Data on caprine tuberculosis in Brazil and its importance for public health are scarce. Therefore, the purpose of this study was to investigate the occurrence and examine epidemiological aspects of tuberculosis in dairy goat herds in the Triângulo Mineiro and Alto Paranaíba mesoregion in the state of Minas Gerais, Brazil. Two hundred and thirty-three goats from seven dairy farms were tuberculinized using the comparative cervical tuberculin test, taking as reference the procedures and interpretation criteria of standard results for this species. Of the animals subjected to the test, 1.29% (3/233) reacted positively, 2.14% (5/233) showed inconclusive reactions, and 96.57% (225/233) showed no reaction. All the goats that tested positive and one showing an inconclusive response were necropsied. Samples of lymph nodes, liver, lung and kidney presenting alterations were collected for a histopathological examination (hematoxylin-eosin). Material removed from abscesses and lymph node fragments were cultured in Stonebrink and Petragnani culture media. An epidemiological inquiry was carried out to analyze risk factors on the farms. The necropsied animals did not exhibit macroscopic lesions suggestive of tuberculosis or any histopathological alterations, nor did the cultures show Mycobacterium sp. growth. The goats prevalence rate to tuberculin reaction was 1.29%, which may represent a zoonotic risk in this mesoregion since these are dairy herds. The interpretation of the epidemiological records, allied to the results of the tuberculinization procedure, indicated positive reagents only on a farm where fresh cow milk was fed to newborn goat kids. / Dados referentes à tuberculose caprina no Brasil e sua importância em saúde pública são escassos. Diante disso, este trabalho teve por objetivos investigar a ocorrência e pesquisar aspectos epidemiológicos da tuberculose em rebanhos leiteiros caprinos na mesorregião do Triângulo Mineiro e Alto Paranaíba, MG. Foram tuberculinizados 233 caprinos provenientes de sete propriedades leiteiras, utilizando-se o teste cervical comparativo, tomando-se como referência os procedimentos e critérios de interpretação de resultados padronizados para esta espécie. Dos animais submetidos ao teste, 1,29% (3/233) reagiram positivamente, 2,14% (5/233) apresentaram reações inconclusivas e 96,57% (225/233) não reagiram. Todos os caprinos positivos e um com resposta inconclusiva foram submetidos à necropsia. Amostras de linfonodos, fígado, pulmão e rim que apresentaram alterações foram colhidos para exame histopatológico (Hematoxilina-Eosina). Material proveniente de abscessos e fragmentos de linfonodos foi submetido à cultura em meio de Stonebrink e Petragnani. Um inquérito epidemiológico foi procedido para análise dos fatores relacionados à ocorrência de tuberculose nas propriedades. Não foram encontradas lesões macroscópicas sugestivas de tuberculose nos animais necropsiados, nem alterações histopatológicas. Também não houve crescimento de micobactérias na cultura. A taxa de reatividade à tuberculina dos caprinos foi de 1,29 %. Por se tratar de rebanhos leiteiros, a ocorrência de tuberculose pode representar risco zoonótico na mesorregião do Triângulo Mineiro e Alto Paranaíba, MG. A interpretação dos registros epidemiológicos, associada aos resultados da tuberculinização constatou reagentes positivos somente na propriedade onde se utilizou leite bovino in natura para alimentar cabritos recém nascidos, porém a ocorrência da tuberculose não pode ser confirmada nesses caprinos. / Mestre em Ciências da Veterinárias

One key to the pathogenic potential of the mycobacteria lies in their capacity to resist destruction by infected macrophages and dendritic cells. Robust host immune responses during mycobacterial infection often involve a potent CD4, CD8 and gamma delta T cell mediated effector responses including lysis of mycobacteria infected host cells, secretion of variety of cytokines like IFN-γ etc. However, pathogenic mycobacteria survives for prolonged periods in the phagasomes of infected macrophages within the host in an asymptomatic, latent state and can reactivate years later if the host’s immune system wanes. One of the most devastating consequences of infection with mycobactreia is the formation of caseating granulomas followed by tissue destruction with liquefaction causing cavity formation. Pathogenic mycobacteria reside in these granulomas, which are formed by the accumulation of monocytes, epithelioid and foamy macrophages as well as cytolytic lymphocytes including CD8 T cells around the infection focus. In this regard, rigid balance as well as modulation of inflammatory immune responses by the host upon infection of pathogenic microbes is one of the crucial steps not only in controlling the spread of pathogen from the site of infection to reminder of host organs, but also in mounting an effective memory response so that future exposures/infections by similar pathogen can be effectively controlled. Significantly, despite this complex host response, it remains unclear, that why the immune response controls mycobacteria but does not eradicate infection. Both human and mouse studies have provided ample evidence that even in the face of an adequate immune response, mycobacteria are able to persist inside macrophages. These findings have suggested series of survival strategies employed by Mycobacterium sp. during its infection of host macrophages/dendritic cells which include, blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, decrease in secretion of proinflammatory cytokines by inducing secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. In view of above-mentioned observations, graulomas in response to pathogenic mycobacterial infections have long been considered host-protective structures formed to contain infection. In this perspective, Matrix metalloproteinase-9 (MMP-9), an important member of Zn2+ and Ca2+ dependent endopeptidases, participates in a significant manner in several aspects of host immune responses to mycobacterial infection such as graunloma formation, matrix (ECM) reorganization, lymphocytes trafficking and infiltrations, inflammation etc. MMP-9 is expressed at various clinical categories of tuberculosis disease like active cavitary tuberculosis, meningitis and pleuritis. Notably, in case of pulmonary tuberculosis, breakdown of ECM by MMP-9 forms an integral part of the granuloma formation. Importantly, Mycobacterium tuberculosis infection in MMP-9 deficient mice revealed defective bacterial proliferation, reduced bacterial burden and reduced lung macrophages recruitment compared to wild-type, in addition, to reduced ability to initiate or maintain well-formed