2008 SOUTHEASTERN NATURALIST 7(1):145–158 Distribution of Mycoplasma agassizii in a Gopher Tortoise Population in South Florida Melissa L. Karlin* Abstract - Gopherus polyphemus (Gopher Tortoise) is a threatened species in Florida and is heralded as a keystone species throughout its range in the southeastern United States. However, this species has faced drastic population declines due mainly to habitat loss, and now disease is threatening the species. Upper respiratory tract disease (URTD) is a highly contagious disease first observed in Gopher Tortoise populations in Florida as early as 1989. URTD may be caused by multiple pathogens, such as Mycoplasma agassizii, which has been documented in Gopher Tortoise populations (Berish et al 2000). The long-term effects of URTD are unknown, as are the effects of the disease on the demographics of the species. In this study, 40 plasma samples were collected from a Gopher Tortoise population to determine the exposure of Mycoplasma agassizii among different age classes and genders. There was not a significant difference in number infected when comparing adult males and adult females. The results suggest adults are exposed to the pathogen at a greater rate than subadults, and exposure may be dependent on age. All subadults tested in this population tested seronegative, indicating they had no previous exposure to Mycoplasma agassizii. Knowledge of the effects of this pathogen is necessary for wildlife management agencies to assess the options available for managing Gopher Tortoise populations. The results of this study suggest a zero known mortality rate due to the pathogen over a 4-year period; however, additional pathological research is required to determine if Mycoplasma agassizii is causing URTD in this population. These results may have implications for the “take” policy in Florida: if Mycoplasma agassizii is not causing URTD in this population, and if the pathogen is not leading to a high mortality rate, then “take” permits based solely on seropositive enzyme-linked immunosorbent assay (ELISA) results may not be justified. Introduction Gopherus polyphemus Daudin (Gopher Tortoise) has been listed as a species of special concern in Florida since 1979 (Diemer 1992, Ernst et al. 1994). In 2006, the Florida Fish and Wildlife Conservation Commission (FFWCC) decided to raise the Gopher Tortoise to a threatened status once a management plan for the species is approved, which is anticipated in mid-2007 (FFWCC 2006a). Studies indicate that the Gopher Tortoise population may have declined by as much as 80% throughout its range in the southeastern United States in the last century due mainly to human activities (Auffenberg and Franz 1982, Diemer 1986). In recent years, *Florida Department of Environmental Protection, Florida Parks Service, 13798 SE Federal Highway, Hobe Sound, FL 33455. Current address - 924 SE 12th Way, Deerfield Beach, FL 33441; Melissa.Karlin@dep.state.fl .us. 146 Southeastern Naturalist Vol.7, No. 1 disease has also become a threat to the species. A serious respiratory disease known as URTD was documented in a Sanibel Island Gopher Tortoise population in South Florida in 1989 (Puckett and Franz 1991). URTD was first documented in the early 1980s in Gopherus agassizii Cooper (Desert Tortoise) in the southwestern United States (Berish et al. 2000) and may have contributed to a significant decline of this species (Jacobson et al. 1991). The disease can be caused by Mycoplasma agassizii, as well as other pathogens, and is transmitted via direct contact between tortoises. The Sanibel Island population reportedly had a 25–50% reduction in breedingage adult tortoises due to URTD-related deaths over a one- to three-year period, and a 30–90% decline over a 10-year period (McLaughlin 1997). Signs of the disease include chronic runny nose, congestion, wheezing, sneezing, coughing, swollen conjunctiva, foamy eyes, watery eyes, and lethargy (Berish et al. 2000; Brown et al. 1999; Doonan and Epperson 2001; Schumacher et al. 1993, 1997). One method for diagnosing Mycoplasma agassizii in a population is with an enzyme-linked immunosorbent assay (ELISA) test which measures the presence of anti-M. agassizii antibodies (Schumacher et al. 1997). Under experimental conditions, clinical signs may occur as early as 2 weeks post infection, while seroconversion takes approximately 8 weeks post infection (Brown et al. 1999). Using an ELISA, tortoises without clinical signs of URTD, which may be silent carriers or may have recovered from a former infection of M. agassizii, can be identified in a population. However, ELISA testing cannot distinguish between an active infection and exposure to M. agassizii in the past. Therefore, a seropositive individual is one that has been exposed to M. agassizii at some point, causing