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Veterinary Microbiology 133 (2009) 43–53

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Bottlenose dolphin (Tursiops truncatus) papillomaviruses:

Vaccine antigen candidates and screening test development
Manuela Rehtanz a,b,*, Gregory D. Bossart a, Bethany Doescher c, Annabel Rector d,
Marc Van Ranst d, Patricia A. Fair e, Alfred B. Jenson b, Shin-Je Ghim b
a
Harbor Branch Oceanographic Institution, Center for Coastal Research, Marine Mammal Research and Conservation,
Fort Pierce, FL 34946, USA
b
James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA
c
Sea Life Park by Dolphin Discovery, Waimanalo 96795, HI, USA
d
Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium
e
National Oceanic and Atmospheric Administration/National Ocean, Service/Center for Coastal Environmental Health and
Biomolecular Research, Charleston, SC 29412, USA
Received 7 March 2008; received in revised form 9 June 2008; accepted 26 June 2008

Abstract

Papillomaviruses (PVs) have been shown as being the etiologic agents of various benign and malignant tumours in many
vertebrate species. In dolphins and porpoises, a high prevalence of orogenital tumours has recently been documented with at
least four distinct novel species-specific PV types detected in such lesions. Therefore, we generated the immunological reagents
to establish a serological screening test to determine the prevalence of PV infection in Atlantic bottlenose dolphins [(Tursiops
truncatus (Tt)]. Using the baculovirus expression system, virus-like particles (VLPs) derived from the L1 proteins of two TtPV
types, TtPV1 and TtPV2, were generated. Polyclonal antibodies against TtPV VLPs were produced in rabbits and their
specificity for the VLPs was confirmed. Electron microscopy and enzyme-linked immunosorbent assay (ELISA) studies
revealed that the generated VLPs self-assembled into particles presenting conformational immunodominant epitopes. As such,
these particles are potential antigen candidates for a TtPV vaccine.
Subsequently, the VLPs served as antigens in initial ELISA tests using sera from six bottlenose dolphins to investigate PV
antibody presence. Three of these sera were derived from dolphins with genital tumour history and showed positive PV ELISA
reactivity, while the remaining sera from lesion-free dolphins were PV antibody-negative. The results suggest that the developed
screening test may serve as a potential tool for determining PV prevalence and thus for observing transmission rates in dolphin
populations as the significance of PV infection in cetaceans starts to unfold.
# 2008 Elsevier B.V. All rights reserved.

Keywords: Papillomavirus prevalence; TtPV vaccine; Serological screening test; Bottlenose dolphin; Tursiops truncatus

* Corresponding author. Current address: New York University, Department of Basic Sciences, Infectious Diseases, 345 East 24th Street 921
D, New York, NY 10010, USA. Tel.: +1 212 998 9326; fax: +1 212 995 3250.
E-mail address: Manuela.Rehtanz@gmx.de (M. Rehtanz).

0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2008.06.017
44 M. Rehtanz et al. / Veterinary Microbiology 133 (2009) 43–53
1. Introduction
Papillomaviruses (PVs) are small, non-enveloped,
double-stranded DNA viruses exhibiting a strong host-
and tissue-specificity (zur Hausen and de Villiers,
1994). Direct contact with an infected individual of the
same species is typically required for virus transmis-
sion. After infecting the basal cells of the cutaneous or
mucosal skin, PVs induce increased proliferation of
the stratified squamous epithelium. Hyperproliferation
of the suprabasal cells, such as the development of
warts, papillomas or condylomas, is induced by viral
protein activity interfering with cellular tumour
suppressor protein pathways (Chow and Broker,
1994; Howley and Lowy, 2001). Human PVs (HPVs)
are the most frequently sexually transmitted viruses
(Garland, 2002). Ten to thirty percent of PV-induced
severe cervical neoplasias lead to invasive cervical
carcinoma, the second most frequent cancer of women
worldwide (Walboomers et al., 1999; Einstein and
Goldberg, 2002). It is generally accepted that PV
infection is necessary for cervical carcinoma devel-
opment in humans, such that all cervical cancer cases
can be attributed to infection (Walboomers et al.,
1999; Parkin and Bray, 2006). Cervical carcinoma also
represents the most common cause of cancer mortality
in women in developing countries (Reynolds, 1998).
