Download New insights into oral fluids as a diagnosis procedure to detect and

New insights into oral fluids as a diagnosis
procedure to detect and determine the prevalence
of PRRSV under field conditions
Nardy ROBBEN, Thermo Fisher Scientific, Bleiswijk, Netherlands
Anne QUIJADA, Thermo Fisher Scientific, Lissieu, France.
Lorenzo José FRAILE SAUCE, University of Lleida, Lleida, Spain.
ABSTRACT
CONCLUSIONS
RESULTS
Diagnostic tests are often used to assess the PRRSV
infection status of pig herds. For routine settings, ELISA
test methods and reversed transcriptase PCR (RT-PCR)
are used to determine antibody titers and detect antigen
on serum/blood and tissue samples, respectively.
Recently, the detection of this virus in oral fluids is
worldwide being used as an alternative technique. Based
on two different experimentations with a PRRSV
European and North American strains, the probability to
detect PRRSV virus in oral fluids is significantly
associated to the PRRSV prevalence in serum (p<0.0001).
If the serum prevalence is higher than 50%, the
probability to detect this virus in oral fluid samples is
close to 1. Therefore, oral fluid sample is a good tool to
detect PRRSV at herd level but it is not suitable to
determine the prevalence of this disease.
Figure 2. Diagnostic agreement of PRRSV infectious level on
blood/serum and oral fluid samples over several days postinfection (dpi) by RT-PCR
Figure 4. Determination of the probability to detect PRRSV
virus in oral fluid taking into account the prevalence of
PRRSV in serum
Figure 2a. Experiment 1 - A challenge with a PRRSV European
strain over 70 days post-infection (70 dpi) analysed by RT-PCR
with LSI VetMAX™ PRRS EU/NA kit
Figure 4a. Logic regression analysis merging data from the North
American and European studies
Oral fluid sample is a good tool to detect PRRSV at herd
level but it is not suitable to determine the prevalence of
this disease.
This sampling is able to detect PRRSV even with a low
prevalence of the disease at herd level that normally
corresponds to an early phase of the disease.
It is necessary to sample a relative low number of pens
compared to a standard number of serum samples for
diagnostic purposes.
Testing the sampling recommendation under field
conditions is requested and these studies are being
carried out.
INTRODUCTION
Experiment 1
Diagnostic tests are often used to assess the PRRSV
infection status of pig herds. For routine settings, ELISA
test methods and reversed transcriptase PCR (RT-PCR)
are used to determine antibody titers and detect antigen.
Until now, RT-PCR on serum/blood and tissue samples is
the most used technique to detect PRRSV antigen,
respectively. Recently, the detection of this virus in oral
fluids is worldwide being used as an alternative
technique. The main goal of this study was to establish
clear recommendations in herd management for swine
practitioners, when oral fluids are used as a diagnosis
procedure depending on the goal: detection of a disease
versus prevalence determination with a focus on PRRSV.
The positivity
positivity per pen (oral fluids analys
analysis)
is) is analys
analysed
ed from day+
day+0
0 to
day+42 post-infection. The positivity in individual samples (blood/serum
analysis) is observed at day+0 to day+56 post-infection. In this study, from
day+0 to day+42 after infection, the oral fluids sample and blood/ serum
samples give the same results.
The probability to detect PRRSV in oral fluid is significantly
associated to the PRRSV prevalence in serum (p=0.0001) and
the determination coefficient of the logistic regression analysis is
82%.
Figure 2b. Experiment 2 - A challenge with a PRRSV North
American strain over 40 days post-infection (40 dpi) analysed by
RTT PCR with TaqMan® NA and EU PRRSV Reagents kit
RT-PCR
Figure 4b. Relation between the prevalence of PRRSV in serum and
the detection in oral fluid
Probability
Prevalence of
P
(0-1)
PRRSV
to detect
(d
(determined
d
in
PRRSV in
serum)
oral fluid
MATERIALS AND METHODS
Raw Data coming from 2 different experiments were
included. In both cases, before starting the studies, oral
fluids and blood/serum samples were collected and
analyzed by RT-PCR and ELISA to confirm PRRSV
negative status.
Experiment 2
Figure 1. Study design
PRRSV negative pigs
(ELISA + PCR results)
Experiment 1
Challenge with an European PRRSV strain
D0
80 pigs - 8 pens
D70
The positivity per pen (oral fluids analysis) and in individual samples
(blood analysis) are similar from day+0 to day+40 post-infection. CT
range in serum samples were from 15 to 30 CT’s in the first 25 days postinfection. Lower CT range in serum samples were between 25 to 40 days
post-infection. CT range in oral fluids were from 30 to 35, with a lower
delta for most of the entire 40 days of the study.
Results obtained per pen (oral fluids analysis) demonstrate an
excellent correlation with results on individual samples (blood/serum
analysis). The presence of PRRSV EU/NA RNA was identified in an
early infectious stage.
PRRSV negative pigs
(ELISA + PCR results)
The evaluation of the sensitivity and specificity of the oral fluid taking into
account the detection of PRRSV in serum as gold standard technique.
It was considered that serum PCR analysis is the gold standard. One pen
is really positive to PRRSV if, at least, one animal is positive to this
technique. This result will be compared with the result obtained from the
oral fluid analysis.
Experiment 2
Table 1a. Sensitivity and Specificity determination on European
study
Challenge with an American PRRSV strain
Gold standard
D0
D40
190 pigs - 12 pens
A logistic regression analysis was carried out in SPSS
15.0 (SPSS Inc., 1989-2006) to calculate the probability
of virus detection in a pen by oral fluid (PCR positive
results) taking into account the prevalence of PRRSV in
serum as independent variable. With this probability, it
was estimated the number of pens necessary to be
sampled in order to detect a disease as previously
published (Thrusfield, 1997) .
