Complex immune responses and molecular reactions to pathogens and disease in a desert reptile (Gopherus agassizii)

Abstract

Immune function plays an important role in an animal's defense against infectious disease. In reptiles, immune responses may be complex and counterintuitive, and diagnostic tools used to identify infection, such as induced antibody responses are limited. Recent studies using gene transcription profiling in tortoises have proven useful in identifying immune responses to various intrinsic and extrinsic stressors. As part of a larger experiment with Mojave desert tortoises (Gopherus agassizii), we facilitated the transmission of the pathogenic bacteria, Mycoplasma agassizii (Myag), to naïve adults and measured innate and induced immune reactions over time. Specifically, we evaluated clinical condition, presence of Myag in the nasal/oral cavity, induced antibody responses specific to Myag, and measured molecular reactions (gene transcript profiles) in 15 captive tortoises classified as naïve, exposed, or infected and 14 wild tortoises for comparison. Myag was confirmed inside the nasal/oral cavity in exposed tortoises within 30-60 days of introduction to infected animals, yet we did not detect Myag specific induced antibody responses in these individuals until 420-595 days post exposure. Surprisingly, we found no overall differences in the gene transcript profiles between our experimental treatment groups throughout this study. This work highlights the complexities in assessing immune function and diagnosing pathogen related infections in tortoises and other reptiles.

Keywords: Mycoplasma agassizii; desert tortoise; immunity; mRNA; transcription.

Figure 1

Proportion of Mojave desert tortoises (Gopherus agassizii) in each treatment group (control, exposed, infected, reference) with positive or suspect laboratory results for the presence of the pathogen Mycoplasma agassizii (Myag via qPCR tests using nasal and oral swabs), Myag antibodies (via ELISA using plasma), and nasal discharge (classified as moderate to severe). Control and reference tortoises yielded negative laboratory results and did not exhibit nasal discharge. Myag was detected in exposed tortoises within 2–4 weeks of exposure, but these animals did not develop Myag antibodies until ~60–85 weeks post exposure. Infected tortoises mostly yielded positive results for the presence of and antibodies to Myag throughout the study. Tortoises include five control, five exposed, and five infected captive adult tortoises and 14 reference wild adult tortoises in Clark County, Nevada, USA

Figure 2

Multivariate, nonmetric multi‐dimensional scaling (NMDS) two‐dimensional plots for (a) normalized cycle threshold (C_T) values for 11 genes of interest in March 2014 (222 days post experiment; a) and the change in C_T values throughout the experiment (222 days–0 days; (b). Blood samples were analyzed for 15 adult captive male Mojave desert tortoises (Gopherus agassizii) at the Desert Tortoise Conservation Center and 12 adult (6M:6F) wild tortoises at Hidden Valley in Clark County, Nevada. Tortoises include five control (red), five exposed (blue), five infected (green), and 12 reference (purple) individuals

Figure 3

Hypotheses explored to explain why changes in transcription (ΔC_T) for genes thought to be involved in antibacterial immunity were not observed in tortoises exposed to and infected with Mycoplasma agassizii throughout our experiment. Evaluation of transcription levels (C_T) for genes following winter dormancy provided an additional hypothesis of a potential synergic effect of seasonality and pathogen load to down‐regulate immunity