the production of antibodies, but may or may not be actively shedding bacteria (Schumacher et al. 1997). The ELISA test alone cannot determine the presence of URTD in a Gopher Tortoise; this can only be determined by additional diagnostics such as histological evaluation of the upper respiratory tract (McLaughlin et al 2000). A study investigating clinical signs of URTD (mucous nasal discharge, palpebral edema, etc.) and the ELISA test results (seropositive, seronegative, or suspect) in Desert Tortoises found a significant positive relationship between these factors (Schumacher et al. 1997). A total of 144 tortoises were tested, 45 of which (31%) had clinical signs, and 72 (50%) of which were seropositive. Of all the clinical signs a tortoise may exhibit, mucous nasal discharge was the most predictive for a positive ELISA test. Confounding the identification of URTD, additional pathogens have also been detected in Desert Tortoises that have clinical signs similar to URTD. A new pathogen, M. testudinis, was isolated from nasal lavages of Desert Tortoises with URTD (Brown et al. 2004). Clinical signs associated with this pathogen that are similar to URTD include chronic rhinitis and conjunctivitis, and the pathogen has also been found in Gopher Tortoises (Brown et al. 2004). The long-term effects of this pathogen are still 2008 M.L. Karlin 147 unknown. A mycoplamsa-like bacterium, Acholeplasma laidlawii, was also isolated from nasal lavages of a Gopher Tortoise with advanced symptoms of URTD (Brown et al. 1995). In one study, a Gopher Tortoise with clinical URTD signs was also diagnosed with an iridovirus using a Feulgen stain and ultrastructural methods (Westhouse et al. 1996), and in another case, a California Desert Tortoise was diagnosed with herpesvirus infections, including Pasteurella testudinis, Streptococcus veridans, and Staphylococcus spp. (Pettan-Brewer et al. 1996). This Desert Tortoise had lesions in the oral cavity, trachea, and lungs, and Pasteurella testudinis, Streptococcus veridans, and Staphylococcus spp. were cultured from specimens of the lung, trachea, tongue, and choanal swab (Jacobson et al. 1991). Signs of this disease have been documented in multiple Florida populations. Combined with habitat destruction and other environmental stressors, URTD has become a factor in the decline of some tortoise populations (Berish et al. 2000, Brown et al. 1999). Annual fl uctuations in temperature, rainfall, forage availability, as well as environmental stressors such as droughts or hurricanes, may cause outbreaks within an infected population (McLaughlin 1997). In many cases, the disease is clinically silent, and although the ease of transmission of M. agassizii under natural conditions is still unknown, direct contact is probably needed (McLaughlin 1997). Under the previous FFWCC regulations for relocation and URTD testing in force until August 2006, URTD-positive tortoises (based solely on ELISA test results) could not be relocated. In cases where ELISA tests indicated exposure to a pathogen such as M. agassizii, the FFWCC often issued a “take” permit, which authorized the entombment of the burrow, plus all the occupants, and destruction of the habitat. However, with the decision to raise the Gopher Tortoise to a threatened status, the FFWCC has recently changed the regulations for relocation and eliminated the mandatory URTD testing requirement. URTD-positive Gopher Tortoises are still not relocated and may be euthanized (FFWCC 2006b). The purpose of this study was to determine if the M. agassizii antibody status of Gopher Tortoises in a fenced preserve correlates with age, clinical signs, or gender. My research hypothesis was that exposure to M. agassizii in the population is dependent on the age and gender of the individual, and I expected that adult males would be exposed to the pathogen in significantly greater numbers than adult females or subadults. This hypothesis was based on the premise that since males have a larger home range and come into more contact with other tortoises (mate seeking, territorial disputes), they are at a greater risk of contracting and spreading M. agassizii (McLaughlin 1997). Methods The study site was known as “Range VIa” in the Abacoa greenway system, located in Jupiter, Palm Beach County, FL. The 9.27-ha (22.9-acre) site 148 Southeastern Naturalist Vol.7, No. 1 consisted of remnant pine fl atwoods, dominated by Serenoa repens (Bartr.) Small (Saw Palmetto) and Pinus elliottii var densa Little and Dorman (South Florida Slash Pine). The population size of Gopher Tortoises in 2005 on this site was estimated at 60, based on field observations and burrow counts (M. Karlin and J. Moore, unpubl. data). In 2001, when record keeping on the number of Gopher Tortoises inhabiting Range VIa began, 79 Gopher