No description available.

No description available.

No description available.

No description available.

No description available.

The cyst nematode, Globodera tabacum tabacum Behrens, and the parasitic angiosperm, Egyptian broomrape, Orobanche aegyptiaca Pers., are obligate root parasites that cause severe yield and quality loss of many important crop hosts. Although these represent two diverse classes of parasites, they have significant similarities in the modes of parasitism and complex interactions with their hosts. Conventional control methods have had limited success in controlling these parasites. The overall objective of this research was to engineer resistance to the cyst nematode and Egyptian broomrape by expressing genes encoding parasite specific toxins under the control of parasite-responsive promoters using tobacco (Nicotiana tabacum L. cv. Xanthi). For nematode resistance, an anti-feeding strategy was employed utilizing the tomato proteinase inhibitor I (PI-I) gene as a nematode specific toxin. Transgenic tobacco plants were generated that expressed genes encoding an intracellarly retained or secreted form of tomato PI-I under the control of the nematode-inducible promoter, derived from tomato (Lycopersicon esculentum L.) Hmg2 gene. Our goals were to determine the effectiveness of local PI-I expression on nematode resistance and to determine if intracellular or extracellular PI-I deposition enhances resistance. Two constructs were generated that contained either the coding region of the tomato PI-I gene, lacking the signal sequence (EM1), or the coding region of PI-I including the signal sequence (EM2), fused to the nematode-responsive Hmg2 promoter. Transgenic PI-I plants were inoculated with G. t. tabacum cysts and evaluated for nematode interactions. Our results suggest that local expression of intercellular of PI-I significantly reduced cyst production when compared to the nontransformed controls. For broomrape resistance, a well characterized R/avr gene pair, the tobacco N resistance gene and the tobacco mosaic virus replicase (TMV) gene, was utilized to create novel gene-for-gene resistance via a N gene-mediated hypersensitive response (HR) to limit broomrape parasitism. The bean (Phaselous vulgaris L.) chalcone synthase 8 (CHS8) promoter has been characterized as a broomrape–responsive promoter. We introduced the CHS8:TMV replicase gene construct into tobacco plants that contains an endogenous N gene. Transgenic tobacco plants were inoculated with O. aegyptiaca seeds and monitored for parasite attachment and development. The expression of the TMV replicase leads to a significant reduction in broomrape parasitism. These genetic engineering strategies show promise in enhancing resistance to these destructive parasites. / Ph. D.

Coccidia are obligate intracellular parasites belonging to the phylum Apicomplexa. Many coccidia are of medical and veterinary importance such as Cystoisospora species and Toxoplasma gondii. The need to discover new anticoccidial therapies has increased due to development of resistance by the parasite or toxicity issues in the patient. The goals of this work were to develop a model for canine coccidiosis while proving that Cystoisospora canis is a true primary pathogen in dogs and to determine the efficacy of a new anticoccidial agent. A canine coccidiosis model would be useful in evaluating new anticoccidial treatments. Oral infections with 5 X 104 (n=2) and 1 X 105 (n=20) sporulated C. canis oocysts were attempted in 22 purpose bred beagle puppies. Clinical signs associated with disease were observed in all dogs. Bacterial and viral pathogens were ruled out by transmission electron microscopy (TEM) and bacterial growth assays. Development of C. canis in cell culture was also evaluated. The efficacy of ponazuril, a new anticoccidial drug, was examined in T. gondii. In vitro studies were conducted to determine the activity of ponazuril on tachyzoites and how this agent affects development of apicomplexcan parasites. The tachyzoite production assay was conducted. Ponazuril at a dose of 1.0 µg/ml had a significant affect on tachyzoite reproduction. Comparisons were made on how ponazuril affects T. gondii and Neospora caninum. Inhibition of T. gondii tachyzoites occurred after the second round of replication and with N. caninum tachyzoites after 4 rounds of replication. Results of TEM revealed ponazuril affects replication of T. gondii and N. caninum differently. The efficacy of ponazuril to prevent and treat acute and chronic toxoplasmosis was investigated. Mice treated prophylactically with ponazuril were completely protected from developing an acute T. gondii infection. Fatal toxoplasmosis was prevented in mice starting treatment 3 and 6 days post infection at a dose of 20 mg/kg. Immunohistochemistry was used to evaluate ponazuril's effect on chronic toxoplasmosis. Sections of brain were scored according to the number of tissue cysts present. Ponazuril also proved to be highly active against toxoplasmic encephalitis in an interferon-gamma knockout mouse model. / Ph. D.

