Age-related development and functional maturation of bronchus-associated lymphoid tissue (BALT) in broiler chickens

Age-related development and functional maturation of bronchus-associated lymphoid tissue (BALT) in broiler chickens | 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 Short Report Age-related development and functional maturation of bronchus-associated lymphoid tissue (BALT) in broiler chickens Shama Shanjida Shaon, Orthi Banik, Md. Abdul Masum, Subrato Biswas, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7584583/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 Around the world, broiler chickens are an essential source of affordable animal protein. But their intensive farming practices make them particularly vulnerable to respiratory infections, which cause significant economic losses. To control these respiratory infections and to establish therapeutic targets against diseases, a deep understanding of the respiratory immune system is essential. Bronchus-associated lymphoid tissue (BALT) is the main entry point of pathogens for chickens, but its development and cellular constituents in broiler chickens under physiological conditions remain poorly understood. Our present study aimed to investigate the age-related development, cellular composition, and role of BALT in the lungs of healthy broiler chickens. BALT was absent in day-old chicken, but small BALT appeared at 15 days of age, which later fully developed at 32 days of age. Cellular analysis revealed that fully developed BALT is composed of B-cells, T-cells, and macrophages. It also contains a well-organized vascular network, including high endothelial venules and lymphatic vessels. BALT shows the presence of macrophage and MHCII⁺ molecules, which indicate that antigen presentation occurs in BALT. In addition, germinal centers are present in mature BALT, indicating the maturation of B-cells and production of plasma cells. Therefore, BALT offers a new immunological gateway for respiratory-related therapeutic research and vaccine development. Bronchus Lymphoid tissue Lung Chicken Figures Figure 1 Figure 2 Figure 3 Introduction Nowadays, broiler chickens are a major source of affordable and superior animal protein in global agriculture compared to other types of animal protein. (Abbas et al., 2025). To achieve a higher production rate poultry farm requires intensive management. Despite vaccination, birds in conventional poultry farms are frequently exposed to different types of pathogens, especially respiratory pathogens such as infectious bronchitis virus, Mycoplasma species, and Newcastle disease virus (Liu et al., 2025). These respiratory diseases have become a potential threat against the developing poultry industry. As birds are highly susceptible to respiratory pathogens, the understanding of respiratory immunity in poultry is very important (Yehia et al., 2023). The immune system in poultry plays a critical role in defending against pathogens and maintaining productivity (Adams et al., 2023). The broiler immune system is comprised of central (primary) lymphoid organs—the thymus and bursa of Fabricius—and peripheral (secondary) lymphoid tissues, including mucosa-associated lymphoid tissue (MALT). MALT maintains the immunological defense at the body's inner and outer mucosal surfaces and is found in different mucosal organs such as the gut (gut-associated lymphoid tissue or GALT) and airways (bronchial-associated lymphoid tissue or BALT) (Knop et al., 2005). BALT might play a central role in antigen uptake, initiating immune responses and lymphoid cell distribution in the respiratory tract. Previous studies show that in normal conditions, BALT is absent in human and mouse lungs; however, it forms in pathological conditions (Randall, 2010; Tango et al., 2000). BALT is present in the lungs of rats and rabbits in both physiological and pathological conditions, regardless of age (Gregson et al., 1979; Seefeld et al., 2024). In addition, cellular characterization of BALT and identification of vascular networks have been done in the case of mice and humans (Randall, 2010; Tango et al., 2000). In disease conditions, BALT plays a significant role by initiating local immune responses in the lungs in various species. Additionally, inducible BALT (iBALT) forms in humans, mice, rats, and rabbits in response to infection or inflammation and aids in removing antigens from the airways, triggering adaptive immunity, and preserving memory cells (Halle et al., 2009; Seefeld et al., 2024). In broiler chicken, age-related development of BALT has been studied using light and electron microscopy, but detailed cellular characterization has not been done (Fagerland & Arp, 1993). Moreover, how cellular components interact and contribute to protective immunity or pathology has not been established. So detailed cellular characterization, antigen presentation, and germinal center identification are necessary to establish the role of BALT in physiological as well as pathological conditions. In our present study, we have characterized the development of BALT from 1 day to 32 days of age and observed