0,72
40
0,91
50
0,97
60
0,99
70
0,99
80
0,99
90
0,99
100
0,99
The probability (0-1) to detect PRRSV in oral fluid samples was 0.15,
0.40, 0.72, 0.91 and 0.97 for a serum prevalence (%) of 10, 20, 30, 40
and 50, respectively.
If the serum prevalence is higher than 50%, the probability to
detect this virus in oral fluid samples is close to 1.
Below, the number of pens to be sampled is calculated in order to detect
at least one positive pen, taking into account the prevalence present in
serum and the information obtained previously in the logistic regression
analysis.
Table 2. Number of pens to be sampled to be able to detect PRRSV in
oral fluids with a confidence level of 95 or 99.99% .
Prevalence of PRRSV
Confidence level of
Confidence level of
(determined in serum)
95%
99.99%
10
19
58
Positives
49
0
20
6
19
Negatives
7
16
30
3
8
Sensitivity :
87.5% (78.8%, 96.2%)
40
1
4
Specificity :
100.0% (100.0%, 100.0%)
50
1
3
Positive Predictive Value :
100.0% (100.0%, 100.0%)
Negative Predictive Value :
69.6% (50.8%, 88.4%)
60
1
2
70
1
2
80
1
2
90
1
1
100
1
1
Oral fluids allow the swine industry to generate a more
accurate and reliable monitoring system, where early
detection can result in faster response time. It is an
opportunity to increase the number of pigs tested while
decreasing the cost of analysis. Therefore, oral fluid
allow to estimate the circulation of pathogens in swine
population for effective herd health monitoring.
Oral fluids sampling is easy to use for farmers and
veterinarians. Animals are less stressed than blood
sampling and have a natural attraction to the rope and
start chewing. It could be an early warning system for
monitoring of herds, on pen level, for example to
estimate the circulation of different swine pathogens
(PRRSV, PCV2 and SIV) as the data generated in this
study clearly support.
REFERENCES
™J. Brouwer, K. Frankena, M.F. De Jong, R. Voets, A.
Dijkhuizen, J.H.M. Verheijden, R.E. Komijn. PRRS: effect
on herd performance after initial infection and risk
analysis Vet. Q., 2 (1994), pp. 95–100
™E. Mattheu, M. Tello, A. Coll, J. Casal, M. Martin.
Comparison of three ELISAs for the diagnosis of porcine
reproductive and respiratory syndrome. Vet. Rec., 159
(2006), pp. 717–718
™E.J. Neumann, J.B. Kliebenstein, C.D. Johnson, J.W.
Mabry, E.J. Bush, A.H. Seitzinger, A.L. Green, J.J.
Zimmermann. Assessment of the economic impact of
porcine reproductive and respiratory syndrome on swine
production in the United States JAVMA, 227 (2005), pp.
385–392
™Thrusfield, M. (2007). Diagnostic testing. In: “Veterinary
Epidemiology”, 3rd Ed. Blackwell publishing, Hong Kong.
™G.J. Wellenberg. Review: diagnostic methods for the
detection of porcine reproductive and respiratory
syndrome
virus
(PRRSV)
infections.
Tijdschr.
Diergeneeskd., 131 (16) (2006), pp. 566–572
Evaluated test
PRRSV experimental infection with a North American strain was
carried out in naïve pigs. Animals were sampled repeatedly over the 40
days post-infection. Individual blood samples and oral fluids samples of
each pen were collected.
In both cases, sample extraction was carried out with
magnetic beads sample extraction (MagMAX™ Viral RNA
Isolation Kits and MagVet™ Universal Isolation Kit 4x 96
tests) and RT-PCR with LSI VetMAX™ PRRS EU/NA and
TaqMan® NA and EU PRRSV Reagents. A CT value over
40 is considered negative.
0,40
30
Healthy
Experiment 1
Diseased
0,15
20
Figure 5. Estimation of the number of pens to be sampled in
order to detect at least one positive one taking into account
the prevalence present in serum
Figure 3. Sensitivity and Specificity determination
A challenge with a PRRSV European strain. After carrying out the
challenge with the virus, a weekly sampling of blood and oral fluid of 80
pigs was carried out during 3 months.
10
DISCUSSION
Table 1b. Sensitivity and Specificity determination on North
American study
Gold standard
Experiment 2
Diseased
Healthy
Positives
96
0
Negatives
0
12
Evaluated test
Sensitivity :
100.0% (100.0%, 100.0%)
Specificity :
100.0% (100.0%, 100.0%)
ACKNOWLEDGEMENTS
™GSP laboratory, Lleida (Spain)
™University of Lleida , Lleida (Spain)
™The veterinary team of the cooperating farm in Spain
™Dr Bob Rowland, Kansas State University
TRADEMARKS/LICENSING
™LSI VetMAX™ PRRSV EU/NA (Ref.: PRRSEUNA)
The number of oral fluids samples to be taken, on herd level, in order to
find, at least, 1 positive PRRSV oral fluid sample was for example 3 for a
serum prevalence 50 % at 99,99 % of confidence level.
If the serum prevalence is higher than 50%, only one pen of 10
animals will be taken for a confidence level of 95 %.
™TaqMan® NA and EU PRRSV Reagents
™MagMAX™ -96 Viral RNA Isolation Kit (PN 4462359)
™MagVet™ Universal Isolation kit (Ref.: MV384)
™TEGO™ Swine Oral Fluid Collection Kit (Ref.: 12329)
© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks
are the property of Thermo Fisher Scientific and its subsidiaries unless
otherwise specified. TaqMan is a registered trademark of Roche
Molecular Systems, Inc. used under permission and licence. TEGO is a
trademark of ITL Animal Healthcare, Ltd
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