Development and evaluation of an indigenous Salmonella gallinarum ghost vaccine for the control of fowl typhoid in poultry
Abstract
Background and Objectives: Salmonella gallinarum, causes fowl typhoid, continues to be a significant limitation to poultry production in developing nations, with continuously rising antimicrobial resistance. The current research was design and test an indigenous S. gallinarum ghost vaccine and to measure its immunogenicity and protective effect on layer birds.
Materials and Methods: Bacteriological culture of suspected clinical cases of fowl typhoid was performed, and isolates were verified by PCR against the 16S rRNA gene, after which they were sequenced, and phylogenetic analysis was performed. To obtain bacterial ghost cells, a representative native isolate was subjected to a detergent-based chemical lysis procedure, followed up by scanning electron microscopy. 120-day-old layer chicks were divided into six groups randomly and immunized at the age of seven days using oral, intramuscular, air-spray, intramuscular with oil adjuvant or eye-drop routes. There was a 1st dose at 26 days of age in all vaccinated groups and the challenge of virulent S. gallinarum at 42 and 93 days of age. ELISA was used to test humoral immune response at different time points.
Results: The highest antibody titers were found in vaccinated groups compared to controls (p < 0.05), and the intramuscular route generated the best and most enduring response in systemic immunity. Mucosal pathways elicited not high but rather significant antibody responses and provided protection against clinical disease.
Conclusion: The research contributes to the feasibility of ghost-based vaccination in the role of a sustainable alternative to the disease control measures that are highly reliant on antibiotics in endemic poultry production systems.
1. Lamichhane B, Mawad AMM, Saleh M, Kelley WG, Harrington PJ 2nd, Lovestad CW, et al. Salmonellosis: An Overview of Epidemiology, Pathogenesis, and Innovative Approaches to Mitigate the Antimicrobial Resistant Infections. Antibiotics (Basel) 2024; 13: 76.
2. Shaji S, Selvaraj RK, Shanmugasundaram R. Salmonella infection in poultry: A Review on the pathogen and control strategies. Microorganisms 2023; 11: 2814.
3. Wales A, Lawes J. JMM Profile: Salmonella enterica serovar Gallinarum, biovars Pullorum and Gallinarum. J Med Microbiol 2023; 72. DOI 10.1099/jmm.0.001653.
4. Farhat M, Khayi S, Berrada J, Mouahid M, Ameur N, El-Adawy H, et al. Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum in poultry: Review of pathogenesis, antibiotic resistance, diagnosis and control in the genomic era. Antibiotics (Basel) 2024; 13: 23.
5. Huang K, Herrero-Fresno A, Thøfner I, Skov S, Olsen JE. Interaction Differences of the Avian Host-Specific Salmonella enterica Serovar Gallinarum, the Host-Generalist S. typhimurium, and the Cattle Host-Adapted S. Dublin with Chicken Primary Macrophage. Infect Immun 2019; 87(12): e00552-19.
6. Shivaprasad HL. Fowl typhoid and pullorum disease. Rev Sci Tech 2000; 19: 405-424.
7. Galán-Relaño Á, Valero Díaz A, Huerta Lorenzo B, Gómez-Gascón L, Mena Rodríguez MÁ, Carrasco Jiménez E, et al. Salmonella and Salmonellosis: An update on public health implications and control strategies. Animals (Basel) 2023; 13: 3666.
8. Jones PJ, Niemi J, Christensen JP, Tranter RB, Bennett RM. A review of the financial impact of production diseases in poultry production systems. Anim Prod Sci 2019; 59: 1585-1597.
9. Koutsoumanis K, Allende A, Alvarez-Ordóñez A, Bolton D, Bover-Cid, S, Chemaly M, et al. Salmonella control in poultry flocks and its public health impact. EFSA J 2019; 17(2): e05596.
10. Hassnain M, Subhan A, Beig M, Akram W, Ahmad F, Mushtaq S, et al (2025). Vaccine utilisation in the prevention of viral diseases in Africa. In: Shenge JA, Abia ALK (eds). Viral Diseases and One Health in Africa. Springer, Cham, pp. 67-82.
11. Usman H, Subhan A, Beig M, Hassnain M, Ahmad W, Arshad A (2025). Challenges and Constraints Regarding Control and Prevention of Dog-Mediated Human Rabies Using the One Health Approach in Africa. In: Shenge JA, Abia ALK (eds). Viral Diseases and One Health in Africa. Springer, Cham, pp. 47-65.