that BALT first appears diffusely at the age of 15 days. We have also done cellular characterization and identified germinal centers in BALT. Materials and methods Management of birds For this study, 120-day-old Lohman Meat (Indian River) unsexed broiler chicks were purchased from Kazi Farms Ltd. Upon arrival at the experimental farm, the chicks were randomly divided into three groups, each comprising 20 birds with two replications per group. For the initial age-group examination, 10-day-old chicks were randomly selected for sample collection from the first age group. The remaining chicks were reared under standard husbandry conditions. Adequate lighting and ventilation were ensured throughout the study period. Feed and water were provided ad libitum to all birds. Sample collection The birds of each age group were sacrificed following a cervical dislocation and the lungs of birds were collected. The collected lungs were trimmed into small pieces and fixed with 10% neutral buffered formalin for 24 hours for histopathological examination. Hematoxylin & Eosin Staining The collected samples were blocked with paraffin and trimmed to 3 µm thickness using a microtome. The samples were stained with hematoxylin & eosin stains for general histopathological analysis. Immunofluorescence Immunodetection of cell markers was performed as in our previous study (Akter et al., 2024) for B cells (Bu-1), T cells (CD3), and macrophages (KUL01). Lung tissues were deparaffinized and treated overnight with primary antibodies, followed by secondary antibodies or Alexa Fluor 546-labeled donkey anti-rabbit antibodies. Nuclei were counterstained with Hoechst 33342, and sections were visualized under a fluorescence microscope. Histoplanimetry The entire lung has been collected, trimmed, and embedded in paraffin. Lung sections were cut to 3 µm thickness at 20–30 µm intervals and stained to confirm the presence of lymphoid clusters. The clusters were detected using a Euromex BS.1153-EPLi microscope (S/N-EC 2221114). Statistical analysis A binary logistic regression was conducted to assess the trend in BALT-positive samples across the three time points (days 0, 15, and 32), with time coded as an ordinal variable (0, 1, and 2). A p-value of < 0.05 was considered statistically significant. Result Clarification of bronchus-associated lymphoid tissue (BALT) in the lungs at different ages of broiler chicken We investigated the presence of bronchus-associated lymphoid tissue (BALT) in broiler chicken lungs at various stages (Fig. 1 ). The day-old chick’s lung tissue shows the normal parenchyma, which had no diffuse or nodular lymphatic clusters (Fig. 1 A). At 15 days of age, some small lymphatic nodule was found (Fig. 1 B). In the later stage (32 days), a distinct, organized lymphoid cluster was found near the bronchus (boxed area), which had a germinal center (circled area) (Fig. 1 C). For BALT, 0, 2, and 6 positive samples were observed at Days 0, 15, and 32, respectively (Fig. 1 D). Binary logistic regression showed an increasing trend in BALT-positive samples over time (p = 0.997). Cellular characterization and vascular network of BALT in lungs Immune cells were characterized in the lung tissue by immunofluorescence using specific cell markers in day-old broiler chickens. Various immune cell populations were identified within BALT, including Bu-1⁺ B-cells, CD3⁺ T-cells, and KUL01⁺ macrophages. The fluorescent green dots dispersed around the germinal center indicate B-cells, while T-cells were more diffusely distributed throughout the tissue (Fig. 2 A- 2 B). Macrophages, marked by KUL01, appear as densely packed clusters, which indicate follicular organization (Fig. 2 C). So, this cluster is considered BALT. We also identified vascular networks in BALT by H&E staining. We observed high endothelial venules (HEV) in BALT, and lymphatic vessels were also observed in BALT (Fig. 2 D, 2 E). All these findings suggest that a well-vascularized network is present in BALT in broiler chicken lungs. Antigen presentation and germinal center formation in BALT We identified antigen presentation in BALT at 32 days of age. We observed KUI01⁺ macrophages in BALT (Fig. 3 A). Next, we observed major histocompatibility complex (MHC) class II⁺ molecules. MHC II⁺ molecules were found throughout the tissue (Fig. 3 B). These findings suggest antigen presentation in BALT in chicken lungs. In the earlier stages (day-old and 15 days), we did not find any germinal center (Fig. 3 C, 3 D), whereas in the case of the later stage (32 days), we found a well-formed germinal center (Fig. 3 E). Discussion Nowadays, broiler chickens are very important globally as a primary source of affordable and high-quality animal protein. In developing countries, broiler meat is not only the most affordable source of animal protein but also the most accessible, making it indispensable for food security (Attia et al., 2022). Under intensive farming systems, the health and productivity of broilers depend heavily on their immune system, which serves as the first line of defense against diseases. It is obvious that a strong immune system