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
Accepted 30 May 2014
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/).
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).
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/
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.,
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
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.,
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,
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).
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
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,
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.,
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.
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.
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
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
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 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.
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.,
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.
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-
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
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.
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
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.
The work on mycoplasmosis of tortoises has been a multidisci-
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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.
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the Nature Conservancy, Nevada Wildlife Department, the Nation-
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