With respect to marine mammals, numerous
reports describe cutaneous or mucosal papillomas,
condylomas, warts and/or malignant tumours in at
least 30 species of the suborders Mysticeti, Odonto-
ceti and Pinnipedia (reviewed in Newman and Smith,
2006). Some of these tumours were shown to be
associated with a PV, whereas others were described
with the suggestion of PV involvement. There have
been several reports of cutaneous and mucosal
squamous cell carcinoma in dolphins (reviewed in
Newman and Smith, 2006). However, the develop-
ment of oral papillomas and squamous cell carcinoma
in the same managed Atlantic bottlenose dolphin
(Tursiops truncatus), suggestive of malignant trans-
formation of the benign papillomatous lesions,
was only recently reported (Bossart et al., 2005). A
high prevalence of orogenital papillomas in free-
ranging cetaceans has also been documented (Van
Bressem et al., 1996; Bossart et al., 2005). There is
rising concern that the recent observation of high
tumour prevalence in marine mammals could reflect
emerging or resurging infectious diseases (Bossart,
2006, 2007).
Increasingly intensive searches for animal PVs
during the last decade have lead to the detection of
more than 50 novel distinct species-specific PV types,
found in cutaneous and mucosal tumours from a wide
variety of vertebrate species. Since 2004, four
cetacean PVs were identified and their genomes
completely sequenced: one Burmeister’s porpoise PV
Phocoena spinipinnis PV type 1 (PsPV1); GenBank
accession number NC_003348] and three Atlantic
bottlenose dolphin PVs [T. truncatus PV types 1, 2 and
3 (TtPV1, -2 and -3); GenBank accession numbers
EU240894, NC_008184 and EU240895]. PsPV1 was
associated with genital warts of two Burmeister’s
porpoises off the Peruvian coast (Van Bressem et al.,
2007). TtPV1 and TtPV3 were detected in genital
tumours of one managed European Atlantic bottlenose
dolphin (Rector et al., 2006), while TtPV2 was
associated with a genital tumour of a free-ranging
Atlantic bottlenose dolphin inhabiting the waters off
the eastern coast of the United States (Rehtanz et al.,
2006). Evidence supporting the existence of a second
PsPV type was also recently furnished (Van Bressem
et al., 2007).
A canine model to develop a vaccine for systemic
immunisation that completely protected against
experimentally induced oral mucosal papillomas
was employed early in PV vaccine research (Suzich
et al., 1995). Virus-like particles (VLPs) derived from
the most prevalent HPV types associated with cervical
carcinoma or benign genital condylomas were also
recently developed as potent vaccines (CERVAR-
IXTM, GlaxoSmithKline; GARDASIL1, Merck &
Co.). These VLPs are not infectious. By design, they
are devoid of viral DNA and consist of the
corresponding self-assembled L1 proteins, the major
capsid proteins of the PVs. In clinical trials, both HPV
vaccines prevented persistent infection of the viral
types included in the vaccine for 100% of vaccinated
women and reduced cervical abnormalities by more
than 90% (Cohen, 2005; Schiller and Lowy, 2006),
proving their protective potential.
Little effort has been made to develop animal PV
vaccines or to investigate the effects of such vaccines
on the progression of PV-related tumours in animals.
In most instances, lingual and genital papillomas in
managed dolphins are not curable by surgery, as such
 M. Rehtanz et al. / Veterinary Microbiology 133 (2009) 43–53
tumours typically recur. Immunising bottlenose
dolphins with PV vaccines could minimise or prevent
horizontal transmission of TtPVs among managed
dolphins held in the same pools as well as vertical
transmission between mothers and calves. Vaccine
studies could also yield useful information on possible
cross-protection against viral types not included in the
vaccine as was seen in human trials (reviewed in
Bonnez, 2007) and/or types not yet discovered.
Moreover, a serological screening test would allow
investigations of PV prevalence and transmission
rates, which has not been pursued in cetacean
populations hitherto. Data from these tests would
further the understanding of PV infection and facilitate
inferences regarding the association between PV
infection, tumour progression and malignant trans-
formation.
We recently developed recombinant animal vac-
cine candidates for horses, Equus caballus PV1 VLPs
(Ghim et al., 2004), which currently are being tested in
animal vaccine trials, and for manatees, Trichechus
manatus latirostris PV1 VLPs (Ghim et al., unpub-
lished data), which are scheduled for vaccine trials.
Here, we describe the development of TtPV1 and
TtPV2 VLPs as vaccine candidates against TtPV1 and
TtPV2 infection as well as the development of a
serological screening test for bottlenose dolphin PVs
(Rehtanz et al., 2007).
