28th Annual Meeting and Symposium of the
Desert Tortoise Council, February 21-23, 2003
Abstracts

Tortoise Mycoplasmas and URTD

Adapted from: APPLICATION OF DIAGNOSTIC TESTS FOR MYCOPLASMAL INFECTIONS OF DESERT AND GOPHER TORTOISES WITH MANAGEMENT RECOMMENDATIONS, In press, Chelonian Conservation and Biology.

Daniel R. Brown¹, Isabella M. Schumacher^2,5, Grace S. McLaughlin^3,6, Lori D. Wendland¹, Mary B. Brown¹, Paul A. Klein⁴ and Elliott R. Jacobson^7
1Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL;
²Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL;
³Department of Wildlife Ecology and Conservation, College of Agriculture, University of Florida, Gainesville, FL; ⁴Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL; 58525 Richland Colony Road, Knoxville, TN; 
⁶NationalWildlife Health Center, Madison, WI;
⁷Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL

URTD refers to one or more clinical signs of illness seen in tortoises, including nasal discharge, palpebral edema, conjunctivitis, and ocular discharge. It is likely that multiple infectious agents may cause similar clinical presentations. To date, Koch's postulates have been fulfilled only for Mycoplasma agassizii, and thus it is the only rigorously confirmed etiologic agent of URTD in desert and gopher tortoises (Brown et al., 1994 and 1999b). Preliminary studies indicate that other pathogens, including other species of Mycoplasma, also may cause overlapping signs (McLaughlin, 1997; Origgi, 2001). M. cheloniae has been isolated from clinically ill, wild desert tortoises and has caused URTD in a limited experimental infection pilot study. An iridovirus was reported in a gopher tortoise with URTD (Westhouse et al., 1996). A wild desert tortoise with signs of respiratory disease was found to have a fungal pneumonia (Homer et al., 1998). In Europe, Mediterranean tortoises (Testudo hermanni and T. graeca) with clinical signs of URTD have been associated with a herpesvirus by electron microscopy and serology (Muller et al., 1990), and cases of herpesvirus infection have been reported in captive desert tortoises (Harper et al., 1982; Pettan-Brewer et al., 1996). However, to date experimental transmission studies resulting in clinical signs compatible with URTD have been performed for a herpesvirus in a limited number of Greek tortoises (Origgi, 2001). The herpesvirus ELISA has been used with sera from desert tortoises, but experimental infection studies and correlation of ELISA test results with histopathology and lesion development need to be done to further validate the assay.

The current evidence supports horizontal transmission of Mycoplasma in desert and gopher tortoises, most likely through direct contact of tortoises. Mycoplasmas lack a cell wall and are susceptible to desiccation; therefore, they do not normally persist in the natural environment for any appreciable length of time. A critical concern is contaminated fomites such as field equipment that might contribute to the spread of the organism, especially when mucous and organic contamination is present and proper disinfection techniques are not used. Fomite transmission does occur for other mycoplasmal infections, particularly in food and fiber animals (McMartin et al., 1987). A limited experimental study suggests that environment transmission does not occur in gopher tortoises (McLaughlin, 1997). Seropositive, clinically ill tortoises were housed in outdoor enclosures, these tortoises were removed, and seronegative gopher tortoises were immediately placed in the enclosures (McLaughlin, 1997). None of these tortoises seroconverted or developed URTD. Vertical transmission of mycoplasma is known to occur in poultry, but the rate of transmission is low (Lin and Kleven, 1982; Yoder, 1984). In a study of a very limited number of infected gopher tortoises, no egg transmission was documented (McLaughlin, 1997). Because the sample size was so small, egg transmission cannot be ruled out. Further, maternal antibody is present in the egg and persists in hatchlings, potentially confounding serologic tests (Schumacher et al., 1999).

Under experimental conditions, the onset of clinical signs in desert and gopher tortoises occurs as early as 2 weeks after infection. However, seroconversion lags behind clinical signs, with reliable detection by 8 weeks after infection. Affected desert and gopher tortoises are assumed to be capable of transmitting Mycoplasma through direct contact during the time between onset of signs and seroconversion, because we have found large numbers of Mycoplasma in the upper respiratory tract and nasal secretions of experimentally inoculated gopher tortoises during this time.