is essential for defending birds against contagious diseases (Casteleyn et al., 2010; Lebedev et al., 2024). Importantly, MALT plays a critical role in maintaining mucosal immunity. MALT in lungs like bronchus-associated lymphoid tissue (BALT), which are strategically located at mucosal surfaces to detect pathogens (Cesta, 2006; Pabst, 2022). These structures are developed to provide effective protection against respiratory diseases. But the development of BALT in physiological conditions has not been fully elucidated yet. Our present study clarified the developments and organization of BALT in the lungs of broiler chickens at different stages of age (day 1 to day 32). MALT is present in different forms in different parts of the body (respiratory, gastrointestinal, and urogenital tracts), which is commonly composed of B-cells, T-cells, and macrophages (Akter et al., 2024). If the MALT is found in a physiological condition, it will be a secondary structure that can grow before birth or after birth, and if it is found in a diseased condition, it will be considered a tertiary structure (Pabst, 2022; Peralta et al., 2017). Previous study reports show that the MALT is found around the bronchus. In our study we examined the development of immune cell clusters at various ages, where at the earlier stage we did not find any clusters around the bronchus, but at the age of 15 days a disorganized small lymphoid aggregation became apparent. At the age of 32 days, a fully well-formed lymphoid cluster was formed. So, we can consider that BALT was not found in the initial stage, but after 15 days it appeared deliberately and fully formed at the later stage, which indicates the probable BALT (Fig. 1 ). As we found the cellular cluster at the later stage, we did cellular characterization using different cell markers to confirm the complex organization of immune cells at the later stage. We found that BALT was composed of B cells, T cells, and macrophages. So, BALT acts like a complete MALT, whereas we found all immune-component cells (Fig. 2 ). The vascular network consists of high endothelial venules (HEVs) and lymphatic vessels. These are crucial for cellular trafficking into lymphoid tissues, which invites the immune cells into the circulation (Fagerland & Arp, 1993). In our study, we examined the vascular network in the fully developed BALT of chicken lungs at the age of 32 days and identified distinct HEV and lymphatic vessels. These findings suggest that BALT in chicken lungs at a later stage has a well-established vascular network (Fig. 2 ). The main functions of BALT are antigen presentation, lymphocyte activation, and maturation (Blanchard & Girard, 2021). In our present study, we examined the antigen presentation in BALT of chicken lungs to clarify its functional activity. We observed macrophages, T-cells, and MHCII⁺ molecules present in BALT. Thus, we can suggest that antigen presentation has occurred in the BALT of broiler chicken lungs (Fig. 3 ). Germinal centers are aggregations of B cells that respond to antigens, particularly following T cell-dependent immune responses. The distinct germinal center indicates B-cell proliferation, which produces active antibodies (Ceccopieri & Madej, 2024). Interestingly, in our study the germinal center formation was absent in the earlier stage but became evident at the later stage (32 days). So, we can consider that the formation of germinal centers within BALT indicates structural maturity and also active humoral immunity at later stages of development (Figs. 2 & 3 ). In conclusion, a lymphoid cluster or BALT is present close to the bronchus in the lungs of broiler chickens, which consists of B-cells, T-cells, and macrophages. BALT has a well-designed vascular network for cellular trafficking (Fig. 4). In addition, antigen presentation and germinal center are present as lymphoid tissue organizers (Fig. 4). So, it shows that BALT is a complete, immunologically active lymphoid tissue. The future study will provide more information about the role of BALT in pathological conditions. Declarations Authors contribution SSS: Writing – review & editing, Writing – original draft, Visualization, Software, Resources, Methodology, Investigation, Data curation. OB: Writing – review & editing, Writing – original draft, Investigation, Software, Methodology, Investigation, Data curation. MAM: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Conceptualization. SB: Writing – review & editing, Writing – original draft, Visualization, Software, Investigation, Formal analysis. SH: Software, Methodology, Investigation, Data curation. MJUR: Writing – review & editing, Validation, Methodology, Investigation. RA: Methodology, Investigation. MAZG: Software, Methodology, Investigation. SM: Software, Methodology, Investigation. Funding None. Acknowledgements None. Data availability No datasets were generated or analyzed during the current study. Declarations Ethical approval This experiment was carried out in accordance with institutional ethical standards and approved by the Animal Welfare and Experimentation Ethics Committee of Sher-e-Bangla Agricultural University (SAU), Dhaka-1207, Bangladesh [Approval No: SAU/AHIPHI/24/846]. Competing interest: The authors declare no competing interests. References Abbas AO, Nassar FS, & Al Ali AM (2025) Challenges of ensuring sustainable poultry meat production and economic resilience under climate change for achieving sustainable food security. Res World Agric Econ, 159-171. https://doi.org/https://doi.org/10.36956/rwae.v6i1.1441 Adams JR, Mehat J, La Ragione et al (2023) Preventing bacterial disease in poultry in the post-antibiotic era: a case for innate immunity modulation as an alternative to antibiotic use. 