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The Veterinary Journal 201 (2014) 257–264

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The Veterinary Journal

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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

a Department of Small Animal Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA b Department of Infectious Disease and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA c Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32610, USA d Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616, USA e US Geological Survey, Western Ecological Research Center, Riverside, CA 92518, USA

ARTICLE INFO ABSTRACT

Article history: Tortoise mycoplasmosis is one of the most extensively characterized infectious diseases of chelonians. Accepted 30 May 2014 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- Keywords: gists and clinicians from multiple institutions and agencies. Electron microscopic studies of affected tor- Tortoise toises revealed a microorganism in close association with the nasal mucosa that subsequently was identified Mycoplasmosis as a new species, Mycoplasma agassizii. Over the next 24 years, a second causative agent, Mycoplasma Mycoplasma agassizii testudineum, was discovered, the geographic distribution and host range of tortoise mycoplasmosis were Mycoplasma testudineum Pathology expanded, diagnostic tests were developed and refined for antibody and pathogen detection, transmis- Serology sion studies confirmed the pathogenicity of the original M. agassizii isolate, clinical (and subclinical) disease PCR 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 1991), led to desert tortoises in the Mojave Desert north and west of the Colorado River being declared threatened (US Fish and Wildlife Respiratory infection of tortoises was first reported in Califor- Service, 1990). A similar disease was seen in both captive (Beyer, nia, USA, in the 1970s in confiscated Agassiz’s desert tortoises 1993) and wild (McLaughlin, 1990; Beyer, 1993) gopher tortoises (Gopherus agassizii) with nasal exudates (Fowler, 1980a), and in the (Gopherus polyphemus) in Florida, USA. A microbial and patholog- UK in the 1980s in captive Greek (Testudo graeca) and Hermann’s ical study (Jacobson et al., 1991) resulted in the identification of a (Testudo hermanni) tortoises with rhinitis (Lawrence and Needham, new mycoplasma, Mycoplasma agassizii (Brown et al., 1995) and the 1985). Viruses (Jackson and Needham, 1983), Mycoplasma spp. confirmation of its causal relationship with URTD in desert (Brown (Fowler, 1980b; Lawrence and Needham, 1985) and Pasteurella et al., 1994) and gopher tortoises (Brown et al., 1999b). testudinis (Snipes and Biberstein, 1982; Snipes et al., 1995)werehy- Tortoise mycoplasmosis has since become one of the most ex- pothesized as possible causes. tensively characterized infectious diseases of chelonians. Seminal In the 1980s, major declines in desert tortoise populations in the research studies include: (1) a description of the anatomy and his- Mojave Desert of California, USA (Berry and Medica, 1995), and an tology of the upper respiratory tract of healthy and affected tor- associated upper respiratory tract disease (URTD; Jacobson et al., 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- * Corresponding author. Tel.: +1 352 3391691. ative agent of URTD (Brown et al., 1994, 1999b); (4) development E-mail address: jacobsone@ufl.edu (E. Jacobson). (Schumacher et al., 1993) and refinement (Wendland et al., 2007) 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/). 258 E.R. Jacobson et al./The Veterinary Journal 201 (2014) 257–264 of an ELISA to determine exposure of tortoises to M. agassizii (Brown Clinical disease and pathology 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) Clinical vs. subclinical infection to detect M. agassizii and Mycoplasma testudineum DNA; and (6) cor- relation of specific antibodies against M. agassizii and M. testudineum Clinical signs of mycoplasmosis in tortoises include palpebral with upper respiratory tract lesions in infected tortoises (Homer et al., edema, conjunctivitis, and nasal and ocular discharges (Jacobson 1998; McLaughlin et al., 2000; Jacobson and Berry, 2012). et al., 1991; McLaughlin et al., 2000; Mathes, 2003; Jacobson and In this article, we review these (and other) key studies and assess Berry, 2012). However, subclinical infection with Mycoplasma spp. new research to update the current state of knowledge on myco- also occurs (Jacobson et al., 1995). Cycles of convalescence and re- plasmal URTD in tortoises and its implications for management and crudescence of clinical signs have been observed in captive and free- conservation of tortoises in the wild. ranging desert and gopher tortoises (Brown et al., 1999a and b).

