Johne’s disease (JD) in cattle is a disease of economic importance caused by Mycobacterium avium subspecies paratuberculosis (MAP). Studies were conducted to estimate the losses due to lower weaning weight of beef calves from MAP test-positive dams, to compare the perceptions of producers and veterinarians on the burden and economic aspects of MAP infection in cow-calf herds, and to evaluate whether testing and culling MAP test-positive cows is economically beneficial. Calves from cows with strong-positive ELISA results were 21.5 kg lighter at weaning compared to calves from ELISA-negative cows. Calves from heavy MAP shedding cows were 58.5 kg lighter, and calves from moderate shedders were 40.8 kg lighter compared to the calves from fecal-culture negative cows. Based on average feeder calf value during 2007 to 2012, these losses corresponded to US $57 per calf for ELISA strong-positive dams, US $157 per calf for heavy fecal shedder dams, and US $109 per calf for a moderate fecal shedder dam. Seedstock producers and the producers enrolled in control programs were more likely to have MAP uninfected herds. The average prevalence reported by producers was 0.8%. Compared to the small herds (<50 head), the average test-positive percentages and estimated prevalences were reported to be higher in medium (50-149) and highest in large (≥150) herds. Veterinarians reported an overall animal level prevalence in their client herds of 5%. Seedstock herds had a lower prevalence and these producers were more likely to enroll in a JD control program. Income lost due to the presence of JD in an infected cattle herd was perceived to be higher by veterinarians. Compared to the veterinarians, seedstock producers were more likely to perceive genetic losses due to culling MAP positive cows. Average annual loss due to JD in a 100 cow herd with a 7% MAP prevalence was $1,644 and $1,747 based on information provided by producers and veterinarians, respectively. Herd level production decreased with increasing prevalence. Compared to test and cull after ELISA or ELISA followed by fecal culture, using fecal culture alone provided the fastest reduction in herd prevalence. Fecal culture was also the least costly alternative based on long-term cumulative costs of an annual test and cull program. Results from the current study suggest that although testing provides faster progress, limiting within herd transmission by sale of all weaned calves and purchasing only low-risk replacements can also reduce prevalence. Results suggest that MAP infection in cows causes significant losses for the calves that are produced. While the knowledge about JD varied between producers and veterinarians, seedstock producers were more enthusiastic about MAP control programs and had lower MAP prevalence in their herds. Overall losses due to MAP infection in the herd might be substantial. It is very costly to control or eliminate MAP once the infection is established in a herd.

Background Mycobacterium tuberculosis isolates such as the Beijing and F15/LAM4/KZN families dominate in patients. The emergence of extensively drug resistant (XDR) M. tuberculosis isolates raises concern. The need to better understand the pathogenesis of M. tuberculosis isolates resulted in this work. Methods M. tuberculosis clinical isolates that belonged to the Beijing and F15/LAM4/KZN families, isolates with unique DNA fingerprints and laboratory strains were used. Isolates were grown in the presence of oxygen and then exposed to A549 alveolar and BBM bronchial epithelial cells. The number of bacilli that adhered to the epithelial cells were viewed and counted using light microscopy. Isolates grown in the presence of oxygen and under oxygen deprivation were used for subsequent assays. Invasion of A549 and BBM cells by isolates grown under these different circumstances was investigated. Based on the results, the remaining assays were performed with A549 cells only. Cytotoxicity was quantified using the Cyto Tox96 Non-Radioactive Cytotoxicity Assay kit. Morphological changes in A549 cells after exposure to the isolates were observed using the scanning electron microscopy (SEM). Real-time quantitative PCR was performed to assess the relative expression levels of four genes potentially associated with virulence (hbhA; mdp1; fdxA; hspX). Results were normalized against 16S rRNA and ftsZ gene transcription and reported as fold difference as compared to H37Rv. Results All isolates adhered to and invaded A549 cells in significantly higher numbers than BBM cells (P<0.0029). Isolates grown under oxygen