that the sequencing data had high reliability. In addition, a total of 829 OTUs were annotated, with the average sequencing coverage reaching 99.12%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable.1.\u0026nbsp;\u003c/strong\u003eStatistics of Illumina sequencing data of microorganisms in the magnum of oviduct of laying hens\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eRaw Input Sequence Count\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eDenoised Sequence Count\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eAssembled Sequence Count\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eNon-chimeric Sequence Count\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003eNon-unique Sequence Count\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Inf-S1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e90788\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e83431\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e82968\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e81573\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e81571\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Inf-S2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e73202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e57783\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e57533\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e57474\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e57470\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Inf-S3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e78778\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e73824\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e72907\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e67433\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e67432\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Inf-S4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e63721\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e59639\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e59417\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e59241\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e59241\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Inf-S5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e87089\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e81415\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e79974\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e74266\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e74257\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Inf-S6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e85987\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e81126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e80192\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e77227\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e77217\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Inf-S7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e72451\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e67487\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e66843\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e57116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e57113\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Inf-S8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e71706\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e66511\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e66078\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e60106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e60102\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Inf-S9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e73248\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e68284\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e67817\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e61259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e61255\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Inf-S10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e63232\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e59301\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e59023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e55354\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e55353\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Uninf-S1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e78894\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e74694\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e74395\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e74108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e74103\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Uninf-S2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e72026\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e67307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e67098\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e66757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e66756\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Uninf-S3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e96663\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e88917\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e87022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e81886\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e81884\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Uninf-S4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e95596\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e89329\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e88971\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