COMPUTED TOMOGRAPHY FOR MEASURING BODY FAT RESERVES IN THE THREATENED MOJAVE DESERT TORTOISE (GOPHERUS AGASSIZII).

Noninvasive methods for measuring fat reserves in both captive and free-ranging animals are important for monitoring individual and population health, but chelonian anatomy and physiology present challenges to accurate measurements. Standard field-based methods for assessing body condition in Mojave desert tortoises (Gopherus agassizii) involve the qualitative body condition score, which relies on the apparent height of the temporalis muscle relative to the sagittal crest (in addition to other characteristics) and quantitative body condition indices that measure relative mass at size. However, it is unclear how these metrics relate to body fat reserves in this species. The aims of this study were to (1) describe the use of noninvasive computed tomography in measuring body fat volume of Mojave desert tortoises, (2) describe the location of fat reserves, (3) investigate relationships between fat reserves and body condition score and body condition index, and (4) explore whether relative temporalis muscle depth, measured via computed tomography, correlates with body condition score. Body condition scores were assessed for eight captive Mojave desert tortoises prior to euthanasia, and computed tomography was performed postmortem to quantify fat volume and measure temporalis muscle depth. At necropsy, the distribution of fat was documented. Fat volume calculated by computed tomography ranged from 2.83 to 145.38 cm3 (0.07-2.5% body volume). Neither qualitative body condition score nor quantitative body condition index was correlated with fat volume. Bladder content did not compromise body condition index. Body condition score was not correlated with relative temporalis muscle depth. Computed tomography is a noninvasive method for successfully identifying fat reserves and estimating total fat volume in Mojave desert tortoises. The lack of a relationship between computed tomography-determined metrics and commonly used body condition metrics indicates that computed tomography fills a critical gap in the health assessment tool kit for captive and free-ranging Mojave desert tortoises.

ASSESSMENT OF DISEASE RISK ASSOCIATED WITH POTENTIAL REMOVAL OF ANTHROPOGENIC BARRIERS TO MOJAVE DESERT TORTOISE (GOPHERUS AGASSIZII) POPULATION CONNECTIVITY.

The Mojave Desert tortoise (Gopherus agassizii), federally listed as threatened, has suffered habitat loss and fragmentation due to human activities. Upper respiratory tract disease (URTD), a documented health threat to desert tortoises, has been detected at the Large-Scale Translocation Study Site (LSTS) in southwestern Nevada, US, a fenced recipient site for translocated animals. Our study aimed to 1) estimate prevalence of URTD and Mycoplasma infection at LSTS and three nearby unfenced sites; 2) assess whether Mycoplasma infection status was associated with developing clinical signs of URTD; and 3) determine whether such an association differed between LSTS and unfenced areas. We sampled 421 tortoises in 2016 to describe the current status of these populations. We evaluated three clinical signs of URTD (nasal discharge, ocular discharge, nasal erosions) and determined individual infection status for Mycoplasma agassizii and Mycoplasma testudineum by quantitative PCR and enzyme-linked immunosorbent assay. In 2016, LSTS had the highest prevalence of M. agassizii (25.0%; 33/132), M. testudineum (3.0%; 4/132), and URTD clinical signs (18.9%; 25/132). Controlling for other factors, clinical sign(s) were positively associated with M. agassizii infection (odds ratio [OR]=7.7, P=0.001), and this effect was similar among study sites (P>0.99). There was no association with M. testudineum status (P=0.360). Of the 196 tortoises in a longitudinal comparison of 2011-14 with 2016, an estimated 3.2% converted from M. agassizii-negative to positive during the study period, and incidence was greater at LSTS (P=0.002). Conversion to positive M. agassizii status was associated with increased incidence of clinical signs in subsequent years (OR=11.1, P=0.018). While M. agassizii and URTD are present outside the LSTS, there is a possibility that incidence of Mycoplasma infection and URTD would increase outside LSTS if these populations were to reconnect. Population-level significance of this risk appears low, and any risk must be evaluated against the potential long-term benefits to population viability through increased connectivity.

Immune and sex-biased gene expression in the threatened Mojave desert tortoise, Gopherus agassizii.