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Veterinary Research, 56(1), 101. https://doi.org/10.1186/s13567-025-01506-y Pabst R (2022) The bronchus-associated-lymphoid tissue (BALT) an unique lymphoid organ in man and animals. Ann of Anat, 240, 151833. https://doi.org/https://doi.org/10.1016/j.aanat.2021.151833 Peralta MF, Magnoli A, Alustiza F et al (2017) Gut-associated lymphoid tissue: A key tissue inside the mucosal immune system of hens immunized with Escherichia coli F4. Front Immunol, 8, 568. https://doi.org/https://doi.org/10.3389/fimmu.2017.00568 Randall TD (2010) Bronchus-associated lymphoid tissue (BALT): structure and function. Adv Immunol, 107, 187-241. https://doi.org/https://doi.org/10.1016/B978-0-12-381300-8.00007-1 Seefeld ML, Templeton EL, Lehtinen JM et al (2024) Harnessing the potential of the NALT and BALT as targets for immunomodulation using engineering strategies to enhance mucosal uptake. Front Immunol, 15, 1419527. https://doi.org/https://doi.org/10.3389/fimmu.2024.1419527 Tango M, Suzuki E, Gejyo F et al (2000) The presence of specialized epithelial cells on the bronchus-associated lymphoid tissue (BALT) in the mouse. Arch Histol Cytol, 63(1), 81-89. https://doi.org/https://doi.org/10.1679/aohc.63.81 Yehia N, Salem HM, Mahmmod Y et al (2023) Common viral and bacterial avian respiratory infections: an updated review. Poult Sci, 102(5), 102553. https://doi.org/https://doi.org/10.1016/j.psj.2023.102553 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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12:51:30","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":36344,"visible":true,"origin":"","legend":"","description":"","filename":"21f5a02bfe634b19ba2f9f317b1829ad1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7584583/v1/cbf7e29b7aaa8eb58b706e8b.xml"},{"id":92084619,"identity":"f3034f31-f5a6-43a9-86f9-605738fa3410","added_by":"auto","created_at":"2025-09-24 12:43:30","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":43732,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7584583/v1/e1402b0e7026d00596eef187.html"},{"id":92084609,"identity":"800883b2-de9b-4984-b04d-7a9da5cda064","added_by":"auto","created_at":"2025-09-24 12:43:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1289794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBALT development in broiler chicken lungs at various ages.\u003c/strong\u003e (A) BALT is absent around the bronchial region in day-old chick’s, lymphoid structures are not yet formed, although scattered immune cells are present in the bronchial wall. (B) At 15 days, increased infiltration of immune cells is observed, with noticeable disorganized lymphoid aggregation forming the early stages of immature BALT (arrows). (C) By 32 days, a well-defined and organized BALT structure becomes prominent, often forming dense lymphoid follicles adjacent to the bronchi (circled area). H\u0026amp;E staining. (D) Binary Percentage of BALT appearance in lungs of broiler chicken at different ages. Chi-square test: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 was considered statistically significant.\u003c/p\u003e\n\u003cp\u003eBALT=Bronchus-Associated Lymphoid Tissue. Bars =100 micrometers.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7584583/v1/bb45e173397029be8906b268.jpg"},{"id":92084607,"identity":"61631c4f-e679-4747-963a-c396b83b54c9","added_by":"auto","created_at":"2025-09-24 12:43:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":547813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular characterization and vascular network identification in BALT in the lungs at 32 days of age.\u003c/strong\u003e (A) Bu-1⁺ B-cell infiltration (yellow arrow) dispersed across the tissue. (B) CD3⁺ T-cell infiltration (yellow arrow) was observed throughout the tissue. (C) KUL01⁺macrophages (yellow arrow) are densely packed with nodular clusters. Immunofluorescence staining. (D-E) The cuboidal shaped high endothelial venules (circled area) were present in lymphoid region and lymphatic vessel (circled area) present in BALT. H\u0026amp;E staining.\u003c/p\u003e\n\u003cp\u003eBALT=Bronchus-associated lymphoid tissue.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7584583/v1/63516499294136995f5e9b23.jpg"},{"id":92084608,"identity":"c4b0e8f1-b70e-4f0d-8f2b-301f8f051e43","added_by":"auto","created_at":"2025-09-24 12:43:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":678838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe function of BALT.