2. Materials and methods
2.1. VLP production
To develop TtPV1 and TtPV2 VLPs, the following
procedures were carried out separately for each viral
type. The whole TtPV1 genomic DNA, which was
previously cloned into pUC18 within its L1 open
reading frame (ORF) (Rector et al., 2006), was
isolated from the vector and religated to amplify the
complete L1 ORF. Using the religated TtPV1 template
and the pUC19-cloned TtPV2 template (Rehtanz
et al., 2006), the corresponding L1 genes were
amplified in specific polymerase chain reactions
(PCRs) with XhoI (bold) restriction site-containing
forward primers [50 -ATGCTCGAGATGCTGCATA-
TACCA-30 (TtPV1); 50 -CCGCTCGAGATGCTG-
CAGCTGCCTCC-30 (TtPV2)] and HindIII
45
(underlined) restriction site-containing reverse pri-
mers [50 -ATGAAGCTTTTACTGTTTAGTACGCCT-
30 (TtPV1); 50 -CCCAAGCTTTTAGGTACGTGT-
CACCTTCC-30 (TtPV2)]. The PCRs were performed
with an MJ Research PTC-200 thermocycler using the
Platinum1 Pfx DNA Polymerase (Invitrogen, Carls-
bad, CA, USA) under the following conditions: one
initial 5.5 min denaturation step at 94 8C was followed
by 40 cycles of denaturation at 94 8C for 45 s,
annealing at 50 8C for 1 min and strand extension at
72 8C for 2 min. A final elongation step at 72 8C for
7 min completed the reaction, and the samples were
kept at 4 8C until further analysis.
The PCR fragments were isolated from agarose
gels and subcloned into the vector PCR14Blunt-
TOPO (Invitrogen) following the manufacturer’s
recommendations. The complete TtPV1 and TtPV2
L1 ORFs were sequenced to confirm their correctness
and both fragments were then inserted into the
multiple cloning site of the baculoviral transfer vector
pBlueBac4.5 (Invitrogen, Carlsbad, CA, USA) down-
stream of the polyhedrin promoter. Recombinant
baculoviruses were prepared using a commercialised
kit (Bac-N-BlueTM Kit; Invitrogen, Carlsbad, CA,
USA). TtPV1- and TtPV2-recombinant baculoviruses
were generated by heterologous recombination
between the recombinant baculovirus transfer plasmid
pBlueBac4.5-TtPV1-L1 or -TtPV2-L1 and linearised
baculovirus DNA in Spodoptera frugiperda (Sf9) cells
as described in the manufacturer’s instructions.
The insect cells were cultured in supplemented
Grace’s medium (Gibco/Invitrogen, Gaithersburg,
MD, USA) containing 10% fetal bovine serum and
3.6 mM glutamine. Using Seaplaque GTG agarose
(BioWhitaker, Rockland, ME, USA), positive recom-
binant baculoviruses were plaque-purified according
to the manufacturer’s recommendations. To rule out
false-positive plaques at an early stage of the VLP
development process due to incomplete recombina-
tion and to determine the presence of the L1 ORFs
in putative recombinant viruses, the isolation of pure,
recombinant plaques was confirmed by PCR analysis.
Forward primer 50 -TTTACTGTTTTCGTAA-
CAGTTTTG-3 and reverse primer 50 -CAACAACG-
0
CACAGAATCTAGC-30 , flanking the polyhedrin
region, were used in a PCR with an initial denaturation
at 94 8C for 2 min, 30 cycles of denaturation at 94 8C
for 1 min, annealing at 55 8C for 2 min and strand
46 M. Rehtanz et al. / Veterinary Microbiology 133 (2009) 43–53
extension at 72 8C for 3 min followed by final strand
extension at 72 8C for 7 min. The reaction mixes were
then kept at 4 8C until further analysis of the PCR
products in a 0.8% agarose gel (Fig. 1).
2.2. VLP purification and detection
TtPV1 and TtPV2 VLPs were purified separately.
Sf9 insect cells were harvested and processed for VLP
purification 72 h post-infection with recombinant
TtPV L1-expressing baculoviruses. Cells were pel-
leted by centrifugation (170  g, 10 min, 4 8C),
resuspended in Dulbecco’s phosphate-buffered saline
(DPBS) (Gibco/Invitrogen, Gaithersburg, MD, USA)
and subjected to Dounce homogenisation (50 strokes)
followed by sonication on ice (3 pulses of 10 s at 3 W)
(Sonics Vibra cell VC130; Sonics & Materials Inc.,
Newtown, CT, USA). Cell debris was pelleted by
Fig. 1. PCR analyses revealed that plaque-purified TtPV1 and
TtPV2 L1-recombinant baculovirus clones were not contaminated
with wild-type baculovirus. The TtPV1 L1-recombinant pBlue-
Bac4.5 vector (lane 2) served as a positive control, while the
pBlueBac4.5 vector itself (lane 1) was included as a control to
determine wild-type contamination levels due to incomplete recom-
bination. Lanes 3 (TtPV1) and 4 (TtPV2) demonstrate the presence
of pure recombinant baculovirus maintaining L1 genes in insect
cells (type 1, 1524 bp; type 2, 1539 bp; each including additional
435 bp contributed by the vector). Lane 5 represents uninfected
insect cell extract as a negative control.
