ArticlePDF AvailableLiterature Review

Mycoplasmosis and upper respiratory tract disease of tortoises: A review and update

Authors:

Abstract

Tortoise mycoplasmosis is one of the most extensively characterized infectious diseases of chelonians. A 1989 outbreak of upper respiratory tract disease (URTD) in free-ranging Agassiz's desert tortoises (Gopherus agassizii) brought together an investigative team of researchers, diagnosticians, pathologists, immunologists and clinicians from multiple institutions and agencies. Electron microscopic studies of affected tortoises revealed a microorganism in close association with the nasal mucosa that subsequently was identified as a new species, Mycoplasma agassizii. Over the next 24 years, a second causative agent, Mycoplasma testudineum, was discovered, the geographic distribution and host range of tortoise mycoplasmosis was expanded, diagnostic tests were developed and refined for antibody and pathogen detection, transmission studies confirmed the pathogenicity of the original M. agassizii isolate, clinical (and subclinical) disease and laboratory abnormalities were characterized, many extrinsic and predisposing factors were found to play a role in morbidity and mortality associated with mycoplasmal infection, and social behavior was implicated in disease transmission. The translation of scientific research into management decisions has sometimes led to undesirable outcomes, such as euthanasia of clinically healthy tortoises. In this article, we review and assess current research on tortoise mycoplasmosis, arguably the most important chronic infectious disease of wild and captive North American and European tortoises, and update the implications for management and conservation of tortoises in the wild.
Review
Mycoplasmosis and upper respiratory tract disease of tortoises:
A review and update
Elliott R. Jacobson a,*, Mary B. Brown b, Lori D. Wendland b, Daniel R. Brown b,
Paul A. Klein c, Mary M. Christopher d,Kristin H. Berry e
aDepartment of Small Animal Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA
bDepartment of Infectious Disease and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA
cDepartment of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32610, USA
dDepartment of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616, USA
eUS Geological Survey, Western Ecological Research Center, Riverside, CA 92518, USA
ARTICLE INFO
Article history:
Accepted 30 May 2014
Keywords:
Tortoise
Mycoplasmosis
Mycoplasma agassizii
Mycoplasma testudineum
Pathology
Serology
PCR
ABSTRACT
Tortoise mycoplasmosis is one of the most extensively characterized infectious diseases of chelonians.
A 1989 outbreak of upper respiratory tract disease (URTD) in free-ranging Agassiz’s desert tortoises (Gopherus
agassizii) brought together an investigative team of researchers, diagnosticians, pathologists, immunolo-
gists and clinicians from multiple institutions and agencies. Electron microscopic studies of affected tor-
toises revealed a microorganism in close association with the nasal mucosa that subsequently was identified
as a new species, Mycoplasma agassizii. Over the next 24 years, a second causative agent, Mycoplasma
testudineum, was discovered, the geographic distribution and host range of tortoise mycoplasmosis were
expanded, diagnostic tests were developed and refined for antibody and pathogen detection, transmis-
sion studies confirmed the pathogenicity of the original M. agassizii isolate, clinical (and subclinical) disease
and laboratory abnormalities were characterized, many extrinsic and predisposing factors were found
to play a role in morbidity and mortality associated with mycoplasmal infection, and social behavior was
implicated in disease transmission.
The translation of scientific research into management decisions has sometimes led to undesirable
outcomes, such as euthanasia of clinically healthy tortoises. In this article, we review and assess current
research on tortoise mycoplasmosis, arguably the most important chronic infectious disease of wild and
captive North American and European tortoises, and update the implications for management and con-
servation of tortoises in the wild.
© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-
ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Introduction
Respiratory infection of tortoises was first reported in Califor-
nia, USA, in the 1970s in confiscated Agassiz’s desert tortoises
(Gopherus agassizii) with nasal exudates (Fowler, 1980a), and in the
UK in the 1980s in captive Greek (Testudo graeca) and Hermann’s
(Testudo hermanni) tortoises with rhinitis (Lawrence and Needham,
1985). Viruses (Jackson and Needham, 1983), Mycoplasma spp.
(Fowler, 1980b;Lawrence and Needham, 1985) and Pasteurella
testudinis (Snipes and Biberstein, 1982;Snipes et al., 1995)werehy-
pothesized as possible causes.
In the 1980s, major declines in desert tortoise populations in the
Mojave Desert of California, USA (Berry and Medica, 1995), and an
associated upper respiratory tract disease (URTD; Jacobson et al.,
1991), led to desert tortoises in the Mojave Desert north and west
of the Colorado River being declared threatened (US Fish and Wildlife
Service, 1990). A similar disease was seen in both captive (Beyer,
1993) and wild (McLaughlin, 1990;Beyer, 1993) gopher tortoises
(Gopherus polyphemus) in Florida, USA. A microbial and patholog-
ical study (Jacobson et al., 1991) resulted in the identification of a
new mycoplasma, Mycoplasma agassizii (Brown et al., 1995) and the
confirmation of its causal relationship with URTD in desert (Brown
et al., 1994) and gopher tortoises (Brown et al., 1999b).
Tortoise mycoplasmosis has since become one of the most ex-
tensively characterized infectious diseases of chelonians. Seminal
research studies include: (1) a description of the anatomy and his-
tology of the upper respiratory tract of healthy and affected tor-
toises (Jacobson et al., 1991); (2) identification and characterization
of two new Mycoplasma spp. (Brown et al., 1995, 2001, 2004); (3)
fulfillment of Koch’s postulates, establishing that M. agassizii is a caus-
ative agent of URTD (Brown et al., 1994, 1999b); (4) development
(Schumacher et al., 1993) and refinement (Wendland et al., 2007)
* Corresponding author. Tel.: +1 352 3391691.
E-mail address: jacobsone@ufl.edu (E. Jacobson).
http://dx.doi.org/10.1016/j.tvjl.2014.05.039
1090-0233/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
3.0/).
The Veterinary Journal 201 (2014) 257–264
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of an ELISA to determine exposure of tortoises to M. agassizii (Brown
et al., 1999a and b); (5) development of a conventional PCR (Brown
et al., 1995, 2004) and a quantitative PCR (qPCR; DuPré et al., 2011)
to detect M. agassizii and Mycoplasma testudineum DNA; and (6) cor-
relation of specific antibodies against M. agassizii and M. testudineum
with upper respiratory tract lesions in infected tortoises (Homer et al.,
1998;McLaughlin et al., 2000;Jacobson and Berry, 2012).
In this article, we review these (and other) key studies and assess
new research to update the current state of knowledge on myco-
plasmal URTD in tortoises and its implications for management and
conservation of tortoises in the wild.
Species of Mycoplasma in tortoises
Two mycoplasmas have been isolated from desert and gopher
tortoises, and characterized: M. agassizii, originally isolated from a
desert tortoise with URTD (2001), and M. testudineum, a genetical-
ly distinct organism (Brown et al., 2004). Both organisms cause
similar lesions in the nasal cavities of tortoises, with those caused
by M. testudineum possibly being less severe (Jacobson and Berry,
2012). A third mycoplasma, Mycoplasma testudinis, was isolated from
the cloaca of a healthy pet Greek tortoise in England (Hill, 1985)
and has not been associated with URTD. Recently, a novel, unnamed,
Mycoplasma sp. was identified by genomic sequencing in a sample
obtained from the phallus of a wild desert tortoise (Wellehan et al.,
2014).