Tortoises were documented. By 2005, at the end of this study, 114 Gopher Tortoises had been recorded on Range VIa. This difference in 2005 population size and number of recorded Gopher Tortoises is attributed to emigration and death. Many Gopher Tortoises originally marked on Range VIa were documented in other parts of the greenway system and other surrounding areas. Road mortality was also a significant issue in this area. Countless Gopher Tortoises were found dead along the roadways in the area, although it was not determined if any of these individuals were from Range VIa. Gopher Tortoises are still added to Range VIa and other parts of the greenway system regularly, as residents and passer-bys have been seen moving Gopher Tortoises from the roadway to the fenced greenways (M. Karlin, pers. observ.). The dimensions of each Gopher Tortoise (plastron length, carapace length, carapace width, and shell height) were taken at each capture and were used to determine if the individual had reached sexual maturity (McRae et al. 1981). Sex of adult individuals could also be determined by examining the plastral concavity (a male has a greater than 5 mm concave depression) (Eubanks et al. 2003). Age of each individual was determined by counting the plastral annuli (Eubanks et al. 2003, McRae et al. 1981). In this study, hatchlings (age 0), young (1 to 7 years), and juveniles (8 years old until sexual maturity) were grouped together as one targeted sample group, called subadults, because of their scarcity in this population and cryptic nature (MacDonald and Mushinksy 1988). I surveyed the population from January 2001 to May 2005 and recorded the presence of URTD-like symptoms (Karlin 2002). From May 2004 to May 2005, I captured Gopher Tortoises for blood draws to determine exposure to M. agassizii and sent the samples to the Mycoplasma Testing Lab at the University of Florida. An ELISA was used to test for exposure to mycoplasma, and results were reported as titers. A titer of less than 32 is seronegative, a titer of 32 to 63 is suspect, and a titer of greater than or equal to 64 is seropositive. I used a chi-square analysis to conduct this assessment, and a P ≤ 0.05 was considered statistically significant. For the analysis of the relationship between URTD clinical signs and ELISA test results, I also used a chi-square test with P ≤ 0.05 considered statistically significant. Based on the presence of these clinical signs in the population and previous research (Schumacher et al. 1997), I expected that a significant number of individuals expressing clinical signs would test seropositive, and a positive correlation would be found between signs and ELISA test results. 2008 M.L. Karlin 149 Results I collected a total of 40 blood samples from 38 different tortoises (Fig. 1). Two Gopher Tortoises were retested after receiving suspect results. There were a total of 15 seropositive tests, or 37.5% of the samples. The seropositive tests were comprised of 9 adult males and 6 adult females. There were a total of 21 seronegative tests, representing 52.5% of the samples. The seronegative tests were comprised of 6 adult males, 9 adult females, and 6 subadults. Four samples returned suspect, or 10%. The suspect tests were comprised of 1 adult male and 3 adult females. Table 1 indicates from which year the Gopher Tortoise was first documented on Range VIa, through year 2005. The majority (27 of the 40) of tested Gopher Tortoises were studied since 2001, allowing ample time to observe clinical signs in the population. An analysis of the number of tortoises exposed to M. agassizii showed that adult males have been exposed in this population in greater numbers than the other categories. A chi-square analysis indicated that at a P = 0.04 (χ2 = 6.377, df = 2), the exposure was dependent on one of the classes, age or gender. An analysis of the number exposed between each category individually revealed that in this population, there was not a statistically significant Figure 1. Distribution of ELISA test results. Table 1. Year each tested Gopher Tortoise was first observed. Seropositive Seronegative Suspect Males Females Males Females Juveniles Males Females 2001 8 6 2 8 - 1 2 2002 - - - - 1 - 1 2003 1 - 2 - - - - 2004 - - 2 - 2 - - 2005 - - - 1 3 - - 150 Southeastern Naturalist Vol.7, No. 1 difference in the number exposed between adult males and adult females (χ2 = 1.2, df = 1, P = 0.27). Therefore, a further comparison of exposure was conducted. An analysis of number exposed between adult males and subadults, and between adult females and subadults, revealed that in both cases the adult population has been exposed to M. agassizii in statistically greater numbers than the subadults (adult males versus subadults: χ2 = 6.3, df = 1, P = 0.01; adult females versus subadults: χ2 = 3.36, df = 1, P = 0.07). These results suggest a relationship between age and exposure to M. agassizii. A depiction of locations of tested Gopher Tortoises is provided in Figure 2. There does not appear to be any consistency in the locations of seropositive individuals. In some instances, seropositive individuals were found at the same location, or within 10 m of each other, but in other instances, seronegative individuals were found at the same location as seropositive. Four of the six subadults tested were found in close proximity to seropositive individuals. These inconsistencies may be explained by lack of direct contact between any of these Gopher Tortoises, since the transmission of M. agassizii most likely requires direct contact. In this population, clinical signs of URTD (mucous nasal discharge, palpebral edema, etc.) were observed in a large number of the Gopher Tortoises tested for M. agassizii (Table 2). As in Schumacher et al. (1997), mucous nasal discharge may be the most predictive clinical sign in the study population. Of 18 Gopher Tortoises with clinical signs, 6 exhibited Figure 2. Location of Gopher Tortoises tested for Mycoplasma agassizii. 2008 M.L. Karlin 151 mucous nasal discharge. An analysis of only this clinical sign as it correlated with test results indicates a significant relationship (P = 0.06, χ2 = 3.5, df = 1; Table 3). Although the most common clinical signs in this population included conjunctivitis and congestion, 4 of the 6 Gopher Tortoises with mucous nasal discharge tested seropositive, suggesting it is a strong predictive clinical sign. Discussion My hypothesis for this research predicted that exposure to M. agassizii is dependent on the class of the individual, and I expected that adult males would be infected in significantly greater numbers than adult females or subadults. An analysis of approximately 63% of this population reveals that there is no difference in number infected between genders. However, further analysis revealed that there is a significant difference in number infected between the age groups, most notably adult males compared to subadults. This has a number of possible implications. In a separate study, male Gopher Tortoises were found to have the largest home-range size across all groups, and in a few cases, left the range for a period during dispersal events (M. Karlin, unpubl. data). This same study also found that males used more burrows than females or subadults, moving across the range and utilizing numerous burrows throughout a year. Many of these movements corresponded with the mating season and resulted in multiple observations of male Gopher Tortoises participating in courting events. Relating these observations may be one possible explanation for the similar Table 2. Clinical signs in Gopher Tortoises tested for Mycoplasma agassizii. Mucous Change in nasal pigmentation Year discharge around nares DehydrationA CongestionB ConjunctivitisC 2001 5 3 1 4 18 2002 1 2 5 6 23 2003 1 2 1 4 9 2004 1 2 8 6 17 2005 1 0 2 9 4 AMajor sign was sunken eye orbitals. BSigns included wheezing and sneezing (without mucous nasal discharge). CSigns included swollen eye lid, glossy eyes, and eye discharge. Table 3. Analysis of mucous nasal discharge and ELISA test results. URTD test results Mucous nasal discharge No mucous nasal discharge Total Seronegative 1 20 21 Seropositive 4 11 15 Total 5 31 36 p value = 0.060984198. 152 Southeastern Naturalist Vol.7, No. 1 number of seropositive male and female tortoises; if males were initially the group infected with M. agassizii at greater densities, their larger home range and courting activities may have caused M. agassizii to spread to the sexually mature female population. However, time of exposure cannot be determined in the present study, as ELISA test results were conducted during the same time frame for all Gopher Tortoises. An analysis of home ranges during the 2001–2004 mating seasons for seropositive males and females is shown in Figure 3. In every instance, a seropositive female home range is overlapped by a seropositive male home range. However, as seen in Figure 4, an analysis of all tested Gopher Tortoises shows that seronegative female home ranges were overlapped or bordered by seropositive males on numerous occasions. Like Figure 2 and the locations of tested individuals, there does not appear to be any consistency with ELISA test results and location at the study site. This result may again be attributed to the need for direct contact between Gopher Tortoises to spread M. agassizii. Although the sample size of individuals from the subadult group was smaller than the adult groups, a comparison was still made between this group and the adult tortoises. All 6 Gopher Tortoises from this group, ranging in age from 1 month to 3 years, tested seronegative. Possible Figure 3. 