Annie Page-Karjian

Numerous toxin-producing harmful algal (HAB) species occur in Florida’s coastal waters. Exposure to these toxins has been shown to have sublethal effects in sea
turtles. The objective of this study was to establish concentrations of 10 HAB toxins in plasma samples from green turtles (Chelonia mydas) foraging in Florida’s Big
Bend. Domoic acid, lyngbyatoxin-A, microcystins, nodularin, and okadaic acid were detected, demonstrating exposure to these HAB toxins, which are also a public
health concern.

Important indicators of population health needed for large-scale sea turtle population
recovery efforts include demographics, disease and mortality trends, condition indices, and
baseline blood data. With this comprehensive health assessment of adult female green sea turtles
Chelonia mydas nesting on Juno Beach, Florida, USA, we (1) established baseline health indices;
(2) identified individuals with evidence of infection by chelonid alphaherpes viruses 5 and 6
(ChHV5, ChHV6), which are implicated in fibropapillomatosis and respiratory and skin disease,
respectively; and (3) compared measured health indices between turtles that did versus those that
did not test positive for ChHV5 and/or ChHV6. All 60 turtles included in the study were in good
body condition with no external fibropapillomatosis tumors. Hematological and biochemical reference
intervals were established. Via quantitative PCR (qPCR), 5/60 turtles (8%) tested positive
for ChHV5, and all turtles were negative for ChHV6. Of 41 turtles tested for antibodies to ChHV5
and ChHV6, 29% and 15% tested positive, respectively, and 10% tested positive for antibodies to
both viruses. Notably, there were no statistically significant differences between health variables
for nesting turtles that tested positive for ChHV5 DNA versus those that tested negative; and also
no differences between turtles that tested positive for ChHV5 or ChHV6 antibodies and those that
did not. This suggests that these viruses are enzootically stable in Florida’s adult green turtles.
This study provides a health profile of nesting green turtles in southeastern Florida applicable to
temporal and spatial investigations of this and other populations.

Background: Free-ranging common bottlenose dolphins (Tursiops truncatus) can become entangled in fishing line
and other marine debris. Infrequently, dolphins can be successfully disentangled, released back into the wild, and
later examined postmortem to better understand the pathology and long-term effects of these entanglements.
Case presentation: An entangled common bottlenose dolphin (Tursiops truncatus) calf was observed in the Indian
River Lagoon, Florida, USA, with monofilament fishing line wrapped tightly around its maxilla. A multi-agency team
successfully disentangled the dolphin for immediate release back into its natural habitat. A year after
disentanglement, photos and observations indicated that the now independent calf showed a decline in body
condition, characterized by grossly visible ribs and a prominent post-nuchal depression. More than 2 years postdisentanglement,
the freshly dead carcass of this juvenile dolphin was recovered with extensive predation wounds.
Despite the forestomach being ~ 50% full of ingesta (fish), the dolphin was emaciated. During postmortem
examination, we collected and evaluated photographs and measurements of the maxillary damage resulting from
the entanglement.
Conclusion: The monofilament entanglement caused permanent, bilateral deformation of the maxillary dental
arcade, including a 4.0–4.2 cm long, 0.5 cm deep linear groove where the entanglement eroded the lateral edges of
the maxilla. There was no evidence of maxillary fracture and the dolphin survived for more than 2 years after
disentanglement. External evidence of propeller scars and a fishing hook discovered embedded in the laryngeal
mucosa at necropsy indicated repeated human interactions.
Keywords: Odontocete, Maxilla, Monofilament, Fisheries interaction