12. Focosi D, Maggi F, Casadevall A. Mucosal vaccines, sterilizing immunity, and the future of SARS-CoV-2 virulence. Viruses 2022; 14: 187.
13. Mahmood N, Rizvi F, Saleemi MK, Aslam MA. Seroprevalence and Immunopathological Studies of Salmonella pullorum in Broiler Birds in District Faisalabad Pakistan. Pak Vet J 2022; 42: 47-52.
14. Adzitey F, Huda N, Ali GR. Molecular techniques for detecting and typing of bacteria, advantages and application to foodborne pathogens isolated from ducks. 3 Biotech 2013; 3: 97-107.
15. Zanetti NS, De Carli S, Souza MN, Lehmann FK, Kipper D, Dias KK, et al. Molecular detection and characterization of Salmonella gallinarum from poultry farms in Brazil. J Appl Poult Res 2019; 28: 1335-1341.
16. Vaid RK, Thakur Z, Anand T, Kumar S, Tripathi BN, Comparative genome analysis of Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum decodes strain specific genes. PLoS One 2021; 16(8): e0255612.
17. Soleymani S, Tavassoli A, Hashemi Tabar G, Kalidari GA, Dehghani H. Design, development, and evaluation of the efficacy of a nucleic acid-free version of a bacterial ghost candidate vaccine against avian pathogenic E. coli (APEC) O78:K80 serotype. Vet Res 2020; 51: 144.
18. Czerwińska-Główka D, Krukiewicz K. Guidelines for a morphometric analysis of prokaryotic and eukaryotic cells by scanning electron microscopy. Cells 2021; 10: 3304.
19. Senevirathne A, Hewawaduge C, Lee JH. Immunization of chicken with flagellin adjuvanted Salmonella enteritidis bacterial ghosts confers complete protection against chicken salmonellosis. Poult Sci 2021; 100: 101205.
20. Jawale CV, Lee JH. Comparative evaluation of Salmonella enteritidis ghost vaccines with a commercial vaccine for protection against internal egg contamination with Salmonella. Vaccine 2014; 32: 5925-5930.
21. Chaudhari AA, Jawale CV, Kim SW, Lee JH. Construction of a Salmonella gallinarum ghost as a novel inactivated vaccine candidate and its protective efficacy against fowl typhoid in chickens. Vet Res 2012; 43: 44.
22. Alzwghaibi AB, Yahyaraeyat R, Fasaei BN, Langeroudi AG, Salehi TZ. Rapid molecular identification and differentiation of common Salmonella serovars isolated from poultry, domestic animals and foodstuff using multiplex PCR assay. Arch Microbiol 2018; 200: 1009-1016.
23. Beig MM, Mehmood S, Farooq HMA, Subhan A (2025). Harnessing AI for green health: Revolutionizing disease diagnosis and Prevention in Ecosystem-Centric Environments. In: Poonia RC, Thakur D, Thakur I (eds). Intelligent Systems for Sustainable Infrastructure: AI Solutions Shaping a Green Future. Springer, Cham, pp. 43-71.
24. Haque AKMZ, Akter MR, Islam SKS, Alam J, Neogi SB, Yamasaki S, et al. Salmonella gallinarum in Small-Scale Commercial Layer Flocks: Occurrence, Molecular Diversity and Antibiogram. Vet Sci 2021; 8: 71.
25. Mu J, Lei L, Zheng Y, Li D, Li J, Fu Y, et al. Comparative study of subcutaneous, intramuscular, and oral administration of bovine pathogenic Escherichia coli bacterial ghost vaccine in mice. Front Immunol 2022; 13: 1008131.
26. Jiao H, Yang H, Zhao D, Chen J, Zhang Q, Liang J, et al. Design and immune characterization of a novel Neisseria gonorrhoeae DNA vaccine using bacterial ghosts as vector and adjuvant. Vaccine 2018; 36: 4532-4539.
27. Landy M, Sanderson RP, Jackson AL. Humoral and cellular aspects of the immune response to the somatic antigen of Salmonella enteritidis. J Exp Med 1965; 122: 483-504.
