EFFECTS OF MYCOPLASMAL UPPER-RESPIRATORYTRACT DISEASE ON MOVEMENT AND THERMOREGULATORY BEHAVIOR OF GOPHER TORTOISES (GOPHERUS POLYPHEMUS) IN GEORGIA, USA Author(s): Jessica L. McGuire, Lora L. Smith, Craig Guyer, and Michael J. Yabsley Source: Journal of Wildlife Diseases, 50(4):745-756. Published By: Wildlife Disease Association DOI: http://dx.doi.org/10.7589/2013-11-306 URL: http://www.bioone.org/doi/full/10.7589/2013-11-306

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DOI: 10.7589/2013-11-306

Journal of Wildlife Diseases, 50(4), 2014, pp. 745–756 # Wildlife Disease Association 2014

EFFECTS OF MYCOPLASMAL UPPER-RESPIRATORY-TRACT DISEASE ON MOVEMENT AND THERMOREGULATORY BEHAVIOR OF GOPHER TORTOISES (GOPHERUS POLYPHEMUS) IN GEORGIA, USA Jessica L. McGuire,1,2,3,5,6 Lora L. Smith,3 Craig Guyer,4 and Michael J. Yabsley1,2 1 Daniel B. Warnell School of Forestry and Natural Resources, University of Georgia, 180 E Green Street, Athens, Georgia 30602, USA 2 Southeastern Cooperative Wildlife Disease Study, Department of Population Health, Wildlife Health Building, College of Veterinary Medicine, University of Georgia, 589 D. W. Brooks Drive, Athens, Georgia 30602, USA 3 Joseph W. Jones Ecological Research Center, 3988 Jones Center Drive, Newton, Georgia 39870, USA 4 Department of Biological Sciences, Auburn University, 331 Funchess Hall, Auburn, Alabama 36849, USA 5 Current address: Georgia Department of Natural Resources, 1602 Lake Alexandria Lane, Thomasville, GA 31792, USA 6 Corresponding author (email: [email protected])

ABSTRACT: From 2011–12, we studied a gopher tortoise (Gopherus polyphemus) population with a historically high prevalence of antibodies to Mycoplasma agassizii to assess long-term effects of upper-respiratory-tract disease (URTD) on tortoise behavior. We radiotracked 30 adult tortoises (16 males, 14 females) from a long-term study site with the use of mark–recapture methods to determine site fidelity and to compare home-range size to that of a study in 1997. An additional 10 tortoises (six males, four females) with severe clinical signs of URTD from elsewhere in the study area were radiotracked and compared to tortoises that were asymptomatic or had only mild clinical signs. We also monitored thermoregulatory behavior of tortoises with the use of data loggers affixed to the carapace. There was no significant difference in home-range size between the asymptomatic tortoises and those with mild symptoms. Home ranges of tortoises with severe URTD were significantly larger than asymptomatic or mildly affected tortoises. Tortoises with severe clinical signs moved long distances over short periods, contradicting a hypothesis that chronically infected tortoises are less likely to emigrate. Prevalence of M. agassizii antibodies was similar among the three groups (98% overall), but prevalence of antibodies to a second pathogen associated with URTD, Mycoplasma testudineum, was lower in the asymptomatic (n514, 7%) and mild-symptoms (n57, 14%) groups than the severe-symptoms group (n58, 50%). Variation in the average carapacial temperatures of tortoises with severe URTD was significantly different from carapacial temperatures of mild and asymptomatic tortoises, suggesting differences in thermoregulatory behavior of severely ill tortoises. Our 15-yr recapture data suggest that, despite high prevalence of M. agassizii, population density has not decreased over time. However, emigration, especially of tortoises with severe clinical disease, may play an important role in dispersal and persistence of pathogens. Key words: Behavior, gopher tortoise, Mycoplasma, telemetry, thermoregulation.

and Mycoplasma testudineum are experimentally confirmed etiologic agents that can cause mortality and are most often associated with disease (Jacobson et al. 1991; Brown et al. 1994). Surveillance for these pathogens has been limited, with most studies in gopher tortoises focused on Mycoplasma spp. in Florida, USA (Wendland 2007; Diemer-Berish et al. 2010; Johnson et al. 2010). Studies have shown that tortoises may maintain subclinical upper respiratory tract infection that results in extensive tissue damage over time (Jacobson et al. 1991; Schumacher et al. 1997; McLaughlin