deprivation displayed higher levels of virulence than their aerobic phenotype. Grouped together, the isolates belonging to the Beijing and F15/LAM4/KZN families of strains showed greater adhesion capacity (28%) than isolates with unique DNA fingerprints (5%) (P<0.05%). Three F15/LAM4/KZN isolates (two XDR-variants), were at least twice as invasive (>33%) as the most invasive Beijing isolate (15%) (P<0.05). The highest cytotoxicity level (35.7%) was produced by an XDR-F15/LAM4/KZN strain. SEM revealed bleb-like structures on bacterial cells grown under oxygen deprivation. Beijing and XDR-F15/LAM4/KZN isolates had the highest number of projections (16+5 per bacillus. The expression levels of all four genes were highest in Beijing and F15/LAM4/KZN isolates grown under oxygen deprivation and exposed to A549 cells. Conclusions Beijing and F15/LAM4/KZN strains are more virulent and their successful spread might be related to their interaction with alveolar epithelium. M. tuberculosis pathogenesis studies should include isolates grown under oxygen deprivation. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

In this study we investigated the impact of exogenous reinfection on genetically resistant and genetically sensitive sub populations. We qualitatively analysed the dynamics of TB by assuming that TB is transmitted in two ways namely homogeneous and heterogeneous modes of transmission. Analytically, we computed the fundamental thresholds used to measure disease persistence; the basic reproduction number R₀; and found that the exogenous reinfection parameters do not appear in the basic reproduction number. Hence, basic reproduction number derived in presence of exogenous reinfection does not adequately predict the course of a TB epidemic. We obtained the exogenous reinfection threshold which indicated that exogenous reinfection complicates TB dynamics. Both analytical and simulation results disclosed that when exogenous reinfection is above exogenous reinfection threshold TB dynamics were governed by a backward bifurcation implying TB may continue to invade the population despite basic reproduction number being less than one. We computed critical value of basic reproduction numbers Rᴄ and found that TB can only be eradicated if basic reproduction number is reduced below critical value Rc. Furthermore, we incorporated TB therapy in heterogeneous model among individuals with clinically active TB and performed sensitivity and uncertainty analysis using Latin Hypercube Sampling. The sensitivity and uncertainty results showed that transmission rates, reactivation rates and proportion that is genetically resistant greatly infuenced outcome variables of our TB model. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Molecular modelling has become widely used in chemistry and biology. The aim of this project is to use a range of molecular modelling techniques to study enzymic actions. This thesis consists of two parts. Part A of this thesis describes computational studies conducted for the calpain-calpastatin system. Calpain is a cysteine protease. Over-expression of calpain is associated with many diseases. Calpastatin is the naturally occurring specific regulator of calpain activity. In this part of the thesis, the dynamic conformational preferences of region B of the inhibitory domain in calpastatin were examined in detail by using molecular dynamics simulations and stochastic dynamic simulations with Monte Carlo sampling. Part B of the thesis explores the structure and function of the enzyme 3-dexoy-D-arabino-heptulosonate 7-phosphate synthase from Mycobacterium tuberculosis (MtuDAH7PS). MtuDAH7PS catalyses the first reaction of the shikimate pathway and is a target for the development of anti-tuberculosis drugs. MtuDAH7PS is found to be synergistically inhibited by combinations of aromatic amino acids (Trp+Phe or Trp+Tyr), but not by any single aromatic amino acids. In this part of the thesis, this unique mechanism of allosteric regulation in MtuDAH7PS was investigated by using a range molecular modelling techniques. Firstly protein crystal structure refinements were conducted and those crystal structures of MtuDAH7PS in complex with various ligand molecules are described in Chapter 4. Secondly, the reaction mechanism and roles of active site residues were investigated in Chapter 5, through docking calculations (both rigid docking and induced fit docking) of a series of designed active site inhibitors. Finally, Chapter 6 discusses the molecular basis of the communication mechanism of allosteric regulation in MtuDAH7PS.

