Evaluation of immunostimulatory potential of synthetic TLR2 agonists in chicken

Evaluation of immunostimulatory potential of synthetic TLR2 agonists in chicken | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Evaluation of immunostimulatory potential of synthetic TLR2 agonists in chicken Maramreddy Darshini, Saravanan Ramakrishnan, Firdous Ahmed, Deshkanwar S Brar, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7833167/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract The ligands of toll like receptor (TLR) have great potential as vaccine adjuvants. TLR activation causes effector responses such as the synthesis of cytokines and chemokines, the generation of interferons etc. Synthetic TLR2 agonists have been successfully used as experimental vaccine adjuvant in both mammals and chicken. However, their low solubility, high cost and detrimental effect towards protein antigen limits its wider application. Present study was aimed to screen out few synthetic TLR2 agonists for its immunostimulatory potential using chicken peripheral blood mononuclear cells (PBMCs). Total of five synthetic lipopeptide based TLR2 agonists (P1, P2, P6, P7 and P8) have been evaluated for their potential to induce the expression of immune response genes in chicken PBMCs. Each agonist significantly upregulated the expression of IL-1β and iNOS at 3h post-treatment with highest expression of IL-1β mRNA was observed with P1. There was a significant up-regulation of IFN-β and IFN-γ by treatment with P1 at 3 and 12h intervals ( p <0.05). Further, IL-4 mRNA was significantly upregulated with P1 and P7 treatment at 12 and 24h post-treatment respectively. Our results demonstrate that among all five agonists, P1 was superior in terms of up-regulation of immune response genes such as both type I and type II interferons along with mixed Th1 and Th2 responses. Adjuvant Agonist chicken immune response Toll like receptor Figures Figure 1 Figure 2 Introduction The innate immune system being the first line of defense, acts by limiting the infection and activate the adaptive immune system. Through pattern recognition receptors (PRRs), vertebrate’s innate immune system identifies structurally conserved pathogen-associated molecular patterns (PAMPs) such as viral ds RNA, bacterial DNA, lipopolysaccharide, flagellin, and peptidoglycan and permits prompt host immune responses to restrict invading microorganisms (Janeway and Medzhitov 2002). Six families of PRRs have been recognized which are located either on the cell surface or in the cytoplasm of immune cells. One of the most extensively studied pattern recognition receptors (PRRs) is the Toll-like receptor (TLR) family. Toll proteins are type 1 transmembrane proteins which has been described in fish, amphibians, reptiles, birds, and mammals. The structure of different TLRs is similar but the location and distribution vary between the cells and tissues. The number of TLRs varies in different species: 13 have been reported in mammals with 10 in humans and 13 in mice, while a total of ten TLRs have been identified in chickens (Temperley et al. 2008). TLR2 identifies gram-positive bacteria’s lipoproteins, peptidoglycans (PGN), and lipoteichoic acids (LTA). TLR agonists are efficient adjuvants and prophylactic agents in both mammals and birds. Poly I: C administered as an adjuvant with avian influenza vaccine increased IFN-γ, IL-6, and IL- 12 expression (Liang et al. 2013). LPS induced a Th1 response in mice, and agonist-treated animals performed much better against Plasmodium yoelii infection (Zhang et al. 2016). Using resiquimod, a TLR7 agonist, as an adjuvant with inactivated Newcastle disease vaccine resulted in increased humoral and cellular responses as well as an increase in interferon gene expression (Sachan et al. 2015). CpG ODN was an effective oral adjuvant with protein antigens and produced both Th1 and Th2 responses (Zhirov et al. 2024). When combined with an avian influenza virus vaccine, CpG induced a greater systemic immune response than squalene (Singh et al. 2015). When Pam 3 Cys-Ser-(Lys) 4 (PCSL) was used as an adjuvant with human serum albumin (HSA), recombinant bovine somatotropin (rBST), and human immunoglobulin G (IgG), it increased antibody production (Erhard et al. 2000). TLR2 agonist LTA reduced infectious laryngotracheitis virus (ILTV) titre in egg embryos when given 24 hours before the challenge (Haddadi et al. 2015). Pam 3 CSK 4 and flagellin have been used as experimental avian influenza vaccine adjuvants (St. Paul et al. 2012). Following lipoteichoic acid (LTA) treatment of the avian macrophage cell line MQ-NCSU (Muquarrab Qureshi-North Carolina State University), elevated iNOS and IL-1 mRNA have been reported. LTA has been shown to increase mRNA expression of innate genes such as MyD88, iNOS, and IL-1β and to decrease ILTV plaques in macrophages (Haddadi et al. 2015). Materials and methods Materials Revertaid™ First strand cDNA synthesis kit (Thermo Scientific, USA) and Quantifast®, RT² SYBR Green qPCR Mastermix (Qiagen, Germany) were used in gene expression study. Pam 3 CSK 4 (synthetic TLR2 agonist) was procured from InvivoGen, California (USA). Lipopeptide based synthetic TLR2 agonists P1, P2, P6, P8 were synthesized using previously reported methods (Kaur et al., 2022; Brar, et. al., 2024). Among these, P7 is a new synthetic TLR2 agonists (see supplementary information for the synthesis and characterization). Experimental birds Four- to six-week-old White leghorn birds (n = 4) were procured from Central Avian Research Institute (CARI), Izzatnagar. The birds