INTRODUCTION

The gopher tortoise (Gopherus polyphemus) is experiencing population declines throughout its range in the southeastern US primarily because of habitat loss (Enge et al. 2006). However, upperrespiratory-tract disease (URTD) is a chronic disease that may impact long-term viability of tortoise populations (e.g., Diemer-Berish et al. 2010; Perez-Heydrich et al. 2012). Pathogens associated with clinical signs of URTD include Mycoplasma, Ranavirus, and Herpesvirus, but among these, Mycoplasma agassizii 745

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et al. 2000). Because antibodies to Mycoplasma are not frequently detected in juveniles, transmission likely occurs as individuals reach sexual maturity (Wendland et al. 2010; Gonynor 2013). Mycoplasma can be transmitted between tortoises through contact with nasal secretions (Brown et al. 1994; Wendland 2007), which is important because tortoises frequently have direct contact during combat or courtship (Johnson et al. 2009). Clinical signs of URTD can include nasal and ocular discharge, nasal cavity obstruction, lethargy, emaciation, periocular and palpebral edema, and conjunctivitis (Jacobson et al. 1991; Schumacher et al. 1997). Populations with high prevalence of antibodies to Mycoplasma can exhibit low incidence of clinical disease if they are experiencing low stress and occur in highquality habitats (Tuberville et al. 2008). The ecology and impact of URTD in free-ranging gopher tortoise populations is debated (Seigel et al. 2003; McCoy et al. 2007; Sandmeier et al. 2009), and the longterm impacts of URTD on the health of free-ranging gopher tortoise populations is unknown (Perez-Heydrich et al. 2012). Clinical expression of disease may be intermittent, making it difficult to detect in a population without frequent monitoring (Brown 2002; Wendland 2007). Furthermore, cycles of convalescence and recrudescence of URTD have been confirmed in captive gopher tortoises (Brown et al. 1999; Feldman et al. 2006), although this phenomenon has not been evaluated in wild populations. Few studies have followed individuals over the long term (Diemer-Berish et al. 2010, 2012), and none have addressed long-term impacts of disease persistence in a population. Mortality events may go unnoticed because tortoises may die above (Diemer Berish et al. 2010) or below ground in burrows (Seigel et al. 2003; DeGregorio et al. 2012). It has been hypothesized that chronically infected tortoises are less likely to emigrate than healthy animals (Ozgul et al. 2009), which may limit the spread of a

pathogen, such as Mycoplasma spp., to other populations. Importantly, URTD might not cause acute mortality in gopher tortoises, but rather, it may cause changes in movement, foraging, basking, and hibernation, which could result in decreased fitness or fecundity (Berry and Christopher 2001). In the late 1990s, studies were initiated to investigate the home-range size and behavior of gopher tortoises at a private research site (Ichauway) in Georgia, USA. Initial studies were focused in an area called Green Grove (GG; Fig. 1; Boglioli et al. 2000; Eubanks et al. 2003). In addition, samples from tortoises across Ichauway, including GG, were tested for antibodies to M. agassizii; 73 of 76 (96%) GG tortoises had detectable antibodies, and prevalence was similar among tortoises captured across the Ichauway site (McGuire et al. 2014). We assessed prevalence of antibodies to Mycoplasma spp. and presence of clinical disease in a tortoise population from a longterm mark-recapture site, and whether long-term (15-yr) exposure to Mycoplasma spp. significantly altered movement patterns of tortoises. We also examined thermoregulatory behavior in antibodypositive, asymptomatic tortoises and in tortoises with severe clinical signs of URTD. We hypothesized that 1) a population with long-term, high antibody prevalence would experience decreased density, site fidelity, and home-range size among tortoises; 2) tortoises with clinical disease, regardless of antibody status, would have limited movements across the landscape; and 3) tortoises with clinical disease, regardless of antibody status, would have significantly different thermoregulatory patterns than asymptomatic tortoises. MATERIALS AND METHODS Study area

This study was conducted at Ichauway, an 11,600-ha, privately owned research site of the Joseph W. Jones Ecological Research Center in Baker County, Georgia (31u13916.880N, 84u28937.810W). Uplands at Ichauway are

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FIGURE 1. Location of the Jones Ecological Research Center (JERC) at Ichauway in Baker County (inset box), Georgia, USA. The inset highlights JERC, with the Green Grove study area hatched.

