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).