Infectious diseases are a significant factor in chelonian conservation programs, especially for release and translocation projects.4,5 Mycoplasma agassizii upper respiratory tract infection has caused widespread morbidity in captive and free-ranging desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises and has been observed in captive spur-thighed (Testudo graeca) and star (Geochelone elegans) tortoises.1,5,6,8,9
A mycoplasma survey of 70 (35 male, 34 female, and 1 not sexed) free-ranging and 34 (14 male, 18 female, and 2 not sexed) captive adult eastern box turtles (Terrrapene carolina carolina) was conducted between June 1995 and October 1997. Free-ranging turtles were collected from six locations in Westchester, Nassau, and Suffolk counties New York. The majority (53) were from one study site.3 The free-ranging turtles were clinically healthy except for six which had aural abscesses. They were collected by hand, held for less than 2 days before sampling, and then released at the original collection site. Captive turtles included a colony of nine turtles (C1) and 25 that were presented to wildlife rehabilitators or the Department of Herpetology (C2). The second group included long term pets, animals donated by members of the public due to illness (upper respiratory tract disease, aural abscesses, overgrown beak, subcutaneous abscesses, and general unthriftiness with weight loss), and turtles of unknown background. These may have been recovered from the wild, but collection location and duration in captivity were unknown.
Heparinized blood samples were obtained from all turtles by jugular, or in a few cases by occipital sinus, venipuncture. Plasma was separated by centrifugation and frozen at -30°C until assayed by a plasma enzyme-linked immunosorbent assay (ELISA) validated for the detection of antibodies to M. agassizii in desert and gopher tortoises.8,9 This assay is cross reactive with antibodies of a range of chelonian species although antibodies of the genus Testudo are poorly cross reactive (D. Brown, unpublished data). An ELISA ratio of sample to negative control was calculated for each sample. Samples with a ratio of ≤2 were interpreted as negative, 2–3 as suspect, and >3 as positive. Those cutoffs were conservatively based on the variance of ELISA ratios of healthy and experimentally infected tortoises, with the objective of minimizing the risk of false negatives.
Free-ranging turtles were frequently seropositive (31.4%, with 18.6% serologically suspect and 50% seronegative). The single large free-ranging population had more seropositive turtles (39.6%, with 22.6% serologically suspect and 37.7% seronegative) than the pooled data from all other collection locations (5.9% seropositive, 5.9% serologically suspect, and 88.2% seronegative).
Significantly fewer captive turtles (C1 and C2) were seropositive (14.7%, with 5.9% serologically suspect and 79.4% seronegative) than were free-ranging turtles (Chi Square Test; p<0.05). The captive colony (C1) had similar numbers of seropositive turtles (22.2% with 77.8% seronegative) as did those presented to wildlife rehabilitators (C2) (12% seropositive, 8% serologically suspect, and 80% seronegative).
Seropositive turtles were not associated with aural abscesses, upper respiratory tract disease, or other illnesses. Two free ranging turtles and 13 captive turtles were sampled multiple times (two to three times during an 8- to 14-month interval). Serologic status remained the same for 10 turtles (nine negative and one positive) and converted from one serologic status to another (negative to positive one time, positive to negative three times, and suspect to negative one time) in five turtles.
Nasal washes were performed with sterile 0.9% saline and frozen at -30°C until analyzed. Samples from seropositive turtles were cultured in SP4 mycoplasma broth and aliquots tested by polymerase chain reaction (PCR) testing utilizing Mycoplasma genus specific reaction conditions.9 No Mycoplasma spp. were isolated in culture or identified by PCR testing of nasal wash samples.
These serologic results suggest that captive and free-ranging eastern box turtles in New York have been exposed to a Mycoplasma spp. Until an organism is isolated it is impossible to determine if it is the same species as that infecting the desert and gopher tortoise. M. agassizii has, however, recently been isolated from a free-ranging Florida box turtle (Terrapene carolina bauri) with upper respiratory tract disease (D. Brown, unpublished data). Although captive turtles are potential candidates for repatriation,2,4,7 novel pathogens may have devastating consequences when introduced into naive populations. Until more is known, seropositive box turtles should not be released or translocated into populations which are seronegative or of unknown serologic status and contact between seropositive turtles and other chelonians should be avoided.
This project was funded, in part, by a grant from the Wildlife Conservation Society’s Freed Foundation Species Survival Fund. The authors thank Andy Sabin for his support of this project and his contributions to the Reptile Conservation Station. The authors appreciate the efforts of volunteers who assisted with box turtle collections and technicians who assisted with sample processing.
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