Assessment of Zoonotic Pathogens in Poultry and Environmental Water Sources Using Duplex PCR in Karbala, Iraq
DOI:
https://doi.org/10.48165/ijapm.2025.41.4.7Keywords:
Salmonella, Vibrio cholerae, Poultry, Water sources, Molecular epidemiology, Duplex PCR, Karbala, Zoonotic pathogens.Abstract
Background The world-wide poultry industry has grown rapidly to provide the increasing demand for low-cost protein. This expansion offers more opportunities for zoonotic diseases to be transmitted, especially Salmonella spp. and Vibrio cholerae that are important pathogens for foodborne illness. Both Salmonella has been reported among poultry flocks in Karbala, Iraq and V. cholerae which is commonly associated with contaminated water may also represent potential zoonotic risk within the settings of poultry production. Culture-based detection is time-consuming, laborious and may fail to detect low levels of contamination; molecular methods like the polymerase chain reaction (PCR) provide fast, sensitive and specific identification of targets that are amenable for large epidemiological surveys. Objective: To investigate the molecular prevalence of Salmonella spp. and Vibrio cholerae in the different samples, their concurrent occurrence, assess associated risk factors and estimate the performance of duplex PCR assay for pathogens monitoring. Methods: Descriptive study of 20 poultry farms in Karbala province was conducted over a period of one year. In total, 82 samples were collected from the hens and cloaca swabs of poultry and feces samples (n = 82) and environmental/water (n = 118). Isolation and detection of Salmonella was based on invA gene and V. cholerae using rtxA/lolB genes by duplex PCR; results were confirmed through agarose gel electrophoresis. Chi-squared tests and logistic regression were used to determine prevalence and associations with potential risk factors, including farm biosecurity and quality of water source. Results: Salmonella spp. was found in 15% of poultry, mainly in cloacal swab indicating extensive carrier status. Vibrio cholerae was detected in 9% of water/ environment samples. Simultaneous presentation of both pathogens was found in 6% of waterborne samples, indicating possible cross-contamination predominately from water. Pathogen occurrence was positively correlated with low biosecurity and use of natural water sources (P < 0.05). The duplex PCR showed excellent sensitivity and specificity to rapid diagnosis. Conclusion: This study provides valuable molecular epidemiological evidence regarding the prevalence and cross-resistance of Salmonella and V. cholerae in Karbala region. The biosecurity and water management should be strengthened, and routine surveillance with duplex PCR is needed to prevent resistance zoonotic risks and public health protection. Serotyping of Salmonella and determination of V. cholerae toxicity need to be added in future studies to further assess public health risks.
References
World Health Organization. (2018). Salmonella (non-typhoidal). https://www.who.int/news-room/fact-sheets/detail/salmonella-(non-typhoidal)
Centers for Disease Control and Prevention. (2021). Salmonella. https://www.cdc.gov/salmonella/
Hasan, T. B. (2021). RAPD fingerprinting and genetic diversity of Salmonella isolates from chicken flocks in Karbala, Iraq. Iraqi Journal of Veterinary Sciences, 35(1), 167–172.
Hasan, T. O. (2023). Application of RAPD-PCR and phylogenetic analysis for discrimination of Salmonella isolated from chicken, feed and drinking water for epidemiological control of salmonellosis in Karbala, Iraq. Iraqi Journal of Veterinary Medicine, 46(1), 1–10.
World Health Organization. (2022). Cholera. https://www.who.int/news-room/fact-sheets/detail/cholera
Igbinosa, I. H., Igumbor, E. U., Aghdasi, F., Tom, M., & Okoh, A. I. (2012). Emerging Aeromonas species infections and their significance in public health. The Scientific World Journal, 2012, Article 625023. https://doi.org/10.1100/2012/625023
Malorny, B., Hoorfar, J., Bunge, C., & Helmuth, R. (2003). Multicenter validation of the analytical accuracy of Salmonella PCR: Towards an international standard. Applied and Environmental Microbiology, 69(1), 290–296.
Phopin, K., Tantawiwat, S., & Ananchaipattana, C. (2024). Duplex PCR–lateral flow immunoassay for rapid and visual detection of Salmonella and Vibrio cholerae. LWT – Food Science and Technology, 198, 116041.
Gal-Mor, O., Boyle, E. C., & Grassl, G. A. (2014). Same species, different diseases: How and why typhoidal and non-typhoidal Salmonella enterica serovars differ. Frontiers in Microbiology, 5, 391.
Gantois, I., Ducatelle, R., Pasmans, F., Haesebrouck, F., Gast, R., Humphrey, T. J., & Van Immerseel, F. (2009). Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiology Reviews, 33(4), 718–738.
Kaper, J. B., Morris, J. G., Jr., & Levine, M. M. (1995). Cholera. Clinical Microbiology Reviews, 8(1), 48–86.
Hounmanou, Y. M. G., Leekitcharoenphon, P., Hendriksen, R. S., Dougnon, T. V., Mdegela, R. H., Olsen, J. E., & Dalsgaard, A. (2019). Surveillance and genomics of toxigenic Vibrio cholerae O1 from fish, phytoplankton and water in Lake Victoria, Tanzania. Frontiers in Microbiology, 10, 901.