\u003c/strong\u003e (A-B) shows antigen presentation in BALT (IF). (A) An increased amount of KUI01⁺ macrophages (box area) within the BALT. (B) MHCII⁺ antigen presenting cells (box area) show an uneven distribution, potentially concentrated in follicular and sub-epithelial zones. Immunofluorescence staining. (C-D) No germinal center was identified on day 1 and day 15. (E) Germinal center of BALT was identified on day 32. H\u0026amp;E staining.\u003c/p\u003e\n\u003cp\u003eBALT=Bronchus-Associated Lymphoid Tissue. Bars =100 micrometers.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7584583/v1/41889e740131304dfc7fdb90.jpg"},{"id":95524643,"identity":"e7073979-f66e-45af-834a-72fbcfd58eaf","added_by":"auto","created_at":"2025-11-10 10:03:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3112686,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7584583/v1/a21d208e-63ac-4cf8-aa03-1bfda3ef0d35.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Age-related development and functional maturation of bronchus-associated lymphoid tissue (BALT) in broiler chickens","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNowadays, broiler chickens are a major source of affordable and superior animal protein in global agriculture compared to other types of animal protein. (Abbas et al., 2025). To achieve a higher production rate poultry farm requires intensive management. Despite vaccination, birds in conventional poultry farms are frequently exposed to different types of pathogens, especially respiratory pathogens such as infectious bronchitis virus, Mycoplasma species, and Newcastle disease virus (Liu et al., 2025). These respiratory diseases have become a potential threat against the developing poultry industry. As birds are highly susceptible to respiratory pathogens, the understanding of respiratory immunity in poultry is very important (Yehia et al., 2023).\u003c/p\u003e\u003cp\u003eThe immune system in poultry plays a critical role in defending against pathogens and maintaining productivity (Adams et al., 2023). The broiler immune system is comprised of central (primary) lymphoid organs\u0026mdash;the thymus and bursa of Fabricius\u0026mdash;and peripheral (secondary) lymphoid tissues, including mucosa-associated lymphoid tissue (MALT). MALT maintains the immunological defense at the body's inner and outer mucosal surfaces and is found in different mucosal organs such as the gut (gut-associated lymphoid tissue or GALT) and airways (bronchial-associated lymphoid tissue or BALT) (Knop et al., 2005). BALT might play a central role in antigen uptake, initiating immune responses and lymphoid cell distribution in the respiratory tract.\u003c/p\u003e\u003cp\u003ePrevious studies show that in normal conditions, BALT is absent in human and mouse lungs; however, it forms in pathological conditions (Randall, 2010; Tango et al., 2000). BALT is present in the lungs of rats and rabbits in both physiological and pathological conditions, regardless of age (Gregson et al., 1979; Seefeld et al., 2024). In addition, cellular characterization of BALT and identification of vascular networks have been done in the case of mice and humans (Randall, 2010; Tango et al., 2000). In disease conditions, BALT plays a significant role by initiating local immune responses in the lungs in various species. Additionally, inducible BALT (iBALT) forms in humans, mice, rats, and rabbits in response to infection or inflammation and aids in removing antigens from the airways, triggering adaptive immunity, and preserving memory cells (Halle et al., 2009; Seefeld et al., 2024). In broiler chicken, age-related development of BALT has been studied using light and electron microscopy, but detailed cellular characterization has not been done (Fagerland \u0026amp; Arp, 1993). Moreover, how cellular components interact and contribute to protective immunity or pathology has not been established. So detailed cellular characterization, antigen presentation, and germinal center identification are necessary to establish the role of BALT in physiological as well as pathological conditions.\u003c/p\u003e\u003cp\u003eIn our present study, we have characterized the development of BALT from 1 day to 32 days of age and observed that BALT first appears diffusely at the age of 15 days. We have also done cellular characterization and identified germinal centers in BALT.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eManagement of birds\u003c/h2\u003e\u003cp\u003eFor this study, 120-day-old Lohman Meat (Indian River) unsexed broiler chicks were purchased from Kazi Farms Ltd. Upon arrival at the experimental farm, the chicks were randomly divided into three groups, each comprising 20 birds with two replications per group. For the initial age-group examination, 10-day-old chicks were randomly selected for sample collection from the first age group. The remaining chicks were reared under standard husbandry conditions. Adequate lighting and ventilation were ensured throughout the study period. Feed and water were provided \u003cem\u003ead libitum\u003c/em\u003e to all birds.