Histopathology Species of Mycoplasma in tortoises Mycoplasmosis in tortoises is typically seen as an URTD, pri- Two mycoplasmas have been isolated from desert and gopher marily affecting the nasal cavity (Jacobson et al., 1991, 1995). Pneu- tortoises, and characterized: M. agassizii, originally isolated from a monia is occasionally seen. Histologically, normal nasal cavities of desert tortoise with URTD (2001), and M. testudineum, a genetical- tortoises consist of a ventral, mucous and ciliated, epithelial mucosa, ly distinct organism (Brown et al., 2004). Both organisms cause and a dorsal, multilayered, olfactory epithelium. In tortoises with similar lesions in the nasal cavities of tortoises, with those caused mycoplasmosis due to M. agassizii, lesions in the nasal cavity may by M. testudineum possibly being less severe (Jacobson and Berry, be focal to diffuse, minimal to severe and may include basal cell hy- 2012). A third mycoplasma, Mycoplasma testudinis, was isolated from perplasia in the mucosa, infiltrates of heterophils and histiocytes, the cloaca of a healthy pet Greek tortoise in England (Hill, 1985) and lymphoid hyperplasia in the submucosa. Depending on the ep- and has not been associated with URTD. Recently, a novel, unnamed, ithelial changes and the extent of the inflammatory response, the Mycoplasma sp. was identified by genomic sequencing in a sample following categories have been used to classify lesions: (1) mild in- obtained from the phallus of a wild desert tortoise (Wellehan et al., flammation; (2) moderate inflammation, and (3) severe inflamma- 2014). tion (Jacobson et al., 1995). In a group of desert tortoises that were serologically positive for Hosts and geographic distribution of mycoplasmas of tortoises M. testudineum, lesions in the nasal cavities were less diffuse and severe than in desert tortoises infected with M. agassizii (Jacobson Evidence of infection with M. agassizii in many species of wild and Berry, 2012). This could indicate that M. testudineum is less and captive tortoises across the world has been determined using pathogenic than M. agassizii, or that the desert tortoises were more serology, PCR and/or culture. Most information for wild tortoises recently infected. pertains to gopher tortoises (Beyer, 1993; Berish et al., 2000, 2010; McLaughlin et al., 2000; Wendland, 2007) in South-Eastern USA, both Serology 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 Serological assays (Gopherus morafkai, formerly G. agassizii; Dickinson et al., 2005; Jones, 2008; Murphy et al., 2011) desert tortoises in South-Western USA, An ELISA was developed to detect antibodies against M. agassizii and the Texas tortoise (Gopherus berlanderi; Guthrie et al., 2013)in in plasma and serum using a monoclonal antibody (MAb HL673) Texas, USA. against the light chain of desert tortoise immunoglobulins IgY and In Europe, mycoplasmas have been identified in wild spur- IgM (Schumacher et al., 1993). The antigen used in the ELISA was thighed tortoises (Testudo graeca graeca) in Morocco, wild Her- derived from M. agassizii PS6, the type strain from a desert tor- mann’s tortoises in France (Mathes et al., 2001; Mathes, 2003), toise with URTD. The reactivity of MAb HL673 was validated by captive Hermann’s and spur-thighed tortoises in France (Mathes et al., Western blot analysis (Schumacher et al., 1993) and reference 2001; Mathes, 2003), wild spur-thighed, Hermann’s and margin- polyclonal T. horsfieldii IgY and IgM antisera were obtained from H. ated (Testudo marginata) tortoises in Italy (Lecis et al., 2011), captive Ambrosius, Leipzig, Germany (Ambrosius, 1976). The Mab HL673- spur-thighed and Russian (Testudo, formerly Agrionemys, horsfieldii) based ELISA was further validated using experimental transmis- tortoises in Spain (Salinas et al., 2011), and captive spur-thighed, sion studies in desert (Brown et al., 1994) and gopher tortoises Hermann’s, Russian and leopard (Stigmochelys, formerly Geochelone, (Brown et al., 1999b). In these studies, reference standards that were pardalis) tortoises in the UK (McArthur et al., 2002; Soares et al., independent of the mycoplasmal diagnostic tests were presence of 2004). Mycoplasmas have also been identified in many captive non- clinical signs and histological lesions (Schumacher et al., 1997; Brown native pet tortoises in the USA (Brown et al., 2002; Wendland et al., et al., 2002). 2006). An ELISA was also developed to determine exposure of gopher M. testudineum was originally isolated from the nasal cavity of and desert tortoises to M. testudineum using M. testudineum CB57 a clinically ill desert tortoise from the Mojave Desert, USA (Brown as the antigen (Jacobson and Berry, 2012). In studies with >1000 et al., 2004). This organism was subsequently identified in three wild tortoises (M. Brown, unpublished data), relatively few serum samples gopher tortoise populations in North-Eastern Florida (Wendland, reacted with both M. agassizii and M. testudineum, and those samples 2007). that reacted with both Mycoplasma spp. were from tortoises in popu- Mycoplasma spp. also have been identified in other chelonians, lations with documented presence of both pathogens. As new species including free-ranging Eastern box turtles (Terrapene carolina caro- of mycoplasmas are isolated from tortoises, validation and stan- lina) with URTD in Virginia, USA (Feldman et al., 2006) and a captive dardization of serological assays will be required. ornate box turtle (Terrapene ornata ornata) in Hungary (Farkas and Whereas the original ELISA results were reported as an enzyme Gál, 2009). M. agassizii has also been identified by PCR in the lungs immunoassay (EIA) ratio (Schumacher et al., 1993), the reporting of red-eared sliders (Trachemys scripta elegans) with pneumonia from system was eventually converted to end-point titers (Wendland et al., Louisiana, USA (J. Roberts and E. Jacobson, unpublished data). 