Abstract
Many disease outbreaks appear to be facilitated by increased stress due to overcrowding, and changing environmental conditions triggered by climate variability and human activities. Currently, the health of populations is typically assessed with the tools of population dynamics: estimations of trends in abundance, mortality, and reproductive rates. However, for populations that have long generation times, this approach is sometimes too slow to provide an early warning about the impact of environmental stressors such as disease, pollution, and anthropogenic activities. We have developed new techniques for detecting chronic physiologic stress and disease in mammals, based on the molecular analysis of the expression patterns of multiple stress-activated proteins and genes. This approach represents a novel tool for health monitoring, and can provide an early warning of increased environmental stress and compromised health in elephants and other mammals. This paper describes a study in progress, in which the molecular analysis of stress is being used to explore correlations between stress level and information regarding population abundance, distribution, habitat needs, human-elephant interactions, and movements of elephants (Loxodonta africana) in the northern Botswana region. This technique will provide a more objective way to assess carrying capacity for African elephants, thus facilitating development of effective management plans for this species.
Introduction
With recent estimates in excess of 120,000 animals (M. Vandewalle, pers. comm.), the elephant populations in northern Botswana comprise the largest contiguous population remaining on the African continent.7 Wildlife management authorities are becoming increasingly concerned about the effects that large elephant herds are having on vegetation1,3,6,13 and the intensifying conflicts with local communities, resulting in pressure to reduce the number of elephants. The increasing numbers of veterinary fences in northern Botswana and the civil conflict in neighboring countries may be contributing to the high elephant concentrations seen in this region, at the same time posing barriers to long-distance elephant movements, and compromising the development of regional elephant conservation plans. Many disease outbreaks appear to be facilitated by increased stress due to overcrowding. Development of novel health-monitoring tools could guide the management of ecosystems and facilitate the conservation of key species like elephants.
The effects of stress occur on a cellular level, and are characterized by altered redox balance, DNA/protein damage and high-energy expenditure.5,8-10,14 Cellular stress triggers molecular stress response (MSR), which is a homeostasis-restoring process that has evolved in all living organisms.2,4,8,11,19,21,23 MSR is triggered within hours after perturbation, during the symptom-free phase, and persists until recovery.4,12,19,20,23 In mammals, MSR recruits many fundamental intracellular and systemic processes including cellular detoxification and oxidative stress defense, DNA/protein repair, cell cycle regulation, programmed cell death, cell adhesion, and responses by the endocrine, immune and nervous systems. These processes are useful, however when prolonged, MSR leads to disease and accelerated aging.
A new technique has been introduced recently that allows detecting MSR in micro specimens of skin.14-17 The technique, termed SRP profiling, is based on measuring expression levels of 40 stress response proteins (SRP) using immunohistochemical staining and image analysis. Large numbers of specimens can be processed simultaneously using a high throughput adaptation of SRP profiling.17 Using 93 reference individuals with known health status, it was shown that SRP profiling effectively discriminated between normal individuals and individuals with a variety of clinical diseases or in severe physiologic stress. The general pattern of stress-associated alteration in SRP expression profiles was conserved across nine mammalian and two avian species, genders, ages, and a broad range of stressors.14-17 Besides skin, SRP profiling can be applied to many other tissues and body fluids (S. Southern, unpublished data). The SRP profiling has already been applied to evaluate the impact of tuna fishery on the spotted dolphins in the Eastern Tropical Pacific17, the effects of coastal pollution on the beluga whales in the St. Lawrence River, and the idiopathic population decline of the North Atlantic right whale population16.
African elephant populations are threatened by habitat loss and/or fragmentation in association with the expansion of human populations and activities related to agriculture, ecotourism, civil war, poaching, and disease. Recognition of elevated cellular stress using SRP profiling in stress-sensitive species, such as African elephants, could provide a direct measure of the biologic impact of these factors, and serve as an early warning of compromised health, and as a guide for monitoring the impact of conservation and management strategies.
Methods
We will collect at least 60 skin samples from elephants in the northern Botswana region and assess SRP levels relative to reference specimens. Importantly, this study will be done in close collaboration with long-term research, coordinated by Conservation International (CI), designed to provide important information on the abundance, distribution, population structure, habitat needs, and transboundary movements of elephants in this area. This information, in combination with a digital land cover map of the region, a spatial elephant metapopulation model, and assessment of human-elephant conflicts, can be correlated with the presence of cellular stress to provide wildlife managers with important tools for developing elephant management programs for Botswana.
