The family Xystodesmidae (Polydesmida) includes 521 species with a center of diversity concentrated in the Appalachian Mountains. Within this family, the genus Cherokia, a monotypic taxon with the type species Cherokia georgiana, is divided into three subspecies. The last revision of this genus was made by Richard Hoffman in 1960. Here, I used morphological and molecular data sets to review the genus, and evaluate whether it is a monophyletic group. I included material from literature records and three natural history collections. Newly collected samples were obtained through a citizen science project. Morphological characters such as the shape of the paranota, body size, and coloration were evaluated. Seven gene loci were used to estimate a molecular phylogeny of the genus, and a species delimitation analysis was used to evaluate the status of the subspecies. The geographical range of Cherokia was expanded to include a newly reported state (Virginia) and ca. 160 new localities compared to the previously known range. Morphological characters such as the shape of the paranota and body size that were historically used to establish subspecies, showed a direct relation with geographical distribution and elevation (clinal variation), but not with the phylogeny. Coloration was variable and did not accord with geography or phylogeny. The phylogeny recovered a monophyletic lineage, and the species delimitation test supports a single species. The molecular and morphological evidence showed that Cherokia is a monotypic genus with the sole species Cherokia georgiana being geographically widespread and highly variable in its morphology / Master of Science in Life Sciences / Millipedes are a mega-diverse group of soil dwelling animals that feed on leaf litter. The Appalachian Mountains has a huge diversity of millipedes, in particular those in the family Xystodesmidae. Within this family, I studied the genus Cherokia, commonly known as "Georgia flat-backed millipedes". The single species in this group, Cherokia georgiana, is divided into three subspecies. The last thorough study of this genus was done by Richard Hoffman in 1960, so a modern analysis with DNA sequencing was needed to test subspecies boundaries. Here, I used hundreds of specimens from three natural history museums, and fresh specimens obtained for DNA sequencing with the help of citizen scientists. I measured the shape and size of the body and coloration patterns to determine if they were related to the geographical distribution of Cherokia. I used DNA sequencing to make an evolutionary tree of the genus. I found Cherokia individuals in Virginia for the first time and found ca. 160 new sites or locations not reported previously. The shape and size of the body was related to millipede location and elevation. Coloration was not related to geography or phylogeny, and in some localities, multiple color patterns co-existed. The genetic information from DNA sequencing indicated that all Cherokia were more closely related to each other than to any other millipede genus. In conclusion, I found that the genus Cherokia is a single species, Cherokia georgiana, that has a wide geographical distribution and a considerable diversity of body shape and color. Diversity of shape and color does not reflect subspecies boundaries but instead reflects intra-population and geographic variation.

Xylella fastidiosa subspecies fastidiosa is the causative agent of Pierce’s disease of grape and has caused significant crop stress and loss in vineyards throughout Texas. While multiple techniques are available to identify subspecies of X. fastidiosa, only simple sequence repeat markers can be used for the differentiation of isolates within individual subspecies. In this research, SSR markers were utilized to demonstrate the diversity of subsp. fastidiosa isolates from within a single vineyard. The distributions of strains defined within subsp. fastidiosa were also compared to epidemiological data to clarify any relationships. Initial results from isolation attempts indicate disease severity to have the largest impact on the success of isolation attempts with 7% of samples rated as ‘Healthy’ and 83% of samples rated as ‘Advanced’ producing successful isolations. A conventional PCR protocol employing 5 SSR markers was used to generate banding profiles for 97 isolates collected from 7 grape varieties planted in 5 blocks throughout a single Texas vineyard. SPSS statistical program was used to execute a hierarchical cluster analysis to produce a dendrogram which grouped isolates into 3 strain groups with 7% or 15% dissimilarity. Of the 3 epidemiological factors analyzed, the distribution of strains showed significant dependence on grape variety while having no dependence on disease severity or location within the vineyard.

