Mycoplasmal Upper Respiratory Tract Disease Across the Range of the Threatened Mojave Desert Tortoise: Associations with Thermal Regime and Natural Antibodies

Most research of upper respiratory tract disease (mycoplasmal URTD) in the threatened Mojave Desert tortoise (Gopherus agassizii) has worked under the hypothesis that the pathogen, Mycoplasma agassizii, has a relatively consistent and predictable effect on tortoise populations across their natural range. In contrast, we hypothesized that multiple factors influence the prevalence of disease and analyzed biological and environmental variables that vary significantly across the Mojave Desert. We used multiple regression models to analyze associations between mycoplasmal URTD and the genetic structure of 24 tortoise populations, levels of natural antibody (NAb) to M. agassizii in tortoises (one component of the innate immune system), precipitation, and colder thermal regimes. We detected a significant, positive association between mean levels of NAb and seroprevalence to M. agassizii. We hypothesized that NAbs may provide tolerance to mycoplasmal infections and that more tolerant populations may act as host reservoirs of disease. We also detected significant associations between colder winters and mycoplasmal URTD, suggesting that colder winters may depress tortoise immune resistance against M. agassizii or enhance conditions for the growth of M. agassizii.     

Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise (Gopherus agassizii)

Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise (Gopherus agassizii), and anti-M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.     

Tracing Genetic Lineages of Captive Desert Tortoises in Arizona

Abstract: We genotyped 180 captive desert tortoises (Gopherus agassizii) from Kingman (n = 45), Phoenix (n = 113), and Tucson (n = 22), Arizona, USA, to determine if the genetic lineage of captives is associated with that of wild tortoises in the local area (Sonoran Desert). We tested all samples for 16 short tandem repeats and sequenced 1,109 base pairs of mitochondrial DNA (mtDNA). To determine genetic origin, we performed assignment tests against a reference database of 997 desert tortoise samples collected throughout the Mojave and Sonoran Deserts. We found that >40% of our Arizona captive samples were genetically of Mojave Desert or hybrid origin, with the percentage of individuals exhibiting the Mojave genotype increasing as the sample locations approached the California, USA, border. In Phoenix, 11.5% were Sonoran–Mojave crosses, and 8.8% were hybrids between desert tortoise and Texas tortoise (G. berlandieri). Our findings present many potential implications for wild tortoises in the Sonoran Desert of Arizona. Escaped or released captive tortoises with Mojave or hybrid genotypes have the potential to affect the genetic composition of Sonoran wild populations. Genotyping captive desert tortoises could be used to inform the adoption process, and thereby provide additional protection to native desert-tortoise populations in Arizona.     

Chronic disease in the Mojave desert tortoise: Host physiology and recrudescence obscure patterns of pathogen transmission

A seminatural, factorial‐design experiment was used to quantify dynamics of the pathogen Mycoplasma agassizii and upper respiratory tract disease in the Mojave desert tortoise (Gopherus agassizii) over 2 years. Groups of initially healthy animals were separated into serologically positive (seropositive), seronegative, and artificially infected groups and paired into 23 pens. We found no evidence of long‐term immune protection to M. agassizii or of immunological memory. Initially seronegative, healthy tortoises experienced an equal amount of disease when paired with other seronegative groups as when paired with seropositive and artificially infected groups—suggesting that recrudescence is as significant as transmission in introducing disease in individuals in this host–pathogen system. Artificially infected groups of tortoises showed reduced levels of morbidity when paired with initially seronegative animals—suggesting either a dilution effect or a strong effect of pathogen load in this system. Physiological dynamics within the host appear to be instrumental in producing morbidity, recrudescence, and infectiousness, and thus of population‐level dynamics. We suggest new avenues for studying diseases in long‐lived ectothermic vertebrates and a shift in modeling such diseases.     