2001–2004 mating seasons home ranges for seropositive adult males and females. 2008 M.L. Karlin 153 explanations for this trend may be the lifestyle of Gopher Tortoises at this age; hatchling, juveniles, and subadults tend to remain socially inactive, since they are not sexually mature, and spend the majority of their time foraging near their burrow. This pattern may prevent tortoises in this age group from coming into frequent contact with the infected adult tortoises until they reach sexual maturity. However, this is a small sample size, and additional testing at other study sites is required to confirm this notion. Of these 6 subadults tested, 3 were hatchlings and approximately 1–2 months old at the time of capture. Based on their age, these 3 Gopher Tortoises probably hatched at the study site. An additional subadult, approximately 3 years old, has been documented at the study site since it was approximately 1–2 months old, and probably hatched at the study site. The remaining 2 subadults sampled were 1–2 years old at first capture, so it is unknown if they hatched at the study site or were added to the study site. Similar to Schumacher et al. 1997, there was a significant relationship between mucous nasal discharge and seropositive ELISA results. This clinical sign continues to be the most positive predictor for a seropositive ELISA test, indicating exposure to M. agassizii. Additionally, seropositive ELISA tests and a significant correlation with the most positive clinical sign predictor, mucous nasal discharge, supports the Figure 4. 2001–2004 mating seasons home ranges for tested adult males and females. 154 Southeastern Naturalist Vol.7, No. 1 notion that URTD may be in this population. Additional testing, such as diagnosing histological changes in the tissues of the upper respiratory tract or culture of M. agassizii from the nasal cavity, is required to make this determination. The large number of seronegative tortoises exhibiting URTD-like signs in this population may be attributed to a number of factors unrelated to M. agassizii exposure. Schumacher et al. (1997) attributes URTD signs in seronegative tortoises to activities such as eating or drinking, or a response to dust or allergens, which may lead to wet nares. The same study also states that clinical signs may precede the actual production of detectable levels of M. agassizii antibodies, suggesting that some of the individuals in the current study may in fact be infected. Retesting these individuals at a later date would determine if this is the case. A positive ELISA test represents exposure to not only M. agassizii, but other similar mycoplasmas (FFWCC 2003). For example, M. testudineum, if widespread and affecting Gopher Tortoise populations, may cause URTD-like signs. The mycoplamsa-like bacterium, Acholeplasma laidlawii (Brown et al. 1995), iridoviruses (Westhouse et al. 1996), and herpesviruses (found in captive desert tortoises) (Pettan-Brewer et al. 1996), could all potentially lead to URTD-like signs; their presence in the current study population has not been tested. Individuals in the current study population have presented URTDlike signs since 2001; however, in the majority of the cases, these signs are intermittent (Table 2). The presence of URTD-like signs may also be attributed to environmental factors, such as habitat condition and environmental disturbances. Incidences of URTD signs, especially wheezing and nasal discharge, occurred most frequently in the dry part of the year, March and April (M. Karlin, unpubl. data). This may be due to the stress induced by the lack of food resources at that time of year. Also, “Range VIa” has not been burned in over 7 years, and the habitat has become overgrown and suboptimal. A high mortality rate associated with a pathogen was not documented in this study after 4 years of research. One explanation is that M. agassizii is not causing URTD in this population. Instead, it is simply causing chronic URTD clinical signs, such as nasal mucous discharge. Additional research is required in this population to determine if M. agassizii is causing URTD. If this can be conclusively proven, then the results of this study may be inconsistent with other URTD studies, which describe mortalities due to URTD over a relatively short time period, such as the Sanibel Island population, which had a 25–50% reduction in breeding-age adult tortoises over a one- to three-year period, and a 30–90% decline over a 10-year period (McLaughlin 1997). The current population has been studied since 2001, and while individuals have shown clinical signs of URTD for years, no deaths have been directly attributed to URTD. However, several tortoises were found dead, 2008 M.L. Karlin 155 and many unaccounted for during this