granulomas. In this context, we explored the signaling events modulated by Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) or its novel cell wall antigens during induced expression of MMP-9 or SPI6 in macrophages. Our studies clearly demonstrate that NO, a product of iNOS activity, is responsible for M. bovis BCG-triggered activation of Notch1 in macrophages through direct regulation of Jagged1 expression as well as in generation of activated Notch1. We present the evidence that iNOS activity is a critical factor in TLR2 mediated Notch1 activation as macrophages derived from iNOS knockout (iNOS-/-), but not from wild-type (WT) mice failed to activate Jagged1 expression as well as Notch1 signaling upon M. bovis BCG infection. The loss of TLR2-mediated Jagged1 expression or Notch1 activation in iNOS-/-macrophages could be rescued by treatment with NO donor 3-morpholinosydnonimine (SIN1) or S-nitroso-Nacetylpenicillamine (SNAP). Signaling perturbations strongly implicated the role for cross talk among members of Notch1-PI3 Kinase and MAPK cascades in M. bovis BCG-TLR2– mediated activation of Notch1 target genes MMP-9 or Hes1. Chromatin immunoprecipitation experiments demonstrate that M. bovis BCG’s ability to trigger increased binding of CSL/RBP-Jk to MMP-9 promoter was severely compromised in macrophages derived from iNOS-/-mice compared to WT mice. These results are consistent with the observation that NO-triggered Notch1 signaling-mediated CSL/RBP-Jk recruitment has a positive regulatory role in M. bovis BCG-induced MMP-9 transcription. We show the correlative evidence that this mechanism operates in vivo by immunohistochemical expression analysis of activated Notch1 or its target gene products Hes1 or MMP-9 in brains of WT or iNOS-/-mice that were intracerebrally infected with M. bovis BCG. Further, activation of Notch1 signaling in vivo could be demonstrated only in granulomatous lesions in brains derived from human patients with tuberculous meningitis (TBM) as opposed to healthy individuals, validating the role of Notch1 signaling in mycobacterial pathogenesis. Briefly, we have identified NO as the pathological link between TLR2 and Notch1 signaling, which regulates the relative abundance of various immunopathological parameters including MMP-9 in macrophages. Synopsis Despite mycobacteria elicits robust host T cell responses as well as production of NO, ROI or cytokines like interferon-γ (IFN-γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. These findings clearly advocate roles for mycobacteria mediated various immune evasion strategies to modulate the signaling cascades thus leading to macrophage activation. Importantly, TLR2 triggering by mycobacteria elicits the activation of divers sets of anti or pro-apototic genes expression, a balance of which will have strong bearing on the overall cell-fate decisions across many cell types. In this regard, a novel granzyme B inhibitor, SPI6/PI9, can exhibit robust resistance to various cells including dendritic cells or tumor cells from lysis by CD8 cytotoxic T cells (CTL). SPI6/PI9 predominantly functions by inhibiting Granzyme B, an effector protease of cytotoxic granules released by CTL upon its TCR recognition of infected cells such as macrophages, dendritic cells etc. In this context, current investigation attempted to investigate molecular details involved in M. bovis BCG triggered SPI6 expression as well as the involvement of TLR2NO-Notch1 signaling axis in driving induced expression of SPI6, akin to that of MMP-9 expression. We demonstrate that M. bovis BCG trigger SPI6 expression in macrophages and requires critical participation of TLR2-MyD88 dependent NO-Notch1 signaling events. More importantly, signaling perturbations data suggest the involvement of cross talk among the members of PI3 Kinase and MAPK cascades with Notch1 signaling in SPI6 expression. In addition, SPI6 expression requires the Notch1 mediated recruitment of CSL/RBP-Jk and NF-κB to the SPI6 promoter. Functional studies strongly attribute critical involvement of SPI6 and MMP-9 in imparting protection to M.bovis BCG infected macrophages from lysis effectuated by CTL. Macrophages are principal mediators of initiation as well as activation of host inflammatory responses to pathogenic mycobacterial infection. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, envelope glycoconjugates like Lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), Trehalose 6,6′dimycolate (TDM; cord factor) etc. are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. A number of biological functions have been credited to PIM2. PIM2 was shown to trigger TLR2 mediated activation of macrophages that resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. In addition to pulmonary granuloma-forming activities, PIM2 was shown to recruit NKT cells into granulomas. Further, surface associated PIM was suggested to act as adhesins mediating attachment of M. tuberculosis bacilli to non-phagocytic cells. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether novel cell surface antigen PIM2 similar to whole M. bovis BCG bacilli can contribute to molecular signaling events leading to MMP-9 expression in macrophages. Our current study provides the evidence that PIM2 driven activation of signaling cascades triggers the expression of MMP-9. TLR stimulation by various agonists has been shown to activate Notch signaling resulting in modulation of diverse target genes involved in pro-inflammatory responses in macrophages. In this regard we demonstrated that PIM2 induced expression of MMP-9 involved Notch1 upregulation and activation of Notch1 signaling pathway in a TLR2-MyD88 manner. Enforced expression of the cleaved Notch1 in macrophages induced the expression of MMP-9. Further, PIM2 triggered significant p65 nuclear factor-κB (NF-κB) nuclear translocation that was dependent on activation of PI3 Kinase or Notch1 signaling. Furthermore, MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NFκB to MMP-9 promoter. Taken together, our observations clearly describe involvement of TLR2/iNOS in activating Notch1 and PI3 Kinase signaling during infection of macrophages with M. bovis BCG, thus effectuating the regulation of specific effector gene expressions, such as SPI6 and MMP-9. These results clearly describe the cross talk of Notch1 signaling with PI3 Kinase and MAPK pathways, thus leading to differential effects of Notch1 signaling. Overall, we believe that our work will extend the current understanding of inflammatory parameters associated with host-mycobacteria interactions which might lead to better design as well as evaluation of therapeutic potential of novel agents targeted at diverse mycobacterial diseases.

