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Title:Immune function alterations in Asian elephants (Elephas maximus) infected with Mycobacterium species
Author(s):Landolfi, Jennifer
Director of Research:Terio, Karen A.
Doctoral Committee Chair(s):Terio, Karen A.
Doctoral Committee Member(s):Maddox, Carol W.; Miller, Michele; Zuckermann, Federico
Department / Program:Pathobiology
Discipline:VMS - Veterinary Pathobiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Elephas maximus
Abstract:Tuberculosis is an important health concern for Asian elephant (Elephas maximus) populations worldwide. Most infections are due to Mycobacterium tuberculosis, the cause of human tuberculosis, though mechanisms underlying elephant susceptibility are unknown. In humans and other studied species, disease progression is dependent on the character of the host immune response. Following infection, leukocytes secrete protein mediators (cytokines) that orchestrate immune responses. Importantly, measurement of TH1 (cell-mediated) and TH2 (humoral) cytokine levels within clinical samples can provide valuable information regarding immune function during health and disease that may elucidate disease susceptibility. Disturbances in the balance between host cell-mediated and humoral components of the immune response are central to tuberculosis pathogenesis. Upon exposure, most humans mount an appropriate and effective TH1 dominant immune response, and consequently only 5-10% of humans become infected and develop active tuberculosis. In these susceptible individuals, inadequate systemic TH1 responses have been documented with progression of disease. Currently, there is a paucity of information available on elephant immune function. Considering altered immune responses to tuberculosis are believed to be instrumental in determining disease susceptibility and influencing pathogenesis in humans, it is plausible that immune function alterations may similarly contribute to tuberculosis in Asian elephants. Therefore, this study was undertaken to investigate systemic and local (pulmonary) immune responses in Asian elephants with tuberculosis. To develop molecular tools for assessment of elephant immune function, partial mRNA sequences for Asian elephant interleukin (IL)-2, IL-4, IL-10, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and beta actin were determined. Sequence information was then utilized to design elephant-specific, quantitative, real time RT-PCR and in situ hybridization assays for measurement of cytokine mRNA within samples. Employment of these molecular techniques utilizing mRNA-based detection systems facilitated sensitive and specific cytokine detection and measurement in samples from a species for which commercial reagents were not available. Real time RT-PCR assays were first used to assess normal variance in baseline cytokine levels both among different elephants and within individual elephants over time. Variance of cytokine levels within multiple samples from individual elephants was shown to be lower than variance in cytokine levels among samples from different elephants, suggesting that normal variation in cytokine levels of individual elephants was not of sufficient magnitude to preclude identification of significant differences between animals. In addition, cytokine level variance among normal Asian elephants was negligible in the cases of most analyzed cytokines. Baseline mRNA levels of IL-4, IL-10, IL-12, IFN-gamma, TNF-alpha, and TGF-beta were then measured using the elephant-specific, real time RT-PCR assays in unstimulated, RNA-preserved whole blood samples from 106 Asian elephants, 15% of which were Mycobacterium tuberculosis complex seropositive. No statistically significant differences were detected between seropositive and seronegative groups for any of the examined cytokines, though trends towards higher baseline levels of TNF-alpha, IFN-gamma and IL-4 and slightly lower levels of TGF-beta, IL-10 and IL-12 were noted in the seropositive group. Because the majority of previous studies evaluating tuberculosis systemic immune responses and cytokine imbalances in humans and other species had utilized mitogen- and/or antigen-stimulated peripheral blood mononuclear cell (PBMC) cultures rather than unstimulated, baseline whole blood samples to assess differences between positive and negative groups, the next series of experiments evaluated proliferative responses and cytokine production in PBMC cultures from 8 tuberculosis negative and 8 positive, captive Asian elephants. Standard PBMC culture techniques were first validated and optimized for use with the elephant samples. Cultures were then stimulated with M. bovis purified protein derivative (PPD-B), M. tuberculosis culture filtrate protein (CFP)-10, and M. avium PPD (PPD-A). Proliferation was assessed via brominated uridine incorporation. Cytokine mRNA was measured in RNA extracted from cultures using the elephant-specific, real time RT-PCR assays. Following stimulation with PPD-B and the mitogen, Concanavalin A, proliferation was higher in samples from positive elephants. Fold differences in tumor necrosis factor (TNF)-alpha and interleukin (IL)-12 values following stimulation with PPD-B and CFP-10 were greater in the samples from positive elephants. Levels of IFN-gamma were also higher in the samples from positive elephants. With mycobacterial infection, local immunity is the first line of defense, and the character of the local immune response will determine disease progression. In human and nonhuman primate latent disease, well-organized granulomas containing relatively low numbers of bacteria are associated with TH1 cytokine expression. In contrast, human/nonhuman primate active disease is associated with poorly organized inflammatory infiltrates, extensive necrosis , and local imbalance in TH1/TH2 cytokine levels. No previous studies had been conducted evaluating the elephant local immune response to tuberculosis or associated histologic lesions. To examine features of local immunity in elephants with tuberculosis, archival, formalin-fixed, paraffin-embedded lung samples from 5 tuberculosis negative and 9 tuberculosis positive, deceased Asian elephants were assessed. Using routine light microscopy, pulmonary lesions were described and compared. Additionally, lymphoid infiltrates within lesions were characterized by CD3 immunolabeling. Finally, expression of TH1 and TH2 cytokines were determined using in situ hybridization (ISH) for cytokine mRNA. In all elephant tuberculosis pulmonary lesions, infection was characterized histologically by granulomatous inflammation, and in most samples, inflammation was widespread and poorly organized with large central regions of necrosis and mineralization, similar to morphologic changes associated with human/non-human primate active disease. In the majority of these lesions, CD3 positive lymphocytes were rare. In a single sample, inflammation consisted of well demarcated, quiescent granulomas with numerous CD3 positive lymphocytes, compatible with latent human/non-human primate tuberculosis pulmonary lesions. Using ISH, IFN-gamma, TNF-alpha, and IL-4 mRNA were detected in a subset of the elephant pulmonary tuberculosis lesions; expression typically occurred in viable portions of granuloma walls. The information on tuberculosis immunopathogenesis gained by this study represents a foundation for investigation of the immune response to this disease in elephants. The results indicated that distinguishing features do exist in systemic immune responses between positive and negative animals and among pulmonary lesions of positive animals. In the case of systemic immune responses assessed in peripheral blood samples, antigenic stimulation was required to produce noted differences in proliferation and cytokine production. The distinction between positive and negative groups was not apparent in evaluated baseline, unstimulated samples. Specifically, proliferative responses and production of TNF-alpha, IL-12, and IFN-gamma in response to stimulation with PPD-B and CFP-10 appeared to distinguish Mycobacterium spp. infected from uninfected individuals. Results suggested these parameters are important to tuberculosis immunopathogenesis in this species and could serve as useful diagnostic markers and/or treatment monitoring tools. Additionally, investigation of elephant pulmonary tuberculosis lesions revealed a spectrum of morphology suggestive of disease stage similar to that documented in humans/nonhuman primates. Results also indicated that both TH1 and TH2 cytokines participate in the local immune response to mycobacterial infection. Though results gained by this work have established a solid foundation for the continued investigation of elephant tuberculosis immunopathogenesis, much remains to be learned. Additional and on-going investigations are necessary to further understanding of tuberculosis pathogenesis in elephants for the benefit of human and individual animal health as well as the long-term conservation of this endangered species.
Issue Date:2013-08-22
Rights Information:Copyright 2013 Jennifer Landolfi
Date Available in IDEALS:2013-08-22
Date Deposited:2013-08

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