2007). Results for ~6000 independent desert and gopher tortoises E.R. Jacobson et al./The Veterinary Journal 201 (2014) 257–264 259 were used to develop a distribution curve of absorbance values. A that acquired, experimentally induced, anti-ovalbumin antibody titers subset of samples (n = 90) that were randomly distributed over the in desert tortoises could persist for over a year. spectrum of absorbance values was then used to determine end- point titers and construct a standard curve. Test refinements sub- Pathogen detection stantially improved assay performance (sensitivity 0.98, specificity 0.99, J = 0.98), and test reliability. The authors considered this to be Culture a clinically more meaningful and reliable diagnostic test than the original test based on EIA values. 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, Natural antibodies and interpretation of serological results 2004). The organism ferments glucose under aerobic conditions, re- sulting in an acid pH shift in the medium. The most common sample Natural antibodies are a function of innate immunity and can cultured is nasal lavage fluid, obtained using 0.5–5.0 mL sterile react with epitopes on multiple unrelated antigens of potentially (±phosphate buffered) saline or 0.5–1.0 mL sterile SP4 broth. Since pathogenic microbes (Marchalonis et al., 2002). The antigen binding bacteria and fungi are normal microbiota of the upper respiratory specificities of some natural antibodies have been characterized tract of tortoises, penicillin, polymyxin B and amphotericin B are (Grönwall et al., 2012). Hunter et al. (2008) reported that desert tor- usually added to SP4 medium to inhibit undesirable growth. To toises have natural antibodies (predominantly IgM) that could con- further minimize microbial contamination, a portion of each sample found ELISA results for anti-mycoplasmal antibodies. However, should be passed through a 0.45 μm filter prior to culture. natural antibodies are generally irrelevant in immunological tests, since sera are usually diluted sufficiently to avoid interference from PCR so-called ‘nonspecific background’ (Ochsenbein and Zinkernagel, 2000). Testing using PCR has many advantages for the diagnosis of Hunter et al. (2008) used Western blot analysis in an attempt mycoplasmosis in tortoises, including high specificity and rapid de- to distinguish between natural and acquired anti-M. agassizii anti- tection. A positive PCR provides direct proof of the presence of my- bodies, and concluded that banding patterns obtained using a single coplasmal genomic material at the time of sampling. Whereas the strain of M. agassizii could distinguish between uninfected tor- PCR product can be used to identify the Mycoplasma sp. accurate- toises with natural antibodies and exposed tortoises with ac- ly, it does not provide information about the viability of the organ- quired antibodies. However, most mycoplasmas, even within an ism at the time of testing. Since primers used in both conventional individual animal with a defined isolate, exhibit extensive intras- PCR and qPCR generally target specific gene sequences of a specif- pecies genotypic and phenotypic variability that is manifested as ic organism, they may not detect closely related species that differ antigenic variation (Simmons and Dybvig, 2007). The ability to vary in genomic content; therefore, specificity should be assessed strin- their antigenic patterns not only allows mycoplasmas to evade gently. Conventional PCR with restriction fragment length poly- immune surveillance, but also to confound analysis of mycoplas- morphism (RFLP) analysis and full 16S sequencing remains an mal immunogen recognition when only a single isolate is used as important testing option, particularly in cases where a new species the source of antigen, especially on Western blot analysis may be involved. Important considerations for diagnostic tests tar- (Kittelberger et al., 2006). The need for multiple strains in Western geting the presence of the pathogen are that the microbial load may blot analysis, but not in ELISA, are consistent with findings for other be decreased when clinical signs are absent and that inadequate sam- mycoplasmal species (Tola et al., 1996; Kittelberger et al., 2006). pling of the upper respiratory passages may lead to false negative Using sera from culture positive gopher tortoises with URTD con- results. Finally, molecular techniques do not provide clinical iso- firmed by histopathological examination, Wendland et al. (2010a) lates for further testing, including determination of antimicrobial demonstrated that mycoplasmal strain variation was responsible sensitivity. for the differences in