Clinical signs of URTD such as nasal discharge and conjunctivitis may reflect non-specific host responses to infection (inflammation, mobilization of phagocytes) as well as specific responses (immune-mediated complement activation, formation of immune complexes), although these have not been investigated. Signs may intensify and then abate in cycles, reflecting the progression of URTD. Based upon our transmission studies, our findings suggest that infection and expression of URTD occurs in the following steps: 1) initial colonization with Mycoplasma; 2) host response which reduces the population of Mycoplasma and simultaneously causes acute illness and signs of disease; 3) progression to chronic disease with intermittent expression of clinical signs and shedding of Mycoplasma. The presence of specific antibody to M. agassizii was associated with clinical signs of URTD in wild desert tortoises (Schumacher et al., 1993). In a desert tortoise population with natural infection, some initial fluctuation in antibody levels were observed but once an animal had a strong seroconversion, the antibody levels persisted for years (Brown et al., 1999a). These findings are typical of many mycoplasmal respiratory infections in other hosts.

Upon re-exposure to Mycoplasma, previously infected gopher tortoises may develop clinical signs of URTD more rapidly than naïve animals. Under experimental conditions, the clinical signs were more severe, higher numbers of Mycoplasma were recovered from nasal flushes, and the antibody response was more rapid than after first exposure (McLaughlin, 1997). Thus upon re-exposure to the pathogen, previously infected tortoises may actually experience exacerbated signs of illness rather than immune protection. This is consistent with the immunopathology associated with most mycoplasmal infections, where the host immune response is a key component in determining disease severity. While this evidence is limited to gopher tortoises, we expect a similar response in other tortoises infected with Mycoplasma. It is not clear if naturally infected tortoises develop a protective immune response. Our limited data in experimentally infected tortoises suggests this most likely does not occur. However, there are populations with seropositive animals and no overt disease. Seronegative animals in these populations do often seroconvert, suggesting that Mycoplasma are still present in the population but the extent of carriers is unknown. At this point, there is insufficient data to determine if a protective immune response exists in tortoises.

Most hosts do not typically clear mycoplasmal infections, however numbers of mycoplasmas may be substantially lowered during chronic phases of infection in the absence of clinical signs. It is possible that this may occur in the tortoise as well. An additional consideration is the particular strain of mycoplasma present in the individual or population. Strains of most mycoplasmal species vary in virulence. We have limited evidence in the gopher tortoise that suggests strains of M. agassizii differ in the minimum dose required to colonize and cause disease. Knowledge of the virulence potential of different strains will be important for risk assessment for future management decisions and also for evaluating the role of the microbe in disease transmission.

Mycoplasma agassizii can cause severe changes in the mucosal epithelium of the upper respiratory tract of desert and gopher tortoises (Jacobson et al., 1991; Homer et al., 1998; McLaughlin et al., 2000). These lesions disrupt the normal epithelial arrangement of the tissues and more than likely compromise their function. We have seen a reduction in appetite and other changes in behavior in experimentally challenged tortoises (McLaughlin, 1997). Experimentally infected desert and gopher tortoises often remained in their burrows for extended periods of time once clinical signs of illness developed. Further, irregular basking and burrowing behaviors have been noted in a limited number of animals in the wild (J. Berish and K. Berry, pers. comm.). The full impact of URTD on behavior and the implications of altered behavior on disease transmission and individual health and survival remains to be determined.

Mycoplasmosis is a complex, possibly multifactorial disease, associated with declines of desert tortoises in the southwest United States. Over the last 10 years much critical information has been gained (Table 1). Koch's postulates have been fulfilled for M. agassizii, validated diagnostic tests are now available for M. agassizii, the lesions associated with URTD have been described, and seroprevalence studies have been conducted for a number of wild and captive populations. However, there are significant gaps in our knowledge of the host response, other potential infectious agents, interaction among infectious agents, noninfectious influences on morbidity and mortality, and population dynamics that need to be addressed in future research studies. Some of the major questions regarding URTD that need to be addressed in future studies are summarized in Table 2. Any summary of current knowledge should be considered dynamic and will need to be updated and revised as additional data from stringent scientific studies are reported in peer-reviewed literature.

Table 1. Summary of major conclusions and current knowledge of URTD.

Table 2. Suggested future directions for URTD research.

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