Almanseekanaa, L. H., & Ogaili, R. H. (2023). Comparison between serological and molecular identification of Vibrio cholerae. Academic International Journal of Pharmaceutical Sciences, 1(2), 41–56. https://aijps.aipublishers.org/index.php/aijps/article/view/P125
Law, J. W. F., Ab Mutalib, N. S., Chan, K. G., & Lee, L. H. (2015). Rapid methods for the detection of foodborne bacterial pathogens: Principles, applications, advantages and limitations. Frontiers in Microbiology, 6, 770.
Yamazaki, W., Ishibashi, M., Kawahara, R., & Inoue, K. (2008). Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus. BMC Microbiology, 8, 163.
Rahn, K., De Grandis, S. A., Clarke, R. C., McEwen, S. A., Galán, J. E., Ginocchio, C., Curtiss, R., & Gyles, C. L. (1992). Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Molecular and Cellular Probes, 6(4), 271–279.
Nandi, B., Nandy, R. K., Mukhopadhyay, S., Nair, G. B., Shimada, T., & Ghose, A. C. (2000). Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the ompW gene. Journal of Clinical Microbiology, 38(11), 4145–4151.
Kadhim, N. J., Al-Karawi, N. J., Almanseekanaa, L. H., & Ogaili, R. H. (2021). Molecular characterization and antibiotic pattern of Klebsiella pneumoniae. Veterinary Practitioner, 22(2).
Almanseekanaa, L. H., Ali Alabbas, A. K., Kadhim, N. J., & Ogaili, R. H. (2021). Molecular study of Pseudomonas aeruginosa isolated from different clinical cases. Biochemical and Cellular Archives, 21(2).
Kadhim, N. J., Mahdi, M. S., & Almanseekanaa, L. H. (2025). Molecular detection of Toxoplasma gondii: Accuracy, prevalence, and standardization of diagnostic protocols. International Journal of Applied and Pure Microbiology, 41(2), 17–21. https://journals.acspublisher.com/index.php/ijapm/article/view/23110
Almanseekanaa, L. H. (2022). Molecular study of enteropathogenic Escherichia coli isolated from clinical samples. Academic International Journal of Medical Sciences, 1(1), 6–14. https://aijms.aipublishers.org/index.php/aijms/article/view/M2022-02
Kadhim, S. J., Kadhim, N. J., Ali, M. R., Ogaili, R. H., Saleh, Y. A., & Mohammed, R. Q. (2025). Hepatitis B and hepatitis C virus prevalence among Iraqi blood donors. Microbes and Infectious Diseases, 6(4), 6252–6259.
Muhammed, H. A. (2024). Concordance between RT-PCR and ELISA techniques for detection of CCHFV in Karbala City. Academic International Journal of Veterinary Medicine, 2(1), 48–55. https://aijvm.aipublishers.org/index.php/aijvm/article/view/V217
Jawad, R. A., Haddawee, R. H., Jabber, E. J., Al-Kafhage, F. A., & Farhood, A. F. (2023). Molecular detection of genotypes in sheep infected with Echinococcus granulosus in Holy Karbala Province. Academic International Journal of Veterinary Medicine, 1(2), 13–19. https://aijvm.aipublishers.org/index.php/aijvm/article/view/V123
Uddin, M. A., Ullah, M. W., & Noor, R. (2012). Prevalence of Vibrio cholerae in human, poultry, animal excreta and compost samples. Stamford Journal of Microbiology, 2(1), 38–41.
Al-Shammri, F. K., Obeid, H. N., Abbas, M. S., Mohammed, A. S., Aleigailly, M. A., Hasan, K. A., & Çelebi, F. V. (2024). Developing healthcare using Internet of Things (IoT): Applications, challenges and future directions. BIO Web of Conferences, 97, 00004.
Al-Ghanimi, B. K., Abd, K. I., Al-Baiati, M. N., Al-Ghanimi, H. H., & Al-Madany, R. A. (2024). Using a novel nano-chitosan ampicillin drug as a treatment for lung cancer cell line (A549). Arab Journal of Medicinal and Aromatic Plants, 10(2), 1–21.
Awlqadr, F. H., Altemimi, A. B., Qadir, S. A., Alkanan, Z. T., Faraj, A. M., Alkaisy, Q. H., & Abedelmaksoud, T. G. (2025). Fast food consumption has a great impact on the aging process: A review. Theory and Practice of Meat Processing, 10(1), 11–31.
Mafe, A. N., Edo, G. I., Ali, A. B., Akpoghelie, P. O., Yousif, E., Isoje, E. F., Igbuku, U. A., Opiti, R. A., Ajiduku, L. A., Owheruo, J. O., & Essaghah, A. E. (2025). Next-generation biopolymers for sustainable food packaging: Innovations in material science, circular economy, and smart technologies. Food and Bioprocess Technology, 1–57.
Al-Huseini, L. M. A., Kadhim, N. J., Mahdi, M. S., Ogaili, R. H., & Al-Hammood, O. (2025). Microbial infection disease diagnosis and treatment by artificial intelligence. Wiadomości Lekarskie, 78(2), 442–447.