Made available in DSpace on 2011-05-04T12:36:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2010 / A tuberculose é uma doença infecciosa grave causada pelo Mycobacterium tuberculosis, podendo acometer todos os órgãos, mas em especial os pulmões. Apresenta grande incidência no mundo e ocorre em maior proporção em populações de baixas condições socioeconômicas. A tuberculose continua sendo uma das doenças crônicas mais importantes da história da humanidade, sobretudo nas áreas mais carentes do mundo com grandes repercussões sociais, pois acomete, predominantemente, a população adulta e produtiva. As estratégias de vigilância e controle da tuberculose incluem fundamentalmente o diagnóstico extemporâneo da doença, o tratamento e acompanhamento dos casos confirmados. O diagnóstico precoce constitui um dos principais problemas, pois, atualmente não há disponíveis testes rápidos, adequados e confiáveis para o diagnóstico em condições de campo e ambulatoriais. Diante disto, este trabalho realizou uma análise comparativa entre os testes sorológicos ELISA com antígenos brutos de PPD e de BCG e o teste imunocromatográfico Hexagon TB com o antígeno determinado A60 para o diagnóstico da tuberculose. Devido a características imunogênicas dos antígenos e da composição da prevalência da doença, a reprodutibilidade do ELISA-BCG foi baixa, bem como os parametros sorológicos de sensibilidade (33 por cento) e especificidade (61 por cento), comprometendo assim, a identificação entre pacientes clinicamente sadios e doentes. Os resultados também mostraram que o kit comercial Hexagon TB possui elevada acurácia (87,5 por cento) e é o mais indicado ao diagnóstico da tuberculose, pois identificou melhor os indivíduos com tuberculose e os indivíduos sadios. / Tuberculosis is a serious infectious disease caused by Mycobacterium tuberculosis, it can affect all organs, especially the lungs. It shows a higher incidence in the world and occurs more often in populations of low socioeconomic conditions. Tuberculosis remains one of the most important chronic diseases in human history, especially in more deprived areas of the world with great social impact, since it affects predominantly adult and productive population. Strategies for surveillance and control of tuberculosis include primarily the belated diagnosis of the disease, the treatment and monitoring the confirmed cases. Early diagnosis is a major problem, since there are no rapid, suitable or reliable tests available for diagnosis in field conditions and outpatients. Thus, this study conducted a comparative analysis of the serological ELISA with crude antigens PPD and BCG and Hexagon TB immunochromatographic test with antigen A60 specific for the diagnosis of tuberculosis. Due to the characteristics of immunogenic antigens and the composition of the desease's prevalence of the, the reproducibility of the ELISA, was low, as well as serological parameters of sensitivity (33%) and specificity (61%), thus compromising the identification between healthy and sick patients. The results also showed that the commercial kit TB Hexagon has high accuracy (87,5%) and is better suited for the diagnosis of tuberculosis, because it identified better the healthy and the sick individuals.

Mycobacteria are multifaceted pathogens capable of causing both acute disease as well as an asymptomatic latent infection. Host immune responses during mycobacterial infection involve potent cell effector functions including that of CD4+, CD8+ and γδT cells, macrophages and dendritic cells (DCs). Further, the critical regulators of protective immunity to mycobacterial infection include IFN-γ, IL-12, IL-23, TNF-α, lymphotoxins, CD40, nitric oxide and reactive oxygen species. However, the success of mycobacterial infection often relies in its ability to evade immune surveillance mechanisms mediated by sentinels of host immunity by modulating host signal transduction pathways and expression of immunoregulatory molecules. Therefore, the key to control mycobacterial growth and limit pathogenesis lies in the understanding the interactions between Mycobacterium and primary responders like macrophages and DCs. In this scenario, the role of pattern recognition receptors (PPRs) in orchestrating host immune responses assumes central importance. The cell surface receptors play crucial role in influencing overall immune responses. Of the PRRs, the Toll-like receptors (TLRs) form key immune surveillance mechanisms in recognition as well as control of mycobacterial infection. Among them, TLR2 is the primary interacting receptor on antigen presenting cells that recognize the invading mycobacteria. Mycobacterial cell wall constituents such as LAM, LM, PIM and 19-kDa protein have been shown to activate TLR2 signaling leading to proinflammatory responses. Recent reports have suggested that PE_PGRS antigens of M. tuberculosis interact with TLR2. For example, RV0754, Rv0978c, RV1917c have been implicated in modulation of human DCs. The 19-kDa lipoprotein, LpqH (Rv3763) and LprG (Rv1411c) utilize TLR2 signaling to inhibit macrophage responsiveness to IFN-γ triggered MHC class II expression and mycobacterial antigen presentation. Interestingly, recognition and amplification of pathogenic-specific signaling events play important roles in not only discriminating the invading microbes, but also in regulating explicit immune responses. In this context, integration of key signaling centers, which modulate host immunity to pathogenic mycobacterial infections, remains unexplored. In accordance to above observations, signal transduction pathways downstream to TLRs play a critical role in modulation of battery of host cells genes in terms of expression and production of immune modulatory cytokines and chemokines, recruitment of cellular machineries to site of infections etc. This suggests the decisive role for TLRs in modulation of host cell fate decisions. However, during the ensuing immunity to invading pathogens, beside TLR signaling pathways, various other signaling molecules are thought to execute specific functions in divergent cellular contexts. Recent studies from our laboratory have clearly demarcated a novel cross talk of TLR2-NOTCH1 and TLR2-Wnt signaling pathways during mycobacterial infections. The current study primary focuses on the broad range of cross talk of TLR2 and Sonic hedgehog (SHH) signaling pathways and its functional significance. The present investigation demonstrates that M. bovis BCG, a vaccine strain, triggers a robust activation of SHH signaling in macrophages compared to infection with diverse Gram-positive or Gram-negative microbes. This observation was further evidenced by the heightened SHH signaling signatures during in vivo scenario in cells /tissues from pulmonary tuberculosis (TB) individuals as well as tuberculous meningitis (TBM) patients. Furthermore, we show that the sustained TNF-α secretion by macrophages upon infection