La paratuberculose bovine, ou maladie de Johne, est une maladie inflammatoire intestinale chronique provoquant d’importantes pertes économiques chez les producteurs de ruminants du monde entier. Que ce soit chez la vache laitière ou de boucherie, ces pertes sont causées majoritairement par une diminution de la capacité de reproduction, la baisse de production laitière et l’amaigrissement des vaches qui perdent ainsi beaucoup de valeur à l’abattage, en plus d’être sujettes à une réforme précoce. Outre les pertes économiques, le potentiel de transmission à l’humain est un facteur non négligeable en plus d’un risque de contamination de la chaîne alimentaire. Cette maladie est causée par une bactérie intracellulaire obligatoire nommée Mycobacterium avium subspecies paratuberculosis (MAP). II n’existe actuellement aucune stratégie efficace pour combattre l’infection chez les animaux atteints. L’évolution lente de la maladie fait en sorte que les signes cliniques apparaissent tardivement, soit plusieurs années (4 à 7 ans) après l’infection initiale. Au cours de cette progression, les animaux infectés commencent à excréter le pathogène dans leur environnement. Les animaux atteints deviennent infectieux et peuvent contaminer d’autres congénères, ainsi que leur propre veau. Afin de permettre aux producteurs d’éliminer les vaches atteintes avant qu’elles n’atteignent ce stade, il s’avère important d’établir un diagnostic précoce. Actuellement, ce n’est qu’en phase sous-clinique avancée que les tests diagnostiques sont plus sensibles, soit 2 à 3 ans après le début des excrétions fécales chez les animaux infectés. L'incompréhension du manque de sensibilité des tests de dépistage et de l'évolution de cette maladie justifient les efforts de recherche dans ce domaine en vue de mieux comprendre les réponses immunitaires impliquées dans cette maladie. En effet, une meilleure connaissance des processus d’inflammation chronique pourrait aider à développer des outils diagnostiques complémentaires. Nos résultats suggèrent une dérégulation de la réponse immunitaire. Ainsi, en étudiant les composantes et caractéristiques du sang provenant de vaches infectées, il nous a été possible d’observer que les niveaux de cytokines plasmatiques telles l’interleukine 17 et l'ostéopontine se trouvent sécrétées à différents niveaux chez les vaches atteintes de paratuberculose bovine. De plus, l'analyse de la capacité de leur sérum à soutenir efficacement la prolifération des cellules mononucléées du sang périphérique révèle que le sérum de vaches infectées interfère pour atténuer la prolifération cellulaire. II semble qu’un constituant du sérum provoque une diminution de la réponse immunitaire chez les vaches malades. Les résultats offrnt une appréciation des dérèglements immunitaires provoqués par la paratuberculose bovine.