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003eThe birds of each age group were sacrificed following a cervical dislocation and the lungs of birds were collected. The collected lungs were trimmed into small pieces and fixed with 10% neutral buffered formalin for 24 hours for histopathological examination.\u003c/p\u003e\n\u003ch3\u003eHematoxylin \u0026 Eosin Staining\u003c/h3\u003e\n\u003cp\u003eThe collected samples were blocked with paraffin and trimmed to 3 \u0026micro;m thickness using a microtome. The samples were stained with hematoxylin \u0026amp; eosin stains for general histopathological analysis.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eImmunodetection of cell markers was performed as in our previous study (Akter et al., 2024) for B cells (Bu-1), T cells (CD3), and macrophages (KUL01). Lung tissues were deparaffinized and treated overnight with primary antibodies, followed by secondary antibodies or Alexa Fluor 546-labeled donkey anti-rabbit antibodies. Nuclei were counterstained with Hoechst 33342, and sections were visualized under a fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eHistoplanimetry\u003c/h3\u003e\n\u003cp\u003eThe entire lung has been collected, trimmed, and embedded in paraffin. Lung sections were cut to 3 \u0026micro;m thickness at 20\u0026ndash;30 \u0026micro;m intervals and stained to confirm the presence of lymphoid clusters. The clusters were detected using a Euromex BS.1153-EPLi microscope (S/N-EC 2221114).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eA binary logistic regression was conducted to assess the trend in BALT-positive samples across the three time points (days 0, 15, and 32), with time coded as an ordinal variable (0, 1, and 2). A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eClarification of bronchus-associated lymphoid tissue (BALT) in the lungs at different ages of broiler chicken\u003c/h2\u003e\u003cp\u003eWe investigated the presence of bronchus-associated lymphoid tissue (BALT) in broiler chicken lungs at various stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The day-old chick\u0026rsquo;s lung tissue shows the normal parenchyma, which had no diffuse or nodular lymphatic clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). At 15 days of age, some small lymphatic nodule was found (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In the later stage (32 days), a distinct, organized lymphoid cluster was found near the bronchus (boxed area), which had a germinal center (circled area) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). For BALT, 0, 2, and 6 positive samples were observed at Days 0, 15, and 32, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Binary logistic regression showed an increasing trend in BALT-positive samples over time (p\u0026thinsp;=\u0026thinsp;0.997).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eCellular characterization and vascular network of BALT in lungs\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eImmune cells were characterized in the lung tissue by immunofluorescence using specific cell markers in day-old broiler chickens. Various immune cell populations were identified within BALT, including Bu-1⁺ B-cells, CD3⁺ T-cells, and KUL01⁺ macrophages. The fluorescent green dots dispersed around the germinal center indicate B-cells, while T-cells were more diffusely distributed throughout the tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Macrophages, marked by KUL01, appear as densely packed clusters, which indicate follicular organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). So, this cluster is considered BALT.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe also identified vascular networks in BALT by H\u0026amp;E staining. We observed high endothelial venules (HEV) in BALT, and lymphatic vessels were also observed in BALT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). All these findings suggest that a well-vascularized network is present in BALT in broiler chicken lungs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAntigen presentation and germinal center formation in BALT\u003c/h2\u003e\u003cp\u003eWe identified antigen presentation in BALT at 32 days of age. We observed KUI01⁺ macrophages in BALT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Next, we observed major histocompatibility complex (MHC) class II⁺ molecules. MHC II⁺ molecules were found throughout the tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These findings suggest antigen presentation in BALT in chicken lungs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the earlier stages (day-old and 15 days), we did not find any germinal center (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), whereas in the case of the later stage (32 days), we found a well-formed germinal center (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNowadays, broiler chickens are very important globally as a primary source of affordable and high-quality animal protein. In developing countries, broiler meat is not only the most affordable source of animal protein but also the most accessible, making it indispensable for food security (Attia et