Hosts and geographic distribution of mycoplasmas of tortoises
Evidence of infection with M. agassizii in many species of wild
and captive tortoises across the world has been determined using
serology, PCR and/or culture. Most information for wild tortoises
pertains to gopher tortoises (Beyer, 1993;Berish et al., 2000, 2010;
McLaughlin et al., 2000;Wendland, 2007) in South-Eastern USA, both
Agassiz’s (Jacobson et al., 1991, 1995;Lederle et al., 1997;Christopher
et al., 2003;Dickinson et al., 2005;Johnson et al., 2006) and Morafka’s
(Gopherus morafkai, formerly G. agassizii;Dickinson et al., 2005;Jones,
2008;Murphy et al., 2011) desert tortoises in South-Western USA,
and the Texas tortoise (Gopherus berlanderi;Guthrie et al., 2013)in
Texas, USA.
In Europe, mycoplasmas have been identified in wild spur-
thighed tortoises (Testudo graeca graeca) in Morocco, wild Her-
mann’s tortoises in France (Mathes et al., 2001;Mathes, 2003),
captive Hermann’s and spur-thighed tortoises in France (Mathes et al.,
2001;Mathes, 2003), wild spur-thighed, Hermann’s and margin-
ated (Testudo marginata) tortoises in Italy (Lecis et al., 2011), captive
spur-thighed and Russian (Testudo, formerly Agrionemys,horsfieldii)
tortoises in Spain (Salinas et al., 2011), and captive spur-thighed,
Hermann’s, Russian and leopard (Stigmochelys, formerly Geochelone,
pardalis) tortoises in the UK (McArthur et al., 2002;Soares et al.,
2004). Mycoplasmas have also been identified in many captive non-
native pet tortoises in the USA (Brown et al., 2002;Wendland et al.,
2006).
M. testudineum was originally isolated from the nasal cavity of
a clinically ill desert tortoise from the Mojave Desert, USA (Brown
et al., 2004). This organism was subsequently identified in three wild
gopher tortoise populations in North-Eastern Florida (Wendland,
2007).
Mycoplasma spp. also have been identified in other chelonians,
including free-ranging Eastern box turtles (Terrapene carolina caro-
lina) with URTD in Virginia, USA (Feldman et al., 2006) and a captive
ornate box turtle (Terrapene ornata ornata) in Hungary (Farkas and
Gál, 2009). M. agassizii has also been identified by PCR in the lungs
of red-eared sliders (Trachemys scripta elegans) with pneumonia from
Louisiana, USA (J. Roberts and E. Jacobson, unpublished data).
Clinical disease and pathology
Clinical vs. subclinical infection
Clinical signs of mycoplasmosis in tortoises include palpebral
edema, conjunctivitis, and nasal and ocular discharges (Jacobson
et al., 1991;McLaughlin et al., 2000;Mathes, 2003;Jacobson and
Berry, 2012). However, subclinical infection with Mycoplasma spp.
also occurs (Jacobson et al., 1995). Cycles of convalescence and re-
crudescence of clinical signs have been observed in captive and free-
ranging desert and gopher tortoises (Brown et al., 1999a and b).
Histopathology
Mycoplasmosis in tortoises is typically seen as an URTD, pri-
marily affecting the nasal cavity (Jacobson et al., 1991, 1995). Pneu-
monia is occasionally seen. Histologically, normal nasal cavities of
tortoises consist of a ventral, mucous and ciliated, epithelial mucosa,
and a dorsal, multilayered, olfactory epithelium. In tortoises with
mycoplasmosis due to M. agassizii, lesions in the nasal cavity may
be focal to diffuse, minimal to severe and may include basal cell hy-
perplasia in the mucosa, infiltrates of heterophils and histiocytes,
and lymphoid hyperplasia in the submucosa. Depending on the ep-
ithelial changes and the extent of the inflammatory response, the
following categories have been used to classify lesions: (1) mild in-
flammation; (2) moderate inflammation, and (3) severe inflamma-
tion (Jacobson et al., 1995).
In a group of desert tortoises that were serologically positive for
M. testudineum, lesions in the nasal cavities were less diffuse and
severe than in desert tortoises infected with M. agassizii (Jacobson
and Berry, 2012). This could indicate that M. testudineum is less
pathogenic than M. agassizii, or that the desert tortoises were more
recently infected.
Serology
Serological assays
An ELISA was developed to detect antibodies against M. agassizii
in plasma and serum using a monoclonal antibody (MAb HL673)
against the light chain of desert tortoise immunoglobulins IgY and
IgM (Schumacher et al., 1993). The antigen used in the ELISA was
derived from M. agassizii PS6, the type strain from a desert tor-
toise with URTD. The reactivity of MAb HL673 was validated by
Western blot analysis (Schumacher et al., 1993) and reference
polyclonal T. horsfieldii IgY and IgM antisera were obtained from H.
Ambrosius, Leipzig, Germany (Ambrosius, 1976). The Mab HL673-
based ELISA was further validated using experimental transmis-
sion studies in desert (Brown et al., 1994) and gopher tortoises
(Brown et al., 1999b). In these studies, reference standards that were
independent of the mycoplasmal diagnostic tests were presence of
clinical signs and histological lesions (Schumacher et al., 1997;Brown
et al., 2002).
An ELISA was also developed to determine exposure of gopher
and desert tortoises to M. testudineum using M. testudineum CB57
as the antigen (Jacobson and Berry, 2012). In studies with >1000
tortoises (M. Brown, unpublished data), relatively few serum samples
reacted with both M. agassizii and M. testudineum, and those samples
that reacted with both Mycoplasma spp. were from tortoises in popu-
lations with documented presence of both pathogens. As new species
of mycoplasmas are isolated from tortoises, validation and stan-
dardization of serological assays will be required.
Whereas the original ELISA results were reported as an enzyme
immunoassay (EIA) ratio (Schumacher et al., 1993), the reporting
system was eventually converted to end-point titers (Wendland et al.,
2007). Results for ~6000 independent desert and gopher tortoises
258 E.R. Jacobson et al./The Veterinary Journal 201 (2014) 257–264
were used to develop a distribution curve of absorbance values. A
subset of samples (n=90) that were randomly distributed over the
spectrum of absorbance values was then used to determine end-
point titers and construct a standard curve. Test refinements sub-
stantially improved assay performance (sensitivity 0.98, specificity
0.99, J=0.98), and test reliability. The authors considered this to be
a clinically more meaningful and reliable diagnostic test than the
original test based on EIA values.
Natural antibodies and interpretation of serological results
Natural antibodies are a function of innate immunity and can
react with epitopes on multiple unrelated antigens of potentially
pathogenic microbes (Marchalonis et al., 2002). The antigen binding
specificities of some natural antibodies have been characterized
(Grönwall et al., 2012). Hunter et al. (2008) reported that desert tor-
toises have natural antibodies (predominantly IgM) that could con-
found ELISA results for anti-mycoplasmal antibodies. However,
natural antibodies are generally irrelevant in immunological tests,
since sera are usually diluted sufficiently to avoid interference from
so-called ‘nonspecific background’ (Ochsenbein and Zinkernagel,
2000).
Hunter et al. (2008) used Western blot analysis in an attempt
to distinguish between natural and acquired anti-M. agassizii anti-
bodies, and concluded that banding patterns obtained using a single
strain of M. agassizii could distinguish between uninfected tor-
toises with natural antibodies and exposed tortoises with ac-
quired antibodies. However, most mycoplasmas, even within an
individual animal with a defined isolate, exhibit extensive intras-
pecies genotypic and phenotypic variability that is manifested as
antigenic variation (Simmons and Dybvig, 2007). The ability to vary
their antigenic patterns not only allows mycoplasmas to evade
immune surveillance, but also to confound analysis of mycoplas-
mal immunogen recognition when only a single isolate is used as
the source of antigen, especially on Western blot analysis
(Kittelberger et al., 2006). The need for multiple strains in Western
blot analysis, but not in ELISA, are consistent with findings for other
mycoplasmal species (Tola et al., 1996;Kittelberger et al., 2006).
