Materials and Methods
Slaughterhouse (SH) sites: sampling occurred at Dongdou SH and Nongduang SH (Vientiane, Laos) during May and June, 2019; and at Mandalay City SH, Mandalay, Myanmar during July and August, 2019 (Figure 1). Animal contact at the slaughterhouses varied – at Dongdou animals were gathered in a chute up to 12 hours prior to slaughter, and secured to a railing with a headrope. At the other slaughterhouses, animals typically arrived in small groups by truck up to 24 hours prior to slaughter and were tethered by a headrope to trucks or within the slaughterhouse, or kept in stalls with animals from one trader together until the time of slaughter. Animals from one trader could represent multiple original villages, districts or townships of origin. Animals were brought by traders to the slaughterhouse in groups, collected from different villages and transported together. For the Myanmar slaughterhouse, animals from 16 traders were sampled over five nights. In Laos, all animals tested at Dongdou were supplied by one trader, but at Nongduang (a more traditional slaughterhouse), animals were supplied by 10 traders over three nights.
Study population: Healthy cattle and buffalo bound for human consumption were sampled opportunistically post-mortem. Complete samples were obtained from 132 animals (84 cattle and 48 buffalo) in Laos, and 130 animals (5 buffalo and 125 cattle) in Myanmar.
Samples: a sample set including serum and swabs from three sites (nasals, oral, and dorsal nasopharyngeal) were collected from each animal. Whole blood was collected in 10 mL red-top (plain) Vacutainer tubes (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) following severing of the jugular vein as part of the normal slaughter process. Tubes were either centrifuged at 1500 x g for 3 minutes, or left to clot within 12 hours of collection). Serum (1 mL) was then collected into a 1.5 mL screw cap tube (Sarstedt) and frozen at -80°C until processing. Plain dry rayon CopanTM swabs (Copan Diagnostics Inc., Murrieta, California USA) were collected from the sites mentioned above during the slaughter process, were immediately inserted into a cryovial containing 1.0 mL of DNA/RNA ShieldTM (Zymo Research), and were kept chilled on ice packs for between 3 to 12 hours until arrival at the local laboratory where they were frozen at -80°C for transport to the World Reference Laboratory for FMD at the Pirbright Institute (Ash Road, Surrey, UK).
Swabs were collected in the following manner: oral swabs were rubbed for approximately 3 s on the hard palate, buccal surfaces, and tongue as possible given the position of the animal, and were inserted up to the length of the 15cm swab; nasal swabs were rubbed on all inside surfaces of both nostrils for 1-2 s each, with the swab inserted maximally into the nasal openings; pharyngeal samples prioritised sampling of the dorsal pharyngeal mucosa, the site of optimal experimental FMDV retrieval for persistently-infected animals (Pacheco et al., 2015) and involved insertion of the swab from a caudal direction through the oesophagus, with blind manual guidance to the dorsal nasopharyngeal mucosa, which was rubbed vigorously. During initial sampling, dissection of a buffalo head confirmed that palpation of landmarks allowed sampling of the target mucosal surface. Where ruminal contamination was present, heads were pre-washed with water from a hose or bucket to minimise contamination of swabs. During collection, field staff employed frequent changing of gloves to prevent cross-contamination. Environmental control samples were collected each 10-15 carcasses by waving swabs through air adjacent to carcass collection sites (air controls) and by immersing swabs in local hose or trough water (water controls).
Data collected at sampling: at the time of swabbing, oral and nasal lesions and any signs of lameness were assessed and recorded. Animal species, age and sex were collected from traders at the time of sampling. At the time of sampling, the external nares and rostral oral cavity were observed for the presence of gross lesions including vesicles, erosions or swellings, and evidence of excessive salivation.
Laboratory assays: The frozen serum and swab samples were maintained at -80°C at the National Animal Health Laboratory of the country of origin, then transported to Pirbright Institute on dry ice (Ash Road, Surrey, UK). Serum was evaluated for FMDV non-structural proteins (NSPs) using the FMD PrioCHECK NSP ELISA as per kit instructions with the exception that two wells were used per sample. Swabs were evaluated for the presence of FMDV RNA by the pan‐serotypic one-step real‐time reverse transcription‐PCR (RT‐PCR) (Callahan et al, 2002). RT-PCR values were determined to be positive if the cycle threshold (CT) value was under 40.
Analysis: analyses were performed in R (R version 4.0.0 (2020-04-24) Copyright (C) 2020 The R Foundation for Statistical Computing).