centrifugation (670  g, 15 min, 4 8C). The super-
natant was brought to a final caesium chloride
concentration of 1.33 g/ml by adding equal volumes
of a 2.66-g/ml ceasium chloride/DPBS solution. The
present VLPs were processed for purification using
differential ultracentrifugation (45,000  g, 18 h,
4 8C) as described previously (Suzich et al., 1995).
Banded material containing the VLPs was dialysed
extensively at 4 8C against DPBS in cassettes (Slide-
A-Lyzer1, Pierce, Rockford, IL, USA). Negative
staining of purified VLPs with 2% tungstophosphoric
acid (pH 6.8) was conducted to determine the
structural quality of the self-assembled VLPs by
transmission electron microscopy (Fig. 2).
2.3. Antibody purification and concentration
Protein A-Sepharose is regularly used to purify
immunoglobulin G (IgG) from serum and has been
shown to be effective with serum derived from several
mammalian species (Goudswaard et al., 1978). IgG
from 5 ml combined serum of two free-ranging
Atlantic bottlenose dolphins not displaying any signs
of disease was purified following standard protein A-
Sepharose (Sigma, St. Louis, MO) chromatography
using Econo Column1 Chromatography Column
(BioRad, Hercules, CA) following the manufacturers’
recommendations. The elution was carried out with
ImmunoPure1 IgG Elution Buffer (Pierce, Rockford,
IL) and the purified IgG was concentrated using
CENTRICON1 plus filter devices with Ultracel PL
membranes (Millipore, Billerica, MA).
The untreated combined dolphin serum and the
purified, concentrated dolphin IgG (4 mg/ml) were
diluted as indicated in Fig. 4 and loaded onto a 10%
SDS polyacrylamide gel. The protein staining was
performed with Coomassie Brilliant Blue (BioRad,
Hercules, CA).
2.4. Production of polyclonal antibodies
in rabbits
To obtain high titre polyclonal antibodies, three
New Zealand White Rabbits (NZWRs) were prebled
and then immunised three times at 2-week intervals
with purified immunogens, which were 100 mg intact
TtPV1 VLPs (NZWR 1), 100 mg intact TtPV2 VLPs
(NZWR 2) and 200 mg concentrated purified dolphin
 M. Rehtanz et al. / Veterinary Microbiology 133 (2009) 43–53
Fig. 2. Transmission electron microscopy pictures of purified, negatively stained TtPV1 (A) and TtPV2 (B) VLPs. The L1 proteins of both viral
types self-assembled into VLPs of the expected PV-characteristic size of approximately 55 nm. Individual capsomers composing the VLPs
(arrows) can be noticed.
IgG (NZWR A). To increase immune responses each
rabbit also received 300 ml adjuvant (TiterMaxTM
Gold, CytRx Corporation, Los Angeles, CA, USA).
Animals were bled 2 weeks after the third immunisa-
tion. The cells were separated from sera by
centrifugation (400  g, 10 min), and the antibody
response was tested by enzyme-linked immunosorbent
assay (ELISA). Polyclonal antibodies raised against
bottlenose dolphin IgG are now commercially avail-
able as well (Bethyl Laboratories, Inc., Montgomery,
TX, USA).
2.5. Pilot ELISA studies
All incubation steps were conducted for 1 h at
37 8C with three to five PBS washes in between. As
previously described (Ghim et al., 2004), ELISA
microplate (Dynatech Laboratories, Inc., Chantilly,
VA) wells were coated with 100 ng TtPV1 or TtPV2
VLPs as intact antigen in PBS or with up to 4 mg
denatured VLPs prepared in ELISA-denaturing
buffer (1% SDS, 0.25 mM 2-mercaptoethanol,
15 mM NaCl, 20 mM Tris, pH 7.4). VLPs prepared
in denaturing buffer were incubated for 5 min at
95 8C before coating to support denaturation of the
VLPs. The coated microplate wells were blocked
with 5% BSA and incubated with rabbit anti-VLP
antibodies as indicated in Fig. 3 (rabbit serum
dilution). Afterwards, alkaline phosphatase (AP)-
coupled goat anti-rabbit antibody (Sigma, St. Louis,
47
MO) diluted 1:5000 was added and the reactivities
were measured at 405 nm (Spectra MRTM, Dynex
Technologies, Chantilly, VA, USA) using 1041 p-
Nitrophenyl Phosphate (Sigma, St. Louis, MO).
