Abstract Book of EAVLD2012 - eavld congress 2012

S1 - O - 11
USING ORAL FLUID FOR THE SEROLOGICAL MONITORIZATION OF PRRSV CIRCULATION IN A
GROUP OF INFECTED GILTS
Martos-Raich, Alba 1 , Coma-Oliva, Ester 1 ; Serra-Martínez, Jordi 2 ; Barrera-Toro, Xavier 3 ; Planasdemunt-Regàs,
Llorenç 3 ; Maldonado-García, Jaime 1 ; Porquet-Garanto, Lourdes 1 ; Rebordosa-Trigueros, Xavier 1 .
1 HIPRA 17170 Amer, Girona, Spain.
2 Biofar Laboratoris, S.L 08261 Cardona, Barcelona, Spain.
3 AVP Planasdemunt i Associats 17400 Breda, Girona, Spain
Introduction
The use of oral fluid-based assays is proving every day that is
well suited for monitoring of different pathogens in farm animals 1 .
In the case of PRRSV different studies have shown that pig oral
fluid (OF) is a suitable sample for monitoring both viremia (PCR)
and sero-conversion (ELISA) after infection 2 . In this study a new
methodology to adapt the existing CIVTEST SUIS PRRS E/S and
A/S ELISA kits (Hipra) to be used with OF matrix instead of
serum was evaluated under field conditions. The ultimate goal of
this study was to compare the performance of the ELISA kits
using individual or pooled serum samples, as well as OF from
groups of animals.
PPRS virus, oral fluid, ELISA, alternative for monitoring
Materials & methods
The study included a single group of 10 gilts coming from a
PRRSV-free farm (as confirmed by PCR and ELISA at the
beginning of the study). The animals were infected in isolation
using a field strain of PRRSV Type 1 on day 0. The study lasted
for 9 weeks (wk) with weekly sampling of blood from each
individual and OF samples from the group. The presence of
antibodies to PRRSV Type 1 and 2 was evaluated by using the
CIVTEST SUIS PRRSV E/S and CIVTEST SUIS PRRS A/S
ELISA kits respectively (Hipra) and also by Western blot based in
a European strain. Individual serum samples were tested for the
presence of viral RNA by Real Time RT-PCR 3 . Serums samples
were analyzed individually and as a pool of 10 (pool/10). For OF
serology different conjugates (anti-IgG1,-IgG2, -IgA) for the
ELISA were evaluated.
Results
The incubation conditions for OF were fixed at 4 ºC of a ½
dilution of the sample. In all cases an IRPC of 20.0 was used as
a cut-off (as defined in CIVTEST for individual serum). As seen in
Table I, results from individual serum showed 80% of positive
animals at wk3 (100% at wk4). The IRPC values for the pool/10
and OF samples showed a value above 20 at wk4. Both, the
mean IRPC value of the group and the IRPC of the pool/10
increased until the end of the study. By contrast, IRPC values
obtained for the OF sample showed a maximum at wk4, and then
dropped down till the end of the trial. No reactivity was observed
with the A/S kit (data not shown). The use of different conjugates
for the analysis of OF samples indicated that the most abundant
type of Ig after infection was IgA (already detectable at wk1 postinfection;
Figure 1). The kinetics of IgG1 showed sero-conversion
around 3-4 weeks after infection. IgG2 levels were nondetectable
(data not shown). The maximum sensitivity (IRPC
above 20 at wk1) was achieved by using a combination of IgG1
plus IgA conjugates.
Figure1. OD450 values obtained with the oral fluid weekly
samplings using conjugates with different antibody-type
specificity
Discussion & conclusions
CIVTEST SUIS PRRS E/S has shown a good adaptation to the
OF matrix. This sample evidenced antibody dynamics similar to
those observed with the pool/10 serum sample, representing a
suitable alternative for monitoring PRRSV circulation in pig herds.
The use of different channels (IgG1 or IgA) for the analysis of the
OF sample would allow to asses maximum earliness in the
detection of infection or detection of the fall in post-infection
immunity.
References
1. Prickett, J.R. et al: 2010, Anim Hlth Res. Rev. 11(2): 207-216.
2. Kittawornrat, A. et al: 2010, Virus Res. Dec;154(1-2):170-176.
3. Martínez, E. et al: 2008, Res. Vet. Sci. 85: 184-193
Table I. Serology results obtained with serum and oral fluid using
the CIVTEST SUIS PRRS E/S at weekly intervals after infection
(IRPC values above 20.0 appear in shaded boxes)
PRRS E/S
% Pos
(ELISA)
wk
0
wk1 wk2 wk3 wk4 wk5 wk6 wk7 wk8 wk9
0% 0% 0% 80% 100% 100% 100% 80% 100% 100%
Mean (IRPC) -1,49 -1,38 8,64 24,32 41,75 46,56 70,47 50,96 72,67 72,00
Pool/10
(IRPC)
-1,80 -2,09 4,19 11,52 23,95 54,09 57,88 48,48 68,97 87,19
OF (IRPC) -5,20 -4,22 0,13 14,08 46,65 31,48 35,19 29,74 20,52 32,66
OF/IgG1+IgA
(IRPC)
4,33 44,02 60,07 71,66 99,72 96,37 97,72 86,04 80,60 99,95