Using sera from culture positive gopher tortoises with URTD con-
firmed by histopathological examination, Wendland et al. (2010a)
demonstrated that mycoplasmal strain variation was responsible
for the differences in observed Western blot binding patterns. Several
URTD-positive gopher tortoises had binding patterns similar to those
reported by Hunter et al. (2008) for plasma samples from URTD-
negative desert tortoises. Western blot analysis using a single antigen
(PS6) failed to detect gopher tortoises known to have URTD in ap-
proximately 25% of cases, whereas an ELISA using the same strain
as an antigen reliably detected all infected tortoises (Wendland et al.,
2010a).
Maternal antibodies and antibody persistence
Female desert tortoises transfer Mycoplasma-specific antibod-
ies (IgG class) to their offspring through the egg and these anti-
bodies are still detectable at 1 year of age (Schumacher et al., 1999).
Antibody titers were substantially lower in offspring than in paired
maternal serum, but higher in the offspring of sick female tor-
toises than healthy female tortoises. Importantly, hatchlings from
Mycoplasma antibody positive tortoises were not infected with My-
coplasma spp. (Schumacher et al., 1999). Residual maternal anti-
body potentially can confound interpretation of ELISA tests and result
in misdiagnosis of M. agassizii infection in juveniles if ELISA tests
are not appropriately validated. The current M. agassizii ELISA has
eliminated this as a problem by appropriate dilution of sera
(Wendland et al., 2007). Similar to the long-term persistence of ma-
ternal antibodies in desert tortoises, Sandmeier et al. (2012) found
that acquired, experimentally induced, anti-ovalbumin antibody titers
in desert tortoises could persist for over a year.
Pathogen detection
Culture
M. agassizii and M. testudineum are fastidious organisms that grow
slowly (2–6 weeks) at 30 °C in SP4 broth or agar (Brown et al., 1995,
2004). The organism ferments glucose under aerobic conditions, re-
sulting in an acid pH shift in the medium. The most common sample
cultured is nasal lavage fluid, obtained using 0.5–5.0 mL sterile
(±phosphate buffered) saline or 0.5–1.0 mL sterile SP4 broth. Since
bacteria and fungi are normal microbiota of the upper respiratory
tract of tortoises, penicillin, polymyxin B and amphotericin B are
usually added to SP4 medium to inhibit undesirable growth. To
further minimize microbial contamination, a portion of each sample
should be passed through a 0.45 μm filter prior to culture.
PCR
Testing using PCR has many advantages for the diagnosis of
mycoplasmosis in tortoises, including high specificity and rapid de-
tection. A positive PCR provides direct proof of the presence of my-
coplasmal genomic material at the time of sampling. Whereas the
PCR product can be used to identify the Mycoplasma sp. accurate-
ly, it does not provide information about the viability of the organ-
ism at the time of testing. Since primers used in both conventional
PCR and qPCR generally target specific gene sequences of a specif-
ic organism, they may not detect closely related species that differ
in genomic content; therefore, specificity should be assessed strin-
gently. Conventional PCR with restriction fragment length poly-
morphism (RFLP) analysis and full 16S sequencing remains an
important testing option, particularly in cases where a new species
may be involved. Important considerations for diagnostic tests tar-
geting the presence of the pathogen are that the microbial load may
be decreased when clinical signs are absent and that inadequate sam-
pling of the upper respiratory passages may lead to false negative
results. Finally, molecular techniques do not provide clinical iso-
lates for further testing, including determination of antimicrobial
sensitivity.
A conventional PCR was developed to detect tortoise mycoplas-
mas in culture medium and nasal lavage samples (Brown et al., 1995;
Wendland et al., 2007). If a band of the correct size was present,
then restriction enzyme digests using AgeI and NciI were used for
speciation. These two restriction enzymes give unique patterns for
mycoplasmal 16S rRNA. In the event that an aberrant pattern is ob-
served, complete sequencing of both strands of the 16S rRNA gene
using a minimum coverage of reads obtained from two primers is
recommended.
Using conventional PCR, 23/146 (15.8%) samples from captive
spur-thighed, Hermann’s, Russian and leopard tortoises in the UK
were positive for M. agassizii (Soares et al., 2004). Russian tor-
toises were more frequently infected than other species tested. In
this study, 8.2% of the samples tested were also positive for chelo-
nian herpesvirus (ChHV). Co-infection of M. agassizii and ChHV was
also found in Mediterranean and one Russian tortoise in Spain
(Salinas et al., 2011). These pathogens may work synergistically,
causing more severe clinical signs when present as co-infections,
or one agent may predispose tortoises to infection and disease as-
sociated with the other agent. Of oral swabs obtained from 30 free-
living spur-thighed, Hermann’s and marginated tortoises temporarily
housed in a wildlife center in Sardinia, Italy, 11 (36.7%) were my-
coplasma PCR positive, with the amplified sequences having close
similarity to M. agassizii (Lecis et al., 2011). Three PCR positive tor-
toises exhibited signs of respiratory disease.
259E.R. Jacobson et al./The Veterinary Journal 201 (2014) 257–264
A qPCR using primers specific for M. agassizii and M. testudineum
was developed to quantify mycoplasmas in samples and to corre-
late microbial burdens with clinical signs (DuPré et al., 2011).
M. agassizii DNA (6–72,962 pg/mL) was detected by PCR in 100% of
20 captive desert tortoises tested. When tested by Western blot anal-
ysis, only 16/20 (80%) qPCR positive tortoises were seropositive. This
was interpreted to mean that tortoises were colonized but not in-
fected (DuPré et al., 2011). However, this study did not include ELISA
results available for these tortoises, in which 19/20 (95%) tortoises
were seropositive. A false negative rate of 25% has been observed
when the Western blot analysis does not include the homologous
M. agassizii strain as a source of antigen (Wendland et al., 2010a).
Although not a diagnostic assay, a method for labeling and quan-
tifying viable M. agassizii using a membrane dye that is converted
to a fluorescent signal by viable cells and then quantified by flow
cytometry has been described (Mohammadpour et al., 2011). This
methodology can be used to determine the number of viable my-
coplasmal cells in a broth culture and may be applicable to exper-
imental studies and antimicrobial testing where the number of
microbes is of interest.
Transmission and host response
Experimental transmission
Experimental challenge studies in adult tortoises have demon-
strated that M. agassizii is a causative agent of URTD in desert and
gopher tortoises (Brown et al., 1994, 1999b). Experimentally chal-
lenged tortoises had lesions in the nasal cavity consistent with those
seen in naturally infected tortoises. Preliminary observations indi-
cated that seronegative hatchlings are at least as susceptible to in-
fection as adults and that the disease progresses more rapidly in
younger tortoises, with high morbidity in the first 6 weeks post-
infection (McLaughlin, 1997).
Under experimental conditions, the onset of clinical signs in
desert and gopher tortoises is as early as 2 weeks post-inoculation
(PI) with M. agassizii (Brown et al., 2002). Seroconversion lagged
behind clinical signs, with reliable detection of antibodies by 8 weeks
PI. In an experimental challenge study involving gopher tortoises,
the clinical sign scores of challenged tortoises (previously exposed
to M. agassizii) at 2 weeks PI were higher than those of naïve animals
(McLaughlin, 1997). ELISA values were also greater for challenged
than naïve tortoises at each time point PI. A significant increase
in serum ELISA values of challenged tortoises was observed at
4 weeks PI.