28. Acevedo R, Callicó A, Aranguren Y, Zayas C, Valdés Y, Pérez O, et al. Immune adjuvant effect of V. cholerae O1 derived Proteoliposome coadministered by intranasal route with Vi polysaccharide from Salmonella Typhi. BMC Immunol 2013; 14 Suppl 1(Suppl 1): S10.
29. Correa VA, Portilho AI, De Gaspari E. Vaccines, adjuvants and key factors for mucosal immune response. Immunology 2022; 167: 124-138.
30. Liu W, Yang Y, Chung N, Kwang J. Induction of humoral immune response and protective immunity in chickens against Salmonella enteritidis after a single dose of killed bacterium-loaded microspheres. Avian Dis 2001; 45: 797-806.
31. Takaya A, Yamamoto T, Tokoyoda K. Humoral Immunity vs. Salmonella. Front Immunol 2020; 10: 3155.
32. Kogut MH, Genovese K, Moyes RB, Stanker LH. Evaluation of oral, subcutaneous, and nasal administration of Salmonella enteritidis-immune lymphokines on the potentiation of a protective heterophilic inflammatory response to Salmonella enteritidis in day-old chickens. Can J Vet Res 1998; 62: 27-32.
33. Kamathewatta KI, Kanci Condello A, Noormohammadi AH, Young ND, Kulappu Arachchige SN, Browning GF, et al. Tracheal transcriptional response to challenge with virulent Mycoplasma gallisepticum in chickens spray vaccinated with the Vaxsafe MG304 live-attenuated vaccine. Vet Microbiol 2026; 320: 111123.
34. Penha Filho RA, de Paiva JB, da Silva MD, de Almeida AM, Berchieri A Jr. Control of Salmonella Enteritidis and Salmonella gallinarum in birds by using live vaccine candidate containing attenuated Salmonella gallinarum mutant strain. Vaccine 2010; 28: 2853-2859.
35. SMITH HW. The use of live vaccines in experimental Salmonella gallinarum infection in chickens with observations on their interference effect. J Hyg (Lond) 1956; 54: 419-432.
36. Rodrigues CMC, Plotkin SA. The influence of interval between doses on response to vaccines. Vaccine 2021; 39: 7123-7127.
37. McWhorter AR, Chousalkar KK. A Long-Term Efficacy Trial of a Live, Attenuated Salmonella Typhimurium Vaccine in Layer Hens. Front Microbiol 2018; 9: 1380.
38. Britto CD, Wong VK, Dougan G, Pollard AJ. A systematic review of antimicrobial resistance in Salmonella enterica serovar Typhi, the etiological agent of typhoid. PLoS Negl Trop Dis 2018; 12(10): e0006779.
39. Jaleta H, Mamo B, Disassa H. Review on bacterial ghost and its application. Int J Microbiol Res 2015; 6: 200-210.
40. Park SY. Chemically induced bacterial ghosts: a novel approach for advancing biomedical applications. Mol Cell Toxicol 2023; 19: 657-665.
2. Shaji S, Selvaraj RK, Shanmugasundaram R. Salmonella infection in poultry: A Review on the pathogen and control strategies. Microorganisms 2023; 11: 2814.
3. Wales A, Lawes J. JMM Profile: Salmonella enterica serovar Gallinarum, biovars Pullorum and Gallinarum. J Med Microbiol 2023; 72. DOI 10.1099/jmm.0.001653.
4. Farhat M, Khayi S, Berrada J, Mouahid M, Ameur N, El-Adawy H, et al. Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum in poultry: Review of pathogenesis, antibiotic resistance, diagnosis and control in the genomic era. Antibiotics (Basel) 2024; 13: 23.
5. Huang K, Herrero-Fresno A, Thøfner I, Skov S, Olsen JE. Interaction Differences of the Avian Host-Specific Salmonella enterica Serovar Gallinarum, the Host-Generalist S. typhimurium, and the Cattle Host-Adapted S. Dublin with Chicken Primary Macrophage. Infect Immun 2019; 87(12): e00552-19.
6. Shivaprasad HL. Fowl typhoid and pullorum disease. Rev Sci Tech 2000; 19: 405-424.
7. Galán-Relaño Á, Valero Díaz A, Huerta Lorenzo B, Gómez-Gascón L, Mena Rodríguez MÁ, Carrasco Jiménez E, et al. Salmonella and Salmonellosis: An update on public health implications and control strategies. Animals (Basel) 2023; 13: 3666.