Rabbit sera prior to immunisation with TtPV VLPs
served as negative controls (preserum). ELISA
values represent the mean of two to four experi-
ments with a seropositive cutoff value defined as an
absorbance of 0.1 after background subtraction,
representing the mean plus a minimum 2-fold
standard deviation.
2.6. Dolphin samples
Dolphins in human care are referred to as
‘‘managed dolphins’’. Sera (n = 6) were obtained
from three managed and three free-ranging Atlantic
bottlenose dolphins (T. truncatus). Managed dolphins
(2–4) were sampled in 2006 at Sea Life Park by
Dolphin Discovery (Waimanalo, HI, USA). Sera from
free-ranging dolphins were obtained from the Atlantic
Bottlenose Dolphin Health and Risk Assessment
(HERA) Project, a collaborative effort between
Harbor Branch Oceanographic Institution (HBOI)
and the National Oceanic and Atmospheric Admin-
istration (NOAA). These dolphins were captured,
examined and released in the estuarine waters near
Charleston (CHS, SC, USA) in August of 2004
(dolphin 1) and in the Indian River Lagoon (IRL, FL,
USA) in June of 2005 (dolphins 5 and 6).
48 M. Rehtanz et al. / Veterinary Microbiology 133 (2009) 43–53

Fig. 3. (A) Rabbit anti-TtPV VLP antibodies only target conformational intact VLP epitopes. In pilot ELISA studies microplate wells were
coated with 0.1 mg intact (grey bars) or up to 4.0 mg denatured (black bars; 4.0 mg shown) VLPs derived from TtPV1 L1 (panels A and B) or
TtPV2 L1 (panels C and D). They were incubated with serum from NZWR 1, immunised with TtPV1 VLPs (panels A and C), or with serum from
NZWR 2, immunised with TtPV2 VLPs (panels B and D), and the relative ELISA reactivity was measured. Preimmune sera (preserum) served as
negative controls. (B) Dilution series of rabbit anti-dolphin VLP antibodies. ELISA microplate wells were coated with intact TtPV1 VLPs,
incubated with dilutions of serum from NZWR 1, immunised with TtPV1 VLPs (panel E) and the ELISA reactivity was measured. Preimmune
rabbit serum (preserum) served as the negative control. Results are expressed as means + standard deviation. The seropositive cutoff of 0.1 is
indicated. DA405 nm, relative absorbance after background subtraction at a wavelength of 405 nm; VLPs, virus-like particles; NZWR, New
Zealand White Rabbit.

2.7. Detection of serum antibodies against TtPVs
Detection of TtPV antibodies in dolphin sera was
carried out as described above with minor changes.
Briefly, ELISA microplate wells were coated with
50 ng TtPV1 or TtPV2 VLPs, blocked and incubated
with animal sera diluted 1:300 in PBS/1% BSA.
Rabbit anti-dolphin IgG diluted 1:3000 in PBS/1%
BSA served as the primary antibody; AP-coupled goat
anti-rabbit antibody was used as the secondary
antibody. Given values represent the mean ELISA
reactivity and their standard deviation resulting from
three experiments with a seropositive cutoff at 0.1 as
defined above (Fig. 5).
 M. Rehtanz et al. / Veterinary Microbiology 133 (2009) 43–53
2.8. Animal use
The use of NZWRs (IACUC #03113M2) and the
acquisition of serum from managed dolphins at Sea
Life Park by Dolphin Discovery (HI, USA) for this
project complied with all relevant federal guidelines
and institutional policies.
3. Results
3.1. Development and detection of TtPV VLPs
Plaque-purified positive TtPV1 and TtPV2 L1-
recombinant baculovirus clones were confirmed for
the presence of an insert in a putative recombinant
virus by PCR analyses (Fig. 1). The pBlueBac4.5
vector itself (lane 1) served as a control to determine
possible wild-type baculovirus contamination in the
positive clones due to incomplete DNA recombina-
tion. The TtPV1 L1-recombinant pBlueBac4.5 vector
(lane 2) was used as a positive control, while extracts
from uninfected insect cells (lane 5) served as the
negative control. Lanes 3 (TtPV1 L1) and 4 (TtPV2
L1) demonstrate plaque-purified recombinant bacu-
lovirus clones without wild-type baculovirus DNA
contamination.