Natural transmission
Based on the behavioral inventory of the desert tortoise (Ruby
and Niblick, 1994), we believe that horizontal transmission by direct
contact (combat or courtship) is the most likely route of transmis-
sion of Mycoplasma spp. between tortoises. Whereas transmission
is more likely to occur when the infected tortoise exhibits clinical
signs, tortoises with subclinical infections may be able to transmit
Mycoplasma spp. under appropriate conditions (Jacobson et al., 1995).
Although aerosol transmission is possible, control gopher tor-
toises housed in pens adjacent to clinically affected tortoises did not
become clinically diseased or seroconvert, suggesting that M. agassizii
did not travel even relatively short distances over low (0.7 m) bar-
riers (McLaughlin, 1997).
In the study by McLaughlin (1997), there was no evidence to
support vertical transmission of M. agassizii in hatchlings derived
from female gopher tortoises that were seropositive for exposure
to M. agassizii. However, due to the small sample size, vertical trans-
mission cannot be ruled out (Brown et al., 2002). Since experimen-
tal transmission of Mycoplasma gallisepticum by fomites has been
reported in American house finches (Carpodacus mexicanus;Dhondt
et al., 2007), fomite transmission may be possible in tortoises, but
has not been demonstrated.
In a 4 year study of dynamics of URTD caused by M. agassizii in
wild populations of gopher tortoises, the force of infection (FOI; prob-
ability per year of a susceptible tortoise becoming infected) and the
effect of URTD on survival in free-ranging tortoise populations were
followed in 10 populations in central Florida (Ozgul et al., 2009).
Sites with high (25%) seroprevalence had higher FOI than sites with
low (<25%) seroprevalence. These findings provided the first quan-
titative evidence that the rate of transmission of M. agassizii is di-
rectly related to seroprevalence.
Age (size) differences in exposure to M. agassizii in gopher tor-
toises may affect the spread of URTD in wild populations. In one
study, adult gopher tortoises had a higher rate of exposure to
M. agassizii than subadults (Karlin, 2008). In a 5 year study of my-
coplasmal URTD, free-ranging adult gopher tortoises were 11 times
more likely to be seropositive than immature tortoises (Wendland
et al., 2010b), suggesting that direct or prolonged interaction between
immature and adult tortoises was minimal.
In a study conducted at the Kennedy Space Center, Florida, from
1995 to 2000, to monitor the impact of URTD on gopher tortoises,
there was an increase in the number of tortoises showing signs of
URTD and, starting in 1998, a sudden increase in numbers of dead
tortoises at the site (Seigel et al., 2003). Sex ratios and body sizes
of dead tortoises were not distinguishable from living tortoises, in-
dicating that mortality was not confined to a single sex or age class.
However, no results of postmortem examinations, histopathology
or PCR for detection of Mycoplasma spp. were reported for any of
the tortoises showing signs of illness. Therefore, a causal relation-
ship between mycoplasmosis and deaths of tortoises at this site was
not established. Gopher and other tortoises are also susceptible to
infection and death from other pathogens such as Ranavirus, which
can result in clinical signs that overlap with URTD (Johnson et al.,
2008).
McCoy et al. (2007) did not demonstrate any correlation between
the presence of anti-M. agassizii antibodies and a decrease in the
numbers of tortoises at several study sites. Furthermore, the pro-
portions of tortoises that were seropositive or had intermediate an-
tibody levels were positively correlated with the number of tortoises
tested at each site. This is in agreement with well-established epi-
demiological principles for determining the number of animals in
a population to sample in order to detect the presence of a disease.
Valid population sampling will depend on the population size, the
true prevalence of infection, and the sensitivity and specificity of
each test (Brown et al., 2002). Sampling inadequate numbers of
animals can lead to inaccurate conclusions regarding the status of
a disease in a population.
McCoy (2008) compared the mean size of male and female tor-
toises found dead (based on the presence of shells) with the mean
size of those found alive at a site (McCoy et al., 2007). The mean
size of males found dead did not differ from the mean size of live
males, but the mean size of dead females was lower than the mean
size of live females. Their findings differed from those of Seigel et al.
(2003), who did not detect differences in the mean sizes of dead
and live tortoises for either sex. The difference in these findings may
be related to differences in size distribution of live animals between
different populations (McCoy, 2008). Although, it was assumed that
URTD was the cause of death of tortoises at both sites, no post-
mortem findings were reported, so the possibility of other causes
of death, such as Ranavirus infection, could not be excluded.
Host response
In many mycoplasmal diseases, the host adaptive immune re-
sponse is dysregulated, often providing limited or no protection
260 E.R. Jacobson et al./The Veterinary Journal 201 (2014) 257–264
(Szczepanek and Silbart, 2014). In tortoises with mycoplasmosis,
pathological studies have revealed an over-exuberant host re-
sponse to Mycoplasma spp., resulting in dysplastic changes to the
nasal mucosa (Jacobson et al., 1991, 1995;McLaughlin et al., 2000).
The response of clinically healthy, seropositive, adult gopher tor-
toises in experimental challenge studies with M. agassizii was more
rapid and severe than in naïve tortoises, suggesting that previous
exposure to the organism may exacerbate disease (McLaughlin,
1997). In contrast, several clinically healthy desert tortoises, which
were culture positive and positive for serum antibodies by ELISA,
had normal nasal cavities (Jacobson et al., 1995). Thus, not all tor-
toises respond to M. agassizii with a severe inflammatory re-
sponse, suggesting that multiple strains of M. agassizii may exist with
variable pathogenicity or that different responses are related to dif-
ferent genotypes.
We suspect that mycoplasmosis in tortoises is characterized by
initial high mortality, followed by low mortality and high morbid-
ity. We have seen several infected tortoises survive in captivity for
many years, with clinical signs varying over time. Since tortoises
use olfaction for finding and selecting food, the histological changes
seen in the nasal cavities of tortoises with URTD suggest that their
ability to locate food and feed would be impaired. To determine the
impact of nasal discharge on a tortoise’s ability to locate food,
Germano et al. (2014) designed a study to determine the re-
sponses of Agassiz’s tortoises with or without nasal discharge, and
positive or negative for M. agassizii antibodies, to a visually hidden
olfactory food stimulus and an empty control. The presence of nasal
discharge was associated with a reduced ability to locate food. This
study also showed that moderate chronic nasal discharge in the
absence of other clinical signs did not affect appetite in desert tor-
toises. We have seen tortoises with experimentally induced URTD
and captive tortoises with URTD continue to feed even with a nasal
discharge.
Impact of mycoplasmosis on tortoises
Factors contributing to mycoplasmal disease in tortoises
Factors that appear to contribute to outbreaks of URTD include
environmental stress, human impacts, exposure to heavy metals and
other toxicants, and the escape or release of captive tortoises
(Jacobson et al., 1991;Brown et al., 2002;Sandmeier et al., 2009,
2013). In the Desert Tortoise Natural Area (DTNA), Kern County, Cal-
ifornia, where mycoplasmosis was first identified in wild desert tor-
toises, mercury (Hg) concentrations in the livers (0.326 parts per
million, ppm) of affected tortoises were significantly higher than
those of controls (0.0287 ppm) (Jacobson et al., 1991). Mercury can
have a variety of toxicological effects, including cellular, cardiovas-
cular, hematological, pulmonary, renal, immunological, neurolog-
ical, endocrine, reproductive and embryonic effects (Rice et al., 2014).
