Abstract Book of EAVLD2012 - eavld congress 2012

S1 - O - 1
ORAL FLUIDS – SAMPLE MATRIX FOR EFFECTIVE HERD HEALTH MONITORING
C.Boss 1 , R. Shah 2 , C. O’Connell 2
1
Life Technologies,Darmstadt, Germany
2
Life Technologies, Austin, USA
Oral Fluids, PRRS, PCV2, SIV
Introduction
The use of oral fluid as a sample matrix in porcine reproductive
and respiratory syndrome virus (PRRSV) surveillance has
increased in many parts of the world over the last few years.
Advantages of using this matrix are ease of collection, reduced
stress to the pigs, the low cost of collection and minimum labor
required. Successful implementation of sample testing through
the use of this sample matrix has extended to the evaluation of its
testing for other swine diseases, including porcine circovirus type
2 (PCV2), swine influenza virus (SIV), and mycoplasma
hyopneumoniae (M. Hyo).
Materials & methods
In a multi-center, collaborative study, cotton ropes were used to
collect samples of oral fluid from pigs that were experimentally
infected with PRRSV and PCV2. Additionally, oral fluid samples
from SIV negative pens were spiked with SIV for evaluation.
Nucleic acid was extracted from oral fluid samples using a
MagMAX based magnetic bead extraction and then amplified
using a TaqMan® based real-time PCR assay for each virus. The
method is semi automated, involving the use of the MagMAX
Express-96 Magnetic Particle Processor for the purification of
nucleic acid.
For PRRSV testing, in collaboration with Kansas State University,
oral fluids were collected from pens on the day that the pigs were
experimentally infected through 40 days post-infection. Serum
was collected from each pig within the pen on the same day that
oral fluids were collected from the pen. Serum samples and oral
fluids were both tested using real-time PCR.
For PCV2 testing, in collaboration with Iowa State University,
twenty-four 21-day-old pigs free of PRRSV and SIV were
assigned to 1 of 4 groups and housed in pens in separate rooms.
Pen number One was the control (None-Challenge) group. Each
pig in the other 3 groups received PCV2a strain at 11 weeks of
age (dpi 0). Six pigs (Pen 3) were re-challenged with PCV2a
strain at 35, 70, and 105 dpi. Each pig in Pen 4 group
alternatively received PCV2a (dpi 0 and 70) and PCV2b (dpi 35
and 105). The two PCV2a strains used were heterologous. Oral
fluids samples were collected, dpi 0, 2, 4, 6, 8, 10, 12, 14, and
weekly thereafter until dpi 140. The results are shown in Figure 2
for the time periods tested using the PCV2 real-time PCR
reagents (dpi 2-98
For SIV testing, in collaboration with Iowa State University, a total
of 180 oral fluid samples were spiked with high, medium and low
copy numbers of SIV. The extracted nucleic acids were also
tested with subtyping reagents for the identification of
hemagglutinin and neuraminidase subtypes.
of ~20 swine, the proposed method of sample preparation and
nucleic acid purification efficiently processes multiple samples,
thereby decreasing screening time.
A major advantage of this high throughput method, combined
with the ease of collection of oral fluid, is that large numbers of
pigs can be tested without increased cost or labor. This provides
a more efficient and cost effective testing environment and the
ability to better curb incidences of infection.
Acknowledgements
Dr. Bob Rowland, Kansas State University
Dr. Tanja Opriessnig, Iowa State University
Dr. Christa Irwin, Iowa State University
Dr. Jeff Zimmerman, Iowa State University
Results
PRRSV nucleic acid was amplified from all oral fluid samples
derived from the experimentally infected pigs. Lower Cts,
indicating a higher copy number of viral RNA, were present in the
serum samples early post-infection; but similar levels were
detected in serum and oral fluid at later collection points. These
results indicate a high level of correlation between PRRSV
detection from serum collected from individual animals and from
the pen or herd based oral fluid samples.
Using the SIV screening real-time PCR reagents, SIV nucleic
acid was detectable in all of the spiked oral fluid samples and at
each concentration level. Additionally, the results of the subtyping
study indicate that all positive samples could be sub-typed.
High titers of PCV2 were detected from day 12 through 28 post
infection, and virus was detectable throughout the entire testing
period (dpi 2 – 98).
Discussion & conclusion
In summary, oral fluid samples were demonstrated to provide
sensitive detection of PRRSV, SIV, and PCV2. As these
samples could be used for the detection of virus in a population