Surveillance of Newcastle Disease Virus in Tanzania

The oropharyngeal (OP) and cloacal (CL) samples used in the current study were part of a consignment of samples from backyard chickens collected at live bird markets (LBMs) in Arusha, Dar es Salaam, Iringa, Mbeya, Morogoro and Tanga in Tanzania, during surveillance of the Newcastle disease virus (NDV) conducted between September 2018 and May 2019. From each bird, one OP and one CL was collected. During the sampling period, the flocks did not present overt clinical signs consistent with avian diseases, and their vaccination statuses or histories were not available. After collection using standard procedures [36 (link)], the samples were shipped to the Southeast Poultry Research Laboratory (SEPRL) in Athens, GA, USA for total RNA extraction using the MagMAX™-96 AI/ND Viral RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) as recently described [37 (link)]. Preparation of sequencing libraries (sequence-independent, single-primer amplification [38 (link)] and Nextera ^TM Flex protocols) and paired-end NGS (500-cycle MiSeq Reagent Kit v3) using the Illumina MiSeq platform were performed as previously described [39 (link)]. For the current study, 20 birds that were NDV-positive by real-time reverse transcription–polymerase chain reaction (rRT–PCR) were selected (n = 40 samples; one OP and one CL from each of the 20 birds).