were maintained under standard managemental practices providing feed and water as per ad libitum. All the experiments on live birds were conducted as per the guidelines of Institute Animal Ethics Committee (IAEC). Isolation of peripheral blood mononuclear cells (PBMCs) and treatment with TLR2 synthetic analogues The PBMCs were collected using density gradient centrifugation. The concentration and viability of cells was determined by trypan blue (0.4%) dye exclusion method. Cell count was adjusted to 2 x 10 6 cells/ml using RPMI-1640 growth medium. The PBMCs were treated with synthetic novel TLR2 agonists (P1, P2, P6, P7, and P8) at concentration of 10µg/ml. Cells were harvested at stipulated time intervals like 0h, 3h, 12h, 24h post-treatment to analyse the expression of immune response genes. Immune response gene expression Total RNA was isolated from harvested PBMCs using Qiazol® following instructions of manufacturer. The total RNA isolated was used for cDNA synthesis by RevertaidTM First strand cDNA Synthesis Kit (Thermo Scientific, USA). Briefly, total RNA (1µg) and 1µl of random hexamer were mixed with desired volume of nuclease free water, incubated at 65 0 C for 5 min followed by incubation at 4 o C for 5 min. Reaction mixture containing 5x buffer, 10mM dNTP mix, Reverse transcriptase enzyme (MMuLV) and Ribolock RNase inhibitor was added to above mixture. The reaction mixture was incubated at 25 o C for 10min followed by incubation at 50 o C for 50 min and finally at 85 o C for 5 min. Quantification of gene expression was carried out by real rime PCR using RT SYBR Green qPCR Master mix (Qiagen, Germany) using gene specific primers. The conditions of real time PCR were 95 o C for 5min of pre-incubation, continued by 40 cycles of 94 o C for 30s, 60 o C for 45s, 72 o C for 45s each. The GAPDH was used as housekeeping gene for mRNA normalization. The results were analyzed through ΔΔCt (Pfaffl, 2001) method for calculation of relative fold change of gene expression. Statistical analysis Data was statistically analysed using SPSSTM software version 26.0 (IBM Corp., SA). One-way analysis of variance (ANOVA) test was employed to determine the statistically significant differences in mean values between the groups. Results were considered statistically significant if p < 0.05. The results are presented as mean + standard error of mean. Results Pam 3 CSK 4 showed significantly higher expression of IL-1β at 3h, iNOS at 12h and IFN-β, IFN-γ and IL-4 at 24h post-treatment ( p < 0.05). All the five agonists induced a significantly higher expression of IL-1β at 3h post-treatment in comparison to control ( p < 0.05) (Fig. 1 a). The P1 agonist significantly up-regulated ( p < 0.05) the IFN-β expression at 3h and 12h post treatment while the P7 and P8 agonists significantly ( p < 0.05) up-regulated the IFN-β expression at 24h post-treatment in comparison with control (0h) (Fig. 1 b). The P1 agonist significantly upregulated the IFN-γ mRNA expression at 3 and 12h intervals in comparison to control ( p < 0.05). There was a significant down-regulation of IFN-γ mRNA expression at 24h post-treatment with P6 agonist (Fig. 1 c). The P8 agonist significantly ( p < 0.05) up-regulated the IFN-γ mRNA expression at 24h post-treatment in comparison to control (Fig. 1 c). The P1 agonist showed significant increase ( p < 0.05) in IL-4 mRNA expression at 12h interval when compared with control (Fig. 1 d). There was a significant up-regulation ( p < 0.05) of IL-4 mRNA expression at 24h interval on stimulation with P7 agonist with respect to control (Fig. 1 d). All the five agonists induced a significantly higher expression ( p < 0.05) of iNOS mRNA at 3h interval in comparison to their respective controls (Fig. 1 e). In contrast, they were unable to up-regulate the expression of iNOS mRNA at either 12 or 24h post-treatment (Fig. 1 e). Discussion TLRs are crucial components of the innate immune system. They play an important role by identifying common molecular structures called pathogen-associated molecular patterns (PAMPs) and inducing immune responses. In particular, TLR2 can identify various bacterial molecular patterns and initiates downstream signaling by MyD88-dependent pathway which culminates into the induction of NF-κB and finally production of pro-inflammatory cytokines (Essalemi et al., 2025). This can eventually lead to the induction of Th1, Th2, or mixed Th1/Th2 responses. Chickens have two isoforms of TLR2, ChTLR2a and ChTLR2b. These isoforms combine to form functional heterodimers with either ChTLR1a or ChTLR1b. These heterodimers help recognize triacylated lipopeptides, such as Pam3CSK4, a synthetic TLR2 agonist (St. Paul et al. 2012). Notably, all heterodimer combinations, except ChTLR2a/ChTLR1a, can recognize Pam3CSK4. Pam3CSK4 has shown promise as an experimental vaccine adjuvant in both mammals and chickens (Abdelaziz et al. 2024). However, a number of drawbacks such as high production costs and the propensity of its cationic surfactant-like characteristics to destabilize protein antigens restrict its wider application (Kaur et al. 2022). These problems highlight the need for novel TLR2 agonists that are less expensive, have a simple structure, and preserve or enhance their capacity to elicit an immune response without compromising the integrity of the antigen. The purpose of this study was to assess the capacity of five new lipopeptide-based TLR2 agonists (P1, P2, P6, P7, and P8) that were made in-house to induce the chicken immune system. Prior research by our team demonstrated that these agonists can stimulate the