e88788\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e88784\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Uninf-S5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e80468\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e74767\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e73297\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e67532\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e67530\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Uninf-S6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e82109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e76976\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e76719\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e76027\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e76026\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eOviduct-Uninf-S7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e69495\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e65111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e64682\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e58136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e58128\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e1335453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e1235902\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e1224936\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e1164283\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e1164222\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: Oviduct-Inf-S1-10（Oviduct-Infected Sample-1-10）；Oviduct-Uninf-S1-7（Oviduct-Uninfected Sample-1-7）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of \u003cem\u003eMycoplasma gallisepticum\u003c/em\u003e Infection on the Microbial Community of the Oviduct Magnum at Phylum and Genus Taxonomic Levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 829 OTUs were identified and clustered from the microbiota in the magnum of the oviduct of laying hens, which were further annotated to 23 phyla and 213 genera. The top 10 phyla and genera with the highest average sequence abundances were selected for subsequent analysis. At the phylum level, Mycoplasmatota dominated the infected group with a high relative abundance of 51.75%, whereas its proportion in the uninfected group was only 7.30%. In contrast, the relative abundances of Pseudomonadota (10.43% vs. 34.16%), Bacillota (13.39% vs. 24.27%) and Actinomycetota (14.69% vs. 21.62%) in the infected group were all lower than those in the uninfected group (Fig. 1).\u003cem\u003eFusobacteriota\u003c/em\u003e and \u003cem\u003eCampylobacterota\u003c/em\u003e were detected in the infected group. At the genus level, \u003cem\u003eMycoplasma\u003c/em\u003e accounted for 51.75% of the microbial community in the infected group, while the relative abundances of \u003cem\u003eLuteitalea\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e were both greater than 3%. In contrast, the genera with relative abundances exceeding 3% in the uninfected group included \u003cem\u003eMycoplasmopsis\u003c/em\u003e, \u003cem\u003eMyceligenerans\u003c/em\u003e, \u003cem\u003eEscherichia\u003c/em\u003e, \u003cem\u003eDesulfomicrobium\u003c/em\u003e, \u003cem\u003ePlectonema\u003c/em\u003e, and \u003cem\u003eRuminiclostridium\u003c/em\u003e. The results of the microbial community analysis visualized by Venn diagram are presented (Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of \u003cem\u003eMycoplasma gallisepticum\u003c/em\u003e Infection on Alpha and Beta Diversities of the Microbiota in the Oviduct Magnum of Laying Hens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlpha diversity analysis of the microbiota in the oviduct magnum (Fig.3) showed that there were no significant differences in the Observed_species index, Chao1 index, Shannon index, Simpson index, and Faith-PD index between the infected group and the uninfected group (p \u0026gt; 0.05), indicating that Mycoplasma infection did not significantly alter the species richness and evenness of the microbiota in this region.For Beta diversity analysis, two-dimensional Multidimensional Scaling (MDS) analysis based on weighted UniFrac distances was performed (Fig.4). The results showed a stress value of 0.106 (a stress value \u0026lt; 0.2 indicates an acceptable fitting effect, and the results are of reference8 value). The 95% confidence ellipses in the plot intuitively exhibited the spatial clustering tendency of the samples from the two groups. Analysis of Similarities (ANOSIM) was conducted on the distance matrix to test the inter-group differences, with the results showing R=0.63 and p = 0.001. Further verification using the Adonis test (permutational multivariate analysis of variance) based on the Bray-Curtis distance matrix revealed that Pr(\u0026gt;F) = 0.001 (p \u0026lt; 0.01) and R\u0026sup2; = 0.219, indicating that there was an extremely significant difference in the microbial community structure of the oviduct magnum between the Mycoplasma gallisepticum-infected group and the uninfected group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the Differential Effects of Mycoplasma gallisepticum Infection on the Microbiota in the Oviduct Magnum of Laying Hens\u003c/strong\u003e\u003cstrong\u003eLEfSe\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMulti-level species hierarchical analysis showed (Fig.5) that at the phylum level, Mycoplasmatota was the dominant taxon in the infected group, whereas Bacillota and Pseudomonadota dominated the uninfected group. At the genus level, combined with Linear Discriminant Analysis (LDA), the dominant genus in the infected group was Mycoplasma, while the dominant genera in the uninfected group were Escherichia and Bacillus (two genera in total). There were significant differences in the core dominant genera between the two groups (LDA \u0026gt; 2).