Quantitative PCR method for detection of mycoplasma spp. DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii)

Mycoplasma agassizii and M. testudineum have been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Because microbiological culture methods have proven difficult to employ in wild desert tortoises, our goal was to develop a sensitive and specific qPCR method for detecting and quantifying mycoplasma DNA in nasal lavage fluid collected in the field. Primers for 16S ribosomal RNA gene sequences specific for M. agassizii and M. testudineum were designed, together with primers that recognize conserved sequences of both microorganisms. Standard curves generated with DNA extracted from known numbers of mycoplasma cells revealed a lower detection limit of approximately 5 fg. The qPCR method did not recognize normal flora DNA, and nasal lavage fluid contained no interfering substances. Nasal lavage samples collected from 20 captive desert tortoises housed at the Desert Tortoise Conservation Center (Clark County, Nevada, USA) revealed the presence of M. agassizii DNA in 100% of the tortoises. Concentrations ranged from a low of 6 pg ml^− 1 to a high of 72,962 pg ml^− 1. Only one of the tortoises was positive for M. testudineum. Interestingly, not all of the qPCR positive tortoises showed evidence of seroconversion, suggesting that they were colonized but not infected. This new quantitative method will provide a critical tool for managing threatened populations of the desert tortoise.     

Defining population structure for the Mojave desert tortoise

We used highly variable microsatellite markers to identify population structure, movement, and biological boundaries for populations of the desert tortoise, Gopherus agassizii, in the Mojave and Colorado Deserts of the southwestern United States. The Mojave desert tortoise (listed as “threatened” by the U.S. Fish and Wildlife Service) has a large geographic range, long generation time, low population densities, and little above-ground activity. Additionally, the dispersal patterns of individual tortoises are virtually unknown, making indirect methods to assess movement among populations valuable. Using Bayesian assignment tests, we detected hierarchical structuring within the Mojave desert tortoise. Three basal groups were identified, and these corresponded to the mitochondrial DNA haplotypes reported in 1989. Additional population structure was evident within each basal unit, and this structure corresponds with major geographic barriers. Our analyses suggest that gene flow among populations was historically high because levels of population differentiation were low across the range. Geographic distance explained a large proportion of variation in genetic distance (68%), which pinpoints that dispersal is limited only on a regional scale. In light of these new analyses of the genetic population structure of the Mojave desert tortoise, we make new recommendations for the number and locations of recovery units for conservation of this species.     

Are captive tortoises a reservoir for conservation? An assessment of genealogical affiliation of captive Gopherus agassizii to local, wild populations

The conservation of tortoises poses a unique situation because several threatened species are commonly kept as pets within their native ranges. Thus, there is potential for captive populations to be a reservoir for repatriation efforts. We assess the utility of captive populations of the threatened Agassiz’s desert tortoise (Gopherus agassizii) for recovery efforts based on genetic affinity to local areas. We collected samples from 130 captive desert tortoises from three desert communities: two in California (Ridgecrest and Joshua Tree) and the Desert Tortoise Conservation Center (Las Vegas) in Nevada. We tested all samples for 25 short tandem repeats and sequenced 1,109 bp of the mitochondrial genome. We compared captive genotypes to a database of 1,258 Gopherus samples, including 657 wild caught G. agassizii spanning the full range of the species. We conducted population assignment tests to determine the genetic origins of the captive individuals. For our total sample set, only 44 % of captive individuals were assigned to local populations based on genetic units derived from the reference database. One individual from Joshua Tree, California, was identified as being a Morafka’s desert tortoise, G. morafkai, a cryptic species which is not native to the Mojave Desert. Our data suggest that captive desert tortoises kept within the native range of G. agassizii cannot be presumed to have a genealogical affiliation to wild tortoises in their geographic proximity. Precautions should be taken before considering the release of captive tortoises into the wild as a management tool for recovery.     

PCR primers for microsatellite loci in the desert tortoise (Gopherus agassizii,Testudinidae)

The desert tortoise, Gopherus agassizii, is a threatened species native to the North American desert southwest and is recognized as having distinct Mojave and Sonoran populations. We identified six polymorphic microsatellite loci in the desert tortoise. All six loci were polymorphic in Sonoran samples. Five of the loci were variable in Mojave samples with varying degrees of amplification success. Two of the loci exhibited low allelic variation (2–3 alleles) while four were highly variable (8–27 alleles).     