production of TNF-α, IL-6, and IL-10 in human PBMCs, confirming their potential as TLR2 agonists (Kaur et al. 2022). We extended the study to include a chicken model in this work. Our objective was to find potential adjuvants for poultry vaccines that could boost the immune system in multiple species. Pro-inflammatory cytokines play a key role in inflammation and innate immunity. They help control the body's immune response to infection. One of these cytokines, interleukin-1 beta (IL-1β), is crucial. It is mainly released by monocytes and macrophages, but non-immune cells, such as fibroblasts and endothelial cells, can also produce it. All five tested agonists significantly upregulated the IL-1β expression at 3 h post-treatment ( p < 0.05), consistent with previous findings (Bashir et al. 2019; Ramakrishnan et al. 2015). Notably, the chorioallantoic membrane (CAM) of embryonated chicken eggs produced more IL-1β after being treated with the Pam3CSK4, a known TLR-2 agonist. This enhanced cytokine response may play a pivotal role in restricting infectious bronchitis virus (IBV) replication, as suggested by earlier study (Sharma et al. 2020). Numerous immune and non-immune cells produce interferon-beta (IFN-β), which is a type I interferon. This cytokine has antiviral and anti-inflammatory actions. By promoting neutrophil apoptosis and directing macrophages toward a pro-resolving phenotype, IFN-β aids in the resolution of inflammation and preservation of tissue architecture. In present study, 24 hours after treatment, the synthetic TLR2 agonist markedly elevated the expression of IFN-β mRNA in chicken PBMCs. This result is consistent with earlier studies that demonstrated TLR2-mediated induction of IFN-β expression (Bashir et al. 2019; Kaur et al. 2022). One well-known synthetic TLR2 agonist that has been demonstrated to increase IFN-β expression both in vitro and in vivo is Pam3CSK4 (a triacylated lipopeptide). This lends more credence to the notion that TLR2 signaling can enhance chickens' antiviral responses (Sharma et al. 2020). These results collectively imply that TLR2 activation can successfully modulate type I interferon pathways in avian immune cells, potentially enhancing antiviral defense systems. Interferon-gamma (IFN-γ) is predominantly synthesized by activated CD4 + and CD8 + T cells, as well as natural killer cells, and serves as a crucial mediator of immune responses. It is especially crucial for macrophage-mediated defense mechanisms, as it enhances the synthesis of proinflammatory and antibacterial cytokines, reactive oxygen species (ROS), and nitric oxide (NO). Earlier findings collectively underscore the critical role of TLR2 in orchestrating IFN-γ production, an important cytokine for antiviral and antibacterial immunity (Tugues et al. 2015). Consistent with current findings, the present study demonstrated a significant upregulation of IFN-γ mRNA expression at 24 h post-treatment, corroborating our earlier observations (Bashir et al. 2019). Present observations are in line with earlier findings which established that TLR2 activation profoundly enhances the production of IFN-γ, IL-2, and TNF-α by activated CD4 + T cells, an effect specifically inhibited by anti-TLR2 antibodies, thereby underscoring the functional specificity of TLR2 in cytokine modulation (Chiang et al. 2022) Interleukin-4 (IL-4) is a well-known cytokine for immunoglobulin class switching and a T cell–derived growth factor which exerts pleiotropic effects on both hematopoietic and non-hematopoietic cells. In both human and avian species, IL-4 is a signature Th2 cytokine, essential for triggering humoral immune responses and conferring protection against helminth infections. Furthermore, by inhibiting IFN-γ activity, IL-4 is known to balance Th1-mediated inflammatory responses, thereby avoiding excessive inflammation and maintaining iimmunological homeostasis. In present study, stimulation with P1 and P7 agonists resulted in a marked up-regulation of IL-4 mRNA expression at 12 and 24 h post-treatment, respectively ( p < 0.05). This indicates that these synthetic agonists may promote a Th2-skewed immune response in chicken PBMCs. In line with these findings, our previous work demonstrated a mixed Th1/Th2 immune response marked by concurrent up-regulation of IFN-γ and IL-4 expression following treatment of chicken PBMCs with Pam3CSK4 (Bashir et al. 2019). This targeted approach to immune modulation holds promise for developing advanced avian vaccines that elicit robust and balanced protective immunity against a range of pathogens, moving beyond the limitations of traditional vaccine formulations. Nitric oxide (NO) plays an important role in the host's defense against infectious threats. Production of NO is mediated by an enzyme, inducible nitric oxide synthase (iNOS). Our group has previously reported that iNOS expression in chicken PBMCs is upregulated at 12 h following stimulation with Pam3CSK4 (Ramakrishnan et al. 2015; Bashir et al. 2019). On the other hand, the current study discovered that chicken PBMCs treated with all five synthetic TLR2 agonists showed an early (3 h) upregulation of iNOS expression. This discrepancy may be caused by variations in agonist structure, receptor binding affinity, or downstream signaling kinetics, though the precise cause is unknown. In conclusion, the P1 agonist caused the greatest up-regulation of all immune response genes analyzed out of all the synthetic TLR2 agonists tested. Among the tested agonists, P1 > P2 > P7 > P8 > P6 was the general order of immune response gene upregulation. Our