Altered levels of thyroid hormones have been detected in western
pond turtles (Emys marmorata) with elevated concentrations of Hg
(Meyer et al., 2014). Further work on the physiological impact of
Hg on desert tortoises is needed.
Environmental perturbations and annual fluctuations in tem-
perature, rainfall, and forage availability may result in activation of
a subclinical infection to a clinical level. Although drought is a natural
part of the desert tortoise’s environment (Henen et al., 1998), it can
contribute to morbidity and mortality if combined with disease or
habitat loss (Peterson, 1996). Clinical signs of URTD and heteropenia
were noted at the time of emergence of desert tortoises from hi-
bernation in years that followed periods of intense drought
(Christopher et al., 2003), suggesting that tortoises entering hiber-
nation in a drought year may be physiologically compromised.
Sandmeier et al. (2013) suggested that cold winters could enhance
conditions for the growth of M. agassizii. However, in a study in Las
Vegas Valley, although Mycoplasma spp. and other bacteria were iso-
lated during the warmer months, few aerobic bacteria and no My-
coplasma spp. were isolated from the nasal cavities of tortoises in
January, the coldest month of the year (Jacobson et al., 1995). In ad-
dition, since the optimal growing temperature of M. agassizii is 30 °C
(Brown et al., 1995), it is unlikely that colder temperatures would
enhance growth of the microbe. However, cold winters could have
an impact on the host immune system at the time of emergence
of tortoises from their burrows in the spring, making them more
susceptible to infection and lowering the infectious dose required
to establish infection.
Human impacts on tortoises and their habitats, whether through
disruption of normal behavior patterns, degradation of habitats
through agriculture, silviculture, mining, land development or pol-
lution, may cause sufficient physiological stress to trigger out-
breaks of mycoplasmal disease. Wild tortoises in remote areas of
the central Mojave Desert, distant from human beings and paved
roads, were significantly less likely to be seropositive for M. agassizii
than those in close proximity to human developments (Berry et al.,
2006). The capture, manipulation and transport of tortoises during
research projects, as well as relocation, restocking and repatria-
tion efforts, also may be sources of stress that result in overt disease
(Berry et al., 2002).
The escape or release of captive tortoises in urban and remote
areas may be a significant factor accounting for URTD in wild popu-
lations. Thousands of captive desert tortoises were released into wild
lands prior to their federal listing as a threatened species in 1990,
and releases have continued in recent years (Berry et al., 1986;Ginn,
1990;Jennings, 1991;Connor and Kaur, 2004;Field et al., 2007;
Murphy et al., 2007;Nussear et al., 2012). The outcome of a survey
of 179 captive desert tortoises around Barstow, California re-
vealed anti-mycoplasmal antibodies in 148 (82.7%) (Johnson et al.,
2006). A statistically significant positive association was found
between severity of clinical signs and serum antibody ELISA status.
Furthermore, adult desert tortoises were more likely to have a pos-
itive serum antibody ELISA result than sub-adults or young adults
of undetermined sex. These findings suggest that captive tortoises
can be a reservoir of infection for wild desert tortoises.
Similarly, Morafka’s desert tortoises (G. morafkai;Murphy et al.,
2011) from suburban Tucson, Arizona, were 2.3 times more likely
to test seropositive for antibodies against M. agassizii than tor-
toises from remote locations (Jones, 2008). In addition, captive tor-
toises were 1.8 times more likely to test seropositive for Mycoplasma
spp. than free-ranging tortoises, even in Arizona counties with high
visitor use. Epizootics of URTD occurred on Sanibel Island, Florida,
following the release of gopher tortoises collected in northern Florida
and southern Georgia for use in tortoise races (Dietlein and Smith,
1979;Beyer, 1993;McLaughlin et al., 2000).
Effects of mycoplasmosis on tortoise populations
The effects of mycoplasmosis on mortality, morbidity and the
long-term health and viability of tortoise populations are poorly un-
derstood. Mortality events could be due to an acute outbreak or the
end result of long term physiological stress combined with an ex-
acerbating extrinsic stressor. In the acute outbreak on Sanibel Island,
up to 50% of adult gopher tortoises at one site died with signs of
URTD (McLaughlin, 1990). A similar acute mortality event oc-
curred at the Desert Tortoise Natural Area, Kern County, California
(Jacobson et al., 1991). At this site, URTD evolved from an acute, epi-
zootic disease with high mortality to a chronic endemic disease with
variable morbidity, low mortality and a high seroconversion rate
for antibodies against M. agassizii (Brown et al., 1999a). In a study
of URTD in gopher tortoises, Ozgul et al. (2009) theorized that se-
ropositive tortoises were those that had survived initial infection
and developed chronic disease.
261E.R. Jacobson et al./The Veterinary Journal 201 (2014) 257–264
While mortality events are easily documented and attract con-
siderable attention, morbidity can be more subtle and difficult to
assess. Abnormal hormone profiles observed in desert tortoises with
mycoplasmosis (Rostal et al., 1996;Homer et al., 1998) could lead
to alterations in foraging and reproductive behavior and de-
creased reproductive potential. Chronic inflammation in nasal and
olfactory tissues of affected tortoises (Jacobson et al., 1991;Homer
et al., 1998) could disrupt olfactory function and affect foraging and
reproductive behavior. Soluble proteins in shell scutes also may be
affected by mycoplasmosis (Homer et al., 2001). Further monitor-
ing with follow-up pathological evaluation is needed to assess the
long-term consequences of mycoplasmosis on tortoise morbidity.
Management implications for wild tortoise populations
Reliance on ELISA results to support management decisions
Antibody ELISA testing has been used to manage gopher and
desert tortoises in parts of their range. Jacobson et al. (1995) dis-
cussed several scenarios for the disposition of seropositive desert
tortoises. Although these authors did not recommend euthanasia
of clinically healthy tortoises that were antibody ELISA positive for
M. agassizii, such a policy was adopted in the state of Nevada. This
policy was terminated in 2007, in part (R. Averill-Murray, person-
al communication) based on the following statement in Brown et al.
(2002):‘There are inadequate scientific data to provide definitive guide-
lines for the disposition of seropositive tortoises’.
Euthanasia of seropositive tortoises results in elimination of
animals that might otherwise provide valuable reproductive and
genetic contributions to wild populations and is not recom-
mended. However, relocation of seropositive tortoises could result
in spread of mycoplasmosis to susceptible animals, with detrimen-
tal impacts on recipient populations. Likewise, healthy tortoises that
have not been exposed to Mycoplasma spp. should not be relo-
cated to populations with extensive clinical disease or those un-
dergoing increased mortality events (Brown et al., 2002;Sandmeier
et al., 2009).
While antibody ELISA testing remains an important tool for
making management decisions, it is critical to first establish clear
goals for the tortoise population of interest. Importantly, antibody
ELISA testing should not be used as the sole means of evaluating
the health of an individual animal; rather, it is only one tool among
many for comprehensive health assessment (Brown et al., 2004;
McCoy et al., 2007;Sandmeier et al., 2009).
Modeling population dynamics
Understanding disease transmission dynamics in the context of
tortoise social behavior is an important consideration for the success
of future conservation programs. The finding that adult gopher tor-
toises were more likely to be seropositive than immature tor-
toises (Karlin, 2008;Wendland et al., 2010b) has broad implications
for disease modeling. During mortality events caused by patho-
gens having minimal environmental transmission, such as M. agassizii,
immature size classes may be spared, providing a pool of tor-
toises for later recruitment. However, a significant limitation of this
hypothesis is that immature size classes constitute a small propor-
tion of the overall population and usually are inadequate to sustain
a population. Managing habitat to increase the successful recruit-
ment of juvenile tortoises would be a valuable strategy in these cir-
cumstances. Alternatively, land managers could target smaller size
classes for augmentation or restocking of depleted populations,
thereby reducing the risk of pathogen introduction.
Population modeling techniques hold promise for understand-
ing the impact of mycoplasmosis on wild populations of infected
tortoises. Mycoplasmal URTD in free-ranging gopher tortoise popu-
lations was used as a model system for studying the effects of chronic
recurring disease epizootics on host population dynamics and per-
sistence (Perez-Heydrich et al., 2012). The findings indicate that the
impact of disease on host population dynamics appears to depend
primarily on how often a population experiences an epizootic, rather
than on how long the epizootic persists. Models such as this will
have more value once validated and tested.
Conclusions
Mycoplasmosis is a complex, multifactorial upper respiratory tract
disease, of captive and wild tortoises. M. agassizii and M. testudineum
are proven etiological agents of URTD. Extrinsic factors that most
likely contribute to outbreaks of URTD include environmental stress,
human impacts, exposure to heavy metals and other toxicants, and
the escape or release of captive ill tortoises. Because M. agassizii has
been isolated from multiple species of tortoises in North America
and Europe, all tortoises should be considered potentially suscep-
tible. Like most respiratory mycoplasmoses, URTD is a chronic and
often subclinical disease. Clinical signs may vary in onset, dura-
tion and severity. Both subclinically and clinically affected animals
have damage to the respiratory and olfactory epithelial surfaces,
which affects their ability to identify food. Direct contact (combat
or courtship) between tortoises is the most likely route of trans-
mission, and transmission rates are directly related to overt clini-
cal signs and seroprevalence. Several diagnostic tests are available
to determine the exposure (serology to determine antibodies) and
infection (direct culture and 16S rRNA PCR) status of individuals and
populations of tortoises. Specific antibodies against Mycoplasma spp.
do not appear to provide protective immunity, and the host’s in-
flammatory response may contribute to the severity of nasal lesions.
Translocation as a management tool should include the health status
of translocated tortoises and those at the recipient site, as well as
long-term monitoring of effects on translocated and recipient
populations.
Conflict of interest statement
None of the authors of this paper has a financial or personal re-
lationship with other people or organizations that could inappro-
priately influence or bias the content of the paper.
Acknowledgements
The work on mycoplasmosis of tortoises has been a multidisci-
plinary effort at the University of Florida and elsewhere, involving
faculty, graduate students, research scientists and laboratory tech-
nicians. The following present and former University of Florida per-
sonnel were involved in this project: I.M. Schumacher, B.L. Homer,
C. McKenna, G.S. McLaughlin, B.C. Crenshaw, L. Green, D. Duke, J.
Hutchison, A. Whitemarsh, M. Lao, D. Bunger, S.J. Tucker, N.T.
Chmielewski, N. Gottdenker and J.E. Berish. This work would not
have been possible without support from a number of agencies in-
cluding the US Bureau of Land Management, US Geological Survey,
the Nature Conservancy, Nevada Wildlife Department, the Nation-
al Training Center, Fort Irwin, the National Science Foundation, the
National Institutes of Health, the Walt Disney Company and mul-
tiple biologists and additional personnel who are dedicated to desert
and gopher tortoise conservation.
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264 E.R. Jacobson et al./The Veterinary Journal 201 (2014) 257–264
... URTD is characterized by rhinitis and oculonasal discharge; histologic lesions include necrosis, erosion, and hyperplasia of the nasal epithelium, mucous gland metaplasia, and mixed inflammation (Rodr´ıguez et al., 2018). Clearance of Mycoplasma infections appears uncommon, and tortoises frequently develop chronic infections characterized by recurrent episodes of URTD interspersed with periods of latency (Jacobson et al., 2014). Mycoplasmosis-associated mortal-ity events have also been documented, with up to a 50% mortality rate (McLaughlin, 1990;Jacobson et al., 2014). ...
... Clearance of Mycoplasma infections appears uncommon, and tortoises frequently develop chronic infections characterized by recurrent episodes of URTD interspersed with periods of latency (Jacobson et al., 2014). Mycoplasmosis-associated mortal-ity events have also been documented, with up to a 50% mortality rate (McLaughlin, 1990;Jacobson et al., 2014). ...
... The bacterium is thought to be a commensal organism that may opportunistically cause disease in bog turtles, and this may be true of other emydid turtles (Ossiboff et al., 2015). Recrudescence of clinical mycoplasmosis has been noted in several emydid and tortoise species (Jacobson et al., 2014;Palmer et al., 2016). Based on this information, Mycoplasma sp. may be a commensal organism in the Chelydridae, with the potential to cause URTD and chronic infection, similar to M. agassizii and M. testudineum Clinical signs of mycoplasmosis in tortoises and turtles include blepharedema, oculonasal discharge, conjunctivitis, anorexia, and lethargy. ...
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The alligator snapping turtle, Macrochelys temminckii, is the largest freshwater turtle species in North America and is classified as state-endangered in Illinois. Head start programs that include health assessments and pathogen detection are being conducted to restore this species to its historic range. Physical examinations and oral/cloacal swabs were collected from 97 head start alligator snapping turtles pre-release and 58 re-captured turtles post-release in 2014. Conventional PCR and sequencing targeting the RNA polymerase beta subunit gene and the 16S-23S ribosomal RNA intergenic spacer detected a single pre-release individual with Mycoplasma sp. with closest relation to Mycoplasma spp. of other freshwater turtles. This individual did not display any clinical signs at the time of release/sampling. Mycoplasmosis has been characterized as a disease of conservation concern in tortoises and may represent a threat to small and fragmented chelonian populations. Findings of this study indicate that Mycoplasma sp. DNA is present in an Illinois alligator snapping turtle and future investigation into the impact of this disease may support conservation efforts.
... Three years prior to translocation (2005)(2006)(2007), research teams fitted several hundred tortoises on the NTC with radio-transmitters (RI-2B, 14 g; Holohil, Carp, Ontario, Canada) and conducted periodic health examinations using established protocols . Briefly, we examined each tortoise for posture, behavior, and clinical signs of upper respiratory tract disease caused by Mycoplasma agassizii and M. testudineum (Jacobson et al. 2014) and oral lesions (Christopher et al. 2003, Jacobson et al. 2012. We drew blood samples for enzyme-linked immunoassays (ELISA) to test for antibodies for pathogens that cause and transmit upper respiratory tract disease (Jacobson et al. 2014. ...
... Briefly, we examined each tortoise for posture, behavior, and clinical signs of upper respiratory tract disease caused by Mycoplasma agassizii and M. testudineum (Jacobson et al. 2014) and oral lesions (Christopher et al. 2003, Jacobson et al. 2012. We drew blood samples for enzyme-linked immunoassays (ELISA) to test for antibodies for pathogens that cause and transmit upper respiratory tract disease (Jacobson et al. 2014. We selected 158 healthy adult tortoises from the several hundred tortoises available for translocation. ...
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Translocation of animals, especially for threatened and endangered species, is a currently popular but very challenging activity. We translocated 158 adult Agassiz's desert tortoises (Gopherus agassizii), a threatened species, from the National Training Center, Fort Irwin, in the central Mojave Desert in California, USA, to 4 plots as part of a long‐distance, hard‐release, mitigation‐driven translocation to prevent deaths from planned military maneuvers. We monitored demographic and behavioral variables of tortoises fitted with radio‐transmitters from 2008 to 2018. By the end of the project, 17.72% of tortoises were alive, 65.82% were dead, 15.19% were missing, and 1.27% were removed from the study because they returned to Fort Irwin. Mortality was high during the first 3 years: >50% of the released animals died, primarily from predation. Thereafter, mortality declined but remained high. After 10.5 years, survival was highest, 37.50% (15/40), on the plot closest to original home sites, whereas from 2.56% to 23.68% remained alive on the other 3 release plots. Surviving tortoises settled early, repeatedly using locations where they constructed burrows, compared with tortoises that died or disappeared. Models of behavioral and other variables indicated that numbers of repeatedly used locations (burrows) were a driver of survival throughout the study, although plot location, size and sex of tortoises, and distance traveled were contributors, especially during early years. Because >50% mortality occurred, we considered this translocation unsuccessful. The study area appeared to be an ecological sink with historical and current anthropogenic uses contributing to habitat degradation and a decline in both the resident and released tortoises. Our findings will benefit design and selection of future translocation areas. In 2008, we translocated 158 adult desert tortoises from a military base in a long‐distance, mitigation‐driven translocation in the central Mojave Desert of California, USA, into a low density, declining resident population. At the end of the 10.5‐year project, few tortoises remained alive, primarily because of predation. Plot location (closer to home site), size and sex of tortoise (large males), and repeatedly used burrows during temperature extremes contributed to survival.
... Some species are considered commensal bacteria of the host (Farkas andGal 2009, Di Ianni et al. 2015), whereas others can cause severe diseases of the upper respiratory tract, with chronic evolution and mortality. Mycoplasma testudinis has been reported as a non-pathogenic agent of the excretory tract of T. graeca, whereas Mycoplasma agassizii and Mycoplasma testudineum have been demonstrated to cause disease (upper respiratory tract disease) in both free-ranging and captive tortoises in the USA and Europe (Brown et al. 1995, Brown et al. 1999, Feldman et al. 2006, Lecis et al. 2011, and they are considered a threat for the management of wild tortoise populations (Jacobson et al. 2014). ...
... Detection of M. agassizii DNA in rescued tortoises only, raises concerns for relocation programs. Mycoplasmoses, especially those caused by M. agassizii and M. testudineum, are considered to have contributed to the decline of some wild populations of tortoises in the USA (Jacobson et al. 2014). In this perspective, the clear difference found in the prevalence of Mycoplasma spp. ...
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Testudo hermanni is included as near‑threatened in the Red List of the International Union for Conservation of Nature, while T. hermanni hermanni is considered endangered in the Italian Red List. Appropriate management of smuggled or seized wild individuals is recommended before their reintroduction into the wild. Accordingly, a health monitoring study was carried out. During 2014‑2016, 133 oral swabs and 121 cloacal swabs were collected from a total of approximately 180 free‑ranging and rescued T. hermanni hermanni from eight different Italian regions to investigate the presence of DNA of Testudinid alphaherpesvirus (TeAHV), Chlamydia spp. and Mycoplasma spp. in the oral cavity, and Salmonella spp. isolates in the cloaca. Mycoplasma spp. was detected in 52 out of 87 (59.77%) of rescued and in 1 out of 46 free‑ranging (2.17%) individuals; 33 out of 53 (62.26%) Mycoplasma spp. positive samples were typed as M. agassizii by PCR. Salmonella spp. was isolated from 45 out of 121 (37.19%) cloacal swabs, typed into 14 serovars, and characterized for complete antimicrobial susceptibility. A significantly different distribution of Salmonella spp. isolates was found in 2016 in comparison with 2014 and 2015, without any difference between free‑ranging and rescued tortoises. All the tested tortoises were negative for TeAHV and Chlamydia spp. These results are considered a baseline information critical to monitor the dynamics of these microorganisms in free‑ranging and rescued populations of T. h. hermanni, and to correctly approach the management of rescued animals and possible relocation programs.
... sex-biased mortality from disease: see Wendland et al. 2010). However, we found no evidence of shell disease, previously reported nearby (Jacobson et al. 1994), or obvious symptoms of upper respiratory tract disease (URTD) (Jacobson et al. 2014) such as mucus exudate from the nares. ...
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We conducted population surveys for desert tortoises Gopherus agassizii at 2 nearby sites in the western Sonoran Desert of California, USA, from 2015-2018, during the driest ongoing 22 yr period (2000-2021) in the southwestern USA in over 1200 yr. We hypothesized that drought-induced mortality would be female-biased due to water and energy losses attributable to egg production during protracted periods of resource limitation. At the higher-elevation, cooler, wetter Cottonwood site from 2015-2016, the sex ratio of live adult tortoises was biased toward males and the sex ratio of tortoises estimated to have died during the intensified drought conditions from 2012-2016 was essentially even. At the lower-elevation, warmer, drier Orocopia site from 2017-2018, the sex ratio of live adult tortoises was biased toward males and the sex ratio of tortoises with estimated times of death from 2012-2016 was biased toward females. High female mortality at the Orocopia site may have resulted from the interaction of drought effects and the bet-hedging reproductive strategy of tortoises wherein they continue to produce clutches of eggs in drought years. Annual reproductive output results in an estimated loss of up to 13.5% of female tortoise body mass including over 0.20 l of water. Combined with dehydration during severe droughts, these losses may compromise their ability to survive droughts lasting more than 2 yr. The low tortoise density and high mortality of females observed may reflect reduced survival of tortoises near the southern edge of their range due to climate change, including protracted and intensified droughts.
... Although habitat loss is the primary driver of declines for gopher tortoises (Smith et al., 2006), disease is also considered a threat (Jacobson, 1994;McLaughlin, 1997). Upper respi-ratory tract disease (URTD) caused by pathogenic bacteria in the genus Mycoplasma (with possible revision to genus Mycoplasmopsis; Gupta et al., 2018), has historically been the focal disease of management concern (Jacobson et al., 2014). Experimental trials confirmed M. agassizii as the etiologic agent of URTD for gopher tortoises (Brown et al., 1999), and a second etiologic agent, M. testudineum, was later identified (Brown et al., 2004). ...
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Translocation, the intentional movement of animals from one location to another, is a common management practice for the gopher tortoise (Gopherus polyphemus). Although the inadvertent spread of pathogens is a concern with any translocation effort, waif tortoises—individuals that have been collected illegally, injured and rehabilitated or have unknown origins—are generally excluded from translocation efforts due to heightened concerns of introducing pathogens and subsequent disease to naïve populations. However, repurposing these long-lived animals for species recovery is desirable when feasible, and introducing waif tortoises may bolster small populations facing extirpation. The objective of this study was to assess the health of waif tortoises experimentally released at an isolated preserve in Aiken County, SC, USA. Our assessments included visual examination, screening for 14 pathogens using conventional or quantitative polymerase chain reaction (qPCR) and haematological evaluation. Of the 143 individuals assessed in 2017 and 2018, most individuals (76%; n = 109 of 143) had no overt clinical evidence of disease and, when observed, clinical findings were mild. In both years, we detected two known tortoise pathogens, Mycoplasma agassizii and Mycoplasma testudineum, at a prevalence of 10.2–13.9% and 0.0–0.8%, respectively. Additionally, we found emydid Mycoplasma, a bacterium commonly found in box turtles (Terrapene spp.), in a single tortoise that showed no clinical evidence of infection. The presence of nasal discharge was an important, but imperfect, predictor of Mycoplasma spp. infection in translocated tortoises. Hemogram data were comparable with wild populations. Our study is the first comprehensive effort to assess pathogen prevalence and hemogram data of waif gopher tortoises following translocation. Although caution is warranted and pathogen screening necessary, waif tortoises may be an important resource for establishing or augmenting isolated populations when potential health risks can be managed.