Transmission electron microscopy proved the
structural quality of the generated, purified recombi-
nant VLPs. Both L1 protein types were expressed in
insect cells and self-assembled into TtPV1 VLPs
(Fig. 2A) and TtPV2 VLPs (Fig. 2B) of the expected
PV-typical size of approximately 55 nm. Individual
capsomers composing the VLPs could be identified
(arrows).
3.2. Determination of anti-TtPV VLP antibody
recognition in ELISA studies
One rabbit (NZWR 1) was immunised with TtPV1
VLPs, while another rabbit (NZWR 2) was immunised
with TtPV2 VLPs. Subsequently, the generated
polyclonal rabbit anti-TtPV1 and anti-TtPV2 VLP
antibodies were used in ELISA studies to determine
whether they target conformational and/or linear
epitopes of the corresponding VLPs (Fig. 3A). ELISA
microplates were coated with purified intact (grey
bars) or denatured (black bars) TtPV1 or TtPV2 VLPs
49
as indicated. NZWR sera prior to immunisation
(preserum) served as negative controls. Hyperimmune
sera obtained from rabbits immunised with TtPV
VLPs were highly reactive with intact particles (grey
bars). Consistent with cross-reactivity, the serum from
NZWR 1 reacted with both type 1 and type 2 VLPs,
but the response to type 1 VLPs was much stronger
(panel A versus panel C). The serum from NZWR 2
reacted similarly to type 1 and type 2 VLPs (panel B
versus panel D). While the specificity of the rabbit
anti-TtPV1 VLP antibodies for type 1 VLPs was
obvious (panel A versus panel B), the rabbit anti-
TtPV2 VLP antibodies recognised both type 1 and
type 2 VLPs similarly with only slightly higher
reactivities with type 2 particles (panel C versus panel
D). Although the plates were coated with up to 40-fold
higher concentrations of denatured VLPs than intact
particles, neither serum reacted with denatured VLPs
(black bars), indicating that the rabbit antibodies
recognised immunodominant intact conformational
epitopes of both VLPs at the expense of linear
epitopes.
To address the reactivity of the NZWR 1 serum
(Fig. 3A, panel A) in more detail, the serum was
diluted serially and shown to be highly reactive with
the VLPs (Fig. 3B, panel E).
3.3. Analysis of TtPV VLP ELISA reactivity using
dolphin serum
SDS gel analyses of purified, concentrated immu-
noglobulin G (IgG) (Fig. 4, lanes 3–6) from combined
serum (lanes 1 and 2) of two free-ranging Atlantic
bottlenose dolphins demonstrated the presence of both
the heavy and light IgG chains and provided
information about protein yield and purity. By
immunising NZWR A with purified and concentrated
dolphin IgG, polyclonal anti-dolphin IgG antibodies
were generated to be used in further ELISA analyses.
Six dolphin sera were investigated to test the
applicability of the developed test (Fig. 5). Serum
derived from individuals merely devoid of orogenital
neoplasia may not represent an adequate negative
control in PV ELISA assays. The absence of clinical
signs does not guarantee a PV exposure-free history
and thus complicates the selection of the negative
control bottlenose dolphin. The youngest animal
tested, dolphin 4 (black bar), was only 4 months of
50 M. Rehtanz et al. / Veterinary Microbiology 133 (2009) 43–53
Fig. 4. Sodium dodecyl sulfate polyacrylamide gel showing purified
and concentrated IgG (lanes 3–6) from combined serum (lanes 1 and
2) of two free-ranging Atlantic bottlenose dolphins not showing any
sign of a disease.
age at the time of serum collection and represented our
best negative control. On the basis of its young age this
dolphin had the fewest chances to become infected
with a PV via horizontal transmission before blood
collection and did not display orogenital lesions of any
kind. Dolphin 1, which inhabits the estuarine waters
Fig. 5. PV ELISA analyses in free-ranging and managed Atlantic
bottlenose dolphins (Tursiops truncatus). Dolphin 1 (inhabiting the
estuarine waters near Charleston, SC, USA) represents the positive
control animal with an active PV-positive genital tumour at the time
of blood collection. Dolphins 2–4 (managed dolphins held at Sea
Life Park, HI, USA) have no orogenital lesion history. Within this
group dolphin #4 represents the negative control animal. Serum
antibodies of these three animals did not react with the VLPs.