results support the development of alternative immunomodulators promoting avian health. This work lays the foundation for future optimization and in vivo validation of modern vaccine adjuvants in the poultry industry by identifying more straightforward and reasonably priced lipopeptide agonists. Declarations Supplementary Information Supplementary data to this article can be found online Acknowledgements Maramreddy Darshini acknowledges ICAR, New Delhi for the Research Fellowship during her master’s programme. The authors thank Director, Indian Veterinary Research Institute, Izatnagar for providing all necessary facilities to carry out this research work. This research work was supported by a research grant from the Science and Engineering Research Board –Department of Science & Technology, Government of India, to S.R. (CRG/2021/005467). Author contributions M. D: investigation, methodology, writing– original draft; M. S, S. R. and D. B. S: conceptualization, funding acquisition, project administration, supervision, writing – review & editing; F. A: methodology, writing– original draft. D. S. B. and A. K: data analysis, writing – review & editing; V. C: writing – data analysis, review & editing. All authors read and approved the final manuscript. Funding This research was supported by grants from the Science and Engineering Research Board –Department of Science & Technology, Government of India, to S.R. (CRG/2021/005467). Data availability The data generated during the current study are available from the corresponding author on reasonable request. Ethics approval This study was approved by the Institutional Animal Ethics Guidelines (IAEC), ICAR-IVRI, with reference F. No. 26-5/2021-22/JD(R)/IAEC dated 15.12.2021. Consent to participate and publish This research did not involve human subjects. Competing Interests The authors declare no conflicts of interest. References Abdelaziz K, Helmy YA, Yitbarek A, Hodgins DC, Sharafeldin T A, Selim MSH (2024). 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08:55:00","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":50656,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7833167/v1/9815aa2ffbeeabb597293e58.html"},{"id":95184833,"identity":"d813fb59-1c22-4503-8037-9f8599525406","added_by":"auto","created_at":"2025-11-05 08:55:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":253462,"visible":true,"origin":"","legend":"\u003cp\u003eRelative fold-change expression of (a) IL-1β, (b) IFN-β, (c) IFN-γ, (d) IL-4 and (e) iNOS genes in chicken PBMCs at 3h, 12h, and 24h post-treatment with synthetic TLR2 agonists (P1, P2, P6, P7, P8) assessed with SYBER Green Quantitative real-time polymerase chain reaction. Asterisk indicate significant differences compared to the control group (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7833167/v1/30ce8545ad4748260db725a6.png"},{"id":95184831,"identity":"929ad46e-c272-4778-949f-d2400cd595f2","added_by":"auto","created_at":"2025-11-05 08:55:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":77862,"visible":true,"origin":"","legend":"\u003cp\u003eStructures of TLR2 agonist (P8)\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7833167/v1/d20c9991539f8af6db3b128b.png"},{"id":95312271,"identity":"9ee146f6-6ae0-475b-9d94-945e42ffffbe","added_by":"auto","created_at":"2025-11-06 15:48:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":790572,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7833167/v1/912011ce-7e92-43fc-afbd-2a66afb01200.pdf"},{"id":95184846,"identity":"b452aa5d-e6a1-4b9d-95e5-5cf1b819a9c9","added_by":"auto","created_at":"2025-11-05 08:55:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2976934,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryChickenVaccineTLR207062025DBDraft1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7833167/v1/ca958556ce95bd3bd265f936.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of immunostimulatory potential of synthetic TLR2 agonists in chicken","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe innate immune system being the first line of defense, acts by limiting the infection and activate the adaptive immune system. Through pattern recognition receptors (PRRs), vertebrate\u0026rsquo;s innate immune system identifies structurally conserved pathogen-associated molecular patterns (PAMPs) such as viral ds RNA, bacterial DNA, lipopolysaccharide, flagellin, and peptidoglycan and permits prompt host immune responses to restrict invading microorganisms (Janeway and Medzhitov 2002). Six families of PRRs have been recognized which are located either on the cell surface or in the cytoplasm of immune cells.\u003c/p\u003e\u003cp\u003eOne of the most extensively studied pattern recognition receptors (PRRs) is the Toll-like receptor (TLR) family. Toll proteins are type 1 transmembrane proteins which has been described in fish, amphibians, reptiles, birds, and mammals. The structure of different TLRs is similar but the location and distribution vary between the cells and tissues. The number of TLRs varies in different species: 13 have been reported in mammals with 10 in humans and 13 in mice, while a total of ten TLRs have been identified in chickens (Temperley et al. 2008). TLR2 identifies gram-positive bacteria\u0026rsquo;s lipoproteins, peptidoglycans (PGN), and lipoteichoic acids (LTA).