dominated by longleaf pine (Pinus palustris) and wiregrass (Aristida stricta) and are managed with prescribed fire. Gopher tortoises occur throughout Ichauway (Smith et al. 2006); however, our telemetry study and long-term comparisons of movements and behavior were primarily focused within GG (Fig. 1). Annual maximum air temperature at Ichauway is 33 C and the minimum air temperature is 2 C, with an annual average of 132.1 cm of rainfall (University of Georgia 2012). Green Grove population survey

In 2011, we surveyed the gopher tortoise population at GG to determine the population density. We replicated the methods in the previous surveys (Boglioli et al. 2000; Eubanks et al. 2003), which included locating all burrows within the 49.5-ha area. Surveys were conducted with one–three observers, who

walked roughly parallel transects searching for burrows. Burrows were marked with numbered identification tags. We inspected burrows with a burrow camera (Sandpiper Technologies, Inc., Manteca, California, USA) to determine the number of tortoises and calculated density (tortoises/ha) to compare to the 1997 study. Pathogen screening, radio telemetry, and temperature monitoring

In May and June 2011, we set cage traps (Tractor Supply, Brentwood, Tennessee, USA, and HavahartH, Lititz, Pennsylvania, USA) at occupied burrows in GG to obtain 30 adult tortoises for radiotelemetry (.230-mm carapace length; Landers et al. 1980; McRae et al. 1981). Additionally, in 2011 and 2012 we opportunistically collected and radiotagged 10 tortoises encountered across the property that displayed severe clinical signs of URTD. All

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FIGURE 2. Gopher tortoise (Gopherus polyphemus) in Georgia, USA with severe clinical signs of upper-respiratory-tract disease. Visible clinical signs include cloudy nasal exudate and conjunctivitis.

tortoises captured were taken to a field laboratory for processing, collection of biological samples, and transmitter attachment. During processing, tortoises were assessed for clinical signs suggestive of URTD (Fig. 2; McGuire et al. 2014), and lesions suggestive of past URTD (e.g., nasal scarring). Tortoises were categorized as asymptomatic (no clinical signs), mild (clinical signs included clear nasal discharge alone), or severe (clinical signs included cloudy nasal exudate; eye swelling or conjunctivitis; lethargy; labored, openmouth breathing; or audible breathing). Morphologic measurements, blood samples, and nasal exudate swabs were collected as described by McGuire et al. (2014). Tortoises were outfitted with radiotransmitters (American Wildlife Enterprises, Monticello, Florida, USA) attached to the right anterior marginal scutes with the use of epoxy putty (Rectorseal, EP-400, Houston, Texas, USA). To assess tortoise thermoregulatory behavior, Thermochron iButtons capable of recording temperatures between 240 C and 85 C (Embedded Data Systems, DSL1922L-F5, Lawrenceburg, Kentucky, USA) were affixed to the carapace of each tortoise with epoxy putty (DeGregorio et al. 2012). Carapace temperature and internal body temperature are strongly correlated in turtles (e.g., Nussear et al. 2007); thus, carapacial temperature readings are a good indication of thermoregulatory behavior. The iButtons were programmed to record temperature once per hour. Combined weight of the transmitter, iButton, and epoxy was ,50 g (,5% of body mass). Tortoises were marked by notching the marginal scutes (Cagle 1939)

with a DremelTM Stylus hand tool (Racine, California, USA). Animals were released within 48 hr of capture at the original capture site. Radiotagged tortoises in GG were located once a week during the active season (June– September 2011 and April–June 2012) by a radio receiver (Communications Specialists Inc., Orange, California, USA) and a threeelement Yagi antenna (Wildlife Materials, Inc., Murphysboro, Illinois, USA). In the inactive season (October 2011–March 2012) tortoises were located at least once every 2 wk. Tortoise locations were recorded with the use of a Trimble Nomad GPS (Trimble Navigation, LTD, Sunnyvale, California, USA). For each location, we noted whether the tortoise was above ground or below ground in a burrow. Incidentally captured tortoises with severe clinical signs of URTD were tracked every 1– 3 days until they settled in the same burrow for at least 2 wk, after which they were located weekly. If the tortoises became moribund they were humanely euthanized by a veterinarian and submitted for necropsy to the Southeastern Cooperative Wildlife Disease Study (Athens, Georgia, USA) or Auburn University diagnostic laboratory, College of Veterinary Medicine (Auburn, Alabama, USA). Plasma samples were submitted for testing for antibodies to Mycoplasma spp. by enzyme-linked immunosorbent assay (ELISA) as described by McGuire et al. (2014). Tortoise capture, handling, sample collection, and euthanasia methods were approved by The University of Georgia’s Animal Care and Use Committee (AUP A2009 6-112) and were conducted under scientific collection permits (29-WBH-09-151and 29-WBH-1046) issued by the Georgia Department of Natural Resources. Data analysis

