Open Journal of Veterinary Medicine, 2012, 2, 113-119 Published Online September 2012 (
Ovine Progressive Pneumonia Virus Is Transmitted More
Effectively via Aerosol Nebu lization than Oral
Lynn M. Herrmann-Hoesing1*, Stephen N. White1,2,3, Liam E. Broughton-Neiswanger1,
Wendell C. Johnson2, Susan M. Noh1,2, David A. Schneider2, Hong Li1,2, Naomi S. Taus1,2,
James Reynolds2, Thomas Truscott2, Rohana P. Dassanayake2, Donald P. Knowles1,2
1Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, USA
2Animal Disease Research Unit, US Department of Agriculture-Agricultural Research Service,
Washington State University, Pullman, USA
3Center for Integrated Biotechnology, Washington State University, Pullman, USA
Email: *
Received June 28, 2012; revised August 1, 2012; accepted August 10, 2012
A new method of experimental infection of ovine progressive pneumonia virus (OPPV), aerosol nebulization (Nb), was
compared to intravenous (IV) and oral (PO) methods of experimental infection. Seven month old lambs were given 3.5 × 107
TCID50 of Dubois OPPV LMH19 isolate using IV, PO, or Nb methods and were monitored for infection using cELISA
and OPPV quantitative (q) PCR for 35 weeks. Four out of four sheep in the IV group, six out of six sheep in the Nb
group, but only two out of six sheep in the PO group became infected by OPPV; whereas the uninoculated controls (n =
2) and a sentinel control (n = 1) remained uninfected during the course of the study. The time to a cELISA or OPPV
qPCR positive result in the Nb group was quicker and statistically different from the time to a cELISA or OPPV qPCR
positive result in the PO group (cELISA P-value = 0.0021 and OPPV qPCR P-value = 0.0007). When the Nb and IV
groups were compared, sheep became cELISA and OPPV qPCR positive at similar times (cELISA P-value = 0.6 and
OPPV qPCR P-value = 0.1). In addition, sheep became OPPV qPCR positive prior to cELISA in both the IV and Nb
groups (IV P-value = 0.027 and Nb P-value = 0.007). Aerosol nebulization is a more natural experimental method of
transmitting OPPV and may be valuable for testing potential vaccines or specific host genetics.
Keywords: Ovine Progressive Pneumonia Virus = OPPV; Visna/Maedi Virus = VMV; Small Ruminant Lentivirus =
SRLV; Caprine Arthritis-Encephalitis Virus = CAEV; Transmission
1. Introduction
The small ruminant lentiviruses (SRLVs) include ovine
progressive pneumonia virus (OPPV), visna/maedi virus
(VMV) and caprine arthritis-encephalitis virus (CAEV).
The SRLVs are part of the family Retroviridae, genus
lentivirus that also includes feline immunodeficiency
virus, equine infectious anemia virus, bovine immunode-
ficiency virus, and human immunodeficiency virus.
OPPV infection at a minimum results in life-long persis-
tent infection of sheep and may cause clinical signs and
histopathological lesions as sheep age. Clinical signs
include mastitis, swollen carpal joints, wasting, dypsnea,
and ataxia, and histopathological lesions are detected in
the congruent tissues such as mammary gland, synovial
membranes, lung, and brain. Since clinical signs are va-
riable and histopathological assessment is performed
post-mortem, antemortem detection of infection is reliant
upon highly sensitive and specific serological and mo-
lecular diagnostic tests [1-3]. Since there is no known
treatment or effective vaccine for SRLVs, annual diag-
nostic testing followed by removal or separation of in-
fected animals has been the main tool in controlling the
number of infected animals.
