Surveillance of Mycoplasma agassizii in Texas tortoises (Gopherus berlandieri) for translocation with emphasis on treatment and recovery

Abstract

Background
Texas tortoises (Gopherus berlandieri) are a Texas-state threatened species. Translocation is often suggested as a mitigation option; however, disease status and the potential for spread must be considered prior to such efforts. Mycoplasma infection of the upper respiratory tract is a concern within tortoise populations, which requires monitoring so translocation efforts do not inadvertently spread the disease.

Objectives
We determined and compared the prevalences of Mycoplasma agassizii in Texas tortoises from donor and recipient sites in southern Texas prior to translocation, treated Mycoplasma agassizii-infected tortoises with danofloxacin, and developed alternate Mycoplasma agassizii treatments for Texas tortoises.

Methods
We collected 171 and 23 Texas tortoises from a 270-ha and a 100-ha donor site and recipient site, respectively. We began a regimen of danofloxacin (6 mg/kg body weight injected subcutaneously every other day for 30 days) for tortoises with clinical signs (N = 20). We noted an additional 10 tortoises began displaying clinical signs of upper respiratory tract disease (URTD) after translocation, so we designed a trial to test tulathromycin (5 mg/kg body weight given intramuscularly once/week for 7 weeks) or oxytetracycline (8 mg/kg body weight given subcutaneously once/day for 14 days) as Mycoplasma treatments for symptomatic tortoises.

Results
Within the donor and recipient sites, 56 (32.7%) and 8 (34.8%), respectively, had antibody titers suggestive of past exposure. Eighteen tortoises from the donor site (10.5%) and 2 from the recipient site (8.7%) displayed clinical signs (i.e., clear serous nasal discharge, conjunctivitis, and palpebral edema) consistent with Mycoplasmal URTD upon initial collection, even though all polymerase chain reaction (PCR) results were negative for active shedding of Mycoplasma agassizii. We ceased treatment after the first dose of danofloxacin due to adverse reactions, which only began to subside after 72 h from the initial dose. Neither tulathromycin or oxytetracycline caused the clinical signs of URTD to subside after a 50-day treatment period.

Conclusion
Mycoplasma is a persistent issue facing Texas tortoises. Stressors, such as translocation, can cause Mycoplasma-seropositive tortoises to display clinical symptoms of URTD, which can abate without treatment, once the stressor subsides.

Implications
Danofloxacin, the recommended treatment for Mycoplasma infection in tortoises, is too potent for Texas tortoises.

Full text

Frontiers in Veterinary Science 01 frontiersin.org
Surveillance of Mycoplasma
agassizii in Texas tortoises
(Gopherus berlandieri) for
translocation with emphasis on
treatment and recovery
ChristinA.Moeller
1, SarenPerales
1, WraithRodriguez
1,
AlynnM.Martin
1, CordB.Eversole
2, SandraRideout-Hanzak
1,
PaulCrump
3, ClaytonD.Hilton
1 and ScottE.Henke
1*
1 Caesar Kleberg Wildlife Research Institute, Texas A&M University–Kingsville, Kingsville, TX,
UnitedStates, 2 Arthur Temple College of Forestry and Agriculture, Stephen F. Austin State University,
Nacogdoches, TX, UnitedStates, 3 Texas Parks and Wildlife Department, Austin, TX, UnitedStates
Background: Texas tortoises (Gopherus berlandieri) are a Texas-state threatened
species. Translocation is often suggested as a mitigation option; however,
disease status and the potential for spread must beconsidered prior to such
eorts. Mycoplasma infection of the upper respiratory tract is a concern within
tortoise populations, which requires monitoring so translocation eorts do not
inadvertently spread the disease.
Objectives: We determined and compared the prevalences of Mycoplasma
agassizii in Texas tortoises from donor and recipient sites in southern Texas
prior to translocation, treated Mycoplasma agassizii-infected tortoises with
danofloxacin, and developed alternate Mycoplasma agassizii treatments for
Texas tortoises.
Methods: We collected 171 and 23 Texas tortoises from a 270-ha and a 100-ha
donor site and recipient site, respectively. Webegan a regimen of danofloxacin
(6 mg/kg body weight injected subcutaneously every other day for 30 days)
for tortoises with clinical signs (N = 20). We noted an additional 10 tortoises
began displaying clinical signs of upper respiratory tract disease (URTD) after
translocation, so wedesigned a trial to test tulathromycin (5 mg/kg body weight
given intramuscularly once/week for 7 weeks) or oxytetracycline (8 mg/kg body
weight given subcutaneously once/day for 14 days) as Mycoplasma treatments
for symptomatic tortoises.
Results: Within the donor and recipient sites, 56 (32.7%) and 8 (34.8%),
respectively, had antibody titers suggestive of past exposure. Eighteen tortoises
from the donor site (10.5%) and 2 from the recipient site (8.7%) displayed clinical
signs (i.e., clear serous nasal discharge, conjunctivitis, and palpebral edema)
consistent with Mycoplasmal URTD upon initial collection, even though all
polymerase chain reaction (PCR) results were negative for active shedding of
Mycoplasma agassizii. Weceased treatment after the first dose of danofloxacin
due to adverse reactions, which only began to subside after 72 h from the initial
dose. Neither tulathromycin or oxytetracycline caused the clinical signs of URTD
to subside after a 50-day treatment period.
Conclusion: Mycoplasma is a persistent issue facing Texas tortoises. Stressors,
such as translocation, can cause Mycoplasma-seropositive tortoises to display
OPEN ACCESS
EDITED BY
Valentina Virginia Ebani,
University of Pisa, Italy
REVIEWED BY
Michael James Murray,
Monterey Bay Aquarium, UnitedStates
Sid Knotek,
Avian and Exotic Animal Clinic, Czechia
*CORRESPONDENCE
Scott E. Henke
scott.henke@tamuk.edu
RECEIVED 08 November 2024
ACCEPTED 27 December 2024
PUBLISHED 17 January 2025
CITATION
Moeller CA, Perales S, Rodriguez W,
Martin AM, Eversole CB, Rideout-Hanzak S,
Crump P, Hilton CD and Henke SE (2025)
Surveillance of Mycoplasma agassizii in Texas
tortoises (Gopherus berlandieri) for
translocation with emphasis on treatment and
recovery.
Front. Vet. Sci. 11:1525179.
doi: 10.3389/fvets.2024.1525179
COPYRIGHT
© 2025 Moeller, Perales, Rodriguez, Martin,
Eversole, Rideout-Hanzak, Crump, Hilton and
Henke. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 17 January 2025
DOI 10.3389/fvets.2024.1525179