\u003c/p\u003e\u003cp\u003eTLR agonists are efficient adjuvants and prophylactic agents in both mammals and birds. Poly I: C administered as an adjuvant with avian influenza vaccine increased IFN-γ, IL-6, and IL- 12 expression (Liang et al. 2013). LPS induced a Th1 response in mice, and agonist-treated animals performed much better against \u003cem\u003ePlasmodium yoelii\u003c/em\u003e infection (Zhang et al. 2016). Using resiquimod, a TLR7 agonist, as an adjuvant with inactivated Newcastle disease vaccine resulted in increased humoral and cellular responses as well as an increase in interferon gene expression (Sachan et al. 2015). CpG ODN was an effective oral adjuvant with protein antigens and produced both Th1 and Th2 responses (Zhirov et al. 2024). When combined with an avian influenza virus vaccine, CpG induced a greater systemic immune response than squalene (Singh et al. 2015). When Pam\u003csub\u003e3\u003c/sub\u003eCys-Ser-(Lys)\u003csub\u003e4\u003c/sub\u003e (PCSL) was used as an adjuvant with human serum albumin (HSA), recombinant bovine somatotropin (rBST), and human immunoglobulin G (IgG), it increased antibody production (Erhard et al. 2000). TLR2 agonist LTA reduced infectious laryngotracheitis virus (ILTV) titre in egg embryos when given 24 hours before the challenge (Haddadi et al. 2015). Pam\u003csub\u003e3\u003c/sub\u003eCSK\u003csub\u003e4\u003c/sub\u003e and flagellin have been used as experimental avian influenza vaccine adjuvants (St. Paul et al. 2012). Following lipoteichoic acid (LTA) treatment of the avian macrophage cell line MQ-NCSU (Muquarrab Qureshi-North Carolina State University), elevated iNOS and IL-1 mRNA have been reported. LTA has been shown to increase mRNA expression of innate genes such as MyD88, iNOS, and IL-1β and to decrease ILTV plaques in macrophages (Haddadi et al. 2015).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eRevertaid\u0026trade; First strand cDNA synthesis kit (Thermo Scientific, USA) and Quantifast\u0026reg;, RT\u0026sup2; SYBR Green qPCR Mastermix (Qiagen, Germany) were used in gene expression study. Pam\u003csub\u003e3\u003c/sub\u003eCSK\u003csub\u003e4\u003c/sub\u003e (synthetic TLR2 agonist) was procured from InvivoGen, California (USA). Lipopeptide based synthetic TLR2 agonists P1, P2, P6, P8 were synthesized using previously reported methods (Kaur et al., 2022; Brar, et. al., 2024). Among these, P7 is a new synthetic TLR2 agonists (see supplementary information for the synthesis and characterization).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExperimental birds\u003c/h3\u003e\n\u003cp\u003eFour- to six-week-old White leghorn birds (n\u0026thinsp;=\u0026thinsp;4) were procured from Central Avian Research Institute (CARI), Izzatnagar. The birds were maintained under standard managemental practices providing feed and water as per ad libitum. All the experiments on live birds were conducted as per the guidelines of Institute Animal Ethics Committee (IAEC).\u003c/p\u003e\n\u003ch3\u003eIsolation of peripheral blood mononuclear cells (PBMCs) and treatment with TLR2 synthetic analogues\u003c/h3\u003e\n\u003cp\u003eThe PBMCs were collected using density gradient centrifugation. The concentration and viability of cells was determined by trypan blue (0.4%) dye exclusion method. Cell count was adjusted to 2 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml using RPMI-1640 growth medium.\u003c/p\u003e\u003cp\u003eThe PBMCs were treated with synthetic novel TLR2 agonists (P1, P2, P6, P7, and P8) at concentration of 10\u0026micro;g/ml. Cells were harvested at stipulated time intervals like 0h, 3h, 12h, 24h post-treatment to analyse the expression of immune response genes.\u003c/p\u003e\n\u003ch3\u003eImmune response gene expression\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from harvested PBMCs using Qiazol\u0026reg; following instructions of manufacturer. The total RNA isolated was used for cDNA synthesis by RevertaidTM First strand cDNA Synthesis Kit (Thermo Scientific, USA). Briefly, total RNA (1\u0026micro;g) and 1\u0026micro;l of random hexamer were mixed with desired volume of nuclease free water, incubated at 65\u003csup\u003e0\u003c/sup\u003eC for 5 min followed by incubation at 4\u003csup\u003eo\u003c/sup\u003eC for 5 min. Reaction mixture containing 5x buffer, 10mM dNTP mix, Reverse transcriptase enzyme (MMuLV) and Ribolock RNase inhibitor was added to above mixture. The reaction mixture was incubated at 25\u003csup\u003eo\u003c/sup\u003eC for 10min followed by incubation at 50\u003csup\u003eo\u003c/sup\u003eC for 50 min and finally at 85\u003csup\u003eo\u003c/sup\u003eC for 5 min.\u003c/p\u003e\u003cp\u003eQuantification of gene expression was carried out by real rime PCR using RT SYBR Green qPCR Master mix (Qiagen, Germany) using gene specific primers. The conditions of real time PCR were 95\u003csup\u003eo\u003c/sup\u003eC for 5min of pre-incubation, continued by 40 cycles of 94\u003csup\u003eo\u003c/sup\u003eC for 30s, 60\u003csup\u003eo\u003c/sup\u003eC for 45s, 72\u003csup\u003eo\u003c/sup\u003eC for 45s each. The GAPDH was used as housekeeping gene for mRNA normalization. The results were analyzed through ΔΔCt (Pfaffl, 2001) method for calculation of relative fold change of gene expression.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData was statistically analysed using SPSSTM software version 26.0 (IBM Corp., SA). One-way analysis of variance (ANOVA) test was employed to determine the statistically significant differences in mean values between the groups. Results were considered statistically significant if \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The results are presented as mean\u0026thinsp;+\u0026thinsp;standard error of mean.