Many early VMV and OPPV studies suggested that
maternal transmission accounted for the majority of nat-
ural transmission within a flock. This was primarily
based upon the fact that if lambs were removed from
their dams prior to suckling and raised artificially and
isolated from the flock, the majority of lambs would re-
main uninfected [4,5]. The few lambs that became in-
fected in these scenarios were either thought to have
suckled briefly or acquired infection in utero [6]. More
recently, field studies in Spanish flocks have shown in-
creased VMV seroprevalence in intensive versus exten-
*Corresponding author.
opyright © 2012 SciRes. OJVM
sive operations suggesting horizontal transmission plays
a large role in transmission [7,8]. Furthermore, a mo-
lecular epidemiology study in dam and daughter sets of a
naturally infected Idaho, USA ewe flock showed that
maternal transmission of OPPV contributed only 10% -
14% of transmission events; whereas, non-maternal trans-
mission, which includes horizontal transmission and pos-
sible paternal transmission during conception, accounted
for the remaining 86% - 90% of transmission [9]. In the
context of the earlier epidemiology studies that relied
only on diagnostic testing of serum, isolation of lambs
from all infected animals probably contributed more to
lowering infection incidence than removal of lambs from
their infected mothers.
Identification of the major sources and routes of natu-
ral horizontal transmission is of great importance. There
have been many studies describing sources of OPPV that
could contribute to natural transmission including the
bronchial alveolar fluid, colostrum/milk, peripheral blood,
and semen. Bronchial alveolar lavage fluid and colos-
trum/milk contain cell-associated and cell-free virus in
SRLV infected animals, which suggests that these sources
infect via both horizontal and maternal modes [10-16].
Peripheral blood of SRLV infected animals contains
cell-associated virus [17,18] and may be a source of in-
fection for: 1) developing fetuses; and 2) naïve sheep
during vaccination needle re-use, tail docking, and shea-
ring. Cell-associated SRLV has been detected in semen
of bucks and rams, and cell-free virus has been detected
in semen of rams that are co-infected with Brucella ovis
[19,20]. This suggests that semen may be a source of hori-
zontal sexual transmission and/or paternal transmission.
The site of entry in the naïve animal also needs to be
considered for effective SRLV transmission from a
source. Currently, the respiratory system is considered
the main entry point for SRLV from either respiratory
secretions or colostrum/milk of other sheep due to the
effectiveness of infection via the experimental intrapul-
monary and intratracheal methods [16,21-28]. The intra-
nasal method is considered a less efficient method of
experimental infection compared to the intratracheal me-
thod since 0.5 × 107 TCID50 was required for intranasal
infection whereas 102 TCID50 was required for intratra-
cheal infection during a titration experiment [28]. In con-
trast, the intravenous method required only 4 TCID50 of
OPPV WLC1 to infect lambs making it a very efficient
and positive control method of infection; however, it
does not represent a natural form of transmission [29]. In
addition, maedi-visna virus was shown to be present in
the epithelium of the ileum of newborn lambs that had
naturally suckled colostrum/milk [30], suggesting that
the gastrointestinal tract could be a possible site of entry
for virus as well. However, 1 × 105 TCID50 of OPPV
infected only 2 out of 10 newborn lambs after oral (PO)
administration via bottle feeding, suggesting aspiration
and/or ingestion of virus is less effective at causing in-
fection than other experimental infection methods [31].
Although the intravenous, intratracheal and intrapulmo-
nary experimental methods guarantee infection, they are
invasive procedures and represent non-natural methods
of exposure. Aerosol nebulization (Nb) is not invasive
and may represent a more natural experimental method
of infection. In this study, we tested aerosol nebulization
(Nb) as a new experimental method of infection and hy-
pothesized that aerosol Nb would infect sheep more ef-
fectively than the PO method of infection in terms of the
numbers of sheep infected and the rate of cELISA or
OPPV qPCR positive results.
2. Materials and Methods
2.1. Animals
Nineteen Suffolk lambs were derived from an OPPV and
ovine herpesvirus 2 free flock. Animals were defined
OPPV free based upon multigenerational OPPV diagnos-
tic testing using CAEV cELISA, western blot analysis
and OPPV quantitative (q) PCR (see below). Lambs were
weaned at 2.5 months and entered the study at seven
months of age. The Washington State University Institute
for Animal Care and Use Committee approved the ani-
mal procedures.