Infectious disease continues to cause significant problems on reproductive efficiency in the cattle industry. The purpose of this project is to evaluate new testing strategies for Tritrichomonas foetus and Campylobacter fetus subsp. venerealis. This thesis describes the result of three studies that evaluated the use of real-time PCR for the identification of Tritrichomonas foetus and Campylobacter fetus subsp. venerealis in carrier bulls. The first study evaluated the specificity of a real-time PCR test for T. foetus in individual culture enriched samples, and the sensitivity of the assay for use in pooled samples of up to 25 bulls. Specificity estimates were 98.8% (95% CI 97-99.4) and 100% (95% CI 98.9-100) for culture and real-time PCR, respectively. The sensitivity of the real-time PCR assay for pooled preputial samples was: 96.8% (83.8-99.4) for pool ratios 1/3 and 1/5; 93.5% (79.3-98.2) for pool ratios 1/2, 1/15, 1/20 and 1/25; and 90.3% (75.1-96.6), and were not significantly different. However, 13 of the 217 pools tested were negative and 9 of these negative testing pools contained the same positive sample. The media in this positive sample showed evidence of contamination and could potentially explain the failure to detect T. foetus. The second study evaluated the sensitivity of a real-time PCR for the detection of T. foetus in individual and pooled direct preputial samples. Sensitivity of individual samples tested by culture, real-time PCR in direct and culture enriched samples were determined from 121 samples obtained from 9 infected bulls. Sensitivity estimates were: 95.0% (95% CI: 89.6% to 97.7%) for culture, 95.9% (95% CI: 90.7 to 98.2) for real-time PCR in cultured enriched samples, and 90.1% (95% CI: 83.5 to 94.2) for direct preputial samples and did not differ (P=0.12). Sensitivity estimates for direct pooled samples in groups of 5 or 10 were: 83.6% (95% CI: 75.6 to 89.4) and 77.3% (95% CI: 68.6-84.1), respectively and were not significantly different (P=0.08). The use of repeat sampling tested in pools by real-time PCR increased the sensitivity to 100% and 96% for 3 consecutive samples (pools of 5 or 10, respectively). The use of pooled direct preputial samples although sensitive, still requires the use of repeated sampling. The third study determined the sensitivity and specificity of a recently developed real-time PCR (qPCR) tests for Cfv. A total of 300 virgin bulls were tested by both culture and qPCR. Specificity estimates were 85% (95% CI: 80.5 to 88.6) for qPCR and 100% (95% CI: 98.7 to 100) for culture, and were significantly different (P<0.01). A total of 4 naturally infected bulls and 9 artificially infected bulls were sampled serially to obtain positive samples for a sensitivity analysis. Sensitivity estimates and 95% confidence intervals are as follows: qPCR (85.4%, 95% CI: 80.6-89.2); direct culture on blood agar (82.3%, 95% CI: 77.2-86.5), DFAT (72.1%, 95% CI: 66.2-77.4), direct culture on Skirrow agar (32.7%, 95% CI: 27.2-38.7), TEM and blood agar (30%, 95% CI: 23.4-37.5), and TEM and Skirrow agar (38.1%, 95% CI: 31-45.9). The sensitivity of the different tests evaluated varied significantly with different ambient temperatures (P<0.01). The sensitivity of the qPCR was significantly higher than any other test when temperatures exceeded 5°C. The use of repeated sampling at weekly intervals significantly improved the sensitivity of the qPCR. The real-time PCR assay for the detection of T. foetus in both individual and pooled samples appears to be highly sensitive and specific. Moreover, the possibility of using direct preputial samples provides a cost-effective diagnostic strategy. Real-time PCR in direct preputial samples for BGC diagnosis in bulls has good sensitivity and specificity. However, the use of repeated sampling maybe needed in order to maximize the ability to detect carrier bulls.

Master of Science / Department of Biomedical Sciences / Sanjeev K. Narayanan / Fusobacterium necrophorum is an anaerobic Gram-negative non spore forming rod shaped bacteria that is a normal inhabitant of the alimentary tract of humans and animals. Two subspecies of F. necrophorum have been recognized- subspecies necrophorum and subspecies funduliforme. Subspecies necrophorum is an opportunistic pathogen in animals causing diseases such as bovine hepatic abscesses and sheep foot rot while as subspecies funduliforme is linked with human oral and hepatic infections such as sore throats, Lemierre’s syndrome and hepatic abscesses. The pathogenic mechanisms of F. necrophorum are complex and are not well understood or defined. Several virulence factors such as leukotoxin, haemolysin, haemagglutinin and adhesin have been described. One of the most important factors in F. necrophorum bacterial pathogenesis is the adhesion of the bacteria to the host cell. The adhesion of the bacteria to the host cell helps it colonize the host tissue and this is followed by intracellular multiplication with dissemination to other tissues, which could ultimately lead to septicemia and death. Bacteria use adhesins which are proteins found in the outer membrane which help them bind with host receptors and this helps with the adhesion of the bacteria to the host cell. Not much is known about F. necrophorum adhesins. Here, we describe and characterize a novel adhesin.