Endometriosis is a reproductive disease affecting women in their prime reproductive years characterized by the presence of endometrial glands and stroma in ectopic locations. Animal models have been proven to be indispensable not only for the study of the disease but also to develop better non-invasive diagnostic imaging modalities. A major limitation with the diagnosis of endometriosis is the lack of a specific and sensitive non-invasive diagnostic test. Our objective was to develop a domestic animal model of endometriosis suitable for serial diagnostic imaging procedures and assessment of therapies. Two major studies were conducted to achieve this objective. First study involved in vitro whole tissue-explant culture and surgical induction of endometriosis in dog, pig and sheep to choose the most suitable model. For in vitro co-culture, dog, pig and sheep endometrium was placed on visceral peritoneum for 24 to 72 h to assess the degree of attachment and adhesion characteristics of endometrium (epithelium, glandular and stromal cells). Surgical induction of endometriosis was tested in dog, sheep (n=5 each) and pig (n=4) using autologous endometrial (n=4 grafts per animal) and fat grafts sutured to visceral (urinary bladder surface in dog and pig, uterus in sheep) and parietal (abdominal wall) peritoneum. Sham surgeries were performed in control group animals (dog and sheep n=5, pig n=3) using fat grafts alone. Animals were euthanized between 80-110 days post-surgery. Size, gross characteristics and histopathologic features of endometriotic lesions were recorded. During in vitro explant culture, surface epithelial, stromal and glandular cells of endometrium were capable of attaching to visceral peritoneum within 24 hours with and without an intact layer of mesothelial lining in dog, pig and sheep. The proportion of successful endometrial attachments were greater at 24h compared to 72h (15/18 vs. 7/18, p=0.008; data combined among species) with intermediate attachment at 48h (12/15). Following surgical induction, there was no difference (p>0.05) in proportion of successful tissue grafts placed on serosal surface of visceral vs. parietal peritoneum in dog (10/10 vs. 10/10), pig (7/8 vs. 8/8) or sheep (7/10 vs. 8/10). A variety of outcomes (endometriotic cysts with sero-sangunous fluid, solid lesions, vesicles, absence of lesions) were found. The proportion of cystic lesions was greater (p<0.01) in dog (19/20 grafts) than in pig (8/16) and sheep (5/20). Further, the area of endometriotic lesions at euthanasia was larger (0.89 ± 0.11 cm2) compared to that at the time of surgery (0.50 ± 0.09 cm2) in dog, whereas, the size of lesions decreased by half or more (p<0.05) in pig and sheep. Combined among grafting sites (visceral and parietal peritoneum) and species, a greater proportion (p=0.015) of surgical sites had adhesions in treatment (12/14) versus control group animals (5/13). The wall of majority of endometrial cysts in dogs were characterized by simple cuboidal/columnar epithelium, endometrial glands (normal, dilated and cystic), subepithelial capillary network and presence of stromal and smooth muscle cells. Hemorrhage and/or hemosiderin-laden macrophages were observed in the cystic lesions in dog. Development of a greater proportion of growing lesions in the form of endometriotic cysts in dogs compared to sheep and pig led us to conclude dog as a better suitable domestic animal model for endometriosis than sheep and pig. Second study involved assessing the usefulness and limitations of ultrasonography, magnetic resonance imaging (MRI) and positron emission tomography-computed tomography (PET-CT) in detecting cystic endometriotic lesions in dog and sheep. Surgical induction of endometriois was performed in dogs (n=5) and sheep (n=5) using autologous endometrial grafts (n=4 grafts per animal) and fat grafts sutured to visceral peritoneum (urinary bladder in dogs, uterus in sheep) and parietal peritoneum (ventral abdominal wall). Weekly ultrasonography was performed from Week 1-9 post-surgery and day of euthanasia (Week 14-15). T1 and T2 weighted weighted MRI images (n=2 each for dog and sheep) and PET-CT (n=3 dog, n=1 sheep) using 18F-fluorodeoxyglucose (18F-FDG) as radiolabel was performed between Week 13-15 post-surgery in dog and sheep. Gray-scale B-mode ultrasonography was able to detect endometriotic cysts (0.25-1.75cm) on urinary bladder and abdominal wall in dogs; endometrial grafts Week 1 post-surgery appeared as a homogenous, hypoechoic masses, following which they grew larger with evidence of cyst formation by Week 5. Cysts were undetectable from Week 10-13, whereas they appeared as homogenous masses with a hypoechoic fluid-filled cavity with diffuse hyperechoic echoes and low vascularisation (Color-Doppler imaging) by Week 14-15. In sheep, endometrial grafts were detected as hypoechoic masses Week 1 post-surgery that became smaller until no detectable lesions were visible beyond Week 6-7. Cysts in dogs and sheep appeared hyperintense on T2 and hypointense on T1 weighted images. 18F -FDG PET-CT did not show hypermetabolic activity in endometriotic cysts in dogs and sheep. In conclusion, MRI appeared to provide the most definitive diagnostic images of endometriotic cysts in dogs and sheep, particularly for lesions in sheep which were not evident by ultrasonography. However, ultrasonography was sufficient to characterize most endometriotic cysts in dogs. Further research needs to be carried out to develop specific PET tracers for endometriosis.