... This study showed that nearly every resident juvenile green turtle in the IRL develops and recovers from FP. While this is the first report of near ubiquity of FP in a group of sea turtles, other pathogens and diseases in wild, captive, and human populations have similarly high incidences with minimal effects on populations, with examples including Mycoplasma in tortoises and birds (Jacobson et al., 2014;Sawicka-Durkalec et al., 2021), Batrachochytrium dendrobatidis in bullfrogs (Garner et al., 2005;Peterson et al., 2007;Schloegel et al., 2009), and herpesviruses in turtles, elephants, and humans (Hardman et al., 2011;Hidalgo-Vila et al., 2020;Prober, 2005). There are likely many other green turtle aggregation sites that are also heavily affected by FP, but do not have available data or data have not yet been analyzed. ...
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Fibropapillomatosis (FP) is a sea turtle disease characterized by benign tumor development on the skin, eyes, and/or internal organs. It primarily affects juvenile green turtles (Chelonia mydas) in coastal foraging sites. The Indian River Lagoon (IRL), Florida, USA, is a coastal green turtle foraging site where the observed FP annual rate averaged 49% between 1983 and 2018. While FP is no longer considered a major cause of sea turtle mortality and most individuals fully recover, the overall dynamics of this disease are poorly understood because prior disease history is unknown for individuals without FP at capture time, and future disease outcome is unknown for individuals with FP at capture time. To better evaluate FP dynamics for green turtles in the IRL, we developed a hierarchical model for predicting disease state change. We used data from 4149 captures of 3700 individual green turtles captured in the IRL. The hierarchical disease state model contained two levels: Level 1 modeled whether an individual would develop FP, and Level 2 modeled disease state progression, including states for pre‐FP affliction, active FP affliction, and full recovery from FP. From the hierarchical model, we estimated 99.8% (95% credibility intervals 99.1%–100%) of juvenile green turtles in the IRL developed FP, indicating that nearly every individual in the IRL is affected by this disease. The model also suggested that turtles quickly developed FP upon recruitment to the IRL and then recovered at different rates, with most completely recovering before emigrating from the IRL as they mature. This is the first analysis of long‐term sea turtle data suggesting nearly every turtle in an aggregation both develops and recovers from FP.
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We assessed the potential for microbial interactions influencing a well-documented host-pathogen system. Mycoplasma agassizii is the known etiological agent of upper respiratory tract disease in Mojave desert tortoises (Gopherus agassizii), but disease in wild animals is extremely heterogeneous. For example, a much larger proportion of animals harbor M. agassizii than those that develop disease. With the availability of a new quantitative PCR assay for a microbe that had previously been implicated in disease, Pasteurella testudinis, we tested 389 previously collected samples of nasal microbes from tortoise populations across the Mojave desert. We showed that P. testudinis is a common commensal microbe. However, we did find that its presence was associated with higher levels of M. agassizii among the tortoises positive for this pathogen. The best predictor of P. testudinis prevalence in tortoise populations was average size of tortoises, suggesting that older populations have higher levels of P. testudinis. The prevalence of co-infection in populations was associated with the prevalence of URTD, providing additional evidence for an indirect interaction between the two microbes and inflammatory disease. We showed that URTD, like many chronic, polymicrobial diseases involving mucosal surfaces, shows patterns of a polymicrobial etiology.
Chapter
At present, turtles, tortoises, and terrapins of the order Testudines comprise 356 species within 14 extant families (Rhodin et al. 2017). They demonstrate significant morphological diversity, occupying a wide array of terrestrial and aquatic habitats and ranging in adult size from 8 cm in length or 140 g in the speckled tortoise (Chersobius signatus) to over 2 m in length or 900 kg in the leatherback sea turtle (Dermochelys coriacea) (Davenport et al. 2009; Loehr 2018). While substantial diversity can be found among the ocular structures of these species, the general anatomy of the chelonian eye shares much in common with other reptilian orders. The majority of ophthalmic disease in these species involves the adnexal structures that support the health of the globe and the ocular surface itself. Trauma, infectious disease and inappropriate husbandry account for most adnexal and ophthalmic disease in these animals.
Chapter
Interpretation of cytological preparations from herptiles is like that of birds; therefore, avian cytodiagnosis is a useful guideline for herptile cytodiagnosis. Normal exfoliation of the superficial cells of the epithelium lining the upper alimentary tract reveals normal, mature squamous epithelial cells. The reptilian cloaca consists of three compartments: the coprodeum, the urodeum, and the proctodeum. The respiratory tract of reptiles varies somewhat among the different species. Heterophilic inflammation of reptiles is represented by a predominance of heterophils in the cytological sample. Macrophagic inflammation consists primarily of macrophages with multinucleated giant cells and lesser numbers of other inflammatory cells. Septic inflammation is indicated by the presence of bacteria within leukocytes. Ovarian cystadenocarcinomas and adenocarcinomas have been reported in reptiles, commonly in the green iguana. Mycoplasmosis is the most important chronic infectious disease of wild and captive North American and European tortoises. Mycobacteriosis is an important cause of morbidity and mortality in amphibians, especially those in captivity.
Preprint
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The Anthropocene is tightly associated with a drastic loss of species worldwide, and consequently, with the disappearance of key ecosystem functions. The ongoing reduction in species functionality is driven by global and local threats. The orders Testudines (turtles and tortoises) and Crocodilia (crocodilians and alligators) contain numerous threatened, long-lived species for which their functional diversity and potential erosion by anthropogenic impacts remains unknown. Here, we examine 259 of the existing 375 species of Testudines and Crocodilia quantifying their life history strategies (i.e., schedules of survival, development, and reproduction) from open-access data on their demography, ancestry, and threats. We find that the loss of functional diversity in simulated extinction scenarios of threatened species is greater than expected by chance. Moreover, the effects of unsustainable local consumption, diseases, and pollution are associated to specific functional strategies. Meanwhile climate change, habitat disturbance, and global trade affect all species independent of their life history strategy. Importantly, the loss of functional diversity for threatened species by habitat disturbance is twice greater than for all other threats. Our findings highlight the key importance of conservation programmes focused on preserving the functional diversity of life history strategies jointly with the phylogenetic representativity of these highly threatened groups.
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• The C-terminal variable region of the mycoplasma variable surface antigen (Vsa) proteins contains up to 60 tandem repeat units, and this variable length proves a major factor affecting adherence properties and shielding these bacteria from host immune responses. • The cell-surface Vsa proteins constitute as much as 10% of total mycoplasma protein, helping to shield cells against environmental factors. • When grown on solid surfaces and producing short Vsa proteins, M. pulmonis forms biofilms that confer partial resistance to immune system components. • Mycoplasmas form biofilm-like aggregates on host tissues, where they might also form tower structures to protect against innate immune system responses.
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Blood samples were collected from 386 gopher tortoises (Gopherus polyphemus) on 53 sites in Florida to determine exposure to a pathogen (Mycoplasma agassizii) that causes upper respiratory tract disease (URTD). One-hundred fourteen (30%) of the tortoises were seropositive, indicating that they had been exposed to mycoplasma and had developed a detectable immune response. Another 20 (5%) were considered suspect. The remaining 252 (65%) tortoises were seronegative. The 114 seropositive tortoises were from 14 locations, 12 of which were public lands. Thirty-nine percent of the 386 tortoises showed one or more clinical signs of URTD. Nasal lavage samples were collected from 89 tortoises on eight of the 53 sites. Four tortoises (4%) from three sites were positive, indicating presence of mycoplasma. A different mycoplasma was indicated on each of the three sites.