Moeller et al. 10.3389/fvets.2024.1525179
Frontiers in Veterinary Science 02 frontiersin.org
clinical symptoms of URTD, which can abate without treatment, once the
stressor subsides.
Implications: Danofloxacin, the recommended treatment for Mycoplasma
infection in tortoises, is too potent for Texas tortoises.
KEYWORDS
danofloxacin, Gopherus berlandieri, lethargy, Mycoplasma, oxytetracycline, runny
nose, stress, Texas tortoise
1 Introduction
e Texas tortoise (Gopherus berlandieri) is the smallest and
most sexually dimorphic of six species of tortoises that are native to
North America (1, 2). Within the UnitedStates, the Texas tortoise is
found in southern Texas, in the region south from Del Rio to San
Antonio to Victoria (2, 3), including the Lower Rio Grande Valley
(LRGV). While their overall range is still intact, their abundance is
thought to have declined and their distribution within the LRGV
specically has become sporadic and more restricted due to
agricultural and urban development. Densities of Texas tortoises have
been estimated to beas high as 35 tortoises/ha on lomas (4) [i.e.,
coastal wind-blown clay dunes (5)]. Studies conducted in grasslands
and shrublands estimate densities as low as 0.26 tortoises/ha (6). Due
to threats from illegal collection and commercial exploitation, Texas
tortoises, were listed as a protected nongame species in Texas in 1977.
Due to additional threats from habitat loss, particularly to high
density loma habitats, the species is still in need of conservation
action and applied management.
Texas tortoises can tolerate a broad range of habitat types, from
lomas to grasslands and thorn scrub (4). ey appear to reach their
highest densities in loma habitat (7) and at lower densities, they can
utilize relatively open-canopied or early successional habitats with
increased light intensity at ground level and high herbaceous plant
diversity (8). Texas tortoises are found in grasslands and shrublands
of southern Texas and appear to tolerate grazing-induced brush
encroachment (8).
However, development in the LRGV has been rapid in the last
half-century. Conversion of large areas of native thorn scrub and
coastal grasslands to agricultural, residential, and energy
infrastructure land uses has occurred. For example, large-scale
industrial development projects, such as SpaceX infrastructure and
liquied natural gas (LNG) terminals, have resulted in the loss of
habitat for Texas tortoises. To oset the loss of tortoise (Gopherus)
habitat in other states, state wildlife agencies oer translocation as
their mitigation strategy, even though results from translocations
for gopher tortoises (G. polyphemus) and desert tortoises
(G. agassizii) have varied (9–11). No formal assessment of the
success of translocation in Texas tortoises has been conducted.
One such obstacle before attempting translocation is determining
the presence of pathogens and parasites within the donor and
recipient populations. Without determining the health status of
tortoises, it is possible to introduce a naïve donor population of
tortoises to a disease-infected recipient population of tortoises, or
vice versa. One such disease of concern in Texas tortoises is upper
respiratory tract disease (URTD), which can becaused by the
highly contagious, bacterial species Mycoplasma agassizii and
Mycoplasma testudineum (12), with a possible third Mycoplasma
bacteria identied by genomic sequencing from a desert
tortoise (13).
Mycoplasma produces a variety of metabolites that cause
dysfunction of the respiratory mucosal epithelial cells, and can
migrate to the lungs and air sacs, leading to lung lesions that can
result in pulmonary eusion (14). e bacteria cause a range of
clinical signs including nasal discharge, swollen eyelids, lethargy, and
a general failure to thrive, which can result in death. Tortoises have
no diaphragm; thus, they cannot cough to expel a buildup of mucous
in their lungs, which makes tortoises susceptible to respiratory
infections (15). Chronic infections of Mycoplasma bacteria can cause
lesions in the nasal cavities of tortoises (16), which can facilitate
emaciation because tortoises locate food by olfaction, and lesions
impair their ability to locate food (17). Mycoplasma infection was
considered so detrimental to a threatened population of desert
tortoises that Nevada Department of Wildlife instituted a euthanasia
protocol for seropositive tortoises as a means to prevent transmission
of the bacteria to naïve populations (18).
Danooxacin is a third-generation animal-specic
uoroquinolone antibiotic that has been used to treat Mycoplasma
infections in tortoises (19, 20). Compared with other antibacterial
drugs, danooxacin has stronger cell permeability, higher drug
concentration in plasma and tissues, and stronger antibacterial
activity that can exhibit an antibacterial eect even when the drug
concentration is low (21). Danooxacin is widely used in the
treatment of respiratory diseases caused by Mycoplasma,
Actinobacillus, and Glaesserella parasuis (22–24). erefore, it is
expected to have wide applications for controlling respiratory tract
disease caused by Mycoplasma spp. (25).
Consequently, as part of a larger study investigating the ecacy of
translocation for Texas tortoises, weexamined the role of Mycoplasma
agassizii in the translocation of Texas tortoises. Specically, our
objectives were to: (1) determine Mycoplasma agassizii prevalence in
Texas tortoises from a donor site; (2) determine Mycoplasma agassizii
prevalence in Texas tortoises at a recipient site; (3) compare
prevalences between donor and recipient sites; (4) treat Mycoplasma
agassizii-infected tortoises with danooxacin; and (5) develop
alternate Mycoplasma agassizii treatments for Texas tortoises.
2 Materials and methods
2.1 Study donor site
Our donor site was a 270-ha property located approximately 8 km
west of Port Isabel, in Cameron County (26 1′38″ N, 97 14′53″ W),
Texas, USA. e property is bordered to the north by Highway 48,
which contains a 1-m tall impenetrable, cement barrier in the center of