The overall objective of this thesis is to provide an enhanced understanding of the epidemiology of Mycobacterium bovis in the Greater Riding Mountain Ecosystem (GRME) and provide a scientific basis for disease management from a systems perspective now and into the future. M. bovis prevalence has been consistently higher in elk compared to white-tailed deer, and higher within a defined Core area compared to areas outside. Prevalence in both species declined significantly between 2003 and 2013. Only one infected elk was detected in 2013; the last infected white-tailed deer was detected in 2009 and the last infected cattle herd was detected in 2008. Parallel interpretation of three blood-based assays resulted in effective selective culling of elk within Riding Mountain National Park (RMNP) with predictive value negative of 100%. A lymphocyte stimulation test (LST) was the most sensitive single blood-based assay, but was difficult to perform under field conditions. Combinations of humoral antibody tests and cell-mediated tests performed better than any single test, likely detecting the broad spectrum of host pathology present. Seven of 14 risk factors were identified for wild cervids testing culture positive with the three being most strongly associated with culture positivity being geographical location (within core area), elk density and year category (sampling phase). Age, sex, and surveillance method were also significant factors, but species was not. A rapid decline in elk density in combination with fencing of hay storage yard and non-selective culling were likely key factors resulting in the M. bovis prevalence decline observed in elk, and an overall decline in prevalence from 1997 for both species. Elk were the primary reservoir species in this episystem, but are now considered a spillover host, while white-tailed deer have always been a spillover host due to lower densities and shorter life expectancy. Very limited strain diversity exists within the GRME with one spoligotype restricted to cattle and associated with a limited outbreak in five herds in the early 1990’s, and three other shared strains between cattle and wildlife. A single monomorphic type was present in white-tailed deer. Significant spatial overlap of wildlife and cattle isolates delineated a core area where management activities are now focused. The relative simplicity of this episystem has allowed significant progress on control and management to be achieved, despite being located within a national park. Wildlife surveillance will need to continue until at least 2022 in order to achieve a 95% probability of freedom using three different surveillance streams. Latent cases are likely to be extremely rare in future and unlikely to result in ongoing transmission as the factors that created this wildlife reservoir no longer exist. Wild cervids should not be considered ideal maintenance hosts for M. bovis in North America but rather facultative hosts; acting either as a reservoir or spillover host dependent on regional/local density and presence/absence of baiting and feeding.

The ESX family of genes (esxA-W) in Mycobacterium tuberculosis (Mtb) encodes 23 effector molecules influencing immunogenicity and pathogenicity. This study was aimed at identifying and evaluating variations in ESX sequence and protein expression profiles in clinical isolates and examining how diversity might influence immune responses. 23 ESX genes from 55 clinical isolates (20 Beijing, 25 KZN and 10 Other) and 3 Laboratory strains (H37Rv, H37Ra and BCG) were sequenced. 482 single nucleotide polymorphisms (SNPs) were identified in 12 ESX genes relative to H37Rv. Majority of the identified 363 nsSNPs occured in Beijing isolates. No mutations were observed in esxA, B, C, E, G, H, J, R, S and T. Six unique nsSNPs were identified in the Beijing isolates: esxI (Q20L), esxO (E52G), 2 in esxP (T3S; N83D), esxU (P63S) and esxW (T2A). Three unique nsSNPs were identified in the KZN isolates: esxK (A58T), esxL (R33S). The esxL polymorphism resulted from a dinucleotide change. ESX gene transcription levels were evaluated using RT-qPCR. Varying expression levels were observed for esxA, B, C, F, M and Q across all clinical isolates with lowest levels seen amongst the Beijing isolates. This correlated with immunoblots with confirmed decreased esxAB protein expression relative to the other strains. The Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) spectral protein profiles were quantitatively compared within and between Mtb clinical and laboratory isolates. Protein spectral profiles within the mass range of the CFP-10 protein with variations in peak intensities were observed across all isolates. QILSS and Mtb9.9 peptides were tested individually for immune responses in TB infected patients. Healthy patients displayed no responses to QILSS and Mtb9.9, strong but variable immune responses were detected for specific regions of QILSS and Mtb9.9 in TB infected patients. These findings demonstrate that differences in sequence, transcriptional profiles and protein expression patterns in ESX secreted proteins exist between clinical isolates, and may translate into differences in human immune responses. Further research is needed to correlate human host immune responses to the phenotype and genotype of the infecting strain of Mtb to determine the consequences of specific variations of the other ESX members. These studies are important for the development of improved immune diagnostics and vaccines. / Thesis (Ph.D.)-University of University of Natal, Durban, 2013.