observed Western blot binding patterns. Several A conventional PCR was developed to detect tortoise mycoplas- URTD-positive gopher tortoises had binding patterns similar to those mas in culture medium and nasal lavage samples (Brown et al., 1995; reported by Hunter et al. (2008) for plasma samples from URTD- Wendland et al., 2007). If a band of the correct size was present, negative desert tortoises. Western blot analysis using a single antigen then restriction enzyme digests using AgeI and NciI were used for (PS6) failed to detect gopher tortoises known to have URTD in ap- speciation. These two restriction enzymes give unique patterns for proximately 25% of cases, whereas an ELISA using the same strain mycoplasmal 16S rRNA. In the event that an aberrant pattern is ob- as an antigen reliably detected all infected tortoises (Wendland et al., served, complete sequencing of both strands of the 16S rRNA gene 2010a). using a minimum coverage of reads obtained from two primers is recommended. Maternal antibodies and antibody persistence Using conventional PCR, 23/146 (15.8%) samples from captive spur-thighed, Hermann’s, Russian and leopard tortoises in the UK Female desert tortoises transfer Mycoplasma-specific antibod- were positive for M. agassizii (Soares et al., 2004). Russian tor- ies (IgG class) to their offspring through the egg and these anti- toises were more frequently infected than other species tested. In bodies are still detectable at 1 year of age (Schumacher et al., 1999). this study, 8.2% of the samples tested were also positive for chelo- Antibody titers were substantially lower in offspring than in paired nian herpesvirus (ChHV). Co-infection of M. agassizii and ChHV was maternal serum, but higher in the offspring of sick female tor- also found in Mediterranean and one Russian tortoise in Spain toises than healthy female tortoises. Importantly, hatchlings from (Salinas et al., 2011). These pathogens may work synergistically, Mycoplasma antibody positive tortoises were not infected with My- causing more severe clinical signs when present as co-infections, coplasma spp. (Schumacher et al., 1999). Residual maternal anti- or one agent may predispose tortoises to infection and disease as- body potentially can confound interpretation of ELISA tests and result sociated with the other agent. Of oral swabs obtained from 30 free- in misdiagnosis of M. agassizii infection in juveniles if ELISA tests living spur-thighed, Hermann’s and marginated tortoises temporarily are not appropriately validated. The current M. agassizii ELISA has housed in a wildlife center in Sardinia, Italy, 11 (36.7%) were my- eliminated this as a problem by appropriate dilution of sera coplasma PCR positive, with the amplified sequences having close (Wendland et al., 2007). Similar to the long-term persistence of ma- similarity to M. agassizii (Lecis et al., 2011). Three PCR positive tor- ternal antibodies in desert tortoises, Sandmeier et al. (2012) found toises exhibited signs of respiratory disease. 260 E.R. Jacobson et al./The Veterinary Journal 201 (2014) 257–264

A qPCR using primers specific for M. agassizii and M. testudineum reported in American house finches (Carpodacus mexicanus; Dhondt was developed to quantify mycoplasmas in samples and to corre- et al., 2007), fomite transmission may be possible in tortoises, but late microbial burdens with clinical signs (DuPré et al., 2011). has not been demonstrated. M. agassizii DNA (6–72,962 pg/mL) was detected by PCR in 100% of In a 4 year study of dynamics of URTD caused by M. agassizii in 20 captive desert tortoises tested. When tested by Western blot anal- wild populations of gopher tortoises, the force of infection (FOI; prob- ysis, only 16/20 (80%) qPCR positive tortoises were seropositive. This ability per year of a susceptible tortoise becoming infected) and the was interpreted to mean that tortoises were colonized but not in- effect of URTD on survival in free-ranging tortoise populations were fected (DuPré et al., 2011). However, this study did not include ELISA followed in 10 populations in central Florida (Ozgul et al., 2009). results available for these tortoises, in which 19/20 (95%) tortoises Sites with high (≥25%) seroprevalence had higher FOI than sites with were seropositive. A false negative rate of 25% has been observed low (<25%) seroprevalence. These findings provided the first quan- when the Western blot analysis does not include the homologous titative evidence that the rate of transmission of M. agassizii is di- M. agassizii strain as a source of antigen (Wendland et al., 2010a). rectly related to seroprevalence. Although not a diagnostic assay, a method for labeling and quan- Age (size) differences in exposure to M. agassizii in gopher tor- tifying viable M. agassizii using a membrane dye that is converted toises may affect the spread of URTD in wild populations. In one to a fluorescent signal by viable cells and then quantified by flow study, adult gopher tortoises had a higher rate of exposure to cytometry has been described (Mohammadpour et al., 2011). This M. agassizii than subadults (Karlin, 2008). In a 5 year study of my- methodology can be used to determine the number of viable my- coplasmal URTD, free-ranging adult gopher tortoises were 11 times coplasmal cells in a broth