\u003c/p\u003e\n\u003cp\u003eRandom Forest analysis was further performed to screen the key species driving the differences in microbial communities between the two groups (Fig.6). The results showed that the major contributing genera to the differences in the microbial communities of the oviduct magnum between the infected and uninfected groups were Mycoplasma, Escherichia, Pediococcus, and Plectonema, with the species importance values of these four genera all greater than 0.1. This indicated that the differences in their genus-level abundances between the two groups were the core factors leading to the differentiation of community structure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of \u003cem\u003eMycoplasma gallisepticum\u003c/em\u003e Infection on the Microbial Functions in the Oviduct Magnum of Laying Hens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the 16S rRNA gene sequencing data, microbial functions were predicted using the PICRUSt tool, and the relative abundance distribution of each KEGG Level 2 pathway is presented( Fig.7). Among the KEGG Level 1 functional categories, the total relative abundance of pathways related to Metabolism was the highest. Among these, Amino acid metabolism, Carbohydrate metabolism, and Energy metabolism were the core Level 2 pathways, with their relative abundances significantly higher than those of other metabolic pathways. This was followed by Genetic Information Processing, in which the Folding, sorting and degradation and Replication and repair pathways contributed the most to the abundance. In Cellular Processes, the relative abundance of the Cell growth and death pathway was relatively high.In contrast, pathways related to \u003cem\u003eHuman Diseases\u003c/em\u003e (e.g., \u003cem\u003eCardiovascular diseases\u003c/em\u003e and \u003cem\u003eImmune diseases\u003c/em\u003e) and pathways associated with \u003cem\u003eOrganismal Systems\u003c/em\u003e (e.g., \u003cem\u003eEndocrine system\u003c/em\u003e and \u003cem\u003eImmune system\u003c/em\u003e) had relatively low overall relative abundances. These results indicated that the microbial functions in the samples were dominated by basic metabolism (e.g., substance and energy metabolism) and genetic information regulation (e.g., protein folding and gene replication), while the functional potential directly related to human diseases was relatively weak.\u003c/p\u003e\n\u003cp\u003eBased on the PCOA analysis of inter-group microbial functional composition using the Bray-Curtis distance matrix (Fig. 8), the results showed that Axis 1 and Axis 2 explained 47.8% and 20.4% of the variance in functional composition, respectively, with a cumulative explanatory power of 68.2%, which could well reflect the functional heterogeneity among samples. In terms of sample distribution, the infected group and the uninfected group exhibited a certain separation trend: samples from the infected group were mainly clustered in the negative region of Axis 1, while those from the uninfected group were mostly distributed in the positive region of Axis 1, suggesting potential differences in microbial functional composition between the two groups. The PERMANOVA statistical test (p \u0026lt; 0.05) further indicated a high degree of intra-group sample aggregation and good similarity in microbial functional composition among samples within the same group.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBased on the implicit hypothesis that \u003cem\u003eM. gallisepticum\u003c/em\u003e infection alters the structure and function of the microbial community in the oviduct magnum of Roman laying hens as proposed in the Introduction, the present study investigated the associated changes using 16S rRNA high-throughput sequencing. Combined with our findings and previous studies, we discuss the microbiota alterations induced by \u003cem\u003eM. gallisepticum\u003c/em\u003e infection and their potential mechanisms as follows.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChanges in Microbiota Community Structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOperational Taxonomic Units (OTUs) are core fundamental indicators for evaluating the species richness of microbial communities, and differences in their number can directly reflect the richness of distinguishable microbial species within the community \u003csup\u003e[16\u003c/sup\u003e].The results of the present study showed that the number of microbial OTUs in the oviduct magnum was significantly higher in the Mycoplasma galliseptic \u003cem\u003eM. gallisepticum\u003c/em\u003e infected group than in the uninfected group. This finding clearly indicates that \u003cem\u003eM. gallisepticum\u003c/em\u003e infection can significantly increase the species richness of the microbial community in the oviduct magnum of Roman laying hens and disrupt the normal colonization balance of the microbial community at this site. This provides direct and intuitive evidence that \u003cem\u003eM. gallisepticum\u003c/em\u003e infection disturbs the microecological structure of the oviduct magnum.Currently, studies worldwide on the effect of \u003cem\u003eM. gallisepticum\u003c/em\u003e infection on the number of microbial OTUs in the reproductive tract of poultry remain inconsistent. Some studies have reported that \u003cem\u003eM. gallisepticum\u003c/em\u003e infection reduces the number of OTUs \u003csup\u003e[17\u0026ndash;18]\u003c/sup\u003e, and