A Comprehensive Pathological and Molecular Investigation of Viral Co-Infections in Ducks in Egypt

Rania I. Mohamed ¹ Samah M. Mosad ² Hanaa S. Ali ¹ Wejdan Oudah Albalawi ³ Hanaa A. Elsamadony ⁴ Neven M. Ramzy ⁵ Aalaa Samir Saad ⁶ Deema Fallatah ⁷ Lina Jamil Abdelhafez ⁸ Ashraf Albrakati ⁹ Ehab Kotb Elmahallawy ^10*

• ¹ Department of Pathology, Animal Health Research institute, Mansoura branch (AHRI), Agricultural Research Center (ARC), Giza, POB: 12618, Egypt
• ² Department of Virology, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt
• ³ Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Al Jouf University, Al-Jouf, Saudi Arabia
• ⁴ Poultry diseases Department, Animal Health Research institute (AHRI), Agricultural Research Center (ARC),, Giza, POB: 12618, Egypt
• ⁵ Virology Department, Animal Health Research institute, Ismailia branch (AHRI), Agricultural Research Center (ARC), Dokki, Giza, POB: 12618, Egypt
• ⁶ Biotechnology Department, Animal Health Research institute (AHRI), Agricultural Research Center (ARC), Giza, POB: 12618, Egypt
• ⁷ Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Prince Sattam bin Abdulaziz University, Al-Kharj, Saudi Arabia
• ⁸ Microbiology and Immunology Department, Faculty of Pharmacy, October 6 University, Cairo, Egypt
• ⁹ Department of Human Anatomy, College of Medicine, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
• ¹⁰ Department of Animal Health, Faculty of Veterinary Medicine, University of Cordoba, Cordoba, Spain

Introduction: Duck production in Egypt plays a significant role in the poultry sector. However, viral infections, such as avian influenza virus (AIV), Newcastle disease virus (NDV), and duck hepatitis A virus (DHAV) pose a significant threat to ducks, leading to substantial economic losses. Despite their impact, data on these Duck pathogens in Egypt remain limited.Methods: In this study, 200 samples from various organs were collected from 20 commercial duck farms and pooled into 20 working samples. Samples of brain, liver, spleen, trachea and lung were analysed to detect DHAV, NDV, H5 and H9 AIV using reverse transcriptase polymerase chain reaction (RT-PCR) then positive samples were subjected for sequencing. Samples from the same organs were also subjected for histopathological examination. Results: Interestingly, the RT-PCR detected DHAV, NDV and H9-AIV and mixed viral infections were confirmed in some farms. The phylogenetic analysis of DHAV 3D gene revealed that both DHAV-1 and DHAV-3 genotypes are circulating in Egyptian duckling with most tested samples contain DHAV-3 genotype, considered the vaccine used in Egypt contain DHAV-1 strain only. All detected NDV strains in this study are clustered in Genotype VII.1.1 with F0 cleavage site (RRQKR ↓ F) of velogenic NDV. On the other hand, our studied H9-AIV strains are aligned in H9.4.1.1 sub- lineage with other Egyptian field and vaccine seed strains. Local Egyptian vaccine seed strains were found closely related to our isolates than imported vaccines. H9.4.1 strains displayed HA0 protein cleavage site motif PARSSR↓GLF of LPAI. All the aligned Egyptian H9-AIV field and local vaccine strains have 168N, 191H, 197T, 224L and 234 L amino residues, indicating that these viruses had the characteristic of receptor specificity like that of human influenza virus increasing the zoonotic risk of such virus. Histopathologically, animals showed characteristic lesions in various organs coherent to the infection by these mentioned pathogens. Conclusions: Collectively, the study provided novel information about viral infections linked to neurological diseases of ducks in Egypt and concluded that local DHAV vaccine needs to be modified to contain both DHAV-1 and DHAV-3 strains.