Upper respiratory tract disease in the gopher tortoise is caused by Mycoplasma agassizii

Abstract

Upper respiratory tract disease (URTD) has been observed in a number of tortoise species, including the desert tortoise (Gopherus agassizii) and the gopher tortoise (Gopherus polyphemus). Clinical signs of URTD in gopher tortoises are similar to those in desert tortoises and include serous, mucoid, or purulent discharge from the nares, excessive tearing to purulent ocular discharge, conjunctivitis, and edema of the eyelids and ocular glands. The objectives of the present study were to determine if Mycoplasma agassizii was an etiologic agent of URTD in the gopher tortoise and to determine the clinical course of the experimental infection in a dose-response infection study. Tortoises were inoculated intranasally with 0.5 ml (0.25 ml/nostril) of either sterile SP4 broth (control group; n = 10) or 10(8) color-changing units (CCU) (total dose) of M. agassizii 723 (experimental infection group; n = 9). M. agassizii caused clinical signs compatible with those observed in tortoises with natural infections. Clinical signs of URTD were evident in seven of nine experimentally infected tortoises by 4 weeks postinfection (p.i.) and in eight of nine experimentally infected tortoises by 8 weeks p.i. In the dose-response experiments, tortoises were inoculated intranasally with a low (10(1) CCU; n = 6), medium (10(3) CCU; n = 6), or high (10(5) CCU; n = 5) dose of M. agassizii 723 or with sterile SP4 broth (n = 10). At all time points p.i. in both experiments, M. agassizii could be isolated from the nares of at least 50% of the tortoises. All of the experimentally infected tortoises seroconverted, and levels of antibody were statistically higher in infected animals than in control animals for all time points of >4 weeks p.i. (P < 0.0001). Control tortoises in both experiments did not show clinical signs, did not seroconvert, and did not have detectable M. agassizii by either culture or PCR at any point in the study. Histological lesions were compatible with those observed in tortoises with natural infections. The numbers of M. agassizii 723 did not influence the clinical expression of URTD or the antibody response, suggesting that the strain chosen for these studies was highly virulent. On the basis of the results of the transmission studies, we conclude that M. agassizii is an etiologic agent of URTD in the gopher tortoise.

FIG. 1

Clinical signs of URTD in gopher tortoises experimentally infected with M. agassizii. Tortoises (n = 9) were infected intranasally with 10⁸ CCU of M. agassizii. No clinical signs were seen for control tortoises (n = 10), which received sterile broth. (A) Results are expressed as the mean cumulative score, which was calculated as the nasal discharge score plus the mean for the three ocular scores. Infected tortoises (●) had greater cumulative signs than control tortoises (○) at all time points p.i. (P = 0.001). (B) Results are expressed as the mean clinical sign score + standard error on a scale of from 0 (none) to 3 (severe). At 4 weeks p.i., nasal (P = 0.004) and ocular (P = 0.01) discharges as well as palpebral edema (P = 0.04) were greater in infected tortoises than in control tortoises. At 8 weeks p.i., nasal discharge (P = 0.01) and palpebral edema (P = 0.004) were greater in infected tortoises than in control tortoises. At 12 weeks p.i., nasal (P = 0.004) and ocular (P = 0.004) discharges as well as palpebral edema (P = 0.04) and conjunctivitis (P = 0.04) were greater in infected tortoises than in control tortoises. No clinical signs were seen for control tortoises (n = 10), which received sterile broth (data not shown). The clusters of four bars for each week represent palpebral edema, conjunctivitis, nasal discharge, and ocular discharge from left to right, respectively.

FIG. 2

Specific antibody to M. agassizii in gopher tortoises experimentally infected with M. agassizii (■) and control gopher tortoises (□). Levels of antibody were higher in infected animals than in control animals for all times >4 weeks p.i. (P < 0.0001).

FIG. 3

Specific antibody to M. agassizii in gopher tortoises experimentally infected with different doses of M. agassizii (, low dose; ▨, medium dose; ■, high dose) and control gopher tortoises (□).

FIG. 4

Photomicrograph of the nasal cavity of a control gopher tortoise. (A) Area of mucous and ciliated epithelial cells (M) overlying a lamina propria submucosa (S) primarily consisting of connective tissue and small vessels. (B) Area of multilayered olfactory mucosa (O) overlying a lamina propria submucosa (S) consisting of connective tissue, vessels, and melanophores. Hematoxylin and eosin stain was used.

FIG. 5

Representative moderate to severe changes observed in the upper respiratory tracts of experimentally infected gopher tortoises. The epithelium (E) is hyperplastic, and there are diffuse accumulations of mixed inflammatory cells (IC) in the lamina propria. Hematoxylin and eosin stain was used.