Moeller et al. 10.3389/fvets.2024.1525179
Frontiers in Veterinary Science 03 frontiersin.org
the highway, to the south by the Brownsville Ship Channel, to the west
by a ooded drainage canal, and to the east by a perennial wetland.
Hence, the donor site was essentially an island; thus, immigration and
emigration was not possible. Eight habitat types, which included open
water, wetland/riparian, coastal ats, shrubland, woodland, grassland,
grassland loma, and evergreen loma, were identied on the property.
Common plants found were Gulf cordgrass (Spartina spartinae),
seacoast bluestem (Schizachyrium littorale), honey mesquite (Neltuma
glandulosa), prickly pear cactus (Opuntia spp.), blackbrush (Acacia
rigidula), coastal live oak (Quercus virginiana), common hackberry
(Celtis occidentalis), Texan goatbush (Castela erecta ssp. texana), and
non-native guineagrass (Urochloa maxima).
2.2 Study recipient site
Our recipient site was located about 200 km to the north-
northwest of the donor site. e site was approximately 10 km south
of Kingsville (27 28′21″N, 97 52′58″W), Kleberg County, Texas,
USA. Both the donor and recipient sites were located in the Gulf Coast
and Marshes ecoregion (26). Common plants found on the 110-ha
recipient site included honey mesquite, huisache (Vachellia
farnesiana), prickly pear cactus, granjeno (Celtis ehrenbergiana),
guineagrass, and Kleberg bluestem (Dichanthium annalatum).
We built three, 2.4-ha enclosures to serve as isolation and so
release sites for translocated tortoises. Weerected a 90-cm tall silt
fence that had a 21 gauge, 2 × 2 cm wire back around the perimeter of
each enclosure. e bottom 30-cm of the silt fence was buried in the
soil and the plastic side faced inward to the enclosure so tortoises
could not dig underneath or become entangled in the wire mesh. To
prepare the recipient site to receive tortoises, wecleared brush and
overgrown vegetation via mechanical removal (i.e., chainsaw and
mowing), followed by prescribed re, to develop a grassland with
several mottes of honey mesquite trees scattered throughout each
enclosure (8). Downed trees, areas of taller grasses around tree bases,
and animal burrows were maintained as refugia for tortoises.
2.3 Duane M. Leach Research Aviary
e 700-m
2
pavilion-style facility included 40, 1.2 × 1.8 × 2.0 m
individual pens (Corners Unlimited®, Kalamazoo, Michigan 49001)
made of 2.0 × 2.0 cm wire mesh, metal roof, and a concrete slab oor.
e facility was open to the outside environment via the wire mesh walls,
but sunlight was diminished due to the roof. erefore, overhead lights
were placed on timers to simulate daylength. Photoperiod of southern
Texas uctuates between 11 and 14 h of daylength with December and
August having the shortest and longest days, respectively.
1
e walls of
each pen were lined with 22 mils vinyl-coated polyester tarp to eliminate
direct contact between tortoises. Alfalfa hay was used as bedding and
also could be eaten by tortoises. Tortoises were provided Mazuri®
tortoise low-starch, pelleted diet (Mazuri Exotic Animal Nutrition, St.
Louis, Missouri 63166) and water ad libitum. Wesubsidized water intake
1 https://weatherspark.com/y/7960/
Average-weather-in-Corpus-Christi-Texas-United-States-Year-Round
by providing diced cucumber and watermelon every 2 days. Each pen
was equipped with a 3-sided 40 (L) × 40 (W) × 20 (H) cm escape box
with a wooden top for added shelter, a Fluker® (Fluker Farms, Port
Allen, Louisiana 70767) 150-W ceramic heat emitter bulb, and a
Reptisun® (Zoo Med Laboratories, Inc., San Luis Obispo, California
93401) UVA UVB Reptile 23 W uorescent lamp. e heat lamps and
UV lights were provided ad libitum and tortoises were free to move
underneath or away from both devices as needed.
2.4 Tortoise collection and sampling
We conducted systematic searches, driving searches, detection
dog searches, and incidental encounters for Texas tortoises from
June – November 2022 at both the donor and recipient sites.
Systematic searches consisted of 3–7 human searchers who walked
from sunrise until 1,200 h and from 1,600 h until sunset, which
coincided with the known activity pattern of Texas tortoises (4, 27).
Driving searches used trucks and all-terrain vehicles (ATVs) along
dirt roads and animal paths searching for tortoises. A detection dog
(i.e., 12-year-old, female Labrador retriever) that was trained to locate
Texas tortoises, was used to nd tortoises at both sites. Lastly,
incidental encounters occurred when wefound a Texas tortoise while
traveling to and from search locations within each site.
Tortoises were sexed, marked with an individualized number by
ling grooves on the marginal scutes according to the methods of
Cagle (28), weighed to the nearest gram, and carapace and plastron
length, width, height, and circumference were measured (mm).
Tortoise sex was determined based on external morphology such as
length of the gular projection, plastral concavity, and the ratio of anal
notch to anal fork width (29, 30). Tortoises whose sex could not
be conrmed because of ambiguous characters were labeled as
“unknown.” Due to diculty in dierentiating between male and
female tortoises of small size based on shell morphology alone,
tortoises <130 mm carapace length were categorized collectively as
“juveniles” (31). Tortoise age was estimated from carapace length using
the regression equations of Hellgren etal. (31) and Kazmaier etal. (6).
Tortoises located on the donor site were transported to the
recipient site because the donor site was scheduled for immediate
development. Tortoise health was visually examined according to
methods of Berry and Christopher (32), especially noting clinical signs
suggestive of URTD (e.g., nasal exudates, conjunctivitis, swollen eyes,
labored/wheezy breathing), lesions suggestive of chronic URTD (e.g.,
nasal scarring and asymmetric nares), and lethargy (e.g., head and
limbs limp, little-to-no resistance to having its head extracted from its
shell, and lack of willingness to move when placed on ground);
werecorded whether each of these clinical signs were either present
or absent. Tortoises displaying signs of URTD were immediately
transported to the Duane M. Leach Research Aviary for isolation.
Approximately 1.0 mL of blood was collected from either the caudal
vein, brachial vein, or the subcarapacial venous sinus of each captured
tortoise and placed in tubes containing lithium heparin (33). Blood
samples were centrifuged, plasma collected, and frozen in vials at
−80°C. Aseptic nasal irrigation was performed by injecting 0.5–1.0 mL
of sterile saline in each naris, collecting the nasal discharge in a sterile
container, and adding 0.5 mL of an enrichment medium (SP4 Glucose
Broth, Remel, Lenexa, Kansas 66204), before freezing the solution at
−80°C. Lastly, sterile rayon swabs (Puritan Medical Products Company

Moeller et al. 10.3389/fvets.2024.1525179
Frontiers in Veterinary Science 04 frontiersin.org
LLC, Guilford, Maine, USA) were used to swab the caudal pharynx of
each tortoise. Swab tips were separated and placed into sterile cryovials
and frozen at −80°C. Frozen samples were shipped to the University of
Florida for enzyme-linked immunosorbent assay (ELISA) testing,
Mycoplasma agassizii culture, and polymerase chain reaction (PCR)
testing as per the methods of Brown etal. (34) and Waites etal. (35).
Mycoplasma species were determined based on a unique restriction
fragment-length polymorphism ngerprint of the PCR amplicon (33,
34). ELISA titers of <1:32, 1:32, and ≥ 1:64 were considered negative,
suspect, and positive, respectively (33). Culture and PCR results were
classied as positive or negative for the presence of Mycoplasma agassizii.
2.5 Danofloxacin treatment
Tortoises (18 from the donor site and 2 from the recipient site for
a total N= 20) that exhibited clinical signs consistent with URTD
(described previously) were taken to the Duane M. Leach Research
Aviary and placed in separate pens as previously described. Tortoises
began a regimen of subcutaneous injections of danooxacin (Advocin,
Zoetis, Parsippany, NJ 07054) at 6 mg/kg every 48 h for 30 days, which
is the documented treatment for chronic mycoplasmosis in Gopherus
spp. tortoises (20). Tortoise daily response to treatment and food
consumption was recorded.
2.6 Alternate Mycoplasma treatment
design
We used 30 Mycoplasma-clinical Texas tortoises and 10 seemingly
healthy tortoises in this study. We used the 20 original tortoises
identied with URTD symptoms from the danooxacin treatment and
allowed those tortoises a 20-day acclimation period to recover from
the danooxacin.
During the acclimation period, weassessed tortoise health within
our so-release enclosures at the recipient site and found 10 additional
translocated tortoises that were displaying clinical signs of URTD. e
10 additional tortoises displaying clinical signs of URTD did so within
45 days of translocation to the recipient site. In addition, wecollected
10 non-symptomatic tortoises to use as controls.
e 30 symptomatic and 10 non-symptomatic tortoises were
placed into separate and isolated pens within the Duane M. Leach
Research Aviary. e non-symptomatic tortoises were placed in pens
at the opposite end of the facility, and tortoise handlers used hand
sanitizer and walked through a boot wash containing 2.6% sodium
hypochlorite bleach solution to reduce the likelihood of contaminating
healthy tortoises with Mycoplasma. e bleach of the boot wash was
discarded and replaced daily.
We randomly divided the 30 symptomatic tortoises into 2 drug
treatment groups and a control group, each containing 10 tortoises.
Treatments were tulathromycin (Draxxin, Pzer, Inc., NewYork, NY
10001) and oxytetracycline (Oxytet, Pzer, Inc., NewYork, NY 10001).
Tulathromycin was given intramuscularly once/week for 7 weeks at a
dosage of 5 mg/kg body weight, and oxytetracycline was given
subcutaneously once/day for 14 days at a dosage of 8 mg/kg body weight.
Stress can cause the onset of Mycoplasma symptoms, and handling
of tortoises can bea stressor. erefore, because of the dierent handling
of tortoises due to the dierent drug prescriptions, wesplit each drug
treatment group into two subgroups of 5 tortoises each. One subgroup
of the tulathromycin group received the drug as recommended, and the
other subgroup received the drug but also was handled as if in the other
drug treatment group. For example, 5 tortoises received 1 tulathromycin
injection once/week for 7 weeks; whereas, another 5 tortoises received
the same tulathromycin injections in addition to being handled as if in
the oxytetracycline group and received saline injections instead of
oxytetracycline. In addition, two sets of control tortoises (i.e., 2 groups
of 5 tortoises each) were used. One control set contained 10 symptomatic
tortoises and the other set contained 10 non-symptomatic tortoises.
Control tortoises were randomly assigned to handling schedule
(Table1). is design allowed us to compare the eects of 2 drugs with
dierent handling schedules on a total of 40 tortoises (Table1).
Serology and pharyngeal swabs were obtained as previously
described within the Tortoise collection and sampling section prior to
the start of the study and at the end of the 50-day trial. Frozen samples
were shipped to the University of Florida for ELISA testing and PCR
testing, as previously described. Tortoise general health and food
consumption was recorded daily during the trial.
2.7 Data analysis
Chi-square analysis was used to compare frequencies of
Mycoplasma agassizii-exposed tortoises between sexes (i.e., males and
females) and sites (i.e., donor and recipient sites). Tests were
considered signicant at p≤ 0.05.
3 Results
3.1 Mycoplasma agassizii prevalence at
donor site
We collected 171 (72 M: 97F: 2 Juveniles) Texas tortoises from the
270-ha donor site (1 tortoise/1.6 ha density), of which 18 (10.5%;
8 M:10F) displayed symptoms of URTDs when rst encountered, and
56 (32.7%; 26 M:30F) had titers suggestive of past exposure. Forty-one
(73.2%), 14 (25.0%), and 1 (1.8%) Texas tortoises had titers that were
1:32, 1:64, and 1:128, respectively. e frequency of males and females
with a history of Mycoplasma agassizii exposure did not dier
(χ
2
= 0.17, df = 1, p = 0.68). Adult tortoises, exclusive of the 2 juveniles
(carapace length of 57 and 85 mm, respectively), had a mean carapace
of 158.8 ± 7.4 mm (range = 134–208 mm) and were estimated to
range from 6 to 19years old (i.e., young adults). Of the 18 original
tortoises that displayed clinical signs of URTD, 9, 5, and 4 displayed
no, suspect, and low positive (1:64) titers, respectively, for M. agassizi.
Of the additional 10 clinical tortoises 5, 3, 1, and 1 displayed no,
suspect, 1:64, and 1:128 titers, respectively, for M. agassizi. All
pharyngeal swabs and nasal irrigation samples were negative by
culture and PCR methods to detect Mycoplasma agassizii bacteria.
3.2 Mycoplasma agassizii prevalence at
recipient site
We collected 23 (16 M:7F) Texas tortoises from the 100-ha recipient
site (1 tortoise/4.8 ha density), of which 2 (8.7%; 1 M:1F) displayed