Dolphins 5 and 6 (inhabiting the Indian River Lagoon, FL, USA)
displayed genital papillomas at the time of blood collection and
tested positive in the bottlenose dolphin ELISA. The mean values of
three experiments + standard deviation are shown. The seropositive
cutoff of 0.1 is indicated. DA405 nm, relative absorbance after back-
ground subtraction at a wavelength of 405 nm.
near Charleston (SC, USA), had an average PV ELISA
reactivity of 0.128 (dark grey bar) and represented our
positive control. Confirmed by PCR and rolling circle
amplification, this dolphin had a PV-positive genital
tumour in 2004 that was biopsied during the same
health exam from which the serum sample was
obtained (Rehtanz et al., 2006). Dolphins 2–4 are
managed animals without a history of orogenital
lesions and are held at Sea Life Park in Waimanalo
(HI, USA). Sera from these three animals did not react
with VLPs in our test (Fig. 5). Dolphins 5 and 6 are
free-ranging individuals inhabiting the Indian River
Lagoon (FL, USA). Both of these animals had genital
sessile papillomas when they were assessed for health.
Serum samples from both dolphins were taken at that
time and tested positive for PV in the ELISA.
Regardless of whether TtPV1 or TtPV2 VLPs were
used as the target antigens, all six sera proved
consistently negative or positive, respectively, in both
ELISA tests (data not shown). For example, the serum
from the positive control animal (dolphin #1) had a
mean ELISA-reactivity of 0.128 with type 1 VLPs,
while the mean was 0.141 with type 2 VLPs, suggesting
cross-reactivity between the two mucosotropic TtPVs.
4. Discussion
Bottlenose dolphins are highly active social
mammals, regularly engaging in sexual contact and
intense play (Booth, 1988; Gowans et al., 2007),
which facilitates horizontal pathogen transmission.
We and others previously reported orogenital tumours
in cetaceans and provided results on a correlation
between these tumours and PV infection (Van Bressem
et al., 1996, 2007; Cassonnet et al., 1998; Bossart
et al., 2005; Rector et al., 2006; Rehtanz et al., 2006).
Although the significance of PV infection in humans
and some animals has been known for decades and
albeit prevalence of orogenital tumours in cetaceans
has been studied for some time (Van Bressem et al.,
1996, 2000, 2006; Bossart et al., 2005), little is known
concerning PV prevalence and the significance of PV
infection in cetaceans. It has been suggested that
sexual engagement may be impeded in severe cases of
genital tumour development and that observed
tumours may compromise reproductive success
(Van Bressem et al., 1999).
 M. Rehtanz et al. / Veterinary Microbiology 133 (2009) 43–53
At least four distinct species-specific PV types have
recently been isolated from mucosal tumours of
cetaceans. Based on DNA sequence characteristics of
these PVs, TtPV2 may represent a so-called high-risk
virus with a stronger oncogenic potential in contrast to
the other cetacean types isolated to date (Rehtanz
et al., 2006). The development of dolphin PV vaccine
candidates may prove useful to protect managed
bottlenose dolphins from the disease, while the
development of a serological test will help to better
understand PV infection in managed as well as free-
ranging cetaceans.
For this study, we generated VLPs of two
bottlenose dolphin PVs, TtPV1 and TtPV2, which
are associated with genital tumours in Atlantic
bottlenose dolphins (Rector et al., 2006; Rehtanz
et al., 2006). When we used these VLPs to produce
hyperimmune sera in rabbits, we observed a stronger
immune response against TtPV1 VLPs. This differ-
ence in the immune response of NZWR 1 compared to
NZWR 2, which was immunised with TtPV2 VLPs,
may be reflective of a higher immunogenicity of type 1
VLPs compared to type 2 VLPs, although immune
responses vary among individuals in antibody
production. The question of whether this variability
typically observed in antibody production could have
accounted for the lower immune response against
TtPV2 VLPs will have to be addressed in the future.
The structural quality of both VLP types is compar-
able. However, the preferred use of TtPV1 VLPs over
TtPV2 in the ELISA is a result of higher TtPV1 L1
protein expression in the insect cell system and thus
higher yields of TtPV1 VLPs after purification.
We chose serum obtained from a dolphin display-
ing a PV-related genital tumour as the positive control
to establish the dolphin PV ELISA screening test.
Serum derived from a 4-month-old bottlenose dolphin
calf without any symptoms of PV infection was chosen
as the best negative control. Despite the extensive
evidence from vaccine studies that young mammals
are immunocompetent, there is no scientific evidence
that reveals at what age a bottlenose dolphin has
developed a fully competent immune system. How-
ever, it is well known that the immune system of young
mammals is immature, though functional (reviewed in
Holt, 1995; Marshall-Clarke et al., 2000). Recently,
Upham et al. (2006) also addressed a number of
studies on the functional immaturity of the immune
51
systems of human and small mammal neonates,
revealing the slow and highly variable kinetics of
maturation over the first few years of life. Thus, in
addition to the young age and disease-free condition of
the negative control bottlenose dolphin calf, its
possibly premature immune system might represent
a third criterion for its serum being a valid negative
control.