2.2. Virus Isolation
A Dubois OPPV LMH19 isolate from colostrum cells
[13] was grown and titered in goat synovial membrane
(GSM) cells using previously published methods [32].
Briefly, GSM cells were infected with a multiplicity of
infection of 0.5 of Dubois OPPV LMH19 for approxi-
mately 1.5 weeks in DMEM with 5% fetal bovine serum.
After GSM monolayer obliteration, the supernatant was
centrifuged at 1450 × g for 15 minutes at 4˚C. The re-
sulting supernatant was then centrifuged at 100,000 × g
for 90 minutes at 4˚C. The viral pellet was resuspended
with Dulbecco’s 1X phosphate buffered saline (PBS),
filtered through a 0.22 micron filter, and stored at 4˚C for
3 weeks until titrations and inoculations were conducted.
2.3. Infection Methods
For these studies, a dose of 3.5 × 107 tissue culture infec-
tious doses at 50% (TCID50) of purified Dubois OPPV
LMH19 in 1X Dulbecco’s PBS was used for inoculations.
A 1 ml volume of 3.5 × 107 TCID50/ml was given intra-
venous (IV) into the jugular vein of 4 sheep or was given
orally to 6 sheep by placing it under the tongue of the
sheep and holding the sheep’s muzzle shut for a few
seconds. One milliliter of purified Dubois OPPV LMH19
containing 3.5 × 107 TCID50/ml in 1X Dulbecco’s PBS
Copyright © 2012 SciRes. OJVM
was diluted with 1 ml of 1X Dulbecco’s PBS and admin-
istered to 6 sheep for 6 minutes by aerosol Nb using a
portable nebulizer compressor (Sunrise PulmoAide Com-
pressor/Nebulizer De Vilbiss model #5650D) and a dis-
posable nebulizer (Sunrise Medical with T-piece, mouth-
piece and tubing model #4650D-621) using previously
published methods [33]. The aerosol generated by the
compressor flowed from a piece of plastic tubing with an
inner diameter of ~1.2 cm and was placed into the top
half of a 2 L plastic bottle. The large end of the plastic
bottle was placed on the muzzle of the sheep, and the
plastic tubing was placed in the narrow opening of the 2
L plastic bottle approximately 2.3 cm away from the
sheep’s nose and mouth. Nebulization was performed
under manual restraint, and a pillowcase was placed over
the sheep’s head to help calm the animal. Two sheep
were used as negative controls where 1 ml of 1X Dul-
becco’s PBS was given orally (PO) and 2 ml of 1X Dul-
becco’s PBS was given by Nb to both sheep. One sheep
was placed with the negative control group as a sentinel.
The negative controls, IV, Nb, and PO groups were
housed in separate isolation rooms or buildings. Siblings
were placed in separate groups to ensure maximum ge-
netic diversity within each experimental group.
2.4. Blood Collection and OPPV Diagnostic Tests
Sheep were bled by venous jugular puncture into 1 - 10
ml vaccutainer tube without anti-coagulant and 2 - 10 ml
vaccutainer tubes with EDTA (Becton-Dickinson) per
time point starting at 3 days prior to infection (prebleed),
1 day post-infection, and every 3 to 4 days for a period
ranging from 239 to 246 days. Peripheral blood leuko-
cytes (PBL) were removed from the 2 EDTA vaccutainer
tubes following centrifugation at 2000 × g for 20 minutes
at 4˚C. PBL were incubated for 2 minutes with 2 ml of
Puregene Red Cell Lysis solution (Qiagen, Inc.), 13 ml
of 1X Dulbecco’s PBS pH 7.0 with 10mM EDTA fol-
lowed by centrifugation at 900 × g for 10 minutes. Su-
pernatant was removed and residual buffer was used to
resuspend the PBL pellet and the pellet was frozen at
–20˚C until DNA isolation could be performed. Serum
was removed from the vaccutainer tube without coagu-
lant after centrifugation at 2000 × g for 15 minutes at 4˚C
and was stored at –20˚C until the cELISA could be per-
formed. DNA was isolated from PBL following manu-
facturer’s directions for 10 million cells using Puregene
technology (Qiagen, Inc.). The concentration of DNA
was determined using a Nanodrop 2000 spectropho-
tometer (Thermo Scientific).