Moeller et al. 10.3389/fvets.2024.1525179
Frontiers in Veterinary Science 05 frontiersin.org
symptoms of URTDs when rst encountered, and 8 (34.8%; 5 M:3F)
had titers suggestive of past exposure. Five (62.5%), 2 (25.0%), and 1
(12.5%) Texas tortoises had titers that were 1:32, 1:64, and 1:128,
respectively. e frequency of males and females with a history of
Mycoplasma agassizii exposure did not dier (χ
2
= 0.01, df = 1, p = 0.94).
All tortoises collected from the recipient site were considered young
adults (mean carapace length = 169.7 ± 5.9; range = 142–197 mm);
estimated ages ranged from 8 to 18years old. Of the two original
tortoises that displayed clinical signs of URTD, 1 and 1 displayed no and
low positive (1:64) titers, respectively, for M. agassizi. All throat swabs
and nasal irrigation samples were negative by culture and PCR methods
to isolate active Mycoplasma agassizii bacteria.
3.3 Mycoplasma comparison between
donor and recipient sites
No dierence in past Mycoplasma agassizii exposure (χ
2
= 0.04,
df = 1, p = 0.85) occurred between the donor (32.7% of 171 tortoises)
and (34.8% of 23 tortoises) recipient sites. Both sites had tortoises that
displayed low titers (≤1:128) of past exposure to Mycoplasma agassizii,
with the majority of tortoises either negative (67 and 65% for the
donor and recipient sites, respectively) or suspect (24 and 22% for the
donor and recipient sites, respectively) of past exposure. Sex ratios
(χ
2
< 6.0, df = 3, p = 0.11) and age structures (χ
2
< 0.2, df = 2, p = 0.89)
were similar between the donor and recipient sites.
3.4 Danofloxacin treatment
Twenty Texas tortoises (18 from the donor site [10.5%] and 2 from
the recipient site [8.7%]) displayed clinical signs (i.e., nasal discharge)
consistent with URTD upon initial collection. ese tortoises were
placed into isolation at the Duane M. Leach Research Aviary where
the signs of rhinorrhea continued. Within 3 h of the initial dose of
danooxacin, 7 of the 20 tortoises (35%) became listless, limbs and
head became limp, eyelids were swollen and closed, and they had
excessive salivation. Aer 20 h from the initial dose, 4 additional
tortoises (i.e., an additional 20%) began displaying similar reactions
to danooxacin; however, the signs were not as severe as with the rst
TABLE1 Drug and handling schedule of 40 Texas tortoises (Gopherus berlandieri) to assess the eectiveness of tulathromycin (“Tula”) and
oxytetracycline (“Oxy”) as treatments for Mycoplasma-induced upper respiratory tract disease.
Mycoplasma study in Texas tortoises (5 tortoises in each group per treatment = 40 tortoises in total)
Group1 (1X/wk × 7 weeks) Group2 (1X/
day × 14 days)
Clinical sign (No trt) No clinical signs (No trt)
Day Group1A1,4 Group1B1,5 Group2A2,4 Group2B2,5 Group3A3,4 Group3B3,5 Group4A3,4 Group4B3, 5
0 Phlebotomy (Ab) and pharyngeal and nasal swabs (PCR)– all tortoises
0Tul a Tul a Oxy Oxy S S S S
1 S Oxy Oxy S S
2 S Oxy Oxy S S
3 S Oxy Oxy S S
4 S Oxy Oxy S S
5 S Oxy Oxy S S
6 S Oxy Oxy S S
7Tul a Tul a Oxy Oxy S S S S
8 S Oxy Oxy S S
9 S Oxy Oxy S S
10 S Oxy Oxy S S
11 S Oxy Oxy S S
12 S Oxy Oxy S S
13 S Oxy Oxy S S
14 Tul a Tu l a S S S S S
21 Tul a Tu l a S S S S S
28 Tul a Tu l a S S S S S
35 Tul a Tu l a S S S S S
42 Tul a Tu l a S S S S S
49 Phlebotomy (Ab) and glottal and nasal swabs (PCR)– all tortoises
1Group1 = Tulathromycin dosage, which was 5 mg/kg body weight given intramuscularly (IM) 1x/week for 7 weeks.
2Group2 = Oxytetracycline dosage, which was 8 mg/kg body weight given subcutaneously (SC) 1x/day for 14 days.
3Group 3 and 4 = No drug given.
4Groups A were handled as directed for the designated drug. Control tortoises in Group A were handled for the tulathromycin prescription.
5Group B were provided the designated drug AND handled as if in both drug treatments but given saline injections (“S”) in place of other drug.