study, and could have died in their burrow and never been detected. Other studies have noted that many of the fatalities thought to be associated with URTD have been found outside of their burrow. Seigel et al. (2003) identified a total of 43 dead tortoises between May 1998 and July 2001; the researchers in this case believed URTD was responsible for this decline in population, although no conclusive evidence of URTD or M. agassizii infection was provided. No Gopher Tortoises showed any signs of predation, and most were found within 10 m of a burrow. Also, there was no difference in gender between the numbers of adult carcasses found, similar to the numbers of males and females infected in the current study. Seropositive ELISA test results and the presence of clinical URTD signs have been documented in this population. If additional research can conclusively prove M. agassizii is causing URTD in this population, then another explanation for the minor effects of URTD may relate to the length of this study and duration of infection. As previously discussed, this population is located on a preserve that up until about 1996 was isolated from other populations and generally from human disturbance. As groundbreaking for Abacoa began in 1996, Gopher Tortoises were relocated to this preserve and other preserves within the greenway system. It was during this time that the Gopher Tortoise populations may have started coming into contact with tortoises from other areas of the 822-ha (2055-acre) development area and mycoplasmas such as M. agassizii. Development is still on-going and, as previously mentioned, Gopher Tortoises are still added to this preserve and others in the greenway system, often by residents or passer-bys wanting to move Gopher Tortoises off of roads (M. Karlin, pers. observ.). What is not well known about URTD is whether there is a delay between the time of the M. agassizii infection and when high mortality rates are experienced. Additionally, as in the Sanibel Island population, the time of infection is unknown. While under experimental conditions, the immune response to URTD is detectable 6 to 8 weeks post exposure (Brown et al. 1999), the time between exposure and mortality in the wild is unknown. If there is a latency period of this disease on the order of 5 to 10 years post infection before this mortality is experienced, it is possible that the study population in Abacoa is still within this period, and high mortality rates may be documented in the future. In the current study, pathological research is still required to conclusively determine if URTD is present in this population. If seropositive ELISA test results are found to correlate positively with the presence of URTD in this population, as they did for clinical signs, then the overall effects of the disease on this population need to be monitored for additional time to determine mortality rates. If additional research finds this not to be the case, it supports the notion that “take” permits may be unjustified and populations should not be decimated based solely on seropositive ELISA results. Also, 156 Southeastern Naturalist Vol.7, No. 1 if during additional research on this population and at other sites, subadults tend to remain seronegative, this may be promising for Gopher Tortoise conservation and management, as these individuals may be recovered from the population for restocking efforts prior to “take” permits being issued. Another question that was not addressed in this study and requires further investigation is whether or not there are multiple strains of M. agassizii, such as pathogenic and non-pathogenic strains, that may be affecting Gopher Tortoises. This possibility should also be taken into consideration when “take” permits are issued based on ELISA test results. In the summer of 2006, the FFWCC agreed that a reclassification of the Gopher Tortoise from “species of special concern” to “threatened” was warranted. This reclassification will take place once a management plan for the species is approved. With this decision in the summer of 2006, mandatory URTD testing prior to relocating Gopher Tortoises was suspended (FFWCC 2006b). This policy change has alleviated some of the “take” permit issues. Additional research on the prevalence of M. agassizii and URTD, and tracking the transmission of and mortality associated with this disease, is critical for the management of this species. Acknowledgments I wish to thank J. Moore, J. Berish, and H. Smith for review comments and contributions to this manuscript. Literature Cited Auffenberg, W., and R. Franz. 1982. The status and distribution of the Gopher Tortoise (Gopherus polyphemus). Pp. 95–126, In R.B. Bury (Ed.). North American Tortoises: Conservation and Ecology. 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