{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T19:03:40Z","timestamp":1791227020861,"version":"4.1.0"},"reference-count":47,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2023,10,17]],"date-time":"2023-10-17T00:00:00Z","timestamp":1697500800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"US poultry and Egg Association Foundation","award":["#F-93"],"award-info":[{"award-number":["#F-93"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Infectious laryngotracheitis (ILT) is an economically important disease in chickens. We previously showed that an in ovo adjuvantation of recombinant herpesvirus of the turkey-Laryngotracheitis (rHVT-LT) vaccine with CpG-oligonucleotides (ODN) can boost vaccine-induced responses in one-day-old broiler chickens. Here, we evaluated the protective efficacy of in ovo administered rHVT-LT + CpG-ODN vaccination against a wild-type ILT virus (ILTV) challenge at 28 days of age and assessed splenic immune gene expression as well as cellular responses. A chicken-embryo-origin (CEO)-ILT vaccine administered in water at 14 days of age was also used as a comparative control for the protection assessment. The results showed that the rHVT-LT + CpG-ODN or the CEO vaccinations provided significant protection against the ILTV challenge and that the level of protection induced by both the vaccines was statistically similar. The protected birds had a significantly upregulated expression of interferon (IFN)\u03b3 or interleukin (IL)-12 cytokine genes. Furthermore, the chickens vaccinated with the rHVT-LT + CpG-ODN or CEO vaccine had a significantly higher frequency of \u03b3\u03b4 T cells and activated CD4+ or CD8+ T cells, compared to the unvaccinated-ILTV challenge control. Collectively, our findings suggest that CpG-ODN can be used as an effective adjuvant for rHVT-LT in ovo vaccination to induce protective immunity against ILT in broiler chickens.<\/jats:p>","DOI":"10.3390\/v15102103","type":"journal-article","created":{"date-parts":[[2023,10,17]],"date-time":"2023-10-17T08:10:19Z","timestamp":1697530219000},"page":"2103","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["In Ovo Vaccination with Recombinant Herpes Virus of the Turkey-Laryngotracheitis Vaccine Adjuvanted with CpG-Oligonucleotide Provides Protection against a Viral Challenge in Broiler Chickens"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2658-123X","authenticated-orcid":false,"given":"Carissa","family":"Gaghan","sequence":"first","affiliation":[{"name":"Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew","family":"Browning","sequence":"additional","affiliation":[{"name":"Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7987-8630","authenticated-orcid":false,"given":"Abdelhamid M.","family":"Fares","sequence":"additional","affiliation":[{"name":"Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohamed Faizal","family":"Abdul-Careem","sequence":"additional","affiliation":[{"name":"Health Research Innovation Center 2C58, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabel M.","family":"Gimeno","sequence":"additional","affiliation":[{"name":"Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1126-7494","authenticated-orcid":false,"given":"Raveendra R.","family":"Kulkarni","sequence":"additional","affiliation":[{"name":"Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,10,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1637\/8018-051007-Review","article-title":"Epizootiology of Infectious Laryngotracheitis and Presentation of an Industry Control Program","volume":"52","year":"2008","journal-title":"Avian Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1016\/j.vetmic.2016.12.023","article-title":"Current and Future Vaccines and Vaccination Strategies against Infectious Laryngotracheitis (ILT) Respiratory Disease of Poultry","volume":"206","year":"2017","journal-title":"Vet. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1637\/11967-090218-Review.1","article-title":"Commercial Vaccines and Vaccination Strategies Against Infectious Laryngotracheitis: What We Have Learned and Knowledge Gaps That Remain","volume":"63","author":"Zavala","year":"2019","journal-title":"Avian Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"348","DOI":"10.2307\/1591188","article-title":"Increased Virulence of Modified-Live Infectious Laryngotracheitis Vaccine Virus Following Bird-to-Bird Passage","volume":"35","author":"Guy","year":"1991","journal-title":"Avian Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1080\/03079459208418820","article-title":"Rapid Diagnosis of Infectious Laryngotracheitis Using a Monoclonal Antibody-Based Immunoperoxidase Procedure","volume":"21","author":"Guy","year":"1992","journal-title":"Avian Pathol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1080\/01652176.1987.9694103","article-title":"Virulence of Five Live Vaccines against Avian Infectious Laryngotracheitis and Their Immunogenicity and Spread after Eyedrop or Spray Application","volume":"9","author":"Hilbink","year":"1987","journal-title":"Vet. Q."