culture and may be applicable to exper- more likely to be seropositive than immature tortoises (Wendland imental studies and antimicrobial testing where the number of et al., 2010b), suggesting that direct or prolonged interaction between microbes is of interest. immature and adult tortoises was minimal. In a study conducted at the Kennedy Space Center, Florida, from Transmission and host response 1995 to 2000, to monitor the impact of URTD on gopher tortoises, there was an increase in the number of tortoises showing signs of Experimental transmission 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 Experimental challenge studies in adult tortoises have demon- of dead tortoises were not distinguishable from living tortoises, in- strated that M. agassizii is a causative agent of URTD in desert and dicating that mortality was not confined to a single sex or age class. gopher tortoises (Brown et al., 1994, 1999b). Experimentally chal- However, no results of postmortem examinations, histopathology lenged tortoises had lesions in the nasal cavity consistent with those or PCR for detection of Mycoplasma spp. were reported for any of seen in naturally infected tortoises. Preliminary observations indi- the tortoises showing signs of illness. Therefore, a causal relation- cated that seronegative hatchlings are at least as susceptible to in- ship between mycoplasmosis and deaths of tortoises at this site was fection as adults and that the disease progresses more rapidly in not established. Gopher and other tortoises are also susceptible to younger tortoises, with high morbidity in the first 6 weeks post- infection and death from other pathogens such as Ranavirus, which infection (McLaughlin, 1997). can result in clinical signs that overlap with URTD (Johnson et al., Under experimental conditions, the onset of clinical signs in 2008). desert and gopher tortoises is as early as 2 weeks post-inoculation McCoy et al. (2007) did not demonstrate any correlation between (PI) with M. agassizii (Brown et al., 2002). Seroconversion lagged the presence of anti-M. agassizii antibodies and a decrease in the behind clinical signs, with reliable detection of antibodies by 8 weeks numbers of tortoises at several study sites. Furthermore, the pro- PI. In an experimental challenge study involving gopher tortoises, portions of tortoises that were seropositive or had intermediate an- the clinical sign scores of challenged tortoises (previously exposed tibody levels were positively correlated with the number of tortoises to M. agassizii) at 2 weeks PI were higher than those of naïve animals tested at each site. This is in agreement with well-established epi- (McLaughlin, 1997). ELISA values were also greater for challenged demiological principles for determining the number of animals in than naïve tortoises at each time point PI. A significant increase a population to sample in order to detect the presence of a disease. in serum ELISA values of challenged tortoises was observed at Valid population sampling will depend on the population size, the 4 weeks PI. true prevalence of infection, and the sensitivity and specificity of each test (Brown et al., 2002). Sampling inadequate numbers of Natural transmission animals can lead to inaccurate conclusions regarding the status of a disease in a population. Based on the behavioral inventory of the desert tortoise (Ruby McCoy (2008) compared the mean size of male and female tor- and Niblick, 1994), we believe that horizontal transmission by direct toises found dead (based on the presence of shells) with the mean contact (combat or courtship) is the most likely route of transmis- size of those found alive at a site (McCoy et al., 2007). The mean sion of Mycoplasma spp. between tortoises. Whereas transmission size of males found dead did not differ from the mean size of live is more likely to occur when the infected tortoise exhibits clinical males, but the mean size of dead females was lower than the mean signs, tortoises with subclinical infections may be able to transmit size of live females. Their findings differed from those of Seigel et al. Mycoplasma spp. under appropriate conditions (Jacobson et al., 1995). (2003), who did not detect differences in the mean sizes of dead Although aerosol transmission is possible, control gopher tor- and live tortoises for either sex. The difference in these findings may toises housed in pens adjacent to clinically affected tortoises did not be related to differences in size distribution of live animals between become clinically diseased or seroconvert, suggesting that M. agassizii different populations (McCoy, 2008). Although, it was assumed that did not travel even relatively short distances over low (0.7 m) bar- URTD was the cause of death of tortoises at both sites, no post- riers (McLaughlin, 1997). mortem findings were reported, so the possibility of other causes In the study by McLaughlin (1997), there was no evidence to of death, such as Ranavirus infection, could not be excluded. support vertical transmission of M. agassizii in hatchlings derived from female gopher tortoises that were seropositive for exposure Host response to M. agassizii. However, due to the small sample size, vertical trans- mission cannot be ruled out (Brown et al., 2002). Since experimen- In many mycoplasmal diseases, the host adaptive immune re- tal transmission of Mycoplasma gallisepticum by fomites has been sponse is dysregulated, often providing limited or no protection E.R. Jacobson et al./The Veterinary Journal 201 (2014) 257–264 261