8. Jones PJ, Niemi J, Christensen JP, Tranter RB, Bennett RM. A review of the financial impact of production diseases in poultry production systems. Anim Prod Sci 2019; 59: 1585-1597.
9. Koutsoumanis K, Allende A, Alvarez-Ordóñez A, Bolton D, Bover-Cid, S, Chemaly M, et al. Salmonella control in poultry flocks and its public health impact. EFSA J 2019; 17(2): e05596.
10. Hassnain M, Subhan A, Beig M, Akram W, Ahmad F, Mushtaq S, et al (2025). Vaccine utilisation in the prevention of viral diseases in Africa. In: Shenge JA, Abia ALK (eds). Viral Diseases and One Health in Africa. Springer, Cham, pp. 67-82.
11. Usman H, Subhan A, Beig M, Hassnain M, Ahmad W, Arshad A (2025). Challenges and Constraints Regarding Control and Prevention of Dog-Mediated Human Rabies Using the One Health Approach in Africa. In: Shenge JA, Abia ALK (eds). Viral Diseases and One Health in Africa. Springer, Cham, pp. 47-65.
12. Focosi D, Maggi F, Casadevall A. Mucosal vaccines, sterilizing immunity, and the future of SARS-CoV-2 virulence. Viruses 2022; 14: 187.
13. Mahmood N, Rizvi F, Saleemi MK, Aslam MA. Seroprevalence and Immunopathological Studies of Salmonella pullorum in Broiler Birds in District Faisalabad Pakistan. Pak Vet J 2022; 42: 47-52.
14. Adzitey F, Huda N, Ali GR. Molecular techniques for detecting and typing of bacteria, advantages and application to foodborne pathogens isolated from ducks. 3 Biotech 2013; 3: 97-107.
15. Zanetti NS, De Carli S, Souza MN, Lehmann FK, Kipper D, Dias KK, et al. Molecular detection and characterization of Salmonella gallinarum from poultry farms in Brazil. J Appl Poult Res 2019; 28: 1335-1341.
16. Vaid RK, Thakur Z, Anand T, Kumar S, Tripathi BN, Comparative genome analysis of Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum decodes strain specific genes. PLoS One 2021; 16(8): e0255612.
17. Soleymani S, Tavassoli A, Hashemi Tabar G, Kalidari GA, Dehghani H. Design, development, and evaluation of the efficacy of a nucleic acid-free version of a bacterial ghost candidate vaccine against avian pathogenic E. coli (APEC) O78:K80 serotype. Vet Res 2020; 51: 144.
18. Czerwińska-Główka D, Krukiewicz K. Guidelines for a morphometric analysis of prokaryotic and eukaryotic cells by scanning electron microscopy. Cells 2021; 10: 3304.
19. Senevirathne A, Hewawaduge C, Lee JH. Immunization of chicken with flagellin adjuvanted Salmonella enteritidis bacterial ghosts confers complete protection against chicken salmonellosis. Poult Sci 2021; 100: 101205.
20. Jawale CV, Lee JH. Comparative evaluation of Salmonella enteritidis ghost vaccines with a commercial vaccine for protection against internal egg contamination with Salmonella. Vaccine 2014; 32: 5925-5930.
21. Chaudhari AA, Jawale CV, Kim SW, Lee JH. Construction of a Salmonella gallinarum ghost as a novel inactivated vaccine candidate and its protective efficacy against fowl typhoid in chickens. Vet Res 2012; 43: 44.
22. Alzwghaibi AB, Yahyaraeyat R, Fasaei BN, Langeroudi AG, Salehi TZ. Rapid molecular identification and differentiation of common Salmonella serovars isolated from poultry, domestic animals and foodstuff using multiplex PCR assay. Arch Microbiol 2018; 200: 1009-1016.
23. Beig MM, Mehmood S, Farooq HMA, Subhan A (2025). Harnessing AI for green health: Revolutionizing disease diagnosis and Prevention in Ecosystem-Centric Environments. In: Poonia RC, Thakur D, Thakur I (eds). Intelligent Systems for Sustainable Infrastructure: AI Solutions Shaping a Green Future. Springer, Cham, pp. 43-71.
24. Haque AKMZ, Akter MR, Islam SKS, Alam J, Neogi SB, Yamasaki S, et al. Salmonella gallinarum in Small-Scale Commercial Layer Flocks: Occurrence, Molecular Diversity and Antibiogram. Vet Sci 2021; 8: 71.