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003ePam\u003csub\u003e3\u003c/sub\u003eCSK\u003csub\u003e4\u003c/sub\u003e showed significantly higher expression of IL-1β at 3h, iNOS at 12h and IFN-β, IFN-γ and IL-4 at 24h post-treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). All the five agonists induced a significantly higher expression of IL-1β at 3h post-treatment in comparison to control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The P1 agonist significantly up-regulated (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the IFN-β expression at 3h and 12h post treatment while the P7 and P8 agonists significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) up-regulated the IFN-β expression at 24h post-treatment in comparison with control (0h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The P1 agonist significantly upregulated the IFN-γ mRNA expression at 3 and 12h intervals in comparison to control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was a significant down-regulation of IFN-γ mRNA expression at 24h post-treatment with P6 agonist (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The P8 agonist significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) up-regulated the IFN-γ mRNA expression at 24h post-treatment in comparison to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The P1 agonist showed significant increase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in IL-4 mRNA expression at 12h interval when compared with control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). There was a significant up-regulation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of IL-4 mRNA expression at 24h interval on stimulation with P7 agonist with respect to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). All the five agonists induced a significantly higher expression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of iNOS mRNA at 3h interval in comparison to their respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). In contrast, they were unable to up-regulate the expression of iNOS mRNA at either 12 or 24h post-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTLRs are crucial components of the innate immune system. They play an important role by identifying common molecular structures called pathogen-associated molecular patterns (PAMPs) and inducing immune responses. In particular, TLR2 can identify various bacterial molecular patterns and initiates downstream signaling by MyD88-dependent pathway which culminates into the induction of NF-κB and finally production of pro-inflammatory cytokines (Essalemi et al., 2025). This can eventually lead to the induction of Th1, Th2, or mixed Th1/Th2 responses. Chickens have two isoforms of TLR2, ChTLR2a and ChTLR2b. These isoforms combine to form functional heterodimers with either ChTLR1a or ChTLR1b. These heterodimers help recognize triacylated lipopeptides, such as Pam3CSK4, a synthetic TLR2 agonist (St. Paul et al. 2012). Notably, all heterodimer combinations, except ChTLR2a/ChTLR1a, can recognize Pam3CSK4.\u003c/p\u003e\u003cp\u003ePam3CSK4 has shown promise as an experimental vaccine adjuvant in both mammals and chickens (Abdelaziz et al. 2024). However, a number of drawbacks such as high production costs and the propensity of its cationic surfactant-like characteristics to destabilize protein antigens restrict its wider application (Kaur et al. 2022). These problems highlight the need for novel TLR2 agonists that are less expensive, have a simple structure, and preserve or enhance their capacity to elicit an immune response without compromising the integrity of the antigen.\u003c/p\u003e\u003cp\u003eThe purpose of this study was to assess the capacity of five new lipopeptide-based TLR2 agonists (P1, P2, P6, P7, and P8) that were made in-house to induce the chicken immune system. Prior research by our team demonstrated that these agonists can stimulate the production of TNF-α, IL-6, and IL-10 in human PBMCs, confirming their potential as TLR2 agonists (Kaur et al. 2022). We extended the study to include a chicken model in this work. Our objective was to find potential adjuvants for poultry vaccines that could boost the immune system in multiple species.\u003c/p\u003e\u003cp\u003ePro-inflammatory cytokines play a key role in inflammation and innate immunity. They help control the body's immune response to infection. One of these cytokines, interleukin-1 beta (IL-1β), is crucial. It is mainly released by monocytes and macrophages, but non-immune cells, such as fibroblasts and endothelial cells, can also produce it. All five tested agonists significantly upregulated the IL-1β expression at 3 h post-treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), consistent with previous findings (Bashir et al. 2019; Ramakrishnan et al. 2015). Notably, the chorioallantoic membrane (CAM) of embryonated chicken eggs produced more IL-1β after being treated with the Pam3CSK4, a known TLR-2 agonist. This enhanced cytokine response may play a pivotal role in restricting infectious bronchitis virus (IBV) replication, as suggested by earlier study (Sharma et al. 2020).