Abstract Book of EAVLD2012 - eavld congress 2012

S3 - P - 19
DETECTION OF SCHMALLENBERG VIRUS IN BOVINE SEMEN BY ONE-STEP REAL-TIME RT-PCR
Remco Dijkman 1 , Jet Mars 1 , Gerard Wellenberg 1
1
Animal Health Service, Deventer, The Netherlands
2
Affiliation Institute name, Department name, City, Country
Schmallenberg virus, semen, real-time PCR
Introduction
For the detection of the Schmallenberg virus (SBV) in serum,
tissues and body fluids, a real-time PCR, based on the detection
of specific regions of the L-segment of the Schmallenberg virus,
was developed (1). Recently, two real-time PCR methods, based
on the detection of S-segment sequences, were developed too.
However, these PCR methods have not been validated for
semen samples.
In this study, we have examined whether the L-PCR, S3-PCR
and an “in-house” S-PCR were suitable for the detection of SBV
in bovine semen.
Materials & methods
Besides the L1 and S3 PCR, which were developed by FLI
(Germany), we also used an “in-house” pan-orthobunyavirus S-
PCR (OBV-S PCR) real-time PCR targetting the orthobunyavirus
S-segment for the detection of SBV in bovine semen samples.
RNA was extracted with the AM1836 extraction kit (Life
Technologies) using the MagMAX express 96 system (Life
Technologies). The AgPath-ID one-step RT-PCR kit (Life
Technologies) was used for amplification. PCR amplification was
performed on a ABI7500 sequence detection system from
Applied Biosystems (Life Technologies).
The validation of the three PCR tests was based on the
determination of the detection limit (lowest detectable amount of
SBV per mL semen) for undiluted (fresh semen) and diluted
semen (straw) samples.
Therefore, ten-fold dilutions from a Schmallenberg virus stock
solution with a known titer (10 4.9 TCID50/mL) were used for
spiking semen samples and subsequently used for analyses.
This study was also focused on spiking experiments to determine
the negative influences of inhibitory compounds in semen
samples as they may lead to false-negative results.
As it is unknown whether SBV is excreted by semen, and natural
SBV infected semen samples were not available, spiking
experiments have been performed with undiluted (fresh semen)
and diluted semen (straw) samples. Undiluted semen from 8
different bulls (bulls A t / H) and diluted semen from 14 bulls
(bulls I t / m V) were used. The recovery of the SBV in undiluted
and diluted semen samples was determined by PCR and
compared with the results of control samples.
Results
In silico analyses showed that the primers and probes of the L-
and S3-PCR are specific for SBV, and that the selected primers
and probe of the OBV S-PCR recognize conserved regions within
the S-segment of SBV, also present in other orthobunyaviruses.
The results of the detection limits of the three real-time PCR
methods are reported in Table 1.
straws were comparable or to a maximum of 4 Ct-values higher
than the Ct-value of the Positive Reference sample (PBS matrix).
The semen samples without spiking were negative in all three
PCR methods (Figure 1).
Figure 1: Ct-values of 1:3 diluted (fresh) semen and diluted
semen (straws)spiked with SBV as obtained by L-, S3- and the
OBV-S PCR.
Discussion & conclusions
This validation study showed that the L-, the S3-, and the OBV-S
PCR are able to detect SBV in 1:3 diluted fresh semen and
diluted (straw) semen samples. The detection limits of the S3-
and the OBV-S PCR are lower compared to the detection limit of
the L-PCR.
In addition, the spiking experiments, to examine the influence of
inhibitory compounds in bovine semen samples, showed that no
negative influences were recorded in 1:3 diluted semen and in
diluted semen (straw) samples. Therefore, fresh semen needs to
be diluted prior to the RNA-extraction and semen straws can be
examined by PCR without further dilution.
The use of an internal control, to check for inhibitory effects of the
matrix, was not examined in this study, but can be the scope for
further validation.
As fresh semen can be diluted 1:3, and semen in straws are
diluted approximately between 1:20 and 1:30, the screening of
fresh semen is more sensitive than straws.
It might be useful to use the OBV-S PCR as a general screening
PCR, and the S3-PCR can be used as a screening and/or
confirmatory PCR.
References
1. Hoffmann B, Scheuch M, Höper D, Jungblut R, Holsteg M, Schirrmeier
H, et al. Novel orthobunyavirus in cattle, Europe, 2011. Emerg Infect Dis
(epub).
Table 1: Detection limits of the three real-time SBV PCR methods
Test method
Detection limit (TCID50/ml)
Undiluted semen Diluted semen
Straws
L-PCR 10 1.9 10 0.9
S3-PCR 10 -0.1 10 -1.1
OBV-S PCR 10 -0.1 10 -0.1
Results showed that inhibitory effects were recorded for undiluted
(fresh) semen, while no inhibitory effects were recorded by PCR
when these semen samples were 1:3 pre-diluted. Ct-values of
the 1:3 diluted fresh semen samples and the diluted semen