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Avian infectious bronchitis (IB) caused by the infectious bronchitis virus (IBV) is one of the most important respiratory diseases in poultry. IBV affects the respiratory, kidney, and reproductive systems in broilers and laying chickens and leads to reduced meat production, mortality, quantitative and qualitative loss of egg production, and economic losses in the poultry industry. Due to the highly contagious nature of the disease, the implementation of preventive measures, including vaccination and biosecurity is essential in controlling the disease. For biosecurity, it is important to identify the entry route of new strains of IBV into an area, and markers such as unique mutations make the viruses traceable. Likewise, for effective immunization, the vaccine strain selection can be based on circulating pathogenic serotypes in the field. In this study, which was performed to determine the circulating genotypes in Khorasan Razavi province, 53 commercial chicken flocks including 32 broilers and 21 laying flocks with respiratory diseases were sampled during 2019-2021. Flocks were located in the cities of Mashhad, Chenaran, Torbat Heydariyeh, Kadkan, Kalat, Gonabad, Quchan, Neishabour, Sabzevar, Kashmar, Shandiz, Sarakhs, Baghcheh, and Khaf. Diagnosis of IBV infection and genotyping were performed using nested PCR and phylogenetic analysis of amplified sequences of S1 gene HVR3. PCR was positive for 28 (52.8%) flocks; 16 IBVs were detected in broiler flocks and 12 IBVs in laying flocks. Phylogenetic analysis showed that eleven viruses were detected in Mashhad (3 flocks), Chenaran (1 flock), Torbat Heydariyeh (3 flocks), Kadkan (1 flock), Gonabad (2 flocks), and Quchan (1 flock) were classified in lineage GI-23 (IS-Var2-type). 20 flocks (37.7%) were also positive for 793B serotype and 10 flocks (18.86%) were positive for Massachusetts serotype. Nucleotide and amino acid analysis of GI-23 viruses showed that although the origins of these viruses are Iranian variant 2 IBVs detected in the 2010s, 10 viruses have unique point mutations, and are located in three different clades in the phylogenetic tree. For 5 viruses, point mutations have led to amino acid changes T270M and A350S, and compared to other variant 2 viruses reported from Iran, these viruses are located in two different clades of the protein phylogenetic tree. Analysis of evolutionary distances showed a high evolutionary distance between the viruses detected in this study with the ones detected in Khorasan Razavi province in 2010, and the prevalent pathogenic IBV has changed from IS720 to variant 2 in 10 years. We also observed that the prevalence of variant 2 IBVs in the province in 2020 and 2021 has decreased significantly compared to 2019. Based on the results of this study, circulating viruses in Khorasan Razavi province have found unique point mutations compared to the previously detected variant 2 viruses in Iran, which could indicate the high circulation of variant 2 viruses in the province during at least 3 years period from 2016. Accordingly, the use of more effective vaccination programs against variant 2 viruses should be considered, which can be as live Massachusetts vaccine in one-day chicks and live vaccine of 793B in the second week. It is also recommended to implement stronger management programs such as biosecurity, flock population with peer chickens and cleaning and disinfecting poultry farms, as well as tools and equipment.
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