All radiotagged tortoises were categorized as asymptomatic (no clinical signs), mild (clinical signs included clear nasal discharge alone), or severe (clinical signs included cloudy nasal exudate, eye swelling or conjunctivitis, lethargy, labored open mouth breathing, or audible breathing). Prevalences of antibodies to Mycoplasma spp. among tortoises categorized as asymptomatic, mild, and severe were compared with the use of a Fisher’s exact test for independence. Home-range size (100% minimum convex polygon [MCP] and 95% MCP; Hayne 1949) was calculated for radiotagged tortoises with the use of Home Range Tools for ArcGIS (Rodgers et al. 2007). We were unable to track

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tortoises with severe clinical disease for a full year; thus, our calculations of MCPs for these individuals was to allow a standard means of comparing among groups. We also calculated 95% MCP using the 1997–98 data for the 14 tortoises recaptured. We recorded burrow use and distances moved by tortoises in ArcGIS with the use of XTools Pro 9 (Esri, Redlands, California, USA). We tested for differences in mean home-range size (95% MCP) and mean number of burrows used between males and females, and between tortoises in 1997 and the current study, with the use of analysis of variance (ANOVA) and t-tests. We used ANOVA to determine if home range size (areas used) and distance moved for tortoises in the current study varied by health status (asymptomatic, mild, or severe) and by sex (SAS version 9.3, SAS Institute, Inc., Cary, North Carolina, USA). Temperature data from iButtons were imported into an Oracle database/SQL (Oracle Corporation, Redwood Shores, California, USA) for analyses. We calculated mean temperature, standard deviation, and frequency of data points collected per hour for all time intervals for which we had temperature records for $10 tortoises. If the temperature reading for a tortoise fell outside one standard deviation of the overall mean temperature, we considered this as ‘‘abnormal.’’ Frequency of abnormal temperatures was reported as a percentage of times each tortoise’s temperature fell outside one standard deviation. We compared burrow use and frequency of abnormal temperatures among asymptomatic, mild, and severe URTD tortoises using a Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparisons test with the use of InStat (GraphPad Software, Inc., La Jolla, California, USA). RESULTS

We observed 103 tortoises in burrows at GG resulting in an estimated density of 2.08 tortoises/ha. We captured 79 tortoises (64 adults, 15 juveniles) at GG and of the 64 adults captured, 76% (n549) were recaptures from the study conducted in 1997 (Eubanks et al. 2003). Across Ichauway, the prevalence of ELISA antibodies to M. agassizii was 92% (n5136) and 40% (n570) for M. testudineum (McGuire et al. 2014). All but one tortoise had detectable antibodies to M. agassizii in GG (asymptomatic and

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TABLE 1. Mycoplasma testudineum enzyme-linked immunosorbent assay (ELISA) results for gopher tortoises (Gopherus polyphemus) radiotracked at Ichauway, Baker County, Georgia, USA, 2011–12. A positive ELISA result (titer $64) indicates that the tortoise had detectable antibodies and had been previously exposed. A negative result (titer ,32) means that there were no detectable antibodies to Mycoplasma at the time of the test. A suspect result (titer532) is inconclusive and requires additional testing. ELISA result

Asymptomatic

Mild

Severe

Positive Suspect Negative Total

1 10 3 14

1 2 4 7

3 2 3 8

mild group combined) and only two GG tortoises were M. testudineum–antibody positive (one in the asymptomatic group and one in the mild group; Table 1). All tortoises in the severe group were M. agassizii–antibody positive and three were positive for M. testudineum antibodies. At GG, there was no statistically significant difference in M. agassizii–antibody prevalence between asymptomatic and mild groups (P50.604), but the M. testudineum–antibody prevalence was significantly higher in the mild group (P5 0.002) and the severe group (P50.028), when compared to the asymptomatic group. Antibody status of one GG recaptured tortoise changed from negative to positive for M. agassizii; this tortoise was a juvenile in 1997. All other recaptured GG tortoises with prior serology (n511) were positive in 1997 and remained positive in the current study. All five (100%) nasal swabs from severe tortoises were PCR positive for Mycoplasma. Five of six (83%) nasal swab samples from tortoises with mild clinical signs were PCR positive. Sequence analysis of these 10 samples showed .97% identity to M. agassizii (GenBank AY80802). Asymptomatic tortoises were not tested for shedding because nasal exudates were not present at the time of sampling. Three severe tortoises were tracked for .300 days and each experienced recrudescence of

