Surveillance of zoonotic and non-zoonotic bovine viruses circulating among cattle in Catalonia and characterization of their excreted virome

Abstract

Zoonotic and non-zoonotic viruses with veterinarian interest can enter the environment through different routes, depending on its shedding mechanism, being able to infect other animals and humans continuously. Following the concept of One-health, it’s important to enhance preparedness and early warning of possible viral outbreaks, with potential to jeopardize the lives of animals and humans while maintaining the environment sustainability. The goal of this work was to study the potential of using collective environmental samples to detect viruses of veterinarian interest and/or with zoonotic potential, circulating among cattle, reducing the need for the collection of individual clinical samples that require the use of invasive methods in the animals. In addition, new SOPs for n(RT)PCRs and (RT)qPCRs to detect these viruses were implemented in the laboratory. Between May and June of 2023, individual saliva and faeces samples together with collective samples such as drinking water and bed straw were collected in 4 cattle farms to compare the detection by (RT)qPCR of selected viruses. These samples were analysed to optimize the methods used in this study. A sampling campaign of collective samples was implemented: 16 lixiviates and 16 aerosol samples from 2 cattle farms were collected from September to April 2023-2024 and 8 wastewater samples from 2 cattle slaughterhouses were collected between February and April 2024. For viral detection, all the collective samples were submitted to a viral concentration procedure followed by nucleic acid extraction. Samples were tested for several bovine viruses by (RT)qPCR: BPyV, RoV-A, BCoV, BRSV, CCHFV, EHDV, BTV and AIV. BPyV seemed to be a good bovine viral marker in lixiviate samples (13 /15) but not in clinical samples (2/99) nor in wastewater samples from slaughterhouses (5/8 with very low concentrations). Overall, RoV-A was detected in 31,25% of the lixiviates from the cow farms and in 75% of the wastewater samples from slaughterhouses and BCoV was detected in both lixiviates and aerosol samples and it was present in 50% of the samples of Sabadell slaughterhouse, proving the circulation of the virus among cattle in Catalonia. The viruses AIV, CCHFV, BRSV, EHDV and BTV weren’t detected in any of the individual and collective samples tested by (RT)qPCR. A nested (RT)PCR for the detection of EHDV (targeting mainly the serotypes 6 and 8) was developed “in house” and it showed to be efficient for concentrations as low as 250GC/rx in biological samples. Furthermore, NGS was performed on the collective environmental samples using an Illumina NextSeq 550 platform coupled with a target enrichment to characterize the virome. The NGS enabled the detection of 16 viral genus related to bovine infection. Other non-bovine viruses were also detected in collective samples including viruses not detected by (RT)qPCR techniques, such as BRSV and BPyV in aerosols. However, the method showed difficulties in capturing dsRNA genomes, as the case of RoV-A, detected in high amounts by (RT)qPCR. In this work, the use of collective environmental samples revealed to be suitable for surveillance of viruses circulating in cattle industry, suggesting it’s potential use as an early detection tool of viral outbreaks of veterinary interest in the cows’ population, or to monitor the circulation of viruses with zoonotic potential.

Date of Award	26 Jul 2024
Original language	English
Awarding Institution	Universidade Católica Portuguesa
Supervisor	Sílvia Bofill Mas (Supervisor) & Xavier Fernandez Cassi (Co-Supervisor)