DOI: 10.7589/2017-12-295
Journal of Wildlife Diseases, 55(1), 2019, pp. 000–000
Ó Wildlife Disease Association 2019
Fibropapillomatosis in a Green Sea Turtle (Chelonia mydas) from the
Southeastern Pacific
Diana M. Cárdenas,1 Roberto V. Cucalón,1 Lex G. Medina-Magües,1 Karina Jones,2 Rubén A. Alemán,3
Alonzo Alfaro-Núñez,4,5 and Washington B. Cárdenas1,6 1Laboratorio para Investigaciones Biomédicas,
Facultad de Ciencias de la Vida, Escuela Superior Politécnica del Litoral, 090920, Guayaquil, Guayas, Ecuador;
College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, Queensland, Australia;
Parque Nacional Machalilla–Ministerio del Ambiente. Garcı́a Moreno y Eloy Alfaro, Puerto López, Manabı́, Ecuador;
4
Section for Evolutionary Genomics, Centre for GeoGenetics, Natural History Museum of Denmark, Copenhagen K,
Denmark; 5School of Biological Science and Engineering, Yachay Tech University, Urcuquı́, Imbabura, Ecuador;
6
Corresponding author (email: wbcarden@espol.edu.ec)
2
3
ABSTRACT:
Fibropapillomatosis is a neoplastic
disease that afflicts sea turtles. Although it is
disseminated worldwide, cases of the disease have
not been reported in the southeastern Pacific
region. We describe a case of fibropapillomatosis
in a green sea turtle (Chelonia mydas) during its
rehabilitation at the Machalilla National Park
Rehabilitation Center, Ecuador. Viral presence
was confirmed by PCR, targeting fragments of the
chelonid alphaherpesvirus 5 (ChHV5) unique
long (UL) genes, UL27, UL28, and UL30. The
amplicons were sequenced and included in a
global phylogenetic analysis of the virus with other
reported sequences from GenBank. Results
showed that the available viral sequences segregated into five phylogeographic groups: western
Atlantic and eastern Caribbean, central Pacific,
western Pacific, Atlantic, and eastern Pacific
groups. The concatenated ChHV5 sequences
from Ecuador clustered with the eastern Pacific
sequences.
Key words: Chelonia mydas, chelonid alphaherpesvirus 5, Ecuador, fibropapillomatosis, phylogenetics, sea turtles, southeastern Pacific.
juvenile turtles to aggregation grounds
through ocean currents, where they come in
contact with a high density of infected sea
turtles (Herbst 1994) and then spread the
disease upon return to their natal beach
(Peare and Parker 1996; Lee et al. 2006;
Patrı́cio et al. 2012). Others suggest that
horizontal transmission occurs by direct contact (Herbst et al. 1995; Work et al. 2014)
through vectors such as marine leeches
(Greenblatt et al. 2004), cleaner fish (Lu et
al. 2000), or by highly infectious superspreader individuals (Work et al. 2014). The
prevalence of FP is associated with environmental factors such as degraded water quality
(Herbst 1994; Foley et al. 2005; dos Santos et
al. 2010), water temperature (Herbst 1994;
Herbst et al. 1995), presence of natural
biotoxins (Landsberg et al. 1999), increased
arginine in turtles’ diets, and eutrophication
(Van Houtan et al. 2010, 2014). Several
studies speculate that environmental factors
may lead to immunosuppression, followed by
disease expression (Landsberg et al. 1999;
Aguirre and Lutz 2004; Van Houtan et al.
2010). It is still largely unknown how the
interaction between the virus, host, and the
environment results in infection and disease
(Jones et al. 2016). Although ChHV5 phylogeographic groups described to date include
the eastern Pacific area (Patrı́cio et al. 2012;
Rodenbusch et al. 2014), neither the disease
nor virus has been reported in the southeastern Pacific region. The present study represents a new report of ChHV5 in this region
and describes its genetic relationship with
ChHV5 sequences around the world.
Sea turtles around the world are afflicted by
fibropapillomatosis (FP). This is a debilitating,
neoplastic disease associated with chelonid
alphaherpesvirus 5 (ChHV5) infection
(Herbst 1994; Quackenbush et al. 1998;
Lackovich et al. 1999). The ChHV5 virus is a
linear, double-stranded DNA virus within the
Alphaherpesvirinae subfamily and the Scutavirus genus, with a genome size of approximately 132 kb (Ackermann et al. 2012). After
its first report in Florida in 1938 (Smith and
Coates 1938), FP has been reported in all sea
turtles species (Williams et al. 2006) and from
every ocean of the world (Herbst 1994). It is
speculated that the propagation of the virus is
due to the dispersal and distribution of
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JOURNAL OF WILDLIFE DISEASES, VOL. 55, NO. 1, JANUARY 2019
FIGURE 1. A green sea turtle (Chelonia mydas) found to be affected with fibropapillomatosis during its
rehabilitation at the Machalilla National Park Rehabilitation Center in Ecuador. The tumor, measuring 0.9 cm in
diameter, was found in the axillary region of the sea turtle and was entirely removed for analysis. Viral presence
was confirmed by PCR, targeting fragments of the chelonid alphaherpesvirus 5 unique long (UL) genes, UL27,
UL28, and UL30.