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TABLE 2. Radiotelemetry data for gopher tortoises (Gopherus polyphemus) with severe clinical signs of upper-respiratory-tract disease. Temperature data were collected with iButton data loggers placed on the carapace of the tortoise. Temperatures (C) ranges are reported with 1 standard deviation (SD). Data were collected at Ichauway, Baker County, Georgia, USA, 2011–12.

Days Tortoise Sex tracked

1083A

F

3

1137A 1149A 1518A 1546A 1670A 2036A 2101A 2104A 2033A

M M M F M F M M F

322 334 122 43 53 311 80 79 64

Days observed above ground (%)

Percentage of time observed above ground with clinical signs

Home range size (ha)

Temperature range (SD)a

3 (100)

100

26.37

NA

9 39 86 67 100 74 100 8 71

0.15 0.09 97.80 344.74 0.65 250.24 8.63 483.69b –

33 23 7 33 2 23 3 5 7

(10.2) (6.8) (5.7) (76.7) (3.7) (7.4) (3.8)

22.09–42.56 11.99–38.16 17.66–45.60 15.00–45.00 21.50–44.51 9.56–42.56 NA 20.7–44.7 1.11–30.19

(3.56) (4.81) (4.23) (4.85) (3.40) (5.9) (3.75) (4.63)

Tortoise fate

Hit by car— euthanized Released Euthanized Euthanized Released Released Released Lost signal Euthanized Died in burrow

a

NA 5 data not available.

b

Tortoise was lost in June 2012 but relocated in July 2012 and the iButton was removed, but not the transmitter.

clinical signs between tracking events (Table 2). For example, three tortoises displayed severe clinical signs at 74%, 67%, and 82% of observations, whereas no clinical signs were visible for the remainder of observations (18–33%; Table 2). Three severe tortoises (1149A, 1518A, and 2104A) were euthanized because clinical signs indicated they were morbidly ill. In all three cases, Mycoplasma-related URTD was determined to be the cause of morbidity based on gross and histologic lesions observed during necropsy (J.L.M. unpubl. data). Radiotagged tortoises in GG (16 males, 14 females) were tracked an average of 32 times from June 2011 through July 2012 (Table 3). Fourteen radiotagged tortoises in this study had been radiotracked by Boglioli et al. (2000) and Eubanks et al. (2003). We found no significant difference between 95% MCP and 100% MCPs (t51.140, df538, P50.261); however, both estimates were calculated to allow comparison of our data to those of Eubanks et al. (2003). Mean home range size (95% MCP) of males was significantly larger than that of females (Table 3). Home-range size differed among tortoises

by health status (F55.60, df52, P50.008); mean 95% MCP of severe tortoises (134.71 ha) was significantly larger than that of the asymptomatic and mild tortoises (post hoc Tukey test; Table 4 and Fig. 3). Additionally, there was considerable variation among individual tortoises across groups (Table 4). The mean distance between locations did not differ between males (31.696 23.78 m) and females (30.00619.65; t50.21, df528, P50.83). Mean home range size (95% MCP) of 14 tortoises tracked in 1997–98 and 2011–12 did not differ (F50.24, df51, P50.628). All 14 tortoises recaptured in our study were found in the same general area within GG as the 1997 study. The 30 telemetered GG tortoises were observed above ground nine times (,1% of observations) during the current study. The 10 tortoises with severe clinical signs of URTD were radiotracked between 3 and 334 days; the tortoise tracked for only 3 days was not included in the analyses (mean 156 days; Table 2). Four tortoises were ultimately euthanized, three due to the severity of clinical signs and a fourth as a result of injuries from being hit

8.460.7 (5.0–15.0) 16 6.9a60.7 (3.0–10.0) 14 a

Statistically significant difference between sexes.