Thesis ( |b(MSc. (Biochemistry)) -- University of Limpopo, 2017 / Medicinal plants have been used as a key source for medication and they remain to provide new therapeutic remedies to date. Extracts of Olea europaea subspecies africana leaves are used extensively in South Africa to treat various diseases traditionally. The diseases have been noted to be associated with free radicals, bacterial infections, and inflammation. However, there is little information about the antioxidant, antibacterial, and anti-inflammatory activities of the leaves of this plant in literature and the cytotoxicity of the leaf extracts is still a concern. The information about the Isolated compounds is also minimal hence this study was aimed at filling in those gaps in relation to the traditional use of the leaves in southern Africa and subsequently isolating and identifying the active compounds using bioassay-guided fractionation. Preliminary screening of the crude extracts for antioxidant, antibacterial and antiinflammatory activities indicated that the extracts possessed all biological activities. The presence of major phytochemicals in the crude extracts was determined through the use of standard chemical methods and TLC analysis. The colorimetric methods (Folin-Ciocalteau and Aluminum chloride) were used for quantification purposes. TLC-DPPH assay was used to screen antioxidant activities of the crude extracts. The observed activity was quantified using the spectrophotometric method of DPPH and reducing power. The antibacterial properties of the leaf extracts were determined by direct bioautography and the serial broth microdilution assay using E. coli, P. aeruginosa, E. faecalis and S. aureus as test bacteria. Screening of the acetone crude extract for anti-inflammatory activities was done using the LPSstimulated RAW 264.7, cells where the inhibition of ROS generation was studied. MTT assay was used to determine the cytotoxicity effects of the leaves. Isolation of bioactive compounds started with serial exhaustive extraction, followed by column chromatography packed with silica gel. NMR analysis was conducted to identify the isolated compound. The results revealed the presence of tannins, terpenoids, steroids and flavonoids with the total phenolic (99.67 ± 2.52 mg of GAE/g) and tannin content (114.33 ± 9.02 mg of GAE/g) found in high amounts. All crude extracts exhibited antioxidant activities and the antioxidant activity quantified via the DPPH assay demonstrated to xxi have EC50 value of 1.05 ± 0.0071 mg/mL. The reducing capacity was found to be dose-dependent and great significance was seen at concentration 0.5 mg/mL to 1 mg/mL that was about 2/3 of that of L-ascorbic acid (standard) at a similar concentration. Screening of the crude extracts for antibacterial activity revealed that all crude extracts except n-hexane and water extracts, inhibited the growth of the tested bacteria on the previously developed TLC plates. The activity was seen as clear zones on the bioautograms. Serial broth microdilution assay indicated that dichloromethane, acetone and ethanol had average MIC values of 0.30, 0.32 and 0.35 mg/mL against all tested bacteria, respectively. Good anti-inflammatory activity of the crude extract was demonstrated at the highest concentration of 0.90 mg/mL. MTT assay indicated that the crude extract had no adverse cytotoxic effects. This was demonstrated by the LC50 values greater than 20 µg/mL and considered non-cytotoxic according to the National Cancer Institute (NCI). Isolation following the bioassay-guided-fractionation resulted in the selection of acetone extract to isolate the bioactive compounds from as it demonstrated good antioxidant and antibacterial activities. Fractionation of the compound by column chromatography yielded three combinations (pools) of fractions and of the three from which only pool 1 was considered for further fractionation. NMR spectra information identified the isolated compound as a mixture of ursolic acid (minor) and oleanolic acid (major). This compound had antibacterial and anti-inflammatory activities and no cytotoxic effects. The leaves of Olea europaea subspecies africana have been proven to possess antioxidant, antibacterial and anti-inflammatory properties. Evaluation of the biological activities of the crude extracts was to validate the use of the leaves traditionally to treat free radical and bacterial-related diseases and potential drug that are safe and has less side effects may be produced from the leaves. / National Research Foundation (NRF)