al., 2022). Under intensive farming systems, the health and productivity of broilers depend heavily on their immune system, which serves as the first line of defense against diseases. It is obvious that a strong immune system is essential for defending birds against contagious diseases (Casteleyn et al., 2010; Lebedev et al., 2024). Importantly, MALT plays a critical role in maintaining mucosal immunity. MALT in lungs like bronchus-associated lymphoid tissue (BALT), which are strategically located at mucosal surfaces to detect pathogens (Cesta, 2006; Pabst, 2022). These structures are developed to provide effective protection against respiratory diseases. But the development of BALT in physiological conditions has not been fully elucidated yet. Our present study clarified the developments and organization of BALT in the lungs of broiler chickens at different stages of age (day 1 to day 32).\u003c/p\u003e\u003cp\u003eMALT is present in different forms in different parts of the body (respiratory, gastrointestinal, and urogenital tracts), which is commonly composed of B-cells, T-cells, and macrophages (Akter et al., 2024). If the MALT is found in a physiological condition, it will be a secondary structure that can grow before birth or after birth, and if it is found in a diseased condition, it will be considered a tertiary structure (Pabst, 2022; Peralta et al., 2017). Previous study reports show that the MALT is found around the bronchus. In our study we examined the development of immune cell clusters at various ages, where at the earlier stage we did not find any clusters around the bronchus, but at the age of 15 days a disorganized small lymphoid aggregation became apparent. At the age of 32 days, a fully well-formed lymphoid cluster was formed. So, we can consider that BALT was not found in the initial stage, but after 15 days it appeared deliberately and fully formed at the later stage, which indicates the probable BALT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAs we found the cellular cluster at the later stage, we did cellular characterization using different cell markers to confirm the complex organization of immune cells at the later stage. We found that BALT was composed of B cells, T cells, and macrophages. So, BALT acts like a complete MALT, whereas we found all immune-component cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe vascular network consists of high endothelial venules (HEVs) and lymphatic vessels. These are crucial for cellular trafficking into lymphoid tissues, which invites the immune cells into the circulation (Fagerland \u0026amp; Arp, 1993). In our study, we examined the vascular network in the fully developed BALT of chicken lungs at the age of 32 days and identified distinct HEV and lymphatic vessels. These findings suggest that BALT in chicken lungs at a later stage has a well-established vascular network (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe main functions of BALT are antigen presentation, lymphocyte activation, and maturation (Blanchard \u0026amp; Girard, 2021). In our present study, we examined the antigen presentation in BALT of chicken lungs to clarify its functional activity. We observed macrophages, T-cells, and MHCII⁺ molecules present in BALT. Thus, we can suggest that antigen presentation has occurred in the BALT of broiler chicken lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGerminal centers are aggregations of B cells that respond to antigens, particularly following T cell-dependent immune responses. The distinct germinal center indicates B-cell proliferation, which produces active antibodies (Ceccopieri \u0026amp; Madej, 2024). Interestingly, in our study the germinal center formation was absent in the earlier stage but became evident at the later stage (32 days). So, we can consider that the formation of germinal centers within BALT indicates structural maturity and also active humoral immunity at later stages of development (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn conclusion, a lymphoid cluster or BALT is present close to the bronchus in the lungs of broiler chickens, which consists of B-cells, T-cells, and macrophages. BALT has a well-designed vascular network for cellular trafficking (Fig.\u0026nbsp;4). In addition, antigen presentation and germinal center are present as lymphoid tissue organizers (Fig.\u0026nbsp;4). So, it shows that BALT is a complete, immunologically active lymphoid tissue. The future study will provide more information about the role of BALT in pathological conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors contribution\u0026nbsp;\u003c/strong\u003eSSS: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Visualization, Software, Resources, Methodology, Investigation, Data curation. OB: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Investigation, Software, Methodology, Investigation, Data curation. MAM: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Visualization, Validation, Supervision, Resources, Methodology, Conceptualization. SB: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Visualization, Software, Investigation, Formal analysis. SH: Software, Methodology, Investigation, Data curation. MJUR: Writing \u0026ndash; review \u0026amp; editing, Validation, Methodology, Investigation. RA: Methodology, Investigation. MAZG: Software, Methodology, Investigation. SM: Software, Methodology, Investigation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eNone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eThis experiment was carried out in accordance with institutional ethical standards and approved by the Animal Welfare and Experimentation Ethics Committee of Sher-e-Bangla Agricultural University (SAU), Dhaka-1207, Bangladesh [Approval No: SAU/AHIPHI/24/846].