1.8761.64 (0.11–5.47) 16 0.8060.73 (0.07–2.54) 14

1.1060.13 (0.0–4.8) 70 0.4060.08 (0.0–3.4) 53 Mean6SE (range) n

Eubanks et al. (2003)

This study Mean6SE (range) n

10.060.5 (2.0–22.0) 70 5.2a60.3 (1.0–13.0) 53

Male BU Female BU Male HR (ha) Female HR (ha) Study

TABLE 3. Mean home range size (HR: 95% minimum convex polygon) and number of burrows used (BU) for gopher tortoises (Gopherus polyphemus) in Green Grove, at Ichauway, Baker County, Georgia, 1997–98 (Eubanks et al. [2003] and 2011–12 [current study]).

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by a car (Table 2). One tortoise (2033A) died in its burrow after 73 days of monitoring. Transmitters and iButtons were removed from the four surviving severe tortoises at the completion of the study and they were released at the site of capture (Table 2). Tortoises with severe clinical signs moved from 165 m to 3,498 m in total, as compared to the GG asymptomatic and mild group, for which the maximum distance moved was 147 m. The longest documented movement was a tortoise with severe clinical signs (1546A) that moved 755 m in 1 day. On average, tortoises with severe clinical signs used significantly fewer burrows (0–4) than asymptomatic (11) and mild tortoises (19; H520.298, df52, P,0.0001). We collected 45 wk of temperature data from 37 radiotagged tortoises (29 asymptomatic and mild tortoises from GG and eight severe tortoises). Average temperatures were calculated for the period 13 June 2011 to 29 January 2012 and 30 April to 22 July 2012). Temperatures of tortoises with severe clinical signs deviated from the average significantly more often than the mild or asymptomatic groups (H517.88, df52, P50.0001; Fig. 4). We found no temperature differences between asymptomatic and mild tortoises (P.0.05; Dunn’s multiple comparisons test); however, tortoises with severe clinical signs had significantly higher temperature variation than asymptomatic (P, 0.001) or mild tortoises (P,0.05). The greatest temperature range experienced by a tortoises with severe clinical signs was from 9.56 C to 42.56 C over the 311 days it was tracked (tortoise 2036A; Table 2). DISCUSSION

Gopher tortoises from a population with consistently high prevalence of antibodies to M. agassizii and that were asymptomatic or had mild clinical signs of URTD exhibited stable home range size over 15 yr.

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TABLE 4. Mean (6standard deviation) of average carapacial temperature and 95% minimum convex polygon (MCP) home-range size for asymptomatic, mildly symptomatic (Mild), and severely symptomatic (Severe) (for upper respiratory tract disease) gopher tortoises (Gopherus polyphemus) at Ichauway, Baker County, Georgia, USA. Ranges are presented in parentheses. Asymptomatic tortoises (n519)

Deviation of temperature 95% MCP (ha)

0.2160.06 (0.10–0.38) 1.1661.23 (0.05–4.98)

However, gopher tortoises from the same property with severe clinical signs of URTD had statistically significantly larger home ranges than asymptomatic and mildly symptomatic tortoises. Additionally, tortoises with severe clinical signs experienced greater deviation in carapacial temperatures than asymptomatic tortoises or mildly symptomatic tortoises, suggesting abnormal thermoregulatory behavior. Based on

Mild tortoises (n511)

Severe tortoises (n58)

0.2860.10 (0.12–0.45) 0.4960.17 (0.28–0.77) 1.7661.61 (0.10–5.47) 172.866275.42 (0.09–344.74)

temperature data, tortoises with clinical signs of URTD, especially when they were severe, spent more time out of their burrows, presumably basking (i.e., fever; Kluger 1986), than tortoises with no clinical signs. Either behavioral change can lead to decreased health, such as weight loss. Our results were consistent with studies that show diseased chelonians can exhibit behavioral changes (Monagas and Gatten

FIGURE 3. Home ranges (95% minimum convex polygon) of gopher tortoises (Gopherus polyphemus) with severe clinical signs of upper-respiratory-tract disease (URTD; light grey) and 30 tortoises from the focal area, Green Grove (inset), that were asymptomatic or showed mild clinical signs of URTD (dark grey). Data were collected at Ichauway, in Baker County, Georgia, USA in 2011 and 2012.