Anthropogenic contaminants in the marine environment often biodegrade slowly,
bioaccumulate in organisms, and can have deleterious effects on wildlife immunity,
health, reproduction, and development. In this study, we evaluated tissue toxicant
concentrations and pathology data from 83 odontocetes that stranded in the
southeastern United States during 2012–2018. Mass spectrometry was used to
analyze blubber samples for five organic toxicants (atrazine, bisphenol-A, diethyl
phthalates, nonylphenol monoethoxylate [NPE], triclosan), and liver samples were
analyzed for five non-essential elements (arsenic, cadmium, lead, mercury, thallium),
six essential elements (cobalt, copper, manganese, iron, selenium, zinc) and one
toxicant mixture class (Aroclor1268). Resultant data considerably improve upon the
existing knowledge base regarding toxicant concentrations in stranded odontocetes.
Toxicant and element concentrations varied based on animal demographic factors
including species, sex, age, and location. Samples from bottlenose dolphins had
significantly higher average concentrations of lead, manganese, mercury, selenium,
thallium, and zinc, and lower average concentrations of NPE, arsenic, cadmium, cobalt,
and iron than samples from pygmy sperm whales. In adult female bottlenose dolphins,
average arsenic concentrations were significantly higher and iron concentrations were
significantly lower than in adult males. Adult bottlenose dolphins had significantly higher average concentrations of lead, mercury, and selenium, and significantly lower average
manganese concentrations compared to juveniles. Dolphins that stranded in Florida had
significantly higher average concentrations of lead, mercury, and selenium, and lower
concentrations of iron than dolphins that stranded in North Carolina. Histopathological
data are presented for 72 animals, including microscopic evidence of Campula spp.
and Sarcocystis spp. infections, and results of Morbillivirus and Brucella spp. molecular
diagnostic testing. Sublethal cellular changes related to toxicant exposure in free-ranging
odontocetes may lead to health declines and, in combination with other factors, may
contribute to stranding.

Fibropapillomatosis is a debilitating tumor disease of sea turtles that is sometimes
fatal. This disease is a key concern for sea turtle rehabilitation facilities due to its infectious nature,
as it is associated with a virus called chelonid alphaherpesvirus 5. This is the first study to analyze
antibodies to this virus in loggerhead sea turtles and represents the most complete dataset on
viral detection in sea turtles encountered in the more northern latitudes of their habitat in the
western Atlantic.

Chelonid alphaherpesviruses 5 and 6 (ChHV5 and ChHV6) are viruses that affect wild
sea turtle populations. ChHV5 is associated with the neoplastic disease fibropapillomatosis (FP),
which affects green turtles (Chelonia mydas) in panzootic proportions. ChHV6 infection is associated
with lung-eye-trachea disease (LETD), which has only been observed in maricultured sea turtles,
although antibodies to ChHV6 have been detected in free-ranging turtles. To better understand
herpesvirus prevalence and host immunity in various green turtle foraging aggregations in Florida,
USA, our objectives were to compare measures of innate and adaptive immune function in relation
to (1) FP tumor presence and severity, and (2) ChHV5 and ChHV6 infection status. Free-ranging,
juvenile green turtles (N = 45) were captured and examined for external FP tumors in Florida’s Big
Bend, Indian River Lagoon, and LakeWorth Lagoon. Blood samples were collected upon capture
and analyzed for ChHV5 and ChHV6 DNA, antibodies to ChHV5 and ChHV6, in vitro lymphocyte
proliferation using a T-cell mitogen (concanavalin A), and natural killer cell activity. Despite an
overall high FP prevalence (56%), ChHV5 DNA was only observed in one individual, whereas 20% of
turtles tested positive for antibodies to ChHV5. ChHV6 DNA was not observed in any animals and
only one turtle tested positive for ChHV6 antibodies. T-cell proliferation was not significantly related
to FP presence, tumor burden, or ChHV5 seroprevalence; however, lymphocyte proliferation in
response to concanavalin A was decreased in turtles with severe FP (N = 3). Lastly, green turtles with
FP (N = 9) had significantly lower natural killer cell activity compared to FP-free turtles (N = 5). These
results increase our understanding of immune system effects related to FP and provide evidence that
immunosuppression occurs after the onset of FP disease.