\u003c/p\u003e\u003cp\u003eNumerous immune and non-immune cells produce interferon-beta (IFN-β), which is a type I interferon. This cytokine has antiviral and anti-inflammatory actions. By promoting neutrophil apoptosis and directing macrophages toward a pro-resolving phenotype, IFN-β aids in the resolution of inflammation and preservation of tissue architecture. In present study, 24 hours after treatment, the synthetic TLR2 agonist markedly elevated the expression of IFN-β mRNA in chicken PBMCs. This result is consistent with earlier studies that demonstrated TLR2-mediated induction of IFN-β expression (Bashir et al. 2019; Kaur et al. 2022). One well-known synthetic TLR2 agonist that has been demonstrated to increase IFN-β expression both in vitro and in vivo is Pam3CSK4 (a triacylated lipopeptide). This lends more credence to the notion that TLR2 signaling can enhance chickens' antiviral responses (Sharma et al. 2020). These results collectively imply that TLR2 activation can successfully modulate type I interferon pathways in avian immune cells, potentially enhancing antiviral defense systems.\u003c/p\u003e\u003cp\u003eInterferon-gamma (IFN-γ) is predominantly synthesized by activated CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells, as well as natural killer cells, and serves as a crucial mediator of immune responses. It is especially crucial for macrophage-mediated defense mechanisms, as it enhances the synthesis of proinflammatory and antibacterial cytokines, reactive oxygen species (ROS), and nitric oxide (NO). Earlier findings collectively underscore the critical role of TLR2 in orchestrating IFN-γ production, an important cytokine for antiviral and antibacterial immunity (Tugues et al. 2015). Consistent with current findings, the present study demonstrated a significant upregulation of IFN-γ mRNA expression at 24 h post-treatment, corroborating our earlier observations (Bashir et al. 2019). Present observations are in line with earlier findings which established that TLR2 activation profoundly enhances the production of IFN-γ, IL-2, and TNF-α by activated CD4\u0026thinsp;+\u0026thinsp;T cells, an effect specifically inhibited by anti-TLR2 antibodies, thereby underscoring the functional specificity of TLR2 in cytokine modulation (Chiang et al. 2022)\u003c/p\u003e\u003cp\u003eInterleukin-4 (IL-4) is a well-known cytokine for immunoglobulin class switching and a T cell\u0026ndash;derived growth factor which exerts pleiotropic effects on both hematopoietic and non-hematopoietic cells. In both human and avian species, IL-4 is a signature Th2 cytokine, essential for triggering humoral immune responses and conferring protection against helminth infections. Furthermore, by inhibiting IFN-γ activity, IL-4 is known to balance Th1-mediated inflammatory responses, thereby avoiding excessive inflammation and maintaining iimmunological homeostasis. In present study, stimulation with P1 and P7 agonists resulted in a marked up-regulation of IL-4 mRNA expression at 12 and 24 h post-treatment, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This indicates that these synthetic agonists may promote a Th2-skewed immune response in chicken PBMCs. In line with these findings, our previous work demonstrated a mixed Th1/Th2 immune response marked by concurrent up-regulation of IFN-γ and IL-4 expression following treatment of chicken PBMCs with Pam3CSK4 (Bashir et al. 2019). This targeted approach to immune modulation holds promise for developing advanced avian vaccines that elicit robust and balanced protective immunity against a range of pathogens, moving beyond the limitations of traditional vaccine formulations.\u003c/p\u003e\u003cp\u003eNitric oxide (NO) plays an important role in the host's defense against infectious threats. Production of NO is mediated by an enzyme, inducible nitric oxide synthase (iNOS). Our group has previously reported that iNOS expression in chicken PBMCs is upregulated at 12 h following stimulation with Pam3CSK4 (Ramakrishnan et al. 2015; Bashir et al. 2019). On the other hand, the current study discovered that chicken PBMCs treated with all five synthetic TLR2 agonists showed an early (3 h) upregulation of iNOS expression. This discrepancy may be caused by variations in agonist structure, receptor binding affinity, or downstream signaling kinetics, though the precise cause is unknown.\u003c/p\u003e\u003cp\u003eIn conclusion, the P1 agonist caused the greatest up-regulation of all immune response genes analyzed out of all the synthetic TLR2 agonists tested. Among the tested agonists, P1\u0026thinsp;\u0026gt;\u0026thinsp;P2\u0026thinsp;\u0026gt;\u0026thinsp;P7\u0026thinsp;\u0026gt;\u0026thinsp;P8\u0026thinsp;\u0026gt;\u0026thinsp;P6 was the general order of immune response gene upregulation. Our results support the development of alternative immunomodulators promoting avian health. This work lays the foundation for future optimization and in vivo validation of modern vaccine adjuvants in the poultry industry by identifying more straightforward and reasonably priced lipopeptide agonists.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data to this article can be found online\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaramreddy Darshini acknowledges ICAR, New Delhi for the Research Fellowship during her master\u0026rsquo;s programme. The authors thank Director, Indian Veterinary Research Institute, Izatnagar for providing all necessary facilities to carry out this research work. This research work was supported by a research grant from the Science and Engineering Research Board \u0026ndash;Department of Science \u0026amp; Technology, Government of India, to S.R. (CRG/2021/005467).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. D: investigation, methodology, writing\u0026ndash; original draft; M. S, S. R. and D. B. S: conceptualization, funding acquisition, project administration, supervision, writing \u0026ndash; review \u0026amp; editing; F. A: methodology, writing\u0026ndash; original draft. D. S. B. and A. K: data analysis, writing \u0026ndash; review \u0026amp; editing; V. C: writing \u0026ndash; data analysis, review \u0026amp; editing. \u003cem\u003eAll authors read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from the Science and Engineering Research Board \u0026ndash;Department of Science \u0026amp; Technology, Government of India, to S.R. (CRG/2021/005467).