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FIGURE 4. Frequency with which individual gopher tortoises (Gopherus polyphemus, n538) exhibited carapacial temperatures .1 standard deviation from the overall mean, Ichauway, Baker County, Georgia, USA, in 2011 and 2012. Asymptomatic gopher tortoises are in light grey (tortoises that did not exhibit clinical signs consistent with upper-respiratory-tract disease); tortoises with mild clinical signs are in dark grey, and those with severe clinical signs are in black.

1983; Amaral et al. 2002; Swimmer 2006). For example, box turtles (Terrapene carolina) experimentally infected with lipopolysaccharides derived from Escherichia coli selected higher temperature gradients at which to bask than uninfected control animals (Amaral et al. 2002). Behavioral fever is thought to increase rates of survivorship in sea turtles affected by fibropapillomatosis (Swimmer 2006). It is possible that clinically ill gopher tortoises use a similar physiologic strategy by basking on cold days in an attempt to attain higher temperatures when clinically ill. The temperature profiles of nearly all of the tortoises with severe URTD grouped together. However, four and two tortoises with mild and asymptomatic URTD, respectively, exhibited temperature profiles that grouped their behavior with the severe group (Fig. 4). These tortoises may have been symptomatic during these periods, potentially providing evidence for recrudescence of clinical signs. Alternatively, tortoises may have been incorrectly categorized because of the absence of clinical signs at time of capture. However, this was not observed in the mild or asymptomatic groups because of the tortoises not being encoun-

tered above ground during the study without trapping. One of our significant findings was that gopher tortoises with severe clinical signs had, on average, statistically significantly larger home ranges than asymptomatic tortoises, despite the fact that they were followed for ,1 yr. However, there was a great deal of variation among individuals and a few severely ill tortoises had very small home ranges. For tortoises, homerange size is a function of resource requirements of individuals, and can vary seasonally because of annual variation in weather parameters such as drought (Duda et al. 1999). Severely ill tortoises with large home ranges moved through unsuitable habitat, such as hardwood bottoms and blackberry (Rubus spp.) patches, often remaining in those areas for days. It is not clear why the majority of our tracked severely ill gopher tortoises used such large areas; however, our findings refute the hypothesis that tortoises with chronic URTD are less likely to emigrate (Ozgul et al. 2009). In fact, our data show that some sick tortoises may exhibit abnormal dispersal behavior as compared with asymptomatic tortoises. This could possibly result in lower detec-

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tion probability of severely ill or dead tortoises at an individual site if sick tortoises are emigrating. Importantly, increased area use or long-distance movements would increase risk of contact between diseased tortoises and healthy animals. Tortoises that move long distances may also be more likely to encounter roads and thus may be more susceptible to motor vehicle injury. Despite high prevalence of Mycoplasma antibodies, tortoise population density increased at the GG site. Some of this difference in tortoise density may be a reflection of the additional buffer incorporated into the 1997 study (total area of 100 ha versus 49.5 ha in our study) or to differences in survey techniques (trapping burrows versus use of a camera scope). The difference may also be a result of immigration into the GG area and recruitment of juveniles into the population. Regardless, it does not appear that the GG area has experienced a population decline related to URTD. Impacts of URTD on the Ichauway population as a whole are unknown and, given the high mortality rate in the severely symptomatic group, further study is warranted. The high recapture rate (76%) of tortoises initially marked in 1997 demonstrates high site fidelity in this population. In contrast, Diemer-Berish et al. (2012) reported only 8% recapture after 27 yr at a site in Florida. We suspect that the difference in recapture rates between the two studies is related to differences in land use. Ichauway is managed for conservation of the long leaf pine and wiregrass ecosystem, with frequent prescribed fire, whereas the Florida site was managed for industrial timber production and consisted of slash pine (Pinus elliottii) plantation and clear cuts. Similar to reports from captive animals, clinical signs of URTD may recrudesce in free-ranging tortoises, which is likely to be more pronounced during stressful conditions such as drought, habitat loss, incompatible land use, or increased population

density (Diemer Berish et al. 2000; Wendland et al. 2010). Tortoises with severe clinical disease are capable of making long-distance movements across the landscape and, as sick tortoises are likely shedding bacteria, they could transmit the pathogen to other tortoises, and potentially other chelonians (Feldman et al. 2006). Gopher tortoise populations with high prevalence of antibodies to Mycoplasma can remain stable over time, which is an important finding; however, emigration of clinically ill tortoises may play an important role in dispersal and persistence of Mycoplasma. ACKNOWLEDGMENTS

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Submitted for publication 22 November 2013. Accepted 30 May 2014.