Routine diagnosis and confirmation
The standard procedure in Germany for the diagnosis of ASF includes an
initial testing in an accredited state laboratory in the affected
federal state. These laboratories regularly participate in an
inter-laboratory comparison test performed by the NRL (see also tasks of
the NRL according to Council Directive 2002/60/EC). Samples with a
positive or inconclusive result are sent to the NRL for confirmation.
Only the diagnosis of the NRL confirms an outbreak or case. Suitable
diagnostic tests are listed in the official method collection published
by the FLI, taking into account the EU Diagnostic Manual (Commission
Decision 2003/422/EC).
Despite the advanced state of decomposition of the first carcass and the
resulting low sample quality, the LLBB detected moderate amounts of
genome sequences of ASF virus. At the FLI, these results were confirmed
with very similar cq-values, despite the use of different cyclers and
conditions. By 24th September 2020, 32 cases were detected at the LLBB
and confirmed by FLI. Sample matrices were 19 bone marrow specimens from
carcasses of varying stages of decomposition, 12 blood swab suspensions,
and 1 blood clot suspension and the corresponding serum. Cycle threshold
values ranged from 18 to 36 in the OIE-recommended PCR by King et al.
(2003) and were comparable between the regional and national laboratory.
As expected, PCRs that amplified short genome fragments performed better
on poor quality samples. A combination of different test systems, also
for internal control, is therefore advisable and has proven to be
effective in the present case. Details are shown in Table 1. Blood swab
suspensions, which had been included into the official method collection
following extensive validation studies under experimental and limited
field conditions at the NRL (Carlson et al., 2018; Petrov et al., 2014),
yielded reliable results also in this outbreak situation. The
hemadsorbing virus causing this outbreak was isolated from the serum
sample mentioned above.
Following the confirmation of the first case, the local veterinary
authority had the remains of the carcass exhumed for further
pathological examination at the LLBB. All samples taken from the
carcass, even muscle and fatty tissues were positive in qPCR (see
Supplementary Table 1). This fact underlines that any sample should be
send if the recommended samples are not available. In this case,
communication should be established with the receiving laboratory.
Positive samples were also obtained when diluting the samples 1:5. The
latter would mimic the pooling options currently provided in the
official method collection.