with M. bovis BCG is a critical necessity for SHH activation. Significantly, perturbation studies implicate a vital role for M. bovis BCG stimulated TLR2/PI3K/PKC/MAPK/NF-κB axis to induce TNF-α, that contributes to enhance SHH signaling. The TNF-α driven SHH signaling downregulates M. bovis BCG induced TLR2 signaling events leading to modulation of battery of genes that regulate various functions of macrophages genes like Vegf-a, Socs-3, Cox-2, Mmp-9 and M1/M2 genes. Importantly, utilizing whole-genome microRNA (miRNA) profiling, roles for specific miRNAs were identified as the molecular regulators that bring about the negative-feedback loop comprising TLR2-SHH signaling events. Thus, the current study illustrates how SHH signaling tightly regulates the kinetics and strengths of M. bovis BCG specific TLR2 responses, emphasizing a novel role for SHH signaling in host immune responses to mycobacterial infections. As described, variety of host factors contributes for ensuing effective host defenses and modulation of host cell fate decisions. Interestingly, avirulent pathogenic mycobacteria, including the vaccine strain M. bovis BCG, unlike virulent M. tuberculosis, cause extensive apoptosis of infected macrophages, which suggests a significant contribution of the apoptosis process to the initiation and subsequent amplification of innate as well as adaptive immune responses. Among various cues that could lead to apoptosis of host cells, the initiation of the apoptotic machinery by posttranscriptional mechanisms assumes significant importance. Among posttranscriptional control mechanisms, miRNAs are suggested to regulate several biological processes including immune responses. Various effectors of host immunity are known to be regulated by several miRNAs, and a prominent one among them, miRNA-155 (miR-155), often exhibits crucial roles during innate or adaptive immune responses. In this perspective, we identified a novel role of miR-155 during M. bovis BCG induced apoptosis of macrophages. The genetic and signaling perturbations data suggested that miR-155 regulates PKA signaling by directly targeting a negative regulator of PKA, protein kinase inhibitor alpha (PKI-α). Enhanced activation of PKA signaling resulted in induced expression of the apoptotic genes as well as Caspase-3 cleavage and Cytochrome c translocation. Thus, augmented PKA signaling by M. bovis BCG-driven miR-155 dictates cell fate decisions of infected macrophages, emphasizing a novel role for miR-155 in host immunity to mycobacterial infections. In perspective of these studies, important directives are often comprised of sequential and coordinated activation of TLR and NLR-driven signal transduction pathways, thus exhibiting foremost influence in determining the overall strength of the innate immune responses. As described, TLR2 exhibits dominant role in sensing various agonists including pathogen-associated molecular patterns (PAMPs) of microbes at the cell surface and generally considered as major effectuator of proinflammatory responses. Interestingly, NLRs like NOD1 or NOD2 often act in contrary, thus regulating anti-inflammatory responses as well as polarization of T cells towards skewed Th2 phenotype. This presents an interesting conundrum to functionality of DCs or macrophages in terms of effector functions during rapidly evolving immunological processes including effects originating from immunosuppressive effectors such as CTLA-4 or TGF-. DCs like macrophages are important sentinels of innate immunity, possesses array of PRRs that include TLRs and NOD-like receptors (NLRs). Signaling events associated with innate sensors like TLRs and NLRs often act as regulatory circuits that modulate the overall functions of DCs in terms of maturation process, cytokine or chemokine production, receptor expression, migration to secondary lymphoid organs for antigen presentation for effectuating Th polarization. TLR2, while acting as sensors for extracellular cues or endocytic network, drives signaling events in response to recognition of PAMPs including mycobacterial antigens like ESAT-6, PE_PGRS antigens, while NOD1 and NOD2 operate as cytosolic sensors initiating signaling pathways upon recognition of diaminopimelic acid (DAP) and muramyl dipeptide (MDP), components of bacterial peptidoglycan. Thus, TLRs or NOD receptors could trigger similar or contrasting immune responses by cooperative or non-cooperative sensing, consequently exhibiting immense complexity during combinatorial triggering of host DCs-PRR repertoire. In view of these observations, our current investigation comprehensively demonstrated that maturation process of human DCs were cooperatively regulated by signaling cascades initiated by engagements of TLR2, NOD1 and NOD2 receptors. Importantly, combined triggering of TLR2 and NOD receptors abolished the TGF-β or CTLA-4-mediated impairment of human DCs maturation, which required critical participation of NOTCH1-PI3K signaling cohorts. Thus, our data delineated the novel insights in modulation of macrophages and DCs effector functions by mycobacterial TLR2 or NOD agonists and broaden our understanding on the signal dynamics and integration of multiple signals from PRRs during mycobacterial infections. Altogether, our findings establish the understanding of conceptual frame work in fine tuning of TLR2 responses by SHH signaling as well as potential co-operativity among TLRs and NODs to modulate NOTCH1 dependent DCs maturation. Importantly, our study provides mechanistic and functional insights into various molecular regulators of macrophage cell fate decisions like miR-31. miR-150 and miR-155, which can fuel the search for attractive and effective drug targets and novel therapeutics to combat diseases of the hour like tuberculosis.

Thesis (MScMedSc (Biomedical Sciences. Medical Biochemistry))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Mycobacterium bovis has a wide host range and infects many wild and domestic animal species as well as humans. African buffalo (Syncerus caffer) is considered to be a wildlife reservoir of M. bovis in certain environments in South Africa, such as in the Kruger National Park (KNP) and Hluhluwe-iMfolozi Park (HiP). A detailed pathological study was conducted on 19 African buffalos (Syncerus caffer) from a herd in the HiP in South Africa. The animals tested positive to the intradermal bovine tuberculin test and were euthanazed during a test-and-cull operation to decrease the prevalence of bovine tuberculosis (bTB) in the park. The superficial, head, thoraxic and abdominal lymph nodes and the lungs were examined grossly for presence of tuberculous lesions and were scored on a 1-5 scale for macroscopic changes. The gross lesions were examined histologically and scored I-IV according to a grading system used for bTB lesions in domestic cattle. Macroscopical lesions were limited to the retropharyngeal, bronchial, and