(Szczepanek and Silbart, 2014). In tortoises with mycoplasmosis, Vegas Valley, although Mycoplasma spp. and other bacteria were iso- pathological studies have revealed an over-exuberant host re- lated during the warmer months, few aerobic bacteria and no My- sponse to Mycoplasma spp., resulting in dysplastic changes to the coplasma spp. were isolated from the nasal cavities of tortoises in nasal mucosa (Jacobson et al., 1991, 1995; McLaughlin et al., 2000). January, the coldest month of the year (Jacobson et al., 1995). In ad- The response of clinically healthy, seropositive, adult gopher tor- dition, since the optimal growing temperature of M. agassizii is 30 °C toises in experimental challenge studies with M. agassizii was more (Brown et al., 1995), it is unlikely that colder temperatures would rapid and severe than in naïve tortoises, suggesting that previous enhance growth of the microbe. However, cold winters could have exposure to the organism may exacerbate disease (McLaughlin, an impact on the host immune system at the time of emergence 1997). In contrast, several clinically healthy desert tortoises, which of tortoises from their burrows in the spring, making them more were culture positive and positive for serum antibodies by ELISA, susceptible to infection and lowering the infectious dose required had normal nasal cavities (Jacobson et al., 1995). Thus, not all tor- to establish infection. toises respond to M. agassizii with a severe inflammatory re- Human impacts on tortoises and their habitats, whether through sponse, suggesting that multiple strains of M. agassizii may exist with disruption of normal behavior patterns, degradation of habitats variable pathogenicity or that different responses are related to dif- through agriculture, silviculture, mining, land development or pol- ferent genotypes. lution, may cause sufficient physiological stress to trigger out- We suspect that mycoplasmosis in tortoises is characterized by breaks of mycoplasmal disease. Wild tortoises in remote areas of initial high mortality, followed by low mortality and high morbid- the central Mojave Desert, distant from human beings and paved ity. We have seen several infected tortoises survive in captivity for roads, were significantly less likely to be seropositive for M. agassizii many years, with clinical signs varying over time. Since tortoises than those in close proximity to human developments (Berry et al., use olfaction for finding and selecting food, the histological changes 2006). The capture, manipulation and transport of tortoises during seen in the nasal cavities of tortoises with URTD suggest that their research projects, as well as relocation, restocking and repatria- ability to locate food and feed would be impaired. To determine the tion efforts, also may be sources of stress that result in overt disease impact of nasal discharge on a tortoise’s ability to locate food, (Berry et al., 2002). Germano et al. (2014) designed a study to determine the re- The escape or release of captive tortoises in urban and remote sponses of Agassiz’s tortoises with or without nasal discharge, and areas may be a significant factor accounting for URTD in wild popu- positive or negative for M. agassizii antibodies, to a visually hidden lations. Thousands of captive desert tortoises were released into wild olfactory food stimulus and an empty control. The presence of nasal lands prior to their federal listing as a threatened species in 1990, discharge was associated with a reduced ability to locate food. This and releases have continued in recent years (Berry et al., 1986; Ginn, study also showed that moderate chronic nasal discharge in the 1990; Jennings, 1991; Connor and Kaur, 2004; Field et al., 2007; absence of other clinical signs did not affect appetite in desert tor- Murphy et al., 2007; Nussear et al., 2012). The outcome of a survey toises. We have seen tortoises with experimentally induced URTD of 179 captive desert tortoises around Barstow, California re- and captive tortoises with URTD continue to feed even with a nasal vealed anti-mycoplasmal antibodies in 148 (82.7%) (Johnson et al., discharge. 2006). A statistically significant positive association was found between severity of clinical signs and serum antibody ELISA status. Impact of mycoplasmosis on tortoises Furthermore, adult desert tortoises were more likely to have a pos- itive serum antibody ELISA result than sub-adults or young adults Factors contributing to mycoplasmal disease in tortoises of undetermined sex. These findings suggest that captive tortoises can be a reservoir of infection for wild desert tortoises. Factors that appear to contribute to outbreaks of URTD include Similarly, Morafka’s desert tortoises (G. morafkai; Murphy et al., environmental stress, human impacts, exposure to heavy metals and 2011) from suburban Tucson, Arizona, were 2.3 times more likely other toxicants, and the escape or release of captive tortoises to test seropositive for antibodies against M. agassizii than tor- (Jacobson et al., 1991; Brown et al., 