On August 2015, five sea turtles, two olive
ridley turtles (Lepidochelys olivacea) and
three green turtles (Chelonia mydas) that
were housed together at the Valdivia Aquarium, were transferred to the Marine Fauna
Rehabilitation Center at Machalilla National
Park, Puerto López, Manabı́ Province, Ecuador. After careful evaluation, a 0.9-cm-diameter cauliflower-like tumor on the axillary
region was observed in one of the green
turtles (Fig. 1), which led to the suspicion of
FP disease. In October 2015, two green
turtles and one olive ridley turtle, still kept
at the Marine Fauna Rehabilitation Center,
were assessed for presence of ChHV5. Skin
samples of approximately 6 mm were biopsied
from the shoulder area from each turtle. All
samples were collected by using sterile
scalpels and forceps disinfected with 10%
chlorine for each sample. The biopsy site was
cleaned with 10% chlorine wipes after sampling each sea turtle. Lidocaine was used as a
local anesthesia, followed by the application of
silver sulfadiazine topical cream to prevent
infections. Skin samples were kept in 100%
ethanol at 10 C until they reached the
laboratory, where they were kept at 20 C
until use. Due to logistic limitations, proper
formaldehyde tissue fixation for histopathology was not possible. The tumor of the
suspected FP affected green sea turtle was
entirely removed and aliquoted into approximately 6-mm subsamples. This turtle weighed
29 kg, with a 63.9-cm curved carapace length
CÁRDENAS ET AL.—SHORT COMMUNICATIONS
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FIGURE 2. Phylogenetic tree of the concatenated unique long (UL) genes, UL27, UL28, and UL30, data set
from a green sea turtle (Chelonia mydas) found to be affected with fibropapillomatosis during its rehabilitation at
the Machalilla National Park Rehabilitation Center in Ecuador. A Bayesian method implemented in the BEAST
program was used to infer the topology of the tree. The highlighted Ecuadorian UL27 (KY419199), UL28
(KY419140), and UL30 (KY419141) sequences were generated in this study. The rest of the sequences were
retrieved from GenBank, with the accession number, location, year, and species described on each name
(Supplementary Material File S6). The numbers denote a posterior probability of each node .80%. Cm ¼
Chelonia mydas; Cc ¼ Caretta caretta; Lk ¼ Lepidochelys kempii; Lo ¼ Lepidochelys olivacea.
and 66-cm curved carapace width. All sampling procedures followed the Guide for the
Care and Use of Laboratory Animals of the
National Institutes of Health (National Research Council 2011) and were conducted
with the Ministry of the Environment of
Ecuador authorization (MAE-CGZ4-DPAM2017-0110-M).
The extraction of DNA, PCR, and sequencing methodologies are available in Supplementary Material File S1. Phylogeny was
inferred by a Bayesian analysis of genetic
sequences by using the BEAST program
(Drummond et al. 2012; Supplementary
Material File S1). The sequences used for
the analysis, the concatenated alignment, and
the Extensible Markup Language output file
are available as Supplementary Material Files
S2–S4 File, respectively. We detected ChHV5
only in the green sea turtle with the bulbous
tumor, both in the tumor and in the clinically
normal skin sample. The PCR resulted in the
expected amplification products for all primer
sets used for diagnosis.
The nested PCR assay on the green sea
turtle tumor tissue readily detected ChHV5
unique long (UL) genes UL27, UL28, and
UL30 but were unable to detect these genes
in tissue biopsies from the other two turtles
that shared the same pool. A negative PCR
diagnosis may indicate undetectable levels of
ChHV5 rather than absence of infection
(Quackenbush et al. 2001; Page-Karjian et
al. 2012; Alfaro-Nuñez and Gilbert 2014), but
for the present study, only positive results are
meaningful.
The Bayesian phylogenetic analysis resulted
in five phylogeographic groups: eastern Pacific
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JOURNAL OF WILDLIFE DISEASES, VOL. 55, NO. 1, JANUARY 2019
(Mexico, San Diego, Costa Rica, and Florida,
variant D), western Pacific (Australia), central
Pacific (Hawaii), Atlantic (Puerto Rico, Gulf
of Guinea, and Southeastern Brazil), and
western Atlantic and eastern Caribbean (Florida variants A, B, and C and sequences from
northeastern Brazil and Barbados). As expected, the Ecuador ChHV5 sequences clustered
with the eastern Pacific region group (Fig. 2).
These sequences distribution broadly
matches previous reports (Patrı́cio et al.
2012; Rodenbusch et al. 2014). In agreement
with a report by Herbst et al. (2004), the
ChHV5 Florida variant D sequences formed a
single lineage within the eastern Pacific group.
The raw output tree data set is provided in
Supplementary Material File S5. After this
initial report, new FP-like cases are being
studied, contributing to the knowledge of the
disease in the region.
The authors acknowledge Annie PageKarjian for her technical support and assistance on sea turtle sampling and chelonid
alphaherpesvirus 5 diagnosis. We thank the
Ecuadorian Ministry of the Environment for
its key support and interest in protecting the
marine wildlife.
SUPPLEMENTARY MATERIAL
Supplementary material for this article is
online at http://dx.doi.org/10.7589/
2017-12-295.
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Submitted for publication 5 December 2017.
Accepted 2 May 2018.