mediastinal lymph nodes and the lungs. The most frequently affected lymph nodes were the bronchial (16/19) and mediastinal (11/19). All four grades of microscopic lesions were observed, although grade II lesions were the most frequent. Acid-fast bacilli were observed only rarely. Bovine tuberculosis was confirmed by PCR analyses. All animals were in good body condition and most of the lesions were in an early stage of development, indicating an early stage of the disease. The absence of lesions in the mesenteric lymph nodes and the high frequency of lesions in respiratory tract associated lymph nodes suggest that the main route of M. bovis infection in African buffalo is inhalatory rather than alimentary. This study presents a systematic evaluation and semiquantification of the severity and stages of development of tuberculous lesions in buffalo. The results may contribute to i) the understanding of the pathogenesis of the disease, ii) the evaluation of experimental models of M. bovis infection in Syncerus caffer, and iii) the interpretation of pathological data from vaccination trials. / AFRIKAANSE OPSOMMING: Mycobacterium bovis het ‘n wye reeks van gashere en dit infekteer verskeie wilde en mak dierespesies, sowel as mense. Die buffel (Syncerus caffer) word beskou as die wild reservoir van M. bovis in sekere dele van Suid Afrika, soos in die Kruger Nasionale Park (KNP) en Hluhluwe-iMfolozi Park (HiP). ‘n Breedvoerige patologiese studie is uitgevoer op 19 buffels afkomstig vanaf ‘n trop in die HiP in Suid Afrika. Die diere het almal positief getoets vir die intradermale beestuberkulin toets en is uitgesit tydens ‘n toets-en-slag operasie met die doel om die voorkoms van beestuberkulose (bTB) in die park te bekamp. Die oppervlakkige, kop, toraks en abdominale limfknope en longe is oorsigtelik ondersoek vir die teenwoordigheid van tuberkulose letsels en was ‘n punt toegeken op ‘n skaal van 1-5 vir die teenwoordigheid van makroskopiese veranderinge. Die opsigtelike letsels is histologies ondersoek en ‘n I-IV punt toegeken volgens die gradering wat gebruik word vir bTB letsels in beeste. Makroskopiese letsels was beperk tot die retrofaringeale, brongiale, en mediastinale limfknope en in die longe. Die brongiale (16/19) en mediastinale (11/19) limfknope was meestal geaffekteerd. Al vier grade van mikroskopiese letsels is gevind, alhoewel graad II letsels die volopste was. Suur-vaste basille is slegs selde waargeneem. Beestuberkulose is bevestig deur PKR analises. Al die diere was in ‘n goeie kondisie en meeste van die letsels was in ‘n vroeë stadium van ontwikkeling, wat aandui op ‘n vroeë fase van die siekte. Die afwesigheid van letsels in die mesenteriese limfknope en die hoë frekwensie van letsels in die lugweg geassosieerde limfkliere dui daarop dat the belangrikste roete van M. bovis infeksie in die buffel deur inaseming geskied eerder as deur opname in die spysverteringskanaal. Hierdie studie bied ‘n stelselmatige evaluering en semi-kwantifisering van die graad van erns en die stadia van ontwikkeling van tuberkulose letsels in buffels. Die resultate kan bydra tot i) die begrip van die patogenese van die siekte, ii) die evaluering van eksperimentele modelle van M. bovis infeksie in Syncerus caffer, en iii) die interpretasie van patologiese data van inentingsproewe.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-05-22T19:22:37Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EstudoClinicoMorfologico.pdf: 2306884 bytes, checksum: 3364fe4e2c0a741056dfa4ce9d06de23 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-05-27T13:57:37Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EstudoClinicoMorfologico.pdf: 2306884 bytes, checksum: 3364fe4e2c0a741056dfa4ce9d06de23 (MD5) / Made available in DSpace on 2013-05-27T13:57:37Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EstudoClinicoMorfologico.pdf: 2306884 bytes, checksum: 3364fe4e2c0a741056dfa4ce9d06de23 (MD5) Previous issue date: 2011 / A dificuldade no diagnóstico definitivo da tuberculose extrapulmonar persiste principalmente devido a baixa resolutividade dos métodos convencionais disponíveis para a detecção do Mycobacterium tuberculosis. Esse estudo teve como objetivos avaliar a contribuição da técnica imuno-histoquímica (IHQ) para a detecção de Mycobacterium spp. em casos de tuberculose pleural e ganglionar com histoquímica negativa, assim como, investigar alguns aspectos clínicos, laboratoriais e morfológicos da doença. Para obtenção desta amostra fez-se a busca dos casos no Núcleo de Vigilância Epidemiológica (NVE) e Divisão de Arquivo Médico e Estatística (DAME) do Hospital Universitário João de Barros Barreto (HUJBB) e no Departamento de Anatomia Patológica da Universidade Federal do Pará (UFPA), selecionando-se aqueles que haviam realizado exame histopatológico para esclarecimento diagnóstico do caso. Foram incluídos 50 pacientes, sendo 25 com diagnóstico presuntivo de tuberculose pleural e 25 de tuberculose ganglionar. Para obtenção dos dados clínicos e laboratoriais os respectivos prontuários foram revisados, e para confirmação dos aspectos morfológicos foi realizada a revisão de todas as lâminas selecionadas. Posteriormente, cada amostra foi submetida à técnica IHQ com anticorpo polyclonal Mycobacterium bovis BCG. Encontrou-se no grupo investigado, maior frequência do sexo masculino, cuja média de idade foi de 33,8 anos (desvio padrão: 14,1) sendo a maioria procedente da cidade de Belém-Pará e com nível de escolaridade de sete ou menos anos de estudo. Os sintomas constitucionais mais frequentes em todo o grupo foram a febre e perda ponderal. Nos pacientes com tuberculose pleural, os sintomas específicos mais encontrados foram tosse, dor torácica e dispneia, e naqueles com a forma ganglionar da doença, o envolvimento da cadeia cervical isolada foi mais frequente. Infecção pelo vírus da imunodeficiência humana (HIV) ou Síndrome da Imunodeficiência Adquirida (Sida) e etilismo foram as condições de risco mais frequentemente associadas. Na tuberculose pleural, 20% dos casos cursaram com derrame pleural associado à lesão parenquimatosa, e em 60% o líquido pleural foi do tipo exsudativo. Enquanto, na forma ganglionar, em 50% dos casos evidenciou-se lesão parenquimatosa à radiografia do tórax. Neste estudo, foi inexpressiva a quantidade de participantes nos quais foi realizada a pesquisa direta e cultura para bacilo álcool - ácido resistente (BAAR) nos diversos espécimes clínicos analisados (líquido pleural, tecido pleural e ganglionar, escarro e lavado broncoalveolar) O padrão morfológico predominante em ambas as formas da doença foi o granuloma tipo tuberculoide com necrose caseosa, independente do status sorológico para o HIV. A técnica IHQ contribuiu para o diagnóstico de tuberculose pleural em 21% (4/19) das amostras de tecido pleural e em 37,5% (9/24) de tecido ganglionar. Um resultado imuno-histoquímico positivo define o diagnóstico de micobacteriose, e