Serologic and molecular evidence for Testudinid herpesvirus 2 infection in wild Agassiz's desert tortoises, Gopherus agassizii

Following field observations of wild Agassiz's desert tortoises (Gopherus agassizii) with oral lesions similar to those seen in captive tortoises with herpesvirus infection, we measured the prevalence of antibodies to Testudinid herpesvirus (TeHV) 3 in wild populations of desert tortoises in California. The survey revealed 30.9% antibody prevalence. In 2009 and 2010, two wild adult male desert tortoises, with gross lesions consistent with trauma and puncture wounds, respectively, were necropsied. Tortoise 1 was from the central Mojave Desert and tortoise 2 was from the northeastern Mojave Desert. We extracted DNA from the tongue of tortoise 1 and from the tongue and nasal mucosa of tortoise 2. Sequencing of polymerase chain reaction products of the herpesviral DNA-dependent DNA polymerase gene and the UL39 gene respectively showed 100% nucleotide identity with TeHV2, which was previously detected in an ill captive desert tortoise in California. Although several cases of herpesvirus infection have been described in captive desert tortoises, our findings represent the first conclusive molecular evidence of TeHV2 infection in wild desert tortoises. The serologic findings support cross-reactivity between TeHV2 and TeHV3. Further studies to determine the ecology, prevalence, and clinical significance of this virus in tortoise populations are needed.     

Habitat Use by Sonoran Desert Tortoises

ABSTRACT The distribution of desert tortoises (Gopherus agassizii) spans a wide range of biotic and abiotic conditions in the southwestern United States and northwestern Mexico, with physical and behavioral differences distinguishing tortoises inhabiting the Mojave Desert from those inhabiting the Sonoran Desert. Relative to tortoise populations in the Mojave Desert, populations in the Sonoran Desert have not been well-studied. To assess how habitat use of desert tortoises in the Sonoran Desert was influenced by topography, vegetation, geomorphology, and soil, we surveyed 40 randomly located 3-ha sites for presence of adult tortoises within a site-occupancy framework. We modeled both occupancy and detection probability as a function of environmental features, and compared those results with a logistic regression model that assumed detection probability was equal to 1. Results from both approaches agreed, suggesting that habitat selection of tortoises in the Sonoran Desert was influenced primarily by topographic and geomorphologic features rather than by vegetation. Specifically, tortoises were more likely to occupy sites that were steep (we detected tortoises on 29% of sites with mean slope <5° and 92% of sites with mean slope >15°) and predominantly east-facing (53% of sites with <5% of site facing E and 92% of sites with >20% facing E), and less likely to occupy north-facing slopes (100% of sites with <10% of site facing N and 14% of sites with >60% facing N). Our results contrast with patterns of habitat use in the Mojave Desert where tortoises primarily occupy valley bottoms. Habitat use of tortoises in Sonoran and Mojave Desert populations differ considerably, contributing to the mounting body of evidence suggesting that these geographically distinct populations may represent separate species.     

Mycoplasmosis in free-ranging desert tortoises in Utah and Arizona

Upper respiratory tract disease (URTD) has been associated with major losses of free-ranging desert tortoises (Gopherus agassizii) in the southwestern United States. This prompted a clinical examination of 63 free-ranging desert tortoises for signs of URTD and sampling for Mycoplasma agassizii, the causative agent of URTD. Tortoises were sampled from three sites in the eastern Mojave Desert (1992-93), and from three sites in the Sonoran Desert (1992-94). Plasma samples were tested for antibodies to M. agassizii using an enzyme-linked immunosorbent assay (ELISA). Nasal aspirate samples from 12 Sonoran tortoises were tested using polymerase chain reaction (PCR) test directed at the 16S rRNA gene of M. agassizii. Nasal aspirate samples from all tortoises were cultured for M. agassizii. In the Mojave Desert, nine tortoises had clinical signs of URTD and eight were seropositive for M. agassizii. In the Sonoran Desert, there were no clinical signs of URTD, but two tortoises were seropositive, and two tortoises had positive PCR results.     

Discriminating patterns and drivers of multiscale movement in herpetofauna: The dynamic and changing environment of the Mojave desert tortoise

Changes to animal movement in response to human‐induced changes to the environment are of growing concern in conservation. Most research on this problem has focused on terrestrial endotherms, but changes to herpetofaunal movement are also of concern given their limited dispersal abilities and specialized thermophysiological requirements. Animals in the desert region of the southwestern United States are faced with environmental alterations driven by development (e.g., solar energy facilities) and climate change. Here, we study the movement ecology of a desert species of conservation concern, the Mojave desert tortoise (Gopherus agassizii). We collected weekly encounter locations of marked desert tortoises during the active (nonhibernation) seasons in 2013–2015, and used those data to discriminate movements among activity centers from those within them. We then modeled the probability of movement among activity centers using a suite of covariates describing characteristics of tortoises, natural and anthropogenic landscape features, vegetation, and weather. Multimodel inference indicated greatest support for a model that included individual tortoise characteristics, landscape features, and weather. After controlling for season, date, age, and sex, we found that desert tortoises were more likely to move among activity centers when they were further from minor roads and in the vicinity of barrier fencing; we also found that movement between activity centers was more common during periods of greater rainfall and during periods where cooler temperatures coincided with lower rainfall. Our findings indicate that landscape alterations and climate change both have the potential to impact movements by desert tortoises during the active season. This study provides an important baseline against which we can detect future changes in tortoise movement behavior.     

Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise (Gopherus agassizii)

Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise (Gopherus agassizii), and anti-M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.     

Nutrition as a major facet of reptile conservation

The importance of nutrition has not received much recognition in conservation biology. However, captive breeding is possible only if nutritional requirements of animals are met, and effective habitat management requires an evaluation of nutritional resources. Three examples involving reptile conservation are presented. The formulation and testing of experimental meal-type diets proved essential for the large-scale rearing of green iguanas (Iguana iguana) in Panama and Costa Rica, thousands of which have been released into the wild. Survival and growth of captive land iguanas (Conolophus subcristatus) in the Galapagos Islands was markedly improved by development of a complete feed based on locally available ingredients; this was essential to continuation of the conservation program in which juvenile iguanas were repatriated to islands where populations had previously been exterminated. Research on the desert tortoise (Gopherus agassizii) in the Mojave Desert has identified nutritional constraints that may limit utilization of potential food plants. Thus, nutritional status of wild tortoises may depend more on availability of plant species of high nutritional quality than on overall amounts of annual vegetation. Federal and local agencies involved in the conservation and management of tortoise habitat have recognized the need to fund research on tortoise nutrition. We contend that nutrition should be given a central role in conservation programs for reptiles and other animals. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Effects of climatic variation on field metabolism and water relations of desert tortoises

We used the doubly labeled water method to measure the field metabolic rates (FMRs, in kJ kg^?1?day^?1) and water flux rates (WIRs, in ml H₂O?kg^?1?day^?1) of adult desert tortoises (Gopherus agassizii) in three parts of the Mojave Desert in California over a 3.5-year period, in order to develop insights into the physiological responses of this threatened species to climate variation among sites and years. FMR, WIR, and the water economy index (WEI, in ml H₂O?kJ^?1, an indicator of drinking of free water) differed extensively among seasons, among study sites, between sexes, and among years. In high-rainfall years, males had higher FMRs than females. Average daily rates of energy and water use by desert tortoises were extraordinarily variable: 28-fold differences in FMR and 237-fold differences in WIR were measured. Some of this variation was due to seasonal conditions, with rates being low during cold winter months and higher in the warm seasons. However, much of the variation was due to responses to year-to-year variation in rainfall. Annual spring peaks in FMR and WIR were higher in wet years than in drought years. Site differences in seasonal patterns were apparently due to geographic differences in rainfall patterns (more summer rain at eastern Mojave sites). In spring 1992, during an El Niño (ENSO) event, the WEI was greater than the maximal value obtainable from consuming succulent vegetation, indicating copious drinking of rainwater at that time. The physiological and behavioral flexibility of desert tortoises, evident in individuals living at all three study sites, appears central to their ability to survive droughts and benefit from periods of resource abundance. The strong effects of the El Niño (ENSO) weather pattern on tortoise physiology, reproduction, and survival elucidated in this and other studies suggest that local manifestations of global climate events could have a long-term influence on the tortoise populations in the Mojave Desert.     

Implications of Anthropogenic Landscape Change on Inter-Population Movements of the Desert Tortoise (Gopherus agassizii)

In the Sonoran Desert of North America, populations of the desert tortoise (Gopherus agassizii) occur in rocky foothills throughout southwestern Arizona and northwestern Mexico. Although tortoise populations appear to be isolated from each other by low desert valleys, individuals occasionally move long distances between populations. Increasingly, these movements are hindered by habitat fragmentation due to anthropogenic landscape changes. We used molecular techniques and radiotelemetry to examine movement patterns of desert tortoises in southern Arizona. We collected blood samples from 170 individuals in nine mountain ranges and analyzed variability in seven microsatellite loci to determine genetic differentiation among populations. Gene flow estimates between populations indicate that populations exchanged individuals historically at a rate greater than one migrant per generation, and positive correlation between genetic and geographic distance of population pairs suggests that the limiting factor for gene flow among populations is isolation by distance. Life history traits of the desert tortoise, a long-lived species with delayed sexual maturity, may severely constrain the ability of small populations to respond to disturbances that increase adult mortality. Historic gene flow estimates among populations suggests that recovery of declining populations may rely heavily on the immigration of new individuals from adjacent mountain ranges. Management strategies compatible with the evolutionary history of gene flow among disjunct populations will help ensure the long-term persistence of Sonoran desert tortoise populations.     