For the CI project, 45 telemetry units will be deployed, and will be distributed across the three study regions with 20 units in the Chobe/Linyanti region, 10 units in the Okavango/Moremi region, and 10 units in the Nxai Pan/Tamafupa region. Adult females in breeding herds will be the primary targets for tagging; however, several adult bull elephants will also be tagged during the project. All elephants will be darted from a helicopter using etorphine (M99: C-vet UK) following the guidelines recommended by Thouless.18 Once the elephant is immobilized and recumbent, it will be fitted with a telemetry unit, and physical and health status measurements taken. The effect of the immobilizing drug will be reversed using diprenorphine (M5050: C-vet UK). Body measurements will be taken on all elephants following the guidelines of Whyte,22 including shoulder height, back length, tusk length, tusk basal circumference and hind foot length.
Skin biopsy samples (approximately 5x15 mm) will be collected from free-ranging elephants within these same study regions (n=20 per region) using a hollow-tipped biopsy dart fired from a CO2-powered projector. Multiple samples will be taken from different sub-groups to examine differences as potentially correlated to environmental variables. All skin samples will be preserved in 10% formalin, and will be assigned code names so that the molecular stress analysis can be performed as a blind study.
The fixed specimens will be processed into multi-tissue histologic blocks and checkerboard slides will be prepared. Slides will also be prepared from six reference animals, which will serve as internal controls in each analysis. The SRP proteins will be visualized by immunohistochemical staining using commercial antibodies as previously described.14,15 The SAP expression levels will be determined both by light microscopy (expert assessment of stained cells) and image analysis (computer-assisted assessment of stained cells). Each animal will be assigned 30 parameters describing the expression pattern of the specific SRP. The SRP expression patterns of the field samples will be compared with the reference patterns (normal and highly perturbed animals) analyzed in the same experiments. Elephants with significantly elevated SRP patterns will be assessed as animals with perturbed physiology indicating increased health risk.
Results
Preliminary results using skin specimens collected from 22 normal and two diseased elephants under known-controlled conditions demonstrated that SRP profiling provided good differentiation between the diseased and the healthy elephants. Collection of skin samples from free-ranging African elephants in the northern Botswana region will occur from June 2002 through February 2005.
Discussion
This project will test the application of a new technique, SRP profiling, which provides a new approach to the detection of perturbed homeostasis in African elephants based on molecular analysis of 41 stress response proteins in field tissue specimens. This technique allows screening large numbers of specimens, as necessary for ecologic studies, to correlate the presence of cellular with various environmental and anthropogenic factors. Detection of MSR, as an indicator of significant physiologic stress impact, has the advantage that no previous knowledge of the encountered stressor is needed.
By applying SRP profiling to the ongoing long-term research coordinated by CI, our results can be correlated with information regarding the abundance, distribution, population structure, habitat needs, human-elephant conflicts, and movements of elephants in the northern Botswana region. For the first time, we will be able to correlate the presence of MSR (signaling compromised health status) in elephants with potential environmental stressors on a regional basis. Many disease outbreaks appear to be facilitated by increased stress due to overcrowding, and changing environmental conditions triggered by climate variability and human activities. Carrying capacity has traditionally been determined by evaluating impact on vegetation and population health status—both relatively subjective methodologies. This technique will provide a more objective way to assess carrying capacity for African elephants, thus facilitating the development of effective management plans for this species. This type of analysis can also be applied in other ways (e.g., studies to examine the effects of ecotourism, high-density population research, and herd health correlations with specific environmental and/or anthropogenic factors).
Acknowledgments
This work is being supported by a grant from the USFWS African Elephant Conservation Fund. The SRP profiling analysis has been supported by National Academy of Sciences Senior NRC Associateship to Sarka Southern. We thank Drs. Anna Whitehouse and John Hanks for providing us with preliminary skin samples to develop the initial laboratory protocol, and Drs. John Hanks, Curtis Griffin and Mr. Michael Chase for the opportunity to work together on this project. We also thank Anne Allen for excellent technical assistance with the SRP profiling.
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