The immune system of ectotherms, particularly non-avian reptiles, remains poorly characterized regarding the genes involved in immune function, and their function in wild populations. We used RNA-Seq to explore the systemic response of Mojave desert tortoise (Gopherus agassizii) gene expression to three levels of Mycoplasma infection to better understand the host response to this bacterial pathogen. We found over an order of magnitude more genes differentially expressed between male and female tortoises (1,037 genes) than differentially expressed among immune groups (40 genes). There were 8 genes differentially expressed among both variables that can be considered sex-biased immune genes in this tortoise. Among experimental immune groups we find enriched GO biological processes for cysteine catabolism, regulation of type 1 interferon production, and regulation of cytokine production involved in immune response. Sex-biased transcription involves iron ion transport, iron ion homeostasis, and regulation of interferon-beta production to be enriched. More detailed work is needed to assess the seasonal response of the candidate genes found here. How seasonal fluctuation of testosterone and corticosterone modulate the immunosuppression of males and their susceptibility to Mycoplasma infection also warrants further investigation, as well as the importance of iron in the immune function and sex-biased differences of this species. Finally, future transcriptional studies should avoid drawing blood from tortoises via subcarapacial venipuncture as the variable aspiration of lymphatic fluid will confound the differential expression of genes.

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

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.

Coupling gene-based and classic veterinary diagnostics improves interpretation of health and immune function in the Agassiz's desert tortoise (Gopherus agassizii).

The analysis of blood constituents is a widely used tool to aid in monitoring of animal health and disease. However, classic blood diagnostics (i.e. hematologic and plasma biochemical values) often do not provide sufficient information to determine the state of an animal's health. Field studies on wild tortoises and other reptiles have had limited success in drawing significant inferences between blood diagnostics and physiological and immunological condition. However, recent research using gene transcription profiling in the threatened Mojave desert tortoise (Gopherus agassizii) has proved useful in identifying immune or physiologic responses and overall health. To improve our understanding of health and immune function in tortoises, we evaluated both standard blood diagnostic (body condition, hematologic, plasma biochemistry values, trace elements, plasma proteins, vitamin A levels) and gene transcription profiles in 21 adult tortoises (11 clinically abnormal; 10 clinically normal) from Clark County, NV, USA. Necropsy and histology evaluations from clinically abnormal tortoises revealed multiple physiological complications, with moderate to severe rhinitis or pneumonia being the primary cause of morbidity in all but one of the examined animals. Clinically abnormal tortoises had increased transcription for four genes (SOD, MyD88, CL and Lep), increased lymphocyte production, biochemical enzymes and organics, trace elements of copper, and decreased numbers of leukocytes. We found significant positive correlations between increased transcription for SOD and increased trace elements for copper, as well as genes MyD88 and Lep with increased inflammation and microbial insults. Improved methods for health assessments are an important element of monitoring tortoise population recovery and can support the development of more robust diagnostic measures for ill animals, or individuals directly impacted by disturbance.

Integrating Gene Transcription-Based Biomarkers to Understand Desert Tortoise and Ecosystem Health.

Tortoises are susceptible to a wide variety of environmental stressors, and the influence of human disturbances on health and survival of tortoises is difficult to detect. As an addition to current diagnostic methods for desert tortoises, we have developed the first leukocyte gene transcription biomarker panel for the desert tortoise (Gopherus agassizii), enhancing the ability to identify specific environmental conditions potentially linked to declining animal health. Blood leukocyte transcript profiles have the potential to identify physiologically stressed animals in lieu of clinical signs. For desert tortoises, the gene transcript profile included a combination of immune or detoxification response genes with the potential to be modified by biological or physical injury and consequently provide information on the type and magnitude of stressors present in the animal's habitat. Blood from 64 wild adult tortoises at three sites in Clark County, NV, and San Bernardino, CA, and from 19 captive tortoises in Clark County, NV, was collected and evaluated for genes indicative of physiological status. Statistical analysis using a priori groupings indicated significant differences among groups for several genes, while multidimensional scaling and cluster analyses of transcription C T values indicated strong differentiation of a large cluster and multiple outlying individual tortoises or small clusters in multidimensional space. These analyses highlight the effectiveness of the gene panel at detecting environmental perturbations as well as providing guidance in determining the health of the desert tortoise.