The diversification of unicellular, auto- and heterotrophic protists and the appearance of multicellular microorganisms is recorded in numerous Tonian age successions worldwide, including the Visingsö Group in southern Sweden. The Tonian Period (1000-720 Ma) was a time of changes in the marine environments with increasing oxygenation and a high input of mineral nutrients from the weathering continental margins to shallow shelves, where marine life thrived. This is well documented by the elevated level of biodiversity seen in global microfossil record. The Visingsö Group contains a taxonomically rich assemblage of cyanobacteria, stromatolites, algal phytoplankton, and vase-shaped microfossils. A new study of organic-walled, phytoplanktic microfossils, which are extracted by palynological method from the Visingsö 1 borehole samples, reveals the presence of morphologically disparate taxa. They are in gross cysts of microalgae (Pterospermopsimorpha, Pterospermella, Cerebrosphaera, Trachysphaeridium, Simia and certain Leiosphaeridia with pylome) and some are of uncertain affinities (acritarchs). Representative taxa of two lineages among green algae, Prasinophyceae and Chlorophyceae, are recognized. Cyanobacterial clusters and filaments are abundant and specimens of multicellular, yet systematically unrecognized taxa are recorded. Taxonomically, the assemblage is similar to some from other successions distributed along the margins of Baltica, Laurentia and Siberia in the Tonian Period. The ecological habitats of those organisms are inferred by comparing with their potential modern analogues and from the sedimentological setting of the upper formation of the Visingsö Group. / Denna studie handlar om biodiversiteten och den biologiska affiniteten av mikrofossil från den neoproterozoiska eran, tonianperioden (1000-720 Ma). De har extraherats från övre formationen av Visingsögruppen i södra Sverige.Mikrofossilen har organiska cellväggar, är encelliga och har förmodats representera algcystor (resistenta reproduktiva strukturer), cyanobakterier, och andra organismer av okänd tillhörighet. Neoproterozoikum har den högsta graden av biologisