Abstract Book of EAVLD2012 - eavld congress 2012

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PRRSV DIAGNOSTICS BY RT-PCR AND DIFFERENT ELISA SYSTEMS IN SERUM AND ORAL FLUID
OF PRRSV VACCINATED PIGS
Tatjana Sattler 1 , Sandra Revilla-Fernández 2 , Adolf Steinrigl 2 , Manfred Berger 2 , Friedrich Schmoll 2
1
University Leipzig, Large Animal Clinic for Internal Medicine, Leipzig, Germany
2
AGES, Institute for Veterinary Disease Control, Mödling, Austria
PRRSV, ELISA, PCR, oral fluid, pig
Introduction
PRRSV infection is widely distributed all over the world. PRRSV
eradication programs have been developed and PRRSV negative
herds and farms have been established. PRRSV diagnostic tools
with high sensitivity and specificity are essential to control the
disease and to guarantee the negative status of the herds (1).
Furthermore, specimens like oral fluid are advantageous in terms
of costs, working time and animal welfare, and therefore have
been recently proposed for disease monitoring (2). The objective
of this study was therefore to evaluate PRRSV RNA and antibody
detection in pig oral fluid by RT-qPCR and commercial ELISA
test systems, respectively. Furthermore, the performance of
different ELISA systems was compared both, in blood serum and
oral fluid of pigs.
Materials & methods
Ten pigs (from 8 to 20 weeks of age) from a PRRSV negative
farm were injected with an EU-type PRRSV life vaccine (Porcilis ®
PRRS, Intervet, Unterschleißheim, Germany). Blood and oral
fluid samples were taken individually from each pig before, and 4,
7, 14 and 21 days after vaccination. After automated RNA
extraction, serum and oral fluid samples were analysed with an
in-house PRRSV RT-qPCR assay (3).
PRRSV antibody detection in serum and oral fluid was performed
with three different ELISA systems: a) IDEXX PRRS X3 Ab-Test
(IDEXX GmbH, Ludwigsburg, Germany), b) INGEZIM PRRS
UNIVERSAL, c) INGEZIM PRRS DR (both Ingenasa, Madrid,
Spain). Serum samples were diluted according to manufacturer’s
instructions, whereas oral fluid samples were tested without
dilution.
Results
In serum samples (table 1), antibodies were detected in seven
out of ten pigs by day 14 after vaccination. One pig
seroconverted at day 21. Two pigs did not seroconvert at all
throughout the study period and also remained RT-qPCR
negative throughout the whole study. Three out of ten pigs were
RT-qPCR positive in serum as early as day 4 after vaccination,
while most pigs (7/10) became positive by day 7. One pig
became RT-qPCR positive as late as day 14, while other pigs
became negative in RT-qPCR at this time-point. This animal was
also the last to seroconvert during the study period.
Table 1: Number of serum samples positive by PRRSV RT-qPCR
and by three different PRRSV antibody ELISA testkits (a, b, c –
see material and methods)
day 0 (no
vaccination)
day 4 after
vaccination
day
7
day
14
day
21
PCR 0/10 3/10 7/10 5/10 3/10
ELISA
a)
ELISA
b)
ELISA
c)
0/10 0/10 0/10 7/10 8/10
0/10 0/10 0/10 7/10 7/10
0/10 0/10 3/10 7/10 8/10
In oral fluid (table 2), both RT-qPCR and ELISA positive results
(in two of the tested ELISA kits) were observed, although
sensitivity was significantly reduced in comparison to serum. The
INGEZIM PRRS DR ELISA (Kit c) did not show positive results at
all in oral fluid.
Table 2: Number of oral fluid samples positive by PRRSV RTqPCR
and by three different PRRSV antibody ELISA testkits (a,
b, c – see material and methods)
day 0 (no
vaccination)
day 4 after
vaccination
day
7
day
14
day
21
PCR 0/10 0/10 2/10 2/10 0/10
ELISA
a)
ELISA
b)
ELISA
c)
0/10 0/10 0/10 2/10 3/10
0/10 0/10 0/10 2/10 4/10
0/10 0/10 0/10 0/10 0/10
Discussion & conclusions
PRRSV-antibodies could be detected in serum of some pigs as
early as day 7 after vaccination with the INGEZIM PRRS DR
ELISA (Kit c), which was the most sensitive of the three ELISA
systems. Results of the other two ELISA systems did not differ
substantially from each other, although one pig remained
antibody negative in serum at day 21 with the INGEZIM PRRS
UNIVERSAL (Kit b), while it was found positive in both other
ELISA systems. A seroconversion in this pig, tested with
INGEZIM PRRS UNIVERSAL ELISA, could be expected later
than day 21 after vaccination.
Interestingly, some animals can remain seronegative despite
vaccination, as was the case in this study in two out of ten pigs.
The most likely reason was failure of the vaccine virus to
replicate in these animals, as indicated by negative RT-qPCR
results at all time-points tested. A similar finding was described in
a previous vaccination study, where one out of 20 vaccinated
gilts remained seronegative (4).
Comparison of RT-qPCR and ELISA results obtained from
matched oral fluid and serum samples suggest that testing of.oral
fluid is much less sensitive. Lower amounts of both PRRSV RNA
and antibodies and/or the presence of inhibitory factors can be
expected in oral fluid (5). No positive results in oral fluid samples
were found with the INGEZIM PRRS DR ELISA (kit c).
It can be concluded that all tested ELISA systems were similarly
able to detect PRRSV antibodies after PRRSV life virus
vaccination, whereas INGEZIM PRRS DR ELISA seems to be
the most sensitive. Reliable PRRSV antibody detection in oral
fluid will require validation of test kits for this medium. RT-qPCR
results confirmed the antibody detection results in both, serum
and oral fluid.
References
1. große Beilage, E, Bätza, HJ. (2007): PRRSV-eradication: An option for
pig herds in Germany? Berl Münch Tierärztl Wochenschr. 120, 11/12, 470-
479.
2. Kittawornrat, A, Prickett, J, Chittick, W, Wang, C, Engle, M, Johnson, J,
Patnayak, D, Schwartz, T, Whitney, D, Olsen, C, Schwartz, K,
Zimmerman, J. (2010): Porcine reproductive and respiratory syndrome
virus (PRRSV) in serum and oral fluid samples from individual boars: will
oral fluid replace serum for PRRSV surveillance Virus Res. 154, 1-2,170-6.
3. Revilla-Fernández S, Wallner B, Truschner K, Benczak A, Brem G,
Schmoll F, Mueller M, Steinborn R.(2005): The use of endogenous and
exogenous reference RNAs for qualitative and quantitative detection of
PRRSV in porcine semen. J Virol Methods. 126, 1-2, 21-30.
4. Sipos, W, Indik, S, Irgang, P, Klein, D, Schuh, M, Schmoll, F (2002):
Transfer of EU-ML-PRRSV (Porcilis PRRS) from vaccinated to nonvaccinated
gilts. Proc 17th IPVS Congress, Ames, Iowa, USA, 418
5. Chittick, WA, Stensland, WR, Prickett, JR, Strait, EL, Harmon, K, Yoon,
KJ, Wang, C, Zimmerman, JJ (2010): Comparison of RNA extraction and
real-time reverse transcription polymerase chain reaction methods for the
detection of Porcine reproductive and respiratory syndrome virus in
porcine oral fluid specimens. J Vet Diagn Invest, 23, 248-53