2002; Sandmeier et al., 2009, toises from remote locations (Jones, 2008). In addition, captive tor- 2013). In the Desert Tortoise Natural Area (DTNA), Kern County, Cal- toises were 1.8 times more likely to test seropositive for Mycoplasma ifornia, where mycoplasmosis was first identified in wild desert tor- spp. than free-ranging tortoises, even in Arizona counties with high toises, mercury (Hg) concentrations in the livers (0.326 parts per visitor use. Epizootics of URTD occurred on Sanibel Island, Florida, million, ppm) of affected tortoises were significantly higher than following the release of gopher tortoises collected in northern Florida those of controls (0.0287 ppm) (Jacobson et al., 1991). Mercury can and southern Georgia for use in tortoise races (Dietlein and Smith, have a variety of toxicological effects, including cellular, cardiovas- 1979; Beyer, 1993; McLaughlin et al., 2000). cular, hematological, pulmonary, renal, immunological, neurolog- ical, endocrine, reproductive and embryonic effects (Rice et al., 2014). Effects of mycoplasmosis on tortoise populations Altered levels of thyroid hormones have been detected in western pond turtles (Emys marmorata) with elevated concentrations of Hg The effects of mycoplasmosis on mortality, morbidity and the (Meyer et al., 2014). Further work on the physiological impact of long-term health and viability of tortoise populations are poorly un- Hg on desert tortoises is needed. derstood. Mortality events could be due to an acute outbreak or the Environmental perturbations and annual fluctuations in tem- end result of long term physiological stress combined with an ex- perature, rainfall, and forage availability may result in activation of acerbating extrinsic stressor. In the acute outbreak on Sanibel Island, a subclinical infection to a clinical level. Although drought is a natural up to 50% of adult gopher tortoises at one site died with signs of part of the desert tortoise’s environment (Henen et al., 1998), it can URTD (McLaughlin, 1990). A similar acute mortality event oc- contribute to morbidity and mortality if combined with disease or curred at the Desert Tortoise Natural Area, Kern County, California habitat loss (Peterson, 1996). Clinical signs of URTD and heteropenia (Jacobson et al., 1991). At this site, URTD evolved from an acute, epi- were noted at the time of emergence of desert tortoises from hi- zootic disease with high mortality to a chronic endemic disease with bernation in years that followed periods of intense drought variable morbidity, low mortality and a high seroconversion rate (Christopher et al., 2003), suggesting that tortoises entering hiber- for antibodies against M. agassizii (Brown et al., 1999a). In a study nation in a drought year may be physiologically compromised. of URTD in gopher tortoises, Ozgul et al. (2009) theorized that se- Sandmeier et al. (2013) suggested that cold winters could enhance ropositive tortoises were those that had survived initial infection conditions for the growth of M. agassizii. However, in a study in Las and developed chronic disease. 262 E.R. Jacobson et al./The Veterinary Journal 201 (2014) 257–264

While mortality events are easily documented and attract con- lations was used as a model system for studying the effects of chronic siderable attention, morbidity can be more subtle and difficult to recurring disease epizootics on host population dynamics and per- assess. Abnormal hormone profiles observed in desert tortoises with sistence (Perez-Heydrich et al., 2012). The findings indicate that the mycoplasmosis (Rostal et al., 1996; Homer et al., 1998) could lead impact of disease on host population dynamics appears to depend to alterations in foraging and reproductive behavior and de- primarily on how often a population experiences an epizootic, rather creased reproductive potential. Chronic inflammation in nasal and than on how long the epizootic persists. Models such as this will olfactory tissues of affected tortoises (Jacobson et al., 1991; Homer have more value once validated and tested. et al., 1998) could disrupt olfactory function and affect foraging and reproductive behavior. Soluble proteins in shell scutes also may be Conclusions affected by mycoplasmosis (Homer et al., 2001). Further monitor- ing with follow-up pathological evaluation is needed to assess the Mycoplasmosis is a complex, multifactorial upper respiratory tract long-term consequences of mycoplasmosis on tortoise morbidity. disease, of captive and wild tortoises. M. agassizii and M. testudineum are proven etiological agents of URTD. Extrinsic factors that most Management implications for wild tortoise populations likely contribute to outbreaks of URTD include environmental stress, human impacts, exposure to heavy metals and other toxicants, and Reliance on ELISA results to support management decisions the escape or release of captive ill tortoises. Because M. agassizii has been isolated from multiple species of tortoises in North America Antibody ELISA testing has been used to manage gopher and and Europe, all tortoises should be considered potentially suscep- desert tortoises in parts of their range. Jacobson et al. (1995) dis- tible. Like most respiratory mycoplasmoses, URTD is a chronic and cussed several scenarios for the disposition of