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003eThis study was approved by the Institutional Animal Ethics Guidelines (IAEC), ICAR-IVRI, with reference F. No. 26-5/2021-22/JD(R)/IAEC dated 15.12.2021.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate and publish\u0026nbsp;\u003c/strong\u003eThis research did not involve human subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbdelaziz K, Helmy YA, Yitbarek A, Hodgins DC, Sharafeldin T A, Selim MSH (2024). \u0026nbsp; \u0026nbsp; \u0026nbsp; Advances in Poultry Vaccines: Leveraging Biotechnology for Improving Vaccine Development, Stability and Delivery. 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Cell 6:805\u0026ndash;20. https://doi: 10.1016/j.cell.2010.01.022.\u003c/li\u003e\n \u003cli\u003eTemperley ND, Berlin S, Paton I, Griffin D, Burt D (2008). Evolution of the Chicken Toll-like Receptor Gene Family: A Story of Gene Gain and Gene Loss. BMC Genomics 9:62. https://doi: 10.1186/1471-2164-9-62.\u003c/li\u003e\n \u003cli\u003eTugues S, Burkhard SH, Ohs I, Vrohlings M, Nussbaum K, Vom Berg J, Kulig P, Becher B (2015). New insights into IL-12-mediated tumor suppression. Cell Death Differ 2:237-246. https://doi: 10.1038/cdd.2014.134.\u003c/li\u003e\n \u003cli\u003eZhang Y, Zhu X, Feng Y, Pang W, Qi Z, Cui L, Cao Y (2016). TLR4 and TLR9 Signals Stimulate Protective Immunity against Blood-Stage Plasmodium Yoelii Infection in Mice. Exp Parasitol 170:73\u0026ndash;81. https://doi: 10.1016/j.exppara.2016.09.003.\u003c/li\u003e\n \u003cli\u003eZhirov AM, Kovalev DA, Kurcheva SA, Ponomarenko DG, Kulichenko AN (2024). CPG Oligonucleotides as Vaccine Adjuvants for Prevention of Infectious Diseases. Mol Gen Microbiol Virol 39:209-218. https://doi: 10.3103/S0891416824700253\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Adjuvant, Agonist, chicken, immune response, Toll like receptor","lastPublishedDoi":"10.21203/rs.3.rs-7833167/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7833167/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe ligands of toll like receptor (TLR) have great potential as vaccine adjuvants. TLR activation causes effector responses such as the synthesis of cytokines and chemokines, the generation of interferons etc. Synthetic TLR2 agonists have been successfully used as experimental vaccine adjuvant in both mammals and chicken. However, their low solubility, high cost and detrimental effect towards protein antigen limits its wider application. Present study was aimed to screen out few synthetic TLR2 agonists for its immunostimulatory potential using chicken peripheral blood mononuclear cells (PBMCs). Total of five synthetic lipopeptide based TLR2 agonists (P1, P2, P6, P7 and P8) have been evaluated for their potential to induce the expression of immune response genes in chicken PBMCs. Each agonist significantly upregulated the expression of IL-1β and iNOS at 3h post-treatment with highest expression of IL-1β mRNA was observed with P1. There was a significant up-regulation of IFN-β and IFN-γ by treatment with P1 at 3 and 12h intervals (\u003cem\u003ep \u003c/em\u003e\u0026lt;0.05). Further, IL-4 mRNA was significantly upregulated with P1 and P7 treatment at 12 and 24h post-treatment respectively. Our results demonstrate that among all five agonists, P1 was superior in terms of up-regulation of immune response genes such as both type I and type II interferons along with mixed Th1 and Th2 responses.\u003c/p\u003e","manuscriptTitle":"Evaluation of immunostimulatory potential of synthetic TLR2 agonists in chicken","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 08:54:55","doi":"10.21203/rs.3.rs-7833167/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-15T19:23:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-15T19:09:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-06T12:27:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-31T20:24:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11828401159017922203473053361645505490","date":"2025-10-29T14:42:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-26T17:21:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62146020495858988416780562682150944446","date":"2025-10-26T14:32:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T21:15:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51719616307833132599411860782313994298","date":"2025-10-24T16:59:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145543343896401116960872485451680923845","date":"2025-10-24T13:40:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58029430383349928048050345116837839448","date":"2025-10-24T08:48:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-24T08:06:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-22T16:43:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-22T13:12:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Research Communications","date":"2025-10-11T08:03:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7104a6bd-e7c1-42cd-b0b3-1055100b18e2","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T21:08:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-05 08:54:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7833167","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7833167","identity":"rs-7833167","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}