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is one of the most prevalent infectious diseases in our world today. In order to survive within the host the bacteria need to sense and respond to changes in the environment; however, signal transduction in this bacterium is poorly understood. PknB is a serine/threonine kinase essential for the in vitro survival of M. tuberculosis and therefore a potential drug target against the bacteria. It is the goal of the current study to elucidate downstream substrates of PknB. We have found that PknB shares in vitro substrates with another serine/threonine kinase, PknH, implying the potential complexity of the signaling pathways in the bacteria. We have also provided the first description of the coupling between serine/threonine kinases PknB and PknH with a two-component system response regulator DevR, and further proposed Ser/Thr phosphorylation as the negative regulator of DevR transcription activator activity based on LC-MS/MS analysis. Finally, we have identified a previously unknown phosphoprotein glyceraldehyde 3-phosphate dehydrogenase encoded by the ORF Rv1436, which demonstrates autophosphorylation activity and which phosphorylation is independent of PknB. Overall, the current study has contributed to advance our understanding of the signal transduction pathways and phosphoproteome in Mycobacterium tuberculosis.

Johne�s disease in ruminants is caused by the pathogenic bacterium Mycobacterium avium subspecies paratuberculosis. An experimental infection model in sheep was developed as a prelude to the testing of new vaccines and the development of improved diagnostic assays for Johne�s disease. The final challenge model developed used four doses of 10⁹ viable organisms given at two to three day intervals. Gross and microscopic lesions were found in a high proportion of sheep (80%) at ten months post challenge. There was considerable variation in immune responses from animals challenged with different strains of M. paratuberculosis. Sheep challenged with a low passage laboratory culture of strain (W) M. paratuberculosis, produced strong lymphocyte transformation responses and Interferon gamma (IFN-γ) production at two months post challenge. Subsequent necropsy and culture from intestinal tissues showed only a low level of infection (25%). In comparison a primary tissue isolate of M. paratuberculosis (JD3) resulted in higher (60-90%) infection rates in orally challenged animals. The immune profile from these animals showed very little reactivity for the first three months post challenge, after which IFN-γ production could be detected. Antibody production and lymphocyte transformation response could not be measured until at least seven months post challenge. Sheep challenged with the primary tissue isolate instilled directly into the tonsil resulted in equivalent levels of Johne�s disease to those obtained with oral challenge. However, intratonsillar challenge resulted in higher levels of immune reactivity than oral challenge. The proprietary Johne�s vaccines; NeoparsecTM and GudairTM and an Aqueous vaccine were tested in sheep. The immunological reactions of the sheep to these vaccines showed some variations between the two separate studies, with the NeoparasecTM and GudairTM vaccines evoking high levels of CMI and humoral reactivity within two months of vaccination. Detailed immunological examination of gut associated lymphoid tissues were carried out on subgroups of animals that were either vaccinated or non-vaccinated and went on to develop disease or were immune to experimental challenge. The results showed that the diseased animals examined had multibacillary lesions and strong CMI and humoral responses. There were decreased proportions of CD4⁺, CD8⁺ and CD25⁺ T cells in peripheral blood and gut associated lymphatics of diseased animals compared with the immune or unchallenged subgroups. Profiles from the immune subgroups showed a stronger lymphocyte transformation response than case matched diseased animals. Tissues from immune animals showed increased proportions of B cells above those seen in diseased or unchallenged animals. This study has resulted in the development of a robust experimental sheep model in which Johne�s disease occurs in a high proportion of challenged animals. Critical time points for the establishment of infection or disease have been determined. It can be used in the future to evaluate protective efficacy of vaccines or to critically chart immunological profiles that are associated with infection, disease or protective immunity. Considerable research is needed to develop improved diagnostic tests to identify patterns of immunity during the early stages of infection or while the animal has subclinical disease.

Thesis (M.S.)--University of Akron, Dept. of Biology, 2007. / "December, 2007." Title from electronic thesis title page (viewed 02/23/2008) Advisor, Francisco Moore; Co-Advisor, Brian Bagatto; Committee members, Peter Niewiarowski, Joel Duff; Department Chair, Bruce Cushing; Dean of the College, Ronald Levant; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.