Immune function alterations in Asian elephants (Elephas maximus) infected with Mycobacterium species

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

Characterization and Comparison of SLAM/CD150 in Free-Ranging Coyotes, Raccoons, and Skunks in Illinois for Elucidation of Canine Distemper Virus Disease

Canine distemper virus (CDV) is a cause of significant disease in canids and increasingly recognized as a multi-host pathogen, particularly of non-canid families within Carnivora. CDV outbreaks in sympatric mesocarnivores are routinely diagnosed in the Forest Preserve District of Cook County, Illinois. CDV is diagnosed more commonly and the disease more severe in raccoons and striped skunks than in coyotes. Research in other species suggests host cell receptors may play a role in variable disease outcome, particularly, the signaling lymphocyte activation molecule (SLAM) located on lymphoid cells. To evaluate receptor differences, partial SLAM genes were sequenced, and predicted amino acid (AA) sequences and structural models of the proposed viral interface assessed. Of 263 aligned nucleotide base pairs, 36 differed between species with 24/36 differences between canid and non-canids. Raccoon and skunk predicted AA sequences had higher homology than coyote and raccoon/skunk sequences and 8/11 residue differences were between coyote and raccoons/skunks. Though protein structure was similar, few residue differences were associated with charge and electrostatic potential surface alterations between canids and non-canids. RNAScope®(Advanced Cell Diagnostics, Silicon Valley, USA) ISH revealed low levels of expression that did not differ significantly between species or tissue type. Results suggest that differences in host receptors may impact species-specific disease manifestation

The Purpose of Schooling: Beliefs and Practices of Educators in British Schools

The overall purpose of this study was to explore what British teachers consider to be the purposes of schooling and how their beliefs impacted their classroom practice. The principal aims of the British National Curriculum informed this study, thus we examined teacher perceptions of schooling along a continuum, from academic to personal/social education. Research methodology included the use of teacher surveys, semi-structured interviews, and classroom observations in four different London schools (two elementary, two secondary). Each London school was ethnically and linguistically diverse and primarily served an economically disadvantaged student population. Our research suggests that overall, an emphasis on standardized testing has led to the exclusion of personal/social education while teachers attempted to meet the academic demands of high stakes testing. Social/personal education was typically only addressed implicitly or in response to behavior management issues. Our implications highlight the severe consequences of such trends for both British and American schools

Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation.

The FoxA family of pioneer transcription factors regulates hepatitis B virus (HBV) transcription, and hence viral replication. Hepatocyte-specific FoxA-deficiency in the HBV transgenic mouse model of chronic infection prevents the transcription of the viral DNA genome as a result of the failure of the developmentally controlled conversion of 5-methylcytosine residues to cytosine during postnatal hepatic maturation. These observations suggest that pioneer transcription factors such as FoxA, which mark genes for expression at subsequent developmental steps in the cellular differentiation program, mediate their effects by reversing the DNA methylation status of their target genes to permit their ensuing expression when the appropriate tissue-specific transcription factor combinations arise during development. Furthermore, as the FoxA-deficient HBV transgenic mice are viable, the specific developmental timing, abundance and isoform type of pioneer factor expression must permit all essential liver gene expression to occur at a level sufficient to support adequate liver function. This implies that pioneer transcription factors can recognize and mark their target genes in distinct developmental manners dependent upon, at least in part, the concentration and affinity of FoxA for its binding sites within enhancer and promoter regulatory sequence elements. This selective marking of cellular genes for expression by the FoxA pioneer factor compared to HBV may offer the opportunity for the specific silencing of HBV gene expression and hence the resolution of chronic HBV infections which are responsible for approximately one million deaths worldwide annually due to liver cirrhosis and hepatocellular carcinoma

Effects of FoxA deletion on liver FoxA1, FoxA2, FoxA3 and FoxO1 transcript levels, liver size and serum HBeAg in adult HBV transgenic mice.

<p>Quantitative analysis of the (A) FoxA1, (B) FoxA2, (C) FoxA3 and (D) FoxO1 transcripts by RT-qPCR in the HBV transgenic mice. The GAPDH transcript was used as an internal control for the quantitation of the FoxA1, FoxA2, FoxA3 and FoxO1 RNAs. The mean relative FoxA1, FoxA2, FoxA3 and FoxO1 transcript levels plus standard deviations derived from male and female FoxA-expressing (HBVFoxA2^fl/flAlbCre(-), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(-) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-)) and FoxA-deleted (HBVFoxA2^fl/flAlbCre(+), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(+) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+)) HBV transgenic mice is shown. The levels of the transcripts which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p<0.05) are indicated with an asterisk (*). Similar quantitative analysis of (E) liver size and (F) serum HBeAg levels is also shown. Average number of mice per group was 6.5±1.9 (Range: 3–9).</p

Histological analysis of liver samples from adult HBV transgenic mice.

<p>Control FoxA-expressing (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-), panels A-C) and FoxA-deleted (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+), panels D-F) HBV transgenic mice are indicated. Immunohistochemical staining indicates the presence of nuclear HBcAg throughout the liver whereas cytoplasmic staining is located primarily in the centrolobular hepatocytes in control mice (panel A) whereas HBcAg is minimally detectable in the FoxA-deleted mice (panel D). Hematoxylin and eosin (H&E) staining indicates biliary epithelial proliferation in the FoxA-deleted mice (panel E) which is absent in the control mice (panel B). Trichrome (TC) staining indicates bridging portal fibrosis in the FoxA-deleted mice (panel F) which is absent in the control mice (panel C). The black size bar is 100 μm.</p

Effect of FoxA-deletion on HBV DNA methylation distribution in adult mouse liver.

<p>The CpG DNA methylation frequency distribution across the 11, 38 and 14 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2131–2441, respectively, from male (A, C and E) and female (B, D and F) FoxA-expressing (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-); M A1A2A3Cre(-) and F A1A2A3Cre(-)) and FoxA-deleted (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+); M A1A2A3Cre(+) and F A1A2A3Cre(+)) HBV transgenic mice is shown.</p