seropositive desert often subclinical disease. Clinical signs may vary in onset, dura- tortoises. Although these authors did not recommend euthanasia tion and severity. Both subclinically and clinically affected animals of clinically healthy tortoises that were antibody ELISA positive for have damage to the respiratory and olfactory epithelial surfaces, M. agassizii, such a policy was adopted in the state of Nevada. This which affects their ability to identify food. Direct contact (combat policy was terminated in 2007, in part (R. Averill-Murray, person- or courtship) between tortoises is the most likely route of trans- al communication) based on the following statement in Brown et al. mission, and transmission rates are directly related to overt clini- (2002):‘There are inadequate scientific data to provide definitive guide- cal signs and seroprevalence. Several diagnostic tests are available lines for the disposition of seropositive tortoises’. to determine the exposure (serology to determine antibodies) and Euthanasia of seropositive tortoises results in elimination of infection (direct culture and 16S rRNA PCR) status of individuals and animals that might otherwise provide valuable reproductive and populations of tortoises. Specific antibodies against Mycoplasma spp. genetic contributions to wild populations and is not recom- do not appear to provide protective immunity, and the host’s in- mended. However, relocation of seropositive tortoises could result flammatory response may contribute to the severity of nasal lesions. in spread of mycoplasmosis to susceptible animals, with detrimen- Translocation as a management tool should include the health status tal impacts on recipient populations. Likewise, healthy tortoises that of translocated tortoises and those at the recipient site, as well as have not been exposed to Mycoplasma spp. should not be relo- long-term monitoring of effects on translocated and recipient cated to populations with extensive clinical disease or those un- populations. dergoing increased mortality events (Brown et al., 2002; Sandmeier et al., 2009). While antibody ELISA testing remains an important tool for Conflict of interest statement making management decisions, it is critical to first establish clear goals for the tortoise population of interest. Importantly, antibody None of the authors of this paper has a financial or personal re- ELISA testing should not be used as the sole means of evaluating lationship with other people or organizations that could inappro- the health of an individual animal; rather, it is only one tool among priately influence or bias the content of the paper. many for comprehensive health assessment (Brown et al., 2004; McCoy et al., 2007; Sandmeier et al., 2009). Acknowledgements

Modeling population dynamics The work on mycoplasmosis of tortoises has been a multidisci- plinary effort at the University of Florida and elsewhere, involving Understanding disease transmission dynamics in the context of faculty, graduate students, research scientists and laboratory tech- tortoise social behavior is an important consideration for the success nicians. The following present and former University of Florida per- of future conservation programs. The finding that adult gopher tor- sonnel were involved in this project: I.M. Schumacher, B.L. Homer, toises were more likely to be seropositive than immature tor- C. McKenna, G.S. McLaughlin, B.C. Crenshaw, L. Green, D. Duke, J. toises (Karlin, 2008; Wendland et al., 2010b) has broad implications Hutchison, A. Whitemarsh, M. Lao, D. Bunger, S.J. Tucker, N.T. for disease modeling. During mortality events caused by patho- Chmielewski, N. Gottdenker and J.E. Berish. This work would not gens having minimal environmental transmission, such as M. agassizii, have been possible without support from a number of agencies in- immature size classes may be spared, providing a pool of tor- cluding the US Bureau of Land Management, US Geological Survey, toises for later recruitment. However, a significant limitation of this the Nature Conservancy, Nevada Wildlife Department, the Nation- hypothesis is that immature size classes constitute a small propor- al Training Center, Fort Irwin, the National Science Foundation, the tion of the overall population and usually are inadequate to sustain National Institutes of Health, the Walt Disney Company and mul- a population. Managing habitat to increase the successful recruit- tiple biologists and additional personnel who are dedicated to desert ment of juvenile tortoises would be a valuable strategy in these cir- and gopher tortoise conservation. cumstances. Alternatively, land managers could target smaller size classes for augmentation or restocking of depleted populations, References thereby reducing the risk of pathogen introduction. Population modeling techniques hold promise for understand- Ambrosius, H., 1976. Immunoglobulins and antibody production in reptiles. In: ing the impact of mycoplasmosis on wild populations of infected Marchalonis, J.J. (Ed.), Comparative Immunology, Blackwell Scientific Publications, tortoises. Mycoplasmal URTD in free-ranging gopher tortoise popu- Oxford, UK, pp. 298–334. E.R. Jacobson et al./The Veterinary Journal 201 (2014) 257–264 263

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