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbas AO, Nassar FS, \u0026amp; Al Ali AM (2025) Challenges of ensuring sustainable poultry meat production and economic resilience under climate change for achieving sustainable food security. Res World Agric Econ, 159-171. https://doi.org/https://doi.org/10.36956/rwae.v6i1.1441\u003c/li\u003e\n\u003cli\u003eAdams JR, Mehat J, La Ragione et al (2023) Preventing bacterial disease in poultry in the post-antibiotic era: a case for innate immunity modulation as an alternative to antibiotic use. Front Immunol, 14, 1205869. https://doi.org/https://doi.org/10.3389/fimmu.2023.1205869 \u003c/li\u003e\n\u003cli\u003eAkter R, Masum MA, Biswas S et al (2024) Clarification of a unique mucosal vaccination route for improved systemic and mucosal immune response in broiler. Heliyon, 10(20). https://doi.org/https://doi.org/10.1016/j.heliyon.2024.e39621 \u003c/li\u003e\n\u003cli\u003eAttia YA, Rahman MT, Hossain MJ et al (2022). Poultry production and sustainability in developing countries under the COVID-19 crisis: Lessons learned. Animals, 12(5), 644. https://doi.org/10.3390/ani12050644 \u003c/li\u003e\n\u003cli\u003eBlanchard L, Girard JP (2021) High endothelial venules (HEVs) in immunity, inflammation and cancer. Angiogenesis, 24(4), 719-753. https://doi.org/10.1007/s10456-021-09792-8 \u003c/li\u003e\n\u003cli\u003eCasteleyn C, Doom M, Lambrechts E et al (2010) Locations of gut-associated lymphoid tissue in the 3-month-old chicken: a review. Avian Pathol, 39(3), 143-150. https://doi.org/https://doi.org/10.1080/03079451003786105 \u003c/li\u003e\n\u003cli\u003eCeccopieri C, Madej JP (2024) Chicken secondary lymphoid tissues\u0026mdash;Structure and relevance in immunological research. Animals, 14(16), 2439. https://doi.org/https://doi.org/10.3390/ani14162439 \u003c/li\u003e\n\u003cli\u003eCesta MF (2006) Normal structure, function, and histology of mucosa-associated lymphoid tissue. 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Poult Sci, 102(5), 102553. https://doi.org/https://doi.org/10.1016/j.psj.2023.102553 \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":"Bronchus, Lymphoid tissue, Lung, Chicken","lastPublishedDoi":"10.21203/rs.3.rs-7584583/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7584583/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAround the world, broiler chickens are an essential source of affordable animal protein. But their intensive farming practices make them particularly vulnerable to respiratory infections, which cause significant economic losses. To control these respiratory infections and to establish therapeutic targets against diseases, a deep understanding of the respiratory immune system is essential. Bronchus-associated lymphoid tissue (BALT) is the main entry point of pathogens for chickens, but its development and cellular constituents in broiler chickens under physiological conditions remain poorly understood. Our present study aimed to investigate the age-related development, cellular composition, and role of BALT in the lungs of healthy broiler chickens. BALT was absent in day-old chicken, but small BALT appeared at 15 days of age, which later fully developed at 32 days of age. Cellular analysis revealed that fully developed BALT is composed of B-cells, T-cells, and macrophages. It also contains a well-organized vascular network, including high endothelial venules and lymphatic vessels. BALT shows the presence of macrophage and MHCII⁺ molecules, which indicate that antigen presentation occurs in BALT. In addition, germinal centers are present in mature BALT, indicating the maturation of B-cells and production of plasma cells. Therefore, BALT offers a new immunological gateway for respiratory-related therapeutic research and vaccine development.\u003c/p\u003e","manuscriptTitle":"Age-related development and functional maturation of bronchus-associated lymphoid tissue (BALT) in broiler chickens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 12:43:25","doi":"10.21203/rs.3.rs-7584583/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":"3aedd7bc-4b7d-4990-a162-3526941988b7","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-06T20:53:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-24 12:43:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7584583","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7584583","identity":"rs-7584583","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}