quando associado aos achados clínicos, laboratoriais e morfológicos torna-se uma ferramenta de grande utilidade para melhorar o diagnóstico da tuberculose extrapulmonar. / The difficulty in definitive diagnosis of extra-pulmonary tuberculosis persists, mainly due to poor solutions available from conventional methods for detection of Mycobacterium tuberculosis. This study aimed to evaluate the contribution of immunohistochemical (IHC) for detection of Mycobacterium spp, in cases of pleural and lymph node tuberculosis with negative staining, as well as investigate some clinical, laboratory and morphological aspects of the disease. To obtain this sample was made in the pursuit of cases through surveillance (NVE) and Division of Medical Archives and Statistics (DAME), University Hospital João de Barros Barreto (HUJBB) and the Department of Pathology, University of Pará (UFPA), selecting those who had performed the histopathological examination for diagnosis of the case. Fifty patients were included, twenty-five with presumptive diagnosis of pleural tuberculosis and twenty-five of lymph node tuberculosis. To obtain the clinical and laboratory data were reviewed their medical records, and to confirm the morphological aspects review was performed of all selected slides. Thereafter, each sample was subjected to IHC with polyclonal Mycobacterium bovis BCG. It was found in the investigated group, more often male, whose average age was 33.8 years (SD: 14.1) with the majority coming from the city of Belem, Pará and education level of seven or fewer years of schooling. Constitutional symptoms more frequent in the whole group were fever and weight loss. In patients with pleural tuberculosis, the most frequent specific symptoms were cough, chest pain and dyspnea, and in those with lymph node involvement of the cervical alone was more frequent. Infection with human immunodeficiency virus (HIV) or Acquired Immune Deficiency Syndrome (AIDS) and alcohol consumption were the risk conditions most frequently associated. In pleural tuberculosis, 20% of cases presenting with pleural effusion associated with parenchymal injury, and 60% of the pleural fluid was exudate type. While in the lymph nodes in 50% of the cases revealed a parenchymal lesion on chest. This study was marginal participants in the amount of which has been held to direct research and culture for bacillus acid – resistant (AFB) in various clinical specimens analyzed (pleural fluid, pleural tissue and lymph node, sputum, and broncho-alveolar lavage). The predominant morphological pattern in both forms of the disease was tuberculous granulomas with caseous necrosis, regardless of serologic status for HIV. The IHC technique contributed to the diagnosis of pleural tuberculosis in 21% (4/19) samples of pleural tissue and 37.5% (9/24) of lymph node tissue. A positive immunohistochemical result defines the diagnosis of mycobacterial disease, and when associated with clinical, laboratory and morphological finds become a valuable tool to improve the diagnosis of extra-pulmonary tuberculosis.

The eco-epidemiology of bovine tuberculosis (Tb) in wild deer (mainly red deer Cervus elaphus) in New Zealand was investigated. Bovine Tb is caused by Mycobacterium bovis. Specific aims were to clarify the likely routes of infection in deer, and to determine the status of deer as hosts of Tb, the likely rates and routes of inter- and intra-species transmission between deer and other wildlife hosts, the role of deer in spreading Tb, and the likely utility of deer as sentinels of Tb presence in wildlife. As the possum (Trichosurus vulpecula) is the main wildlife host of Tb, the research also included some investigation of transmission routes in possums. Patterns of infection were measured in 994 deer killed between 1993 and 2003. Tb prevalence varied between areas (range 8–36%). Few deer had generalised infection, with 21–68% of infected deer having no visible lesions, depending on the area. The retropharyngeal lymph nodes and oropharyngeal tonsils were commonly infected. No dependent fawns less than 0.75 years old were infected, indicating intra-species transmission is rare in wild deer. Where possums were not controlled, the net (cumulative) force of infection in young (1–4 y) deer was 0.10–0.24 per year in males and 0.09–0.12 per year in females, but much lower in older deer (less than 0.05 per year). Possum control reduced the net force of infection quickly, and eventually to zero. However, Tb persisted in possum-controlled areas through immigration of infected deer and, for almost a decade, through the survival of resident deer infected before possum control. Tb was lost from infected deer at an exponential rate of 0.13 per year, mostly as a result of deer recovering from infection rather than dying from it. Wild deer do die of Tb, but there was no discernible effect on age structure. The occurrence of infection in deer was not linked to the local deer or possum density at their kill sites (i.e. in their home range), but the area-wide prevalence of Tb in deer was closely correlated with Tb levels in possums, which were in turn correlated with area-wide measures of possum density. For wild deer in New Zealand, Tb is a persistent but usually inconsequential disease of the lymphatic system. It is acquired mainly by young independent deer, usually orally via the tonsils, and probably as a result of licking infected possums. Many species fed on deer carrion, including possums. Most possums encountering carrion did not feed on it, but a few fed for long periods. Other scavengers such ferrets (Mustela furo), hawks (Circus approximans), and weka (a hen-sized flightless native bird; Gallirallus australis) fed in a way that probably increased the infectivity of carrion to possums. Commercial deer hunting may have facilitated the historical establishment of Tb in possums. Scavenging (including cannibalism) and interactions with dead and dying possums are identified for the first time as potentially important routes for transmission of Tb to possums, and I develop new hypotheses involving peri- and post-mortem transmission in possums that explain many of the epidemiological patterns that are characteristic of the disease in possum. In continuous native forest, deer home range size averaged 250 hectares for six young females, and over twice that for two males. Over 90% of infected deer are likely to die within 2 km (females) or 6 km (males) of where they acquired Tb, but deer could occasionally carry Tb up to 30 km. Deer will be useful as sentinels, but only where other sentinels are rare, because the force of infection for a deer with a single infected possum in its home range is only 0.004 per year, compared to greater than 0.2 per year for deliberately released pigs. Deer are occasionally capable of initiating new cycles of infection in wildlife, but deer control is not essential to eradicate Tb from wildlife.