Moeller et al. 10.3389/fvets.2024.1525179
Frontiers in Veterinary Science 06 frontiersin.org
seven tortoises. Aer 24 h following the initial dose, the reaction to
danooxacin by the 11 reactive tortoises had not subsided. ose
tortoises were placed into a shallow tub (i.e., 40 × 40 × 2 cm)
containing water to aid against dehydration due to the excessive
salivation. e remaining 9 tortoises (45%) did not appear to have the
severe reaction to danooxacin; however, all tortoises stopped eating
aer the initial dose. By 48 h aer the initial dose, reaction to
danooxacin had not diminished, so it was decided to terminate the
danooxacin treatment rather than continue with the second dose. By
72 h aer the initial dose, excessive salivation began to clear and the
tortoises’ eyelids were less swollen. Also, tortoise heads and limbs
retracted back into their shells when stimulated. However, all
treatment group tortoises appeared unable or unwilling to move. By
the h day aer the single dose of danooxacin, tortoises began
moving within their pens, and 16 of 20 began to eat their pelleted diet.
Mortality caused by the single danooxacin dose was not observed.
3.5 Alternate Mycoplasma treatment
At the beginning of the trial, morbidity was apparent within the 30
tortoises in the symptomatic groups because all tortoises were displaying
rhinorrhea and raspy breathing, while the 10 tortoises within the
non-symptomatic group continued to appear healthy. However, by the
end of the drug trial, 22 tortoises were displaying signs of morbidity that
were consistent with URTD, of which 8, 4, 6, and 4 tortoises were in the
tulathromycin, oxytetracycline, symptomatic control, and
non-symptomatic control groups, respectively (Table2). Twelve of the
previously symptomatic tortoises (40%) stopped displaying clinical signs
during the drug trial; whereas, 4 (40%) of the non-symptomatic tortoises
began displaying signs. One mortality occurred in a tortoise within the
oxytetracycline treatment group that was additionally handled as a
tulathromycin tortoise (Table2).
By the end of the 50-day trial, titers increased (11/20, 55%) or
remained stable (8/20, 40%) for each drug treatment, with the exception
of one tortoise whose titer decreased (5%) from Suspect to Negative
aer treatment of oxytetracycline but experienced the handling of both
drugs (Table3). By contrast, 3, 2, and 5 of the symptomatic control
tortoises improved (30%), remained stable (20%), and had increasing
titers (50%), respectively, while 0, 4, and 6 of the non-symptomatic
control tortoises improved (0%), remained stable (40%), and had
increasing titers (60%), respectively, by the trial end (Table3).
No dierences were observed (χ
2
= 8.34, df = 6, p = 0.21) between the
frequencies of tortoises with decreasing, stable, or increasing titers within
the drug treatments and control groups. Nearly 50% of the χ
2
-value was
due to a greater-than-expected number of tortoises within the
symptomatic control group that experienced a decreasing titer from the
initial to the end-of-trial serology. Prior to the drug trial, 22 (55%), 16
(40%), and 2 (5%) tortoises exhibited negative, suspect, or positive titers,
respectively, which frequencies of tortoises with negative, suspect, and
positive titers did not dier (χ2 = 11.6, df = 6, p = 0.07) between treatment
groups. At the end of the trial, 9, 23, 6, and 1 tortoises exhibited negative
and suspect titers. e frequencies of tortoises with negative, suspect, and
positive titers did dier (χ
2
= 13.9, df = 6, p = 0.03) between treatment
groups. Nearly 50% of the χ2-value was due to fewer-than-expected within
the tulathromycin group and more than expected within the
oxytetracycline group having negative and positive titers, respectively.
Tortoise consumption of food varied by individual, but all
tortoises ate pelleted feed during the trial and produced normal-
appearing feces every 3 days, on average. Tortoises remained active
TABLE2 Number of Texas tortoises (Gopherus berlandieri), randomly assigned to a drug treatment group, that demonstrated various titers to past
Mycoplasma-exposure before and after the drug treatment.
Group A1Group B2
Titers3Negative Suspect Positive Negative Suspect Positive
Drug: Tulathromycin4
Initial 2 3 0 5 0 0
Trial end 0 5 0 0 5 0
Drug: Oxytetracycline5
Initial 0 3 2 3 2 0
Trial end 0 1 46371 0
Clinical sign control: Tulathromycin handling Oxytetracycline handling
Initial 2 3 0 2 3 0
Trial end 2 2 1 2 2 1
No clinical sign control
Initial 4 1 0 4 1 0
Trial end 2 2 1 1 3 1
1Group A were handled as directed for the designated drug. If a control tortoise, Group A were handled for the tulathromycin prescription.
2Group B were provided the designated drug AND handled as if in both drug treatments but given saline injections in place of other drug.
3Titers were considered Negative if < 1:32, Suspect if 1:32, and Positive if ≥ 1:64. Titers did not exceed 1:128.
4Tulathromycin dosage was 5 mg/kg body weight given intramuscularly (IM) 1x/week for 7 weeks.
5Oxytetracycline dosage was 8 mg/kg body weight given subcutaneously (SC) 1x/day for 14 days.
6One positive titer reached the 1:128 dilution level.
7One tortoise that was initially Negative died during oxytetracycline treatment.