25. Mu J, Lei L, Zheng Y, Li D, Li J, Fu Y, et al. Comparative study of subcutaneous, intramuscular, and oral administration of bovine pathogenic Escherichia coli bacterial ghost vaccine in mice. Front Immunol 2022; 13: 1008131.
26. Jiao H, Yang H, Zhao D, Chen J, Zhang Q, Liang J, et al. Design and immune characterization of a novel Neisseria gonorrhoeae DNA vaccine using bacterial ghosts as vector and adjuvant. Vaccine 2018; 36: 4532-4539.
27. Landy M, Sanderson RP, Jackson AL. Humoral and cellular aspects of the immune response to the somatic antigen of Salmonella enteritidis. J Exp Med 1965; 122: 483-504.
28. Acevedo R, Callicó A, Aranguren Y, Zayas C, Valdés Y, Pérez O, et al. Immune adjuvant effect of V. cholerae O1 derived Proteoliposome coadministered by intranasal route with Vi polysaccharide from Salmonella Typhi. BMC Immunol 2013; 14 Suppl 1(Suppl 1): S10.
29. Correa VA, Portilho AI, De Gaspari E. Vaccines, adjuvants and key factors for mucosal immune response. Immunology 2022; 167: 124-138.
30. Liu W, Yang Y, Chung N, Kwang J. Induction of humoral immune response and protective immunity in chickens against Salmonella enteritidis after a single dose of killed bacterium-loaded microspheres. Avian Dis 2001; 45: 797-806.
31. Takaya A, Yamamoto T, Tokoyoda K. Humoral Immunity vs. Salmonella. Front Immunol 2020; 10: 3155.
32. Kogut MH, Genovese K, Moyes RB, Stanker LH. Evaluation of oral, subcutaneous, and nasal administration of Salmonella enteritidis-immune lymphokines on the potentiation of a protective heterophilic inflammatory response to Salmonella enteritidis in day-old chickens. Can J Vet Res 1998; 62: 27-32.
33. Kamathewatta KI, Kanci Condello A, Noormohammadi AH, Young ND, Kulappu Arachchige SN, Browning GF, et al. Tracheal transcriptional response to challenge with virulent Mycoplasma gallisepticum in chickens spray vaccinated with the Vaxsafe MG304 live-attenuated vaccine. Vet Microbiol 2026; 320: 111123.
34. Penha Filho RA, de Paiva JB, da Silva MD, de Almeida AM, Berchieri A Jr. Control of Salmonella Enteritidis and Salmonella gallinarum in birds by using live vaccine candidate containing attenuated Salmonella gallinarum mutant strain. Vaccine 2010; 28: 2853-2859.
35. SMITH HW. The use of live vaccines in experimental Salmonella gallinarum infection in chickens with observations on their interference effect. J Hyg (Lond) 1956; 54: 419-432.
36. Rodrigues CMC, Plotkin SA. The influence of interval between doses on response to vaccines. Vaccine 2021; 39: 7123-7127.
37. McWhorter AR, Chousalkar KK. A Long-Term Efficacy Trial of a Live, Attenuated Salmonella Typhimurium Vaccine in Layer Hens. Front Microbiol 2018; 9: 1380.
38. Britto CD, Wong VK, Dougan G, Pollard AJ. A systematic review of antimicrobial resistance in Salmonella enterica serovar Typhi, the etiological agent of typhoid. PLoS Negl Trop Dis 2018; 12(10): e0006779.
39. Jaleta H, Mamo B, Disassa H. Review on bacterial ghost and its application. Int J Microbiol Res 2015; 6: 200-210.
40. Park SY. Chemically induced bacterial ghosts: a novel approach for advancing biomedical applications. Mol Cell Toxicol 2023; 19: 657-665.
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| Issue | Vol 18 No 4 (2026) | |
| Section | Original Article(s) | |
| Keywords | ||
| Salmonella gallinarum ghost Fowl typhoid Vaccine Chicken Efficacy | ||
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How to Cite
1.
Ghalib A, Hussain S, Shahzad W, Mir R, Muneer B. Development and evaluation of an indigenous Salmonella gallinarum ghost vaccine for the control of fowl typhoid in poultry. Iran J Microbiol. 2026;18(4):574-583.