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1637\/8011-041907-REGR.1","article-title":"Replication and Transmission of Live Attenuated Infectious Laryngotracheitis Virus (ILTV) Vaccines","volume":"51","author":"Oldoni","year":"2007","journal-title":"Avian Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1080\/03079457.2011.588196","article-title":"Replication of Recombinant Herpesvirus of Turkey Expressing Genes of Infectious Laryngotracheitis Virus in Specific Pathogen Free and Broiler Chickens Following in ovo and Subcutaneous Vaccination","volume":"40","author":"Gimeno","year":"2011","journal-title":"Avian Pathol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1637\/10966-103014-Reg","article-title":"Evaluation of the Protection Efficacy of a Serotype 1 Marek\u2019s Disease Virus-Vectored Bivalent Vaccine Against Infectious Laryngotracheitis and Marek\u2019s Disease","volume":"59","author":"Gimeno","year":"2015","journal-title":"Avian Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1080\/03079457.2019.1644449","article-title":"Evaluation of Vaccination against Infectious Laryngotracheitis (ILT) with Recombinant Herpesvirus of Turkey (RHVT-LT) and Chicken Embryo Origin (CEO) Vaccines Applied Alone or in Combination","volume":"48","author":"Maekawa","year":"2019","journal-title":"Avian Pathol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1080\/03079457.2011.631983","article-title":"Protection Induced by Commercially Available Live-Attenuated and Recombinant Viral Vector Vaccines against Infectious Laryngotracheitis Virus in Broiler Chickens","volume":"41","author":"Vagnozzi","year":"2012","journal-title":"Avian Pathol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1016\/j.smim.2006.12.004","article-title":"TLR Signaling","volume":"19","author":"Kawai","year":"2007","journal-title":"Semin. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3163","DOI":"10.1016\/j.molimm.2009.06.002","article-title":"Chicken TLR21 Acts as a Functional Homologue to Mammalian TLR9 in the Recognition of CpG Oligodeoxynucleotides","volume":"46","author":"Brownlie","year":"2009","journal-title":"Mol. Immunol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1586\/erv.10.174","article-title":"CpG DNA as a Vaccine Adjuvant","volume":"10","author":"Bode","year":"2011","journal-title":"Expert. Rev. Vaccines"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1016\/j.ijmm.2004.07.005","article-title":"CpG-DNA as Immune Response Modifier","volume":"294","author":"Dalpke","year":"2004","journal-title":"Int. J. Med. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"909","DOI":"10.1586\/14760584.2014.920236","article-title":"Toll-like receptor-based adjuvants: Enhancing the immune response to vaccines against infectious diseases of chicken","volume":"13","author":"Gupta","year":"2014","journal-title":"Expert. Rev. Vaccines"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1209","DOI":"10.1099\/jgv.0.000787","article-title":"Toll-like Receptor (TLR)21 Signaling-Mediated Antiviral Response against Avian Influenza Virus Infection Correlates with Macrophage Recruitment and Nitric Oxide Production","volume":"98","author":"Amarasinghe","year":"2017","journal-title":"J. Gen. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Abdul-Cader, M.S., De Silva Senapathi, U., Nagy, E., Sharif, S., and Abdul-Careem, M.F. (2018). Antiviral Response Elicited against Avian Influenza Virus Infection Following Activation of Toll-like Receptor (TLR)7 Signaling Pathway Is Attributable to Interleukin (IL)-1\u03b2 Production. BMC Res. Notes, 11.","DOI":"10.1186\/s13104-018-3975-4"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1038\/s41598-018-36588-6","article-title":"Synthetic CpG-ODN Rapidly Enriches Immune Compartments in Neonatal Chicks to Induce Protective Immunity against Bacterial Infections","volume":"9","author":"Gunawardana","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"16370","DOI":"10.1038\/s41598-018-34760-6","article-title":"The Effects of in ovo Administration of Encapsulated Toll-like Receptor 21 Ligand as an Adjuvant with Marek\u2019s Disease Vaccine","volume":"8","author":"Bavananthasivam","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"813","DOI":"10.1637\/7194-041204R","article-title":"Protection of Neonatal Chicks against a Lethal Challenge of Escherichia Coli Using DNA Containing Cytosine-Phosphodiester-Guanine Motifs","volume":"48","author":"Gomis","year":"2004","journal-title":"Avian Dis."