The sera derived from two dolphins with similar
genital tumours exhibited highly positive dolphin PV
ELISA values, indicating these animals could have
had a more recent infection compared to the positive
control animal. However, positive ELISA values do
not represent proof for a correlation of a present
tumour with PV infection, but serve to possibly
determine whether the animal had contact with a PV in
the past and to suggest a present lesion may be
associated with a PV, which would require further
research. In this initial small study, a correlation
between positive PV ELISA reactivity and the
presence of genital papillomas, one of which was
confirmed to be PV-associated, could be observed.
Future screenings of dolphin sera are also likely to
provide information on which animals, without
obvious signs of disease, are PV-positive. Thus, the
presented test may help elucidate over time whether
there is a protective anti-PV antibody titre in cetaceans
and which animals may be at risk of developing a
related tumour. Further studies with additional sera
that include repeated sampling as well as long-term
observations of animal contacts will be necessary to
validate the assay and to identify transmission
pathways, prevalence rates and the significance of
PV infection for tumour development.
Taken together, TtPV VLPs were shown to carry
conformational immunodominant epitopes and to be
highly immunogenic in rabbits. As has been shown in
humans, these particles may thus be potential antigen
vaccine candidates for bottlenose dolphins. Future
VLP vaccine trials in managed dolphins would
provide information about preventing tumour devel-
opment and possible cross-protection. Vaccinations
would be suitable for seronegative dolphins as a
protection, but may also be beneficial to support the
immune response of seropositive animals while
dealing with an existing infection or of animals
having cleared their infection, as was suggested in
humans (Bonnez, 2007). Although the human
52 M. Rehtanz et al. / Veterinary Microbiology 133 (2009) 43–53
vaccines have been shown to reduce cervical
abnormalities by more than 90% (Cohen, 2005), a
remaining question is whether such a vaccine could
be applied to clinically affected dolphins displaying
PV-related tumours to investigate chances of inducing
or supporting regression of lesions. In case of a
multivalent or bivalent vaccine, e.g. against TtPV1
and TtPV2, immunisations could still protect against
the type a clinically affected animal is not infected
with and/or against types not covered by the vaccine
but which share immunodominant epitopes with
the covered viral types. However, the PV VLPs’
general effects on advanced neoplasia await further
investigation.
The generated VLPs were also shown to be of
potential use to determine PV prevalence in bottlenose
dolphins as they were recognised by serum antibodies
derived from dolphins with genital papillomatous
lesions and by serum derived from a confirmed PV-
positive dolphin. PVs develop with their hosts and are
species-specific. They display greatest nucleotide and
amino acid similarities/homologies with PV types that
infect the same tissues of the same species or their
immediate relatives. Thus, each PV genotype usually
corresponds to its own serotype. However, it has been
widely recognised that evolutionary relatives across
PV types share cross-neutralising epitopes (Bonnez
et al., 1996; Touze et al., 1998). Therefore, a dolphin
PV screening test may also detect PV infection in other
cetacean or marine mammal species, which requires
further research.
Acknowledgements
This work was supported by a postdoctoral
fellowship of Harbor Branch Oceanographic Institu-
tion (HBOI) to Manuela Rehtanz. Lesions from free-
ranging dolphins were collected under National
Marine Fisheries Service Scientific Research Permit
No. 998-1678 issued to Gregory D. Bossart as part of
the Bottlenose Dolphin Health and Risk Assessment
(HERA) project conducted in the Indian River
Lagoon, FL, USA and the estuarine waters of
Charleston, SC, USA. Funding for this study was
provided in part by the Florida Protect Wild Dolphins
License Plate, Florida Ocean Initiatives Grant and
NOAA’s Fisheries Marine Mammal Health and
Stranding Response Program and the National Ocean
Service. Annabel Rector was supported by a
postdoctoral fellowship of the Research Fund K.U.
Leuven.
Sincere appreciation is extended to Zoya B.
Kurago, John S. Reif, Daniel Malamud, William R.
Abrams and David N. Levy for their critical review of
the manuscript and helpful discussions. Special thanks
are also extended to A.C. Marsh and R.H. Defran for
their editing assistance. We further sincerely thank
Renato Lenzi, Jeff Pawloski, Tammy Goodreau,
Andrew Scullion, Philip Browning, Alfonso Lopez
and the Animal Training Staff from Sea Life Park,
Waimanalo, Hawaii, for animal handling and for
providing dolphin samples.
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