Estimating the probability of illegal desert tortoise collection in the Sonoran Desert

The expansion of road networks in desert tortoise (Gopherus agassizii) habitat in the Sonoran Desert has raised questions concerning appropriate mitigation to reduce impacts at the population level. Although some effects, namely road-kill and habitat loss, have been well documented, illegal tortoise collection has been insufficiently addressed. It has become increasingly important for wildlife and land-use managers to understand the cumulative impacts of roads on tortoises and the effect that those impacts have on population persistence. We estimated the probability of desert tortoise detection and collection along 2-lane paved, maintained gravel, and non-maintained gravel roads to evaluate whether collection probabilities were related to road type. Although collection probability did not vary by road type, the probability of desert tortoise detection by passing motorists was greatest on maintained gravel roads and fewest on non-maintained gravel and paved roads. These results have implications for effectively mitigating the impacts of roads on desert tortoises. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.     

Reference intervals and physiologic alterations in hematologic and biochemical values of free-ranging desert tortoises in the Mojave Desert

Desert tortoise (Gopherus agassizii) populations have experienced precipitous declines resulting from the cumulative impact of habitat loss, and human and disease-related mortality. Evaluation of hematologic and biochemical responses of desert tortoises to physiologic and environmental factors can facilitate the assessment of stress and disease in tortoises and contribute to management decisions and population recovery. The goal of this study was to obtain and analyze clinical laboratory data from free-ranging desert tortoises at three sites in the Mojave Desert (California, USA) between October 1990 and October 1995, to establish reference intervals, and to develop guidelines for the interpretation of laboratory data under a variety of environmental and physiologic conditions. Body weight, carapace length, and venous blood samples for a complete blood count and clinical chemistry profile were obtained from 98 clinically healthy adult desert tortoises of both sexes at the Desert Tortoise Research Natural area (western Mojave), Goffs (eastern Mojave) and Ivanpah Valley (northeastern Mojave). Samples were obtained four times per year, in winter (February/March), spring (May/June), summer (July/August), and fall (October). Years of near-, above- and below-average rainfall were represented in the 5 yr period. Minimum, maximum and median values, and central 95 percentiles were used as reference intervals and measures of central tendency for tortoises at each site and/or season. Data were analyzed using repeated measures analysis of variance for significant (P < 0.01) variation on the basis of sex, site, season, and interactions between these variables. Significant sex differences were observed for packed cell volume, hemoglobin concentration, aspartate transaminase activity, and cholesterol, triglyceride, calcium, and phosphorus concentrations. Marked seasonal variation was observed in most parameters in conjunction with reproductive cycle, hibernation, or seasonal rainfall. Year-to-year differences and long-term alterations primarily reflected winter rainfall amounts. Site differences were minimal, and largely reflected geographic differences in precipitation patterns, such that results from these studies can be applied to other tortoise populations in environments with known rainfall and forage availability patterns.     

Spatially explicit decision support for selecting translocation areas for Mojave desert tortoises

Spatially explicit decision support systems are assuming an increasing role in natural resource and conservation management. In order for these systems to be successful, however, they must address real-world management problems with input from both the scientific and management communities. The National Training Center at Fort Irwin, California, has expanded its training area, encroaching U.S. Fish and Wildlife Service critical habitat set aside for the Mojave desert tortoise (Gopherus agassizii), a federally threatened species. Of all the mitigation measures proposed to offset expansion, the most challenging to implement was the selection of areas most feasible for tortoise translocation. We developed an objective, open, scientifically defensible spatially explicit decision support system to evaluate translocation potential within the Western Mojave Recovery Unit for tortoise populations under imminent threat from military expansion. Using up to a total of 10 biological, anthropogenic, and/or logistical criteria, seven alternative translocation scenarios were developed. The final translocation model was a consensus model between the seven scenarios. Within the final model, six potential translocation areas were identified.