A cELISA and a real time OPPV quantitative PCR
(qPCR) were utilized to determine OPPV infection status.
Serum was tested for OPPV antibodies using a previ-
ously validated cELISA [34]. A sheep was considered
cELISA positive if the cELISA measured greater than
20.9% inhibition on two consecutive time points. Meth-
ods for the real time OPPV qPCR have been published
[35]. The primers and Taqman probe were designed
against a conserved region of envelope that encodes the
transmembrane protein. One microgram of DNA from
each animal was tested in triplicate within each run in the
OPPV qPCR. OPP provirus levels were obtained from
the mean OPPV qPCR copy number from the triplicates.
OPP levels of greater than 10 copies of envelope/μg
DNA at two consecutive time points were considered
OPPV qPCR positive for the presence of OPP provirus.
Western blot analysis of sera was performed as previ-
ously described using OPPV WLC1 under reduced con-
ditions [36]. Sheep with antibodies to 2 of 3 of the fol-
lowing OPPV proteins: capsid (CA), transmembrane
protein (TM), and the surface envelope glycoprotein (SU)
were considered OPPV western blot positive. Western
blot analyses were performed on pre-infection and 211
days post-infection sera. A sheep was considered OPPV
infected if both the cELISA and OPPV qPCR were posi-
tive on two consecutive time points and if western blot
analysis was positive at 211 days post-infection.
2.5. Sequencing of OPPV LTR
DNA from PBL was used to sequence OPPV LTR. Me-
thods for sequencing OPPV LTR have been previously
described [9].
2.6. Statistical Analyses
Survival curve analyses were used to compare the per-
centage of remaining cELISA and OPPV qPCR negative
sheep over time between and within the various infection
method groups. Survival curves were generated and
compared using a logrank test and chi-squared analyses
using Prism 4.0 c (GraphPad Software Inc.).
3. Results
Six sheep were aerosol nebulized with 3.5 × 107 TCID50
of Dubois OPPV LMH19 and all six became OPPV in-
fected based on a positive result by cELISA and OPPV
qPCR at two consecutive time points, and a positive
western blot result at 211 days post-infection. This Du-
bois OPPV LMH19 isolate was chosen because it was
able to naturally infect horizontally in a previous study
[9], and the high dose was chosen to ensure infection in
at least one of the different routes. In contrast, only two
out of six sheep PO administered with Dubois OPPV
LMH19 became OPPV infected as determined by cE-
LISA, OPPV qPCR, and western blot analysis. All four
sheep administered Dubois OPPV LMH19 by IV became
infected with OPPV as determined by cELISA, OPPV
Copyright © 2012 SciRes. OJVM
qPCR, and western blot analysis. Neither of the two neg-
ative controls nor the sentinel sheep became infected
with OPPV. To confirm that the sheep were infected by
Dubois OPPV LMH19 isolate and not by some other
isolate, OPPV LTR was successfully sequenced from
PBL of ten infected sheep at 211 days post-infection.
Sequence data showed that OPPV LTR was 98.7% -
100% identical amongst the sheep and the original Du-
bois OPPV LMH19 provirus indicating that the sheep
were infected with Dubois OPPV LMH19 isolate.