Pathogenic Mycobacteria are among the most unrelenting pathogens known to mankind as one-third of the world population is latently infected with Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis. Despite many species of mycobacteria elicits robust host T cell responses as well as production of cytokines like interferon-γ (IFN- γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. One potential mechanism by which mycobacteria may achieve a state of long-term persistence amid a robust host immune response is by modulating the signaling cascades leading to macrophage activation. Activation of proinflammatory responses by the host macrophages upon infection with mycobacteria requires the involvement of a variety of signaling events. Studies have indicated that macrophages infected with pathogenic mycobacteria produce significantly less tumor necrosis factor (TNF)-α and other proinflammatory molecules compared with infection with nonpathogenic mycobacteria, which likely play a role in enhancing mycobacterial survival in vivo. Furthermore, macrophages infected with mycobacteria become refractory to many cytokines including IFN-γ and modulation of host cell signaling responses is critical for the suppression of a generalized inflammatory response which might influence the persistence of mycobacteria within the host. In this context, Suppressor of cytokine signaling (SOCS) 3, a member of SOCS family function as negative regulators of multiple cytokine and toll like receptor induced signaling. The SOCS3 has been shown to specifically inhibit signaling by IFN-γ, IL-6 family of cytokines and can act as a negative regulator of inflammatory responses. In this regard, many species of mycobacteria including M. bovis BCG triggers the inducible expression of SOCS3. Further, it has been suggested that M. bovis BCG triggered SOCS3 and SOCS1 proteins leads to the inhibition of IFN- γ stimulated JAK/STAT signaling in macrophages. Albeit JAK/STAT signaling pathway is generally believed to be involved, STAT-independent signals are suggested to take part in the induction of SOCS proteins in many systems signifying the involvement of multiple signal pathways in regulation of SOCS expression. Further little is known about the early, receptor proximal signaling mechanisms underlying mycobacteria-mediated induction of SOCS3. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, many cell wall antigens like LAM, PIM, TDM, PE family antigens etc are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extra cellular environment in the form of exocytosed vesicles. In this context, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. PIM are suggested to be the common anchor of LM and LAM as PIM, LM, and LAM originate from identical biosynthetic pathway. PIM are present in virulent M. tuberculosis H37Rv as well as in M. bovis BCG and a number of biological functions have been recently credited to PIM2. PIM2 is suggested to trigger the activation of cells via Toll like receptor (TLR)-2 and stimulation resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. PIM2 induces proinflammatory stimuli such as TNF-α and IL-12 in murine and human macrophages in a TLR2 dependent manner. PIM exhibited pulmonary granuloma-forming activities as well as was shown to be responsible for the recruitment of NKT cells to granulomas. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether M. bovis BCG or novel cell surface antigens like PIM2 or Rv0978c, a PE-PGRS protein with unknown function can contribute to M. bovis BCG triggered molecular signaling events leading to SOCS3 expression in macrophages. Our studies clearly demonstrated that M. bovis BCG can trigger SOCS3 expression in macrophages. The inception of signaling by M. bovis BCG is TLR2-MyD88 dependent, but not TLR4 dependent. The perturbation of TLR2 signaling and the downregulation of MyD88 resulted in significant decrease in SOCS3 expression implicating the role of TLR2-MyD88 axis in M. bovis BCG triggered signaling. Experiments with cycloheximide and neutralizing antibodies to IL-10 evinced that M. bovis BCG triggered SOCS3 expression is a primary response and requires direct activation of signaling cascades. In the current study, we show for the first time that infection of macrophages with M. bovis BCG activates NOTCH1 signaling events, which leads to expression of SOCS3. The perturbation of NOTCH signaling in infected macrophages either by siRNA mediated down regulation of NOTCH1 or RBP-Jk or by inhibition with pharmacological inhibitor gamma secretase-I, resulted in the marked reduction in the expression of SOCS3. Further, the enforced expression of the NOTCH1 intracellular domain (NICD) in RAW264.7 macrophages induces the expression of SOCS3, which can be further potentiated by M. bovis BCG. Furthermore, the inhibition of TLR2 signaling by a TLR2 dominant-negative construct resulted in inhibition of NOTCH1 activation. Additionally, our results demonstrates for the first time that physical association of TLR2 with both Phosphoinositide-3 Kinase (PI3K) and NOTCH1, which suggest the significant role of TLR2 triggering by of M. bovis BCG in the activation of PI3K and NOTCH1. More importantly, signaling perturbations data suggest the involvement of cross-talk among the members of PI3K and MAPK cascades with NOTCH1 signaling in SOCS3 expression. In addition, SOCS3 expression requires the NOTCH1 mediated recruitment of CSL/RBP-Jk and Nuclear Factor-B (NF-B) to the SOCS3 promoter. A number of biological functions triggered by mycobacteria are often attributed to many of the cell wall antigens. As part of our current investigation, we explored whether two novel cell wall associated antigens namely PIM2 and a PE-PGRS antigen, Rv0978c could play as significant or crucial cell wall ingredients which imparts ability to M. bovis BCG to trigger activation of NOTCH signaling leading to SOCS3 expression. Akin to M. bovis BCG, PIM2 activates NOTCH1 signaling resulting NICD formation which leads to the expression of SOCS3 in a TLR2-MyD88 dependent manner. PIM2 mediated NOTCH1 activation, both directly influences the SOCS3 expression by serving as coactivator in RBP-Jk complex and indirectly triggers SOCS3 expression by activating PI3K-MAPK-NF-κB cascade. One important outcome of the genome sequencing project of M. tuberculosis was the discovery of two new multigene families designated PE and PPE, named for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. Many PE and PPE proteins are composed only of PE or PPE homologous domains. However, in other proteins, the PE domain is often linked to a unique domain of various lengths that is rich in alanine and glycine amino acids, termed the PGRS domain (PE-PGRS subfamily). PE family genes were suggested to play roles in the virulence of the pathogen and many members of PE family proteins are reported be localized on the surface of M. tuberculosis bacilli. Some of the PE proteins may play a role in immune evasion and antigenic variation or may be linked to virulence. Additionally, it has been suggested that the PE-PGRS subfamily of PE genes is enriched in genes with a high probability of being essential for M. tuberculosis. The uniqueness of the PE genes is further illustrated by the fact that these genes are restricted to mycobacteria. However, despite their abundance in mycobacteria, very little is known regarding the expression or the functions of PE family genes. In this context, we have chosen to study Rv0978c as a typical member of PE-PGRS family based on the following observations. Rv0978c was upregulated in TB bacilli upon infection of macrophages. Rv0978c was demonstrated to be a member of a group of genes called in vivo-expressed genomic island, which were shown to be upregulated in M. tuberculosis bacilli during infection of mice. Rv0978c was also shown to be upregulated, at least eightfold, in human brain microvascular endothelial cell-associated M. tuberculosis infection, suggesting a role for endothelial cell invasion and intracellular survival. In the current investigation, we have demonstrated that Rv0978c is hypoxia responsive gene based on promoter analysis and upregulated in M. tuberculosis during the infection of macrophages. Further, Rv0978c is associated with cell wall and is exposed outside the surface of the bacterium suggesting the possible access to intracellular compartments of the infected macrophages. In this perspective, our results clearly demonstrate that Rv0978c triggers SOCS3 expression by activating PI3K-ERK1/2-NF-B cascade in mouse macrophages. Additionally, Rv0978c elicited humoral antibody reactivities in a panel of human sera or in cerebrospinal fluid samples obtained from different clinical categories of tuberculosis patients. DNA immunizations experiments in mice clearly suggested that Rv0978c is an immunodominant antigen demonstrating significant T cell and humoral reactivites. These observations clearly advocate that Rv0978c protein is expressed in vivo during active infection with M. tuberculosis and that the Rv0978c is immunogenic. These results clearly describe the cross-talk of NOTCH1 signaling with signaling pathways like PI3K and MAPK pathways during infection of macrophages with M. bovis BCG eventually resulting in regulation of specific gene expressions, such as SOCS3. These observations lead to a possibility of differential effects of NOTCH1 signaling activated upon infection by an intracellular bacillus, which could be involved in modulation of macrophage functions depending on a local immunological milieu. Taken together, our findings suggest that, induction of Suppressors of Cytokine Signaling 3 molecule by M. bovis BCG or by its cell wall antigens represents a crucial immune subversion mechanism in order to suppress or attenuate host responses to cytokines to generate the conditions that favor survival of the mycobacteria.