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Frontiers in Veterinary Science 07 frontiersin.org
during the trial, and no tortoise experienced excessive salivation aer
any injection of drug or saline.
Mycoplasma agassizii bacteria was not cultured from the throat
swabs that were collected during the initial sampling or from the
50-day end-of-trial sampling. All Mycoplasma agassizii PCR results
were negative.
4 Discussion
Clinical mycoplasmosis appears to have recently emerged as a
problem in wild Texas tortoises in southern Texas. A wild population
(N = 39) of Texas tortoises sampled from southern Texas during 2004
were all seronegative for Mycoplasma agassizii exposure; whereas 80%
(12/15) of captive Texas tortoises housed at a rehabilitation facility in
southern Texas were seropositive (36). Although seropositive
tortoises were deemed non-releasable, the actual outcome history of
the seropositive tortoises was not documented by Tristan (36).
Guthrie etal. (37) documented that 11/40 (28%) and 3 additional
(8%) Texas tortoises sampled from southern coastal Texas were
antibody positive and suspect, respectively, for Mycoplasma agassizii
exposure. Weitzman etal. (38) documented that 5 of 56 (9%) Texas
tortoises from southern Texas were previously exposed to both
M. agassizii and M. testudineum, 13 (23%) tortoises were only
serapositive to M. testudineum, while the remaining tortoises (38 of
56, 68%) did not display previous exposure to Mycoplasma bacteria.
us, mycoplasmosis may bea fairly new occurrence (i.e., within a
decade) in Texas tortoises. Prevalence of past Mycoplasma agassizii
exposure, titer levels, and percent of tortoises displaying clinical signs
were consistent between our donor and recipient groups. erefore,
it does not appear that translocation of tortoises would cause the
spread of Mycoplasma agassizii to a naïve population. However, it is
prudent to assess prevalence and monitor current Mycoplasma
agassizii outbreaks before translocation occurs.
Mycoplasma testudineum also has been documented to cause
URTD symptoms. Due to nancial constraints, wedid not test for this
species. Unfortunately, the most likely explanation is that the
Mycoplasma symptomatic tortoises that tested negative for M. agassizii
were infected by M. testudineum. Weitzman etal. (38) documented
that Texas tortoises from southern Texas were exposed to both
M. agassizii and M. testudineum; thus, there is precedent for such
a situation.
We recognize that other causes, inclusive of viruses, colonic
obstructions, foreign bodies, and trauma to the carapace, may have
created the signs of URTD weobserved. However, gastric reux and
foreign bodies were ruled out because tortoises did not display
distended bowels, lacked green to brown saliva discharging from their
mouths and nares, and were able to pass normal-appearing stools (39).
Virus, such as herpes virus, ranavirus, adenovirus, reovirus, and
paramyxovirus, can cause similar signs as Mycoplasma infection;
however, tortoises oen die quickly due to such viruses (40), which
was not the case during our study. Coccidiosis caused by a protozoan
parasite also can cause similar URTD signs in tortoises (39), but our
tortoises displayed normal-appearing stools (i.e., no diarrhea);
therefore, coccidiosis was ruled unlikely. Tortoises were examined
upon capture and visual trauma to the carapace was not evident; thus,
physical injury to the carapace was not involved in URTD signs
displayed by tortoises during our study.
Even though we were unable to culture Mycoplasma agassizii
bacteria via PCR, mycoplasmosis was still considered the most likely
cause of our observed clinical signs. A negative PCR nasal ush does not
necessarily mean that a tortoise is Mycoplasma agassizii-free (39). Both,
M. agassizii and M. testudineum, grow slowly (2–8 weeks) at 30°C; thus,
a tortoise may exhibit clinical signs of infection before high burdens of
bacteria are present, limiting our ability to detect the presence of either
pathogen using molecular methods, despite infection (12).
Although danooxacin is oered as the current therapy to combat
Mycoplasma infection in Gopherus spp., it does not appear to
beappropriate for Texas tortoises. is may bebecause Texas tortoises
are the smallest of the North American tortoise species, weighing half
to one quarter, on average, of the gopher and desert tortoises,
respectively (41). Although the dosage for danooxacin is based on
mg of drug per kg of body weight of tortoise, and the fact that Texas
tortoises should have had a higher basal metabolic rate being a smaller
tortoise, the severe reaction to a single dose of danooxacin was
concerning. Typical treatment would constitute 15 injections given
every other day (19); however, tortoises required 3 days of recovery
from the rst dose for excessive salivation to cease. Because Texas
tortoises are a threatened species (42), wefeared excessive salivation
for 30+ days would cause Texas tortoises to become dehydrated and
TABLE3 Number of Texas tortoises (Gopherus berlandieri) that displayed rising titers to Mycoplasma (i.e., increased), or displayed stable or decreasing
titers to Mycoplasma by the end of the 50-day trial.
Tulathromycin1Oxytetracycline2Clinical sign (No trt)3No clinical sign (No trt)4
Serology5Group1A6Group1B7Group2A Group2B Group3A Group3B Group4A Group4B
Increased 2 5 3 1*3 2 2 4
Stable 3 0 2 3 0 2 3 1
Decreased 0 0 0 1 2 1 0 0
Tot a l 5 5 5 5 5 5 5 5
1Tulathromycin dosage was 5 mg/kg given intramuscularly (IM) 1x/week for 7 weeks.
2Oxytetracycline dosage was 8 mg/kg given subcutaneously (subQ) 1x/day for 14 days.
3Clinical sign (No trt) were tortoises that displayed runny noses but were given saline injections to simulate treatments of tulathromycin (Group A) and oxytetracycline (Group B).
4No clinical signs (No trt) were tortoises that appeared healthy but were given saline injections to simulate treatments of tulathromycin (Group A) and oxytetracycline (Group B).
5Blood was acquired from the jugular vein of tortoises. An ELISA test was conducted to determine titers to Mycoplasma. Titers that remained stable or decreased from beginning to trial end
were considered as potentially successful treatments.
6Group A tortoises were handled as directed for their designated treatment AND as directed for the tulathromycin prescription.
7Group B tortoises were handled as directed for their designated treatment AND as directed for the oxytetracycline prescription.
*Tortoise died on Day 38 of the 50-day drug trial.