Both OPPV qPCR and cELISA were utilized on a
daily basis to monitor the sheep infection status of the
sheep in the various groups. One first question was
which assay, OPPV qPCR or cELISA, first detected
OPPV positive sheep within the temporal study. Prior to
this study, no one had examined these two specific as-
says side by side temporally. The Nb and IV groups were
utilized to answer this question since all 6 nebulized
sheep and all 4 IV dosed sheep became OPPV infected.
Figure 1 shows that the OPPV qPCR was positive prior
to the cELISA in both the Nb and IV groups, and this
was statistically significant (P values = 0.007 and 0.029,
respectively). The median times to an OPPV qPCR posi-
tive result in the Nb and IV groups were 29.5 and 47
days post infection, respectively, and the median times to
a cELISA positive result in the Nb and IV groups were
73 and 125.5 days post infection, respectively.
To assess whether Nb of OPPV produced a cELISA or
OPPV qPCR positive test result more quickly than the
other methods of infection, the percent negative sheep in
the Nb group over time were compared to the percent
negative sheep over time in the IV and PO groups. Fig-
ure 1 shows that the Nb and IV groups do not statisti-
cally vary in terms of the percent OPPV qPCR or cE-
LISA negative sheep over time (P-values = 0.1 and 0.6,
respectively). However, sheep in the Nb group became
050100 150 200 250
Percent negative
Days P o st Infect ion
Figure 1. Percent cELISA and OPPV qPCR negative sheep
plotted as a function of days post infect ion for aerosol nebu-
lization (Nb), intravenous (IV) and oral (PO) groups. A
positive test is defined as a cELISA or OP PV qPCR positive
result at 2 consecutive time points.
OPPV qPCR and cELISA positive more quickly as
compared to the PO administered group (P values =
0.0007 and 0.0021, respectively). The median times to an
OPPV qPCR positive result in the Nb and PO groups
were 29.5 and >204 days post infection, respectively, and
the median times to a cELISA positive result in the Nb
and PO groups were 73 and 204 days post infection, re-
4. Discussion
Nb of 3.5 × 107 TCID50 Dubois OPPV LMH19 was
shown to infect more seven month-old lambs than the PO
method of delivery, and the rate at which the sheep be-
came cELISA and OPPV qPCR positive was faster in the
Nb group versus the PO group. Therefore, Nb is a more
effective experimental infection method than PO admini-
stration of OPPV under these dosing and specific virus
conditions. In addition, Nb of OPPV was equally effec-
tive in infecting lambs as IV administration within the
3.5 × 107 TCID50 infectious dose. However, in the future,
a dose dilution should be performed to precisely evaluate
the IV and Nb delivery methods. Nontheless, these re-
sults indicate that Nb is a good experimental infection
method to evaluate vaccine candidates and genetic mark-
ers that associate with OPPV control.
One reason why Nb is an effective method of OPPV
transmission is that sheep are insufflated with aerosolized
viral particles for 6 minutes through mostly the nose,
depositing viral particles not only in the upper respiratory
tract (nasal and pharyngeal mucosa) but also in the lower
respiratory tract, including bronchiolar and alveolar epi-
thelium. One study following intranasal installation of
VMV commented that virus isolations from nasal mu-
cous samples were less frequent than from peripheral
leukocytes [37]. The fact that they successfully isolated
virus from nasal mucous samples suggests that virus may
be present in nasal epithelia; however there is no evi-
dence in the literature that OPPV or VMV infects cells of
the nasal or pharyngeal mucosa. In contrast, the lower
lung is known to be a more permissive site for VMV
entry in vivo as compared to the trachea [25]. And, be-
sides generally infecting monocytes, macrophages, and
dendritic cells [17,18,38], OPPV and VMV replicate and
integrate in alveolar macrophages of the lung of naturally
infected sheep [12,15]. After nebulization, cell free
OPPV may be insufflated to the lower respiratory tract
and may directly infect alveolar macrophages. Others
have shown that lower respiratory tract alveolar epithelial
cells are first to be infected with ovine herpesvirus 2 after
aerosol nebulization, and this virus is larger in size than
OPPV [39]. Evaluation of tissues and fluids from the
upper and lower respiratory tracts for both cell free and
cell-associated OPPV early after Nb may help to under-
Copyright © 2012 SciRes. OJVM
stand the cell types involved in initial infection and dis-
The Dubois OPPV LMH19 isolate was originally de-
rived from colostrum cells of an OPPV infected and lac-
tating ewe. The finding that a mammary derived virus
passaged through GSM cells can transmit experimentally
using aerosol Nb, and the fact that horizontal transmis-
sion comprises accounts for the majority of natural infec-
tion amongst ewes [7-9] suggests that mammary derived
viruses may transmit horizontally by aerosols to naïve
ewes in a natural lambing situation. Further experimental
OPPV infections using aerosol Nb of colostrum and milk
needs to be conducted on naïve post-partum ewes. In
addition, a larger molecular epidemiology study may
help with elucidating if naïve post-partum ewes are at
highest risk to infection during the lambing season.