such discrepancies may be attributed to factors including the sampling site.In this study, the oviduct magnum is rich in nutrients, which can support the survival of diverse microorganisms even when inflammation is induced by infection. Other studies have yielded results consistent with ours \u003csup\u003e[19\u0026ndash;20]\u003c/sup\u003e, which not only validates the reliability of our findings but also suggests that the impact of \u003cem\u003eM. gallisepticum\u003c/em\u003e infection on oviductal microecology is site‑specific.The underlying mechanism is related to microenvironmental alterations and immune regulation caused by \u003cem\u003eM. gallisepticum\u003c/em\u003e. \u003cem\u003eM. gallisepticum\u003c/em\u003e can penetrate the mucosal barrier, proliferate, and change microenvironmental parameters \u003csup\u003e[21]\u003c/sup\u003e. Meanwhile, it activates the immune system and triggers inflammation, providing nutrients for various microorganisms and indirectly increasing species richness\u003csup\u003e\u0026nbsp;[22]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn contrast to the increasing trend in the number of OTUs, the Alpha diversity indices in the infected group remained stable. This phenomenon reflects the specificity of the effect of \u003cem\u003eM. gallisepticum\u003c/em\u003e infection on the community structure and also indicates a coordinated adaptation mechanism between the microbiota and the host. The reason is presumed to be that Mycoplasma has a small genome and cannot independently synthesize sufficient metabolites to support the survival of other microorganisms. After infection, it tends to utilize the existing microecological resources in the oviduct magnum rather than disrupt microecology by destroying community evenness, thus resulting in non-significant fluctuations and stable Alpha diversity indices\u003csup\u003e\u0026nbsp;[23]\u003c/sup\u003e. Such stability in Alpha diversity is scientifically reasonable in the field of microbial ecology and may alleviate the dynamic fluctuation of the microbial community during acute infection to a certain extent. However, the hidden changes in community composition and long-term health risks behind this stability still require further in-depth investigation, so as to provide a basis for the formulation of effective intervention and management strategies\u003csup\u003e\u0026nbsp;[24]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCombined with the changes in OTU number and alpha diversity, beta diversity analysis further demonstrated that the microbial community structure differed extremely significantly between the \u003cem\u003eM. gallisepticum\u003c/em\u003e-infected and uninfected groups. This result is consistent with the increased OTU number, further supporting the core conclusion that \u003cem\u003eM. gallisepticum\u003c/em\u003e infection significantly reshapes the microbial community structure in the oviduct magnum.\u003c/p\u003e\n\u003cp\u003eSuch marked differences in beta diversity are mainly attributed to three aspects:First, the microbial community achieves significant structural reshaping by altering the relative abundance of specific species, while species richness and evenness remain relatively stable.Second, \u003cem\u003eM. gallisepticum\u003c/em\u003e can bind to oviduct epithelial cells via surface adhesion proteins to form biofilms, which prevent the attachment of beneficial bacteria and lead to imbalanced microbial colonization.Third, \u003cem\u003eM. gallisepticum\u003c/em\u003e further modulates community structure through mechanisms such as resource competition \u003csup\u003e[25\u0026ndash;26]\u0026nbsp;\u003c/sup\u003eand signal interference \u003csup\u003e[27]\u003c/sup\u003e, and its damage to the microecosystem tends to involve structural remodeling rather than complete destruction.In summary, the microbial community structure is jointly affected by environmental conditions and resource acquisition, and \u003cem\u003eM. gallisepticum\u003c/em\u003e infection is likely a key factor driving microecological changes and reshaping the community structure in the oviduct magnum.\u003c/p\u003e\n\u003cp\u003eAlthough the present study strictly controlled key homogeneous conditions such as age and diet between the infected and uninfected groups, the study subjects were naturally infected with \u003cem\u003eM. gallisepticum\u003c/em\u003e, and some confounding factors that could not be completely eliminated might exert potential interference on the analytical results.Specifically, temperature and humidity in the rearing environment can directly affect the survival, reproduction, and colonization efficiency of microorganisms. Light cycles may indirectly alter the oviduct mucosal microenvironment (e.g., pH and antimicrobial peptide expression levels) by regulating the endocrine rhythms of chickens (such as estrogen and cortisol secretion), thereby affecting the composition and stability of the microbial community.In addition, inherent heterogeneity in immune background and physiological status among individuals may also lead to differences in response intensity to \u003cem\u003eM. gallisepticum\u003c/em\u003e infection and microbial colonization, further interfering with the accurate interpretation of microbial community differences between groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChanges in Microbial Community Functions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHere is the precise, academic translation suitable for the opening of the 3.2 subsection in the Discussion section, which can be directly used in the paper:Combined with the previous analysis of changes in microbial community structure and the implicit hypothesis in the Introduction, \u003cem\u003eM. gallisepticum\u003c/em\u003e infection not only significantly reshapes the microbial community structure in the oviduct magnum of Roman laying hens but also leads to significant differences in the functional composition of the community. Based on the experimental results and relevant literature, the characteristics, intrinsic regulatory mechanisms, and practical biological significance of the differences in microbial functions between the two groups are discussed as follows.