The gopher tortoise (Gopherus polyphemus), a keystone species, is declining throughout its geographic range. Lack of
knowledge with respect to the potential infectious diseases present within wild populations creates a dilemma for wildlife
biologists, conservationists and public policy makers. The objective of this study was to conduct a health assessment of two
previously unstudied gopher tortoise aggregations located at two sites in southeastern FL. Samples were collected from
91 tortoises (48 adults, 35 juveniles, 8 hatchlings) captured at Florida Atlantic University’s Harbor Branch Oceanographic
Institute, in Fort Pierce, FL, USA in 2019, and Loggerhead Park in Juno Beach, FL, USA, during 2018–2019. Samples of blood,
nasal swabs and oral/cloacal swabs were analyzed for hematology, plasma protein electrophoretic profiles and infectious
disease testing including Mycoplasma spp. serology and polymerase chain reaction (PCR) assays for Ranavirus, Herpesvirus and
Anaplasma spp. Hematological and plasma protein electrophoresis reference intervals are presented for adult and juvenile
tortoises from both sites combined. Clinical signs consistent with upper respiratory tract disease (URTD) were observed in
18/91 (20%) tortoises, and antibodies to Mycoplasma agassizii were detected in 33/77 (42.9%) tortoises. Adult tortoises were
significantly more likely than juveniles to have URTD clinical signs, and statistically significant, positive relationships were
observed between the presence of antibodies to Mycoplasma spp. and carapace length, packed cell volume and plasma
globulin concentrations. Anaplasma spp. inclusions were observed in 8/82 (10%) tortoises, but PCR detected Anaplasma sp.
in 21/83 (25%) tortoises. Herpesvirus and Ranavirus were not detected in any blood or swab samples. This work contributes
important baseline information on the health of gopher tortoises toward the southern end of the species’ range.

Chelonid alphaherpesvirus 5 (ChHV5) is strongly associated with fibropapillomatosis, a
neoplastic disease of sea turtles that can result in debilitation and mortality. The objectives of this
study were to examine green (Chelonia mydas), hawksbill (Eretmochelys imbricata), and leatherback
(Dermochelys coriacea) sea turtles in Grenada, West Indies, for fibropapillomatosis and to utilize
ChHV5-specific PCR, degenerate herpesvirus PCR, and serology to non-invasively evaluate the
prevalence of ChHV5 infection and exposure. One-hundred and sixty-seven turtles examined from
2017 to 2019 demonstrated no external fibropapilloma-like lesions and no amplification of ChHV5
DNA from whole blood or skin biopsies. An ELISA performed on serum detected ChHV5-specific IgY
in 18/52 (34.6%) of green turtles tested. In 2020, an adult, female green turtle presented for necropsy
from the inshore waters of Grenada with severe emaciation and cutaneous fibropapillomas. Multiple
tumors tested positive for ChHV5 by qPCR, providing the first confirmed case of ChHV5-associated
fibropapillomatosis in Grenada. These results indicate that active ChHV5 infection is rare, although
viral exposure in green sea turtles is relatively high. The impact of fibropapillomatosis in Grenada is
suggested to be low at the present time and further studies comparing host genetics and immunologic
factors, as well as examination into extrinsic factors that may influence disease, are warranted.

The spreading global sea turtle fibropapillomatosis (FP) epizootic is threatening some of
Earth’s ancient reptiles, adding to the plethora of threats faced by these keystone species. Understanding
this neoplastic disease and its likely aetiological pathogen, chelonid alphaherpesvirus 5
(ChHV5), is crucial to understand how the disease impacts sea turtle populations and species and
the future trajectory of disease incidence. We generated 20 ChHV5 genomes, from three sea turtle
species, to better understand the viral variant diversity and gene evolution of this oncogenic virus.
We revealed previously underappreciated genetic diversity within this virus (with an average of
2035 single nucleotide polymorphisms (SNPs), 1.54% of the ChHV5 genome) and identified genes
under the strongest evolutionary pressure. Furthermore, we investigated the phylogeny of ChHV5 at
both genome and gene level, confirming the propensity of the virus to be interspecific, with related
variants able to infect multiple sea turtle species. Finally, we revealed unexpected intra-host diversity, with up to 0.15% of the viral genome varying between ChHV5 genomes isolated from different
tumours concurrently arising within the same individual. These findings offer important insights
into ChHV5 biology and provide genomic resources for this oncogenic virus.