Our study is the first to show OPPV infection resulting
from oral feeding of older or seven month-old lambs.
Previous PO experimental CAEV and OPPV infection
studies have utilized only neonatal lambs due to the fact
that the gastrointestinal tract of neonatal lambs is only
permeable to macromolecules such as immunoglobulins
and leukocytes for approximately 48 hours post-parturition
[40-42]. Bottle-feeding oral infection studies using 2 ×
107 TCID50 of a Florida CAEV isolate resulted in two out
of three (66%) neonatal kids becoming infected [43], and
bottle-feeding of 3.8 × 106 TCID50 of CAEV-63 or
CAEV-Co resulted in 17 out of 17 (100%) neonatal kids
and 15 out of 18 (83%) neonatal kids becoming infected,
respectively [44]. Monitoring of VMV 72 hours follow-
ing natural colostrum suckling of neonatal lambs showed
that provirus and viral capsid were detected in ileum epi-
thelial cells [30]. A previous PO experimental OPPV
infection study using 105 TCID50 of OPPV given to ten
newborn lambs resulted in only two sheep (or 20%) be-
coming infected over 19 months [31]. In the 7-month old
lambs, it is possible but somewhat unlikely that OPPV
survives through the four compartment stomach. A more
distinct possibility is that virus was aspirated to the upper
and lower respiratory tracts after being placed under the
tongue. In addition, if there were open wounds or scars in
the mouth, the virus could have directly entered the pe-
ripheral blood circulation, or alternatively, the virus
could have entered the tonsils, submandibular or parotid
lymph nodes where it could disseminate through the
lymph and eventually the blood. Again, a temporal eva-
luation of the tissues and cells during early OPPV PO
infection could help establish which mucosal site was the
site of entry for OPPV in 7-month old sheep.
5. Conclusion
In conclusion, Nb is as effective as IV administration of
OPPV in causing infection, and Nb is more effective than
PO administration of OPPV in causing infection under
these dosing and viral isolate conditions. This new me-
thod of experimental infection provides a less invasive
approach as compared to intravenous, intrapulmonary
and intratracheal methods, which require sedation in
some cases and surgical procedures. Future experiments
are planned to examine another Dubois OPPV isolate
along with a titration of virus using Nb to determine the
efficiency of the experimental method. This experimental
infection method can be utilized to test new OPPV vac-
cines or host genetic markers of OPPV control.
6. Acknowledgements
We thank Nic Durfee for technical assistance. We thank
Alisha Ewing, Evan McQuirk, Ashley Trtek, Emma Ka-
rel, and Lori Fuller for routine animal care, handling and
bleedings. We thank Dr. Steven Parish, Dr. George Bar-
rington and the WSU large animal clinical residents for
their assistance in monitoring the health of the sheep.
This work is supported by ARS CWA #5348-32000-
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