Phosphofructokinase (PFK) from the lactic acid bacterium Lactobacillus delbrueckii subspecies bulgaricus (LbPFK) is a non-allosteric PFK with weak binding affinity for both the allosteric ligands phospho(enol)pyruvate (PEP) and magnesium adenosine diphosphate (MgADP). PEP and MgADP bind to the same allosteric binding site but exhibit opposite effects, PEP acting as an inhibitor and MgADP an activator. In 2005, Parichatttanakul, et al. solved the first crystal structure of LbPFK to 1.87 A resolution and allowed for a structural comparison of LbPFK to the allosteric forms of PFK from E. coli (EcPFK) and Bacillus stearothermophilus (BsPFK). Two additional structures of LbPFK have been determined with the first having phosphates bound at the four active sites and four allosteric sites solved to 2.20 A resolution. The second structure solved to 1.83 A resolution contains phosphates at all eight sites with the addition of the substrate fructose-6-phosphate (F6P) in the active sites. These structures are similar to the published sulfate-bound LbPFK structure. Overall, the secondary, tertiary and quaternary structure is conserved with the exception of the residues in the allosteric site. E55, H59, S211, D214, H215 and G216, as well as the long cassettes of residues 52-61 (PFKs1) and 206-218 (PFKs2) were mutated to the corresponding residue/residues in Thermus thermophilus PFK (TtPFK). PFKs1 and PFKs1 were also combined to form PFKs1s2. The single mutations along with PFKs1 and PFKs2 showed no enhancement in PEP binding, but PFKs1s2 enhanced PEP binding 10-fold with no change in MgADP binding compared to LbPFK. D12, located along the active site interface 15 A away from the allosteric site, was mutated to an alanine and exhibited enhanced binding 9-fold for both PEP and MgADP to the allosteric binding site. A crystal structure of D12A was solved to 2.30 A resolution with sulfate bound to all eight binding sites, and showed no major changes in secondary, tertiary or quaternary structure when compared to the sulfate-bound wild-type LbPFK structure. Combining D12A with PFKs1s2 (PFKs1s2/D12A) further enhanced PEP binding with a 21-fold tighter binding compared to LbPFK with MgADP binding being similar to D12A. PEP inhibition was also quantitated in PFKs1s2/D12A with a Q_ay = 0.007 plus/minus 0.0008. Coupling between PEP and F6P in PFKs1s2D12A is 2-fold stronger than the coupling measured in EcPFK and 7-fold stronger than the coupling measured in BsPFK. The coupling measured in PFKs1s2D12A is the first measured in any of the LbPFK variants.

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.

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 near-threatened southern African hedgehog, Atelerix frontalis (A. Smith, 1831) is divided into two subspecies based on its disjunct distribution of two allopatric populations. This is despite reservations because its nature and extent of geographic variation remains virtually unknown. The present study, therefore, represents the first analysis of geographic variation within A. frontalis and is based on a multidisciplinary approach involving traditional and two-dimensional geometric morphometric analysis of the cranium and mandible, and molecular data in order to test the validity of the subspecies designations. The results of all univariate and multivariate analyses of both traditional and geometric morphometric data were congruent and provide evidence for a north-westerly–south-easterly clinal pattern of variation with cranial configuration being positively correlated with both latitude and longitude. These results are supported by Neighbour-joining, Maximum Likelihood, and Maximum Parsimony analyses of Cyt-b and ND2 data that revealed no variation across a 377 bp and 1034 bp region sequenced for each gene, respectively, while a 377 bp control region sequenced revealed low levels of variation between representatives of the two recognized subspecies (0.54 % pairwise sequence divergence). These results together with the lack of pronounced steps in the clinal pattern of variation suggest that the recognition of subspecies within A. frontalis may be untenable such that its disjunct distribution may represent a recent divergence event. If this is the case, then the results in this study may have implications in the conservation management strategies for A. frontalis, since it could be argued that one disjunct population could act as a source population for the other. However, it is recommended that prior to the implementation of conservation management plans for the species, further studies involving a wide range of alternative systematic techniques need to be undertaken first in order to gain a better understanding of the nature and extent of geographic variation within A. frontalis. These suggested studies should focus on comprehensive sampling and analyses involving a range of environmental and/or climatic variables in an attempt to identify factors that may explain the disjunct distribution and the clinal pattern of variation within the southern African hedgehog. / Dissertation (MS)--University of Pretoria, 2011. / Zoology and Entomology / unrestricted

No description available.