Moeller et al. 10.3389/fvets.2024.1525179
Frontiers in Veterinary Science 08 frontiersin.org
die. Although wedid not continue the danooxacin treatment beyond
the rst dose; therefore, wecannot state with condence that tortoises
would have died if treatment continued. However, the apparent risk
appeared too great for a threatened species.
Our attempt to develop a dierent treatment for Mycoplasma
infection in Texas tortoises was unsuccessful. Oxytetracycline is a
broad-spectrum tetracycline antibiotic used to treat infections caused
by Mycoplasma organisms. It interferes with the ability of bacteria to
produce essential proteins, without which, the bacteria cannot grow
and multiply (43). Tulathromycin is a long-acting macrolide antibiotic
that binds to the 50S ribosomal subunit within the RNA, which
prevents bacteria from making vital proteins, and thus, keeps bacteria
from multiplying (44). Tulathromycin has demonstrated ecacy
against a diversity of respiratory pathogens in a variety of species,
including reptiles (45).
A single mortality occurred during our alternate treatment
method of oxytetracycline injections and additionally handled as
within the tulathromycin group. is tortoise displayed typical URTD
signs during treatment; however, did not appear to experience an
extreme adverse reaction as did the tortoises given danooxacin.
Although only speculative, perhaps the extra handling (i.e.,
oxytetracycline injections plus additional saline injections of
tulathromycin handling schedule) overtly stressed the tortoise,
causing it to succumb to its URTD infection.
Our treatment regimens did not cease the clinical signs associated
with URTD. However, we cannot fully state that treatment was
ineective because wewere not able to culture sucient bacteria to
receive positive PCR results from our initial sampling. Initial positive
PCR results followed by negative results aer treatment would
beindicative of an eective treatment. Interestingly, tortoises may not
always shed bacteria, yet still display clinical signs, and may
besubclinical yet beseropositive (45). Hence, weobtained potentially
conicting results (e.g., some tortoises displayed clinical signs and had
rising titers for Mycoplasma, yet their PCR tests were negative). It is
worth noting that tortoises within our study displayed very low titers
(i.e., 1:32 [suspect] to 1:128), so cross-reactivity between the two
species of Mycoplasma bacteria (i.e., M. agassizi and M. testudineum)
could be possible to explain rising, but low, titers, yet samples
benegative for PCR.
Some Texas tortoises displayed clinical signs of Mycoplasma
infection aer translocation, but the number of tortoises that did was
lower than the number of tortoises found in the wild at the donor site
with clinical signs of Mycoplasma infection. More studies are necessary
to understand the reason for this. Possible explanations include the
potential for a delayed response from additional translocated tortoises
to develop symptoms, stressors at the site that would increase the
frequency of symptomatic tortoises, or that translocation of tortoises
is not any more likely to cause development of symptoms than the
background symptom rate. Tortoises rarely clear Mycoplasma
infections, and clinical signs can intensify and abate cyclically (45).
us, the potential stressors of translocation and placement into an
enclosure, placement into an isolated pen, and frequent handling by
humans for treatment created situations that could have brought about
clinical signs in some, but not all, tortoises. Although
anthropomorphic, webelieve our study demonstrates that Texas
tortoises perceive and cope with various stressors dierently. Hence,
some seropositive tortoises remained subclinical throughout the
study, while others displayed clinical signs throughout each aspect of
the study.
In summary, Mycoplasma is an apparently common and
persistent issue facing Texas tortoises. Stressors, such as
translocation, can cause seropositive, but subclinical, tortoises to
display clinical signs of URTD, but that rate was lower than the
background rate observed in tortoises at the donor site. Webelieve
it prudent to test for both M. agassizii and M. testudineum in Texas
tortoises prior to translocation at both the donor and recipient sites.
In addition, webelieve it judicious to monitor the health of Texas
tortoises aer translocation to determine possible stress-related
URTD eects. Wecaution against the use of antibiotics to combat
mycoplasma infection in Texas tortoises, unless a strict monitoring
plan is in place to oset potential side eects. Instead, weadvocate
that Mycoplasma clinical signs appear cyclic and can abate without
treatment once the stressor subsides or the tortoise suciently copes
with stress.
Data availability statement
e raw data supporting the conclusions of this article will
bemade available by the authors, without undue reservation.
Ethics statement
e animal study was approved by the Texas A&M University–
Kingsville IACUC #Henke-S-2021-03-08. e study was conducted in
accordance with the local legislation and institutional requirements.
Author contributions
CM: Data curation, Investigation, Methodology, Supervision,
Writing– original dra. SP: Methodology, Writing– original dra.
WR: Methodology, Writing– original dra. AM: Writing– review
& editing. CE: Conceptualization, Funding acquisition,
Methodology, Resources, Writing– original dra, Writing– review
& editing. SR-H: Methodology, Validation, Writing – review &
editing. PC: Conceptualization, Funding acquisition, Writing –
review & editing. CH: Writing – review & editing. SH:
Conceptualization, Data curation, Formal analysis, Funding
acquisition, Investigation, Methodology, Project administration,
Resources, Supervision, Visualization, Writing – original dra,
Writing– review & editing.
Funding
e author(s) declare nancial support was received for the
research, authorship, and/or publication of this article. Funding was
received from the Welder Wildlife Foundation and Next Decade. is
is manuscript contribution no. 744 of the Welder Wildlife Foundation
and manuscript no. 24-121 of the Caesar Kleberg Wildlife Research
Institute. is work was conducted under a TPWD Permit for
Scientic Research (SPR-0620-085).

Moeller et al. 10.3389/fvets.2024.1525179
Frontiers in Veterinary Science 09 frontiersin.org
Acknowledgments
We thank the Welder Wildlife Foundation and Next Decade for
nancial support of the project, and Rio Grande LNG and Texas A&M
University–Kingsville for property access.
Conflict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
e author(s) declared that they were an editorial board member
of Frontiers, at the time of submission. is had no impact on the peer
review process and the nal decision.
Generative AI statement
e authors declare that no Gen AI was used in the creation of
this manuscript.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their aliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may beevaluated in this article, or claim
that may bemade by its manufacturer, is not guaranteed or endorsed
by the publisher.
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