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Gaghan, C., Browning, M., Cortes, A.L., Gimeno, I.M., and Kulkarni, R.R. (2023). Effect of CpG-Oligonucleotide in Enhancing Recombinant Herpes Virus of Turkey-Laryngotracheitis Vaccine-Induced Immune Responses in One-Day-Old Broiler Chickens. Vaccines, 11.","DOI":"10.3390\/vaccines11020294"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1637\/aviandiseases-D-19-00097","article-title":"Evaluation of Factors That Influence Dose Variability of Marek\u2019s Disease Vaccines","volume":"63","author":"Abad","year":"2019","journal-title":"Avian Dis."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Roy, J.G., McElhaney, J.E., and Verschoor, C.P. (2020). Reliable Reference Genes for the Quantification of MRNA in Human T-Cells and PBMCs Stimulated with Live Influenza Virus. BMC Immunol., 2.","DOI":"10.1186\/s12865-020-0334-8"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-\u0394\u0394CT Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e45","DOI":"10.1093\/nar\/29.9.e45","article-title":"A New Mathematical Model for Relative Quantification in Real-Time RT-PCR","volume":"29","author":"Pfaffl","year":"2001","journal-title":"Nucleic Acid. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1089\/vim.2017.0195","article-title":"Reduction of Marek\u2019s Disease Virus Infection by Toll-Like Receptor Ligands in Chicken Embryo Fibroblast Cells","volume":"31","author":"Bavananthasivam","year":"2018","journal-title":"Viral Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Senapathi, U.D.S., Abdul-Cader, M.S., Amarasinghe, A., van Marle, G., Czub, M., Gomis, S., and Abdul-Careem, M.F. (2018). The in ovo Delivery of CpG Oligonucleotides Protects against Infectious Bronchitis with the Recruitment of Immune Cells into the Respiratory Tract of Chickens. Viruses, 10.","DOI":"10.3390\/v10110635"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1251","DOI":"10.1637\/9401-052310-Reg.1","article-title":"Protection against Infectious Laryngotracheitis by in ovo Vaccination with Commercially Available Viral Vector Recombinant Vaccines","volume":"54","author":"Johnson","year":"2010","journal-title":"Avian Dis."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Korsa, M.G., Browning, G.F., Coppo, M.J.C., Legione, A.R., Gilkerson, J.R., Noormohammadi, A.H., Vaz, P.K., Lee, S.W., Devlin, J.M., and Hartley, C.A. (2015). Protection Induced in Broiler Chickens Following Drinking-Water Delivery of Live Infectious Laryngotracheitis Vaccines against Subsequent Challenge with Recombinant Field Virus. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0137719"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1080\/03079457.2019.1568389","article-title":"Long-Term Protection against a Virulent Field Isolate of Infectious Laryngotracheitis Virus Induced by Inactivated, Recombinant, and Modified Live Virus Vaccines in Commercial Layers","volume":"48","author":"Zavala","year":"2019","journal-title":"Avian Pathol."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Contreras, A.P., van der Meer, F., Checkley, S., Jospeh, T., King, R., Ravi, M., Peters, D., Fonseca, K., Gagnon, C.A., and Provost, C. (2020). Analysis of Whole-Genome Sequences of Infectious laryngotracheitis Virus Isolates from Poultry Flocks in Canada: Evidence of Recombination. Viruses, 12.","DOI":"10.3390\/v12111302"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1016\/j.dci.2013.03.022","article-title":"Immune Responses to Infectious Laryngotracheitis Virus","volume":"41","author":"Coppo","year":"2013","journal-title":"Dev. Comp. Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Beltr\u00e1n, G., Hurley, D.J., Gogal, R.M., Sharif, S., Read, L.R., Williams, S.M., Jerry, C.F., Maekawa, D.A., and Garc\u00eda, M. (2019). Immune Responses in the Eye-Associated Lymphoid Tissues of Chickens after Ocular Inoculation with Vaccine and Virulent Strains of the Respiratory Infectious Laryngotracheitis Virus (ILTV). Viruses, 11.","DOI":"10.3390\/v11070635"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1080\/03079457.2021.1989379","article-title":"Assessing the Infiltration of Immune Cells in the Upper Trachea Mucosa after Infectious Laryngotracheitis Virus (ILTV) Vaccination and Challenge","volume":"50","author":"Maekawa","year":"2021","journal-title":"Avian Pathol."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Maekawa, D., Riblet, S.M., Whang, P., Hurley, D.J., and Garcia, M. (2021). Activation of Cytotoxic Lymphocytes and Presence of Regulatory T Cells in the Trachea of Non-Vaccinated and Vaccinated Chickens as a Recall to an Infectious Laryngotracheitis Virus (ILTV) Challenge. Vaccines, 9.","DOI":"10.3390\/vaccines9080865"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1046\/j.1365-3083.1998.00441.x","article-title":"Costimulatory Function of CD28 in Avian \u0393\u03b4 T Cells Is Evolutionarily Conserved","volume":"48","author":"Koskela","year":"1998","journal-title":"Scand. J. Immunol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1016\/j.immuni.2009.10.011","article-title":"CD44 Regulates Survival and Memory Development in Th1 Cells","volume":"32","author":"Baaten","year":"2010","journal-title":"Immunity"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2779","DOI":"10.1007\/s00018-019-03117-1","article-title":"Progress on Chicken T Cell Immunity to Viruses","volume":"76","author":"Dai","year":"2019","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1182\/blood-2010-06-288456","article-title":"A CpG-Loaded Tumor Cell Vaccine Induces Antitumor CD4+ T Cells That Are Effective in Adoptive Therapy for Large and Established Tumors","volume":"117","author":"Goldstein","year":"2011","journal-title":"Blood"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1080\/03079457.2018.1492090","article-title":"Cytokine Gene Transcription in the Trachea, Harderian Gland, and Trigeminal Ganglia of Chickens Inoculated with Virulent Infectious Laryngotracheitis Virus (ILTV) Strain","volume":"47","author":"Vagnozzi","year":"2018","journal-title":"Avian Pathol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2636","DOI":"10.3389\/fimmu.2018.02636","article-title":"Tissue Adaptations of Memory and Tissue-Resident Gamma Delta T Cells","volume":"9","author":"Khairallah","year":"2018","journal-title":"Front. Immunol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1016\/S0145-305X(03)00035-1","article-title":"Differential Nitric Oxide Production by Chicken Immune Cells","volume":"27","author":"Crippen","year":"2003","journal-title":"Dev. Comp. Immunol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Hao, X., Zhang, F., Yang, Y., and Shang, S. (2021). The Evaluation of Cellular Immunity to Avian Viral Diseases: Methods, Applications, and Challenges. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.794514"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Elshafiee, E.A., Isham, I.M., Najimudeen, S.M., Perez-Contreras, A., Barboza-Solis, C., Ravi, M., and Abdul-Careem, M.F. (2022). Host Responses Following Infection with Canadian-Origin Wildtype and Vaccine Revertant Infectious Laryngotracheitis Virus. Vaccines, 10.","DOI":"10.3390\/vaccines10050782"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"11396","DOI":"10.1128\/JVI.02151-15","article-title":"Downregulation of Toll-Like Receptor 9 Expression by Beta Human Papillomavirus 38 and Implications for Cell Cycle Control","volume":"89","author":"Pacini","year":"2015","journal-title":"J. Virol."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Li, H., Li, T., Guo, Y., Li, Y., Zhang, Y., Teng, N., Zhang, F., and Yang, G. (2018). Molecular Characterization and Expression Patterns of a Non-Mammalian Toll-like Receptor Gene (TLR21) in Larvae Ontogeny of Common Carp (Cyprinus carpio L.) and upon Immune Stimulation. BMC Vet. Res., 14.","DOI":"10.1186\/s12917-018-1474-4"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/10\/2103\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T21:08:18Z","timestamp":1760130498000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/10\/2103"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,10,17]]},"references-count":47,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2023,10]]}},"alternative-id":["v15102103"],"URL":"https:\/\/doi.org\/10.3390\/v15102103","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,10,17]]}}}