Many mammals are social. The most basic social behaviour is when the actions of one conspecific are directed toward another, what we call the dyadic interaction. Both intrinsic and extrinsic factors may affect an individuals propensity to interact with other members of a population. I used a social cervid, elk (Cervus elaphus), as a model species to test the importance of intrinsic and extrinsic factors of sociality on dyadic interactions. Dyadic interactions not only form the basis for social structure and information transfer within a population, but are also routes of pathogen transmission. My objective in this thesis was thus twofold: to improve our understanding of sociobiology, but also to gain insight into how sociality may underlie the transmission of communicable wildlife disease. I used a hierarchical, autecological approach from DNA, through individual, dyad, group, subpopulation, and ultimately population to explore the effects of intrinsic factors (e.g., sex and pairwise genetic relatedness) and extrinsic factors (e.g., season, conspecific density, habitat, and elk group size) on sociality. Elk in Riding Mountain National Park (RMNP), Manitoba, Canada, are exposed to the causal agent of bovine tuberculosis (Mycobacterium bovis; TB); however, spatial variation in apparent disease prevalence suggests that TB can only persist in one subpopulation within the Park. Using the natural RMNP system and a captive elk herd that I manipulated, I explored factors that influence interaction rates and durations (as a proxy for pathogen transmission) among elk. Sexual segregation in elk results in seasonal and sex-based differences in interaction rate and duration; with interactions peaking in autumn-winter for both sexes. Female-female dyads interact more frequently than male-male dyads. However, male-male dyads interact for longer durations than do female-female dyads. Interaction rate and duration did not covary with pairwise relatedness. Conspecific density also had sex-specific results for interaction rate and duration. Whereas male-male dyadic interaction rates increase with density, female-female dyads increase until they reach a threshold and subsequently reduce their interaction rates at high density. I observed density dependence in interaction rates in experimental trials and from field data. Furthermore, social networks revealed that social familiarity (i.e., heterogeneity of interactions) can be both frequency- and- density dependent depending on the strength of the relationship (i.e., number of repeat interactions). Density also affected the likelihood that an interaction would occur; however, this was modified by vegetation association used by elk. My results reveal several ecological and evolutionary implications for information transfer and pathogen transmission. In particular, I show that seasonal inter-sex routes of transfer may exist and that transfer is likely to be density-dependent. Finally, I conclude that such transfer is modified by available resources.

Many mammals are social. The most basic social behaviour is when the actions of one conspecific are directed toward another, what we call the dyadic interaction. Both intrinsic and extrinsic factors may affect an individuals propensity to interact with other members of a population. I used a social cervid, elk (Cervus elaphus), as a model species to test the importance of intrinsic and extrinsic factors of sociality on dyadic interactions. Dyadic interactions not only form the basis for social structure and information transfer within a population, but are also routes of pathogen transmission. My objective in this thesis was thus twofold: to improve our understanding of sociobiology, but also to gain insight into how sociality may underlie the transmission of communicable wildlife disease. I used a hierarchical, autecological approach from DNA, through individual, dyad, group, subpopulation, and ultimately population to explore the effects of intrinsic factors (e.g., sex and pairwise genetic relatedness) and extrinsic factors (e.g., season, conspecific density, habitat, and elk group size) on sociality. Elk in Riding Mountain National Park (RMNP), Manitoba, Canada, are exposed to the causal agent of bovine tuberculosis (Mycobacterium bovis; TB); however, spatial variation in apparent disease prevalence suggests that TB can only persist in one subpopulation within the Park. Using the natural RMNP system and a captive elk herd that I manipulated, I explored factors that influence interaction rates and durations (as a proxy for pathogen transmission) among elk. Sexual segregation in elk results in seasonal and sex-based differences in interaction rate and duration; with interactions peaking in autumn-winter for both sexes. Female-female dyads interact more frequently than male-male dyads. However, male-male dyads interact for longer durations than do female-female dyads. Interaction rate and duration did not covary with pairwise relatedness. Conspecific density also had sex-specific results for interaction rate and duration. Whereas male-male dyadic interaction rates increase with density, female-female dyads increase until they reach a threshold and subsequently reduce their interaction rates at high density. I observed density dependence in interaction rates in experimental trials and from field data. Furthermore, social networks revealed that social familiarity (i.e., heterogeneity of interactions) can be both frequency- and- density dependent depending on the strength of the relationship (i.e., number of repeat interactions). Density also affected the likelihood that an interaction would occur; however, this was modified by vegetation association used by elk. My results reveal several ecological and evolutionary implications for information transfer and pathogen transmission. In particular, I show that seasonal inter-sex routes of transfer may exist and that transfer is likely to be density-dependent. Finally, I conclude that such transfer is modified by available resources.