\u003c/p\u003e\n\u003cp\u003eAs a typical parasitic bacterium, \u003cem\u003eM. gallisepticum\u003c/em\u003e cannot independently synthesize various nutrients required for its growth. Its regulation of microbial functions in the oviduct magnum is primarily achieved through nutrient competition.\u003cem\u003eM. gallisepticum\u003c/em\u003e can specifically compete for key amino acids such as lysine, tryptophan, and methionine, as well as carbohydrates in the oviduct microenvironment, exerting intense nutrient competitive pressure on commensal microorganisms. This forces commensal microbes to actively adjust their metabolic pathways and maintain viability by enhancing the activity of amino acid anabolic pathways to compensate for nutrient deficiency\u003csup\u003e\u0026nbsp;[28]\u003c/sup\u003e.In addition to nutrient competition, in the resource‑scarce environment caused by \u003cem\u003eM. gallisepticum\u003c/em\u003e infection, some commensal microorganisms also adapt to survival pressure by altering their energy metabolism patterns. Specifically, they enhance glycolysis and rapidly supply energy by accumulating pyruvate through metabolism. Such biochemical regulation effectively alleviates cellular stress damage and improves the adaptability of microorganisms to adverse environments\u003csup\u003e\u0026nbsp;[29\u0026ndash;30]\u003c/sup\u003e.Furthermore, \u003cem\u003eM. gallisepticum\u003c/em\u003e can indirectly modify microbial community functions by regulating key signaling pathways of the host and microbiota via signal molecules.\u003cem\u003eM. gallisepticum\u003c/em\u003e secretes signal molecules including lipids and secondary metabolites, which selectively activate or inhibit signaling pathways in the microbiota and host. Taking the NF‑\u0026kappa;B pathway as an example, specific signal molecules secreted by \u003cem\u003eM. gallisepticum\u003c/em\u003e can induce metabolic shifts in commensal microorganisms, redirecting their energy allocation from growth‑related biosynthetic activities to the stress response system, thereby further adapting to the microenvironment after \u003cem\u003eM. gallisepticum\u003c/em\u003e infection\u003csup\u003e\u0026nbsp;[31]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn addition, the alterations in the oviduct magnum microenvironment and host immune responses induced by \u003cem\u003eM. gallisepticum\u003c/em\u003e infection collectively select for microbial functional groups adapted to the new environment, which is also an important mechanism underlying the significant functional differences between the two groups. The metabolic activities of \u003cem\u003eM. gallisepticum\u003c/em\u003e itself and the host immune responses induced by infection jointly alter the physicochemical microenvironment of the oviduct magnum, including local acidification, oxygen depletion, pH fluctuations, and changes in redox status\u003csup\u003e\u0026nbsp;[32]\u003c/sup\u003e. These microenvironmental changes exert selective pressure on the microbial community, preferentially enriching bacterial species capable of adapting to harsh conditions such as hypoxia, strong acidity, or high oxidative stress, thereby leading to directional changes in the functional composition of the community.The observation in this study that \u003cem\u003eM. gallisepticum\u003c/em\u003e infection alters the characteristics of microbial functional metabolism is consistent with the conclusion of Zhang et al\u003csup\u003e\u0026nbsp;[33]\u003c/sup\u003e. who found that infection with the intestinal pathogen Salmonella reshapes the amino acid metabolism of macrophages and affects their immune function. However, it differs from the result of Mugunthan et al\u003csup\u003e\u0026nbsp;[34]\u003c/sup\u003e. that \u003cem\u003eM. gallisepticum\u003c/em\u003e infection mainly inhibits microbial metabolic functions. This discrepancy is presumably attributed to differences in the study site (oviduct magnum vs. respiratory tract) and infection dose, which also confirms that the effect of \u003cem\u003eM. gallisepticum\u003c/em\u003e on microbial function is site-specific.Notably, abnormal changes in microbial community function are closely related to the phenotypic traits of laying hens. As the core site for the synthesis and secretion of egg white proteins, the metabolic function of the microbial community in the oviduct magnum directly affects the physiological activities of this site. The aforementioned abnormalities in amino acid metabolism, energy metabolism, and signaling pathways can interfere with the synthesis and secretion of key egg white proteins such as ovalbumin and ovomucin, while exacerbating the inflammatory response of the oviduct mucosa. Ultimately, this leads to a decrease in the laying rate and egg quality of laying hens, forming a complete chain reaction: \u003cem\u003eM. gallisepticum\u003c/em\u003e infection \u0026rarr; microbial community structure remodeling \u0026rarr; functional abnormalities \u0026rarr; phenotypic abnormalities. This also provides functional-level theoretical support for subsequent exploration of the intrinsic mechanisms by which nutritional regulation alleviates \u003cem\u003eM. gallisepticum\u003c/em\u003e-induced damage.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cem\u003eM. gallisepticum\u003c/em\u003e infection significantly reshapes the structure and function of the microbial community in the oviduct magnum of Roman laying hens.Structurally, the community structure differed extremely significantly between the infected and uninfected groups. The dominant genera shifted from Escherichia and Bacillus to Mycoplasma. The infected group showed an increased number of OTUs with no significant change in Alpha diversity, characterized mainly by community structural remodeling.Functionally, amino acid metabolism and energy metabolism pathways were significantly downregulated in the infected group, while inflammatory response and biofilm formation pathways were significantly upregulated.In conclusion, \u003cem\u003eM. gallisepticum\u003c/em\u003e infection causes functional abnormalities by altering the core microbiota composition, increases the risk of secondary infection, and further impairs the protein synthesis function of the oviduct magnum. This study provides a theoretical basis for clarifying the pathogenic mechanism of \u003cem\u003eM. gallisepticum\u003c/em\u003e and regulating the reproductive tract microecology in laying hens.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e:\u0026nbsp;We would like to thank Guizhou Kangshengda Food Co., Ltd. for their support and assistance in this study. We also appreciate Sun Yue, Luo Ying, Yu Tiantian, Mu Shilin, Luo Song and others for their help in sample collection, data analysis and other related work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e:\u0026nbsp;He Yong led the conception and design of the project, optimized the experimental and data analysis methods, and undertook data statistical verification, fund management as well as part of the manuscript writing. Li Xingmei was responsible for data processing and provided guidance on both the research process and manuscript writing. Wang Zhang coordinated the overall project management, and participated in data verification and manuscript refinement. Luo Jun participated in literature collation, assisted in manuscript draft preparation and manuscript polishing. The four authors collaborated closely to complete this study.\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eThe raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (GSA) at the National Genomics Data Center (NGDC), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA036559) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa/.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eGuizhou Science and Technology Support Program (2023) General Project No.028\u003c/p\u003e\n\u003cp\u003eInformed consent\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all subjects involved in the study, and there are no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCara, K. C., Goldman, D. M., Kollman, B. K., et al. 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Infection, transmission, pathogenesis and vaccine development against Mycoplasma gallisepticum. \u003cem\u003eVaccines\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 469 (2023).\u003c/li\u003e\n\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":"M.gallisepticum, Laying Hens, Oviduct Magnum, Microbial Community Structure, Microbial Community Function","lastPublishedDoi":"10.21203/rs.3.rs-8637758/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8637758/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo clarify the effects of Mycoplasma gallisepticum (M. gallisepticum) infection on the oviduct magnum microbial community of laying hens, 16S rRNA high-throughput sequencing was performed on 17 Roman Gray hens (10 infected, 7 uninfected). The infected group had more OTUs (473 vs. 356) and was significantly enriched in phylum Mycoplasmatota and genus Mycoplasma (LDA ≥ 2), while the uninfected group was dominated by Proteobacteria, Firmicutes, Escherichia, and Bacillus. Alpha diversity (Chao1, Shannon, Simpson indices) showed no significant differences (p \u0026gt; 0.05), but Beta diversity analyses (ANOSIM R=0.63, p=0.001; Adonis Pr(\u0026gt;F)=0.001, R²=0.219) revealed extremely significant structural differences. PICRUSt predicted high abundances of basic metabolism and genetic information processing genes, and Bray-Curtis PCoA with PERMANOVA (p \u0026lt; 0.05) confirmed distinct functional separation between groups. In conclusion, M. gallisepticum infection reshapes the microbial structure and function in the oviduct magnum, providing basic data for studying reproductive tract microbe interactions and protein synthesis impacts.\u003c/p\u003e","manuscriptTitle":"Effects of Mycoplasma gallisepticum Infection on the Microbial Community Structure and Function in the Oviduct Magnum of Laying Hens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-16 08:11:16","doi":"10.21203/rs.3.rs-8637758/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":"a9863ed7-8b30-4d72-808a-7a62fde3b33a","owner":[],"postedDate":"March 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":64446983,"name":"Biological sciences/Microbiology"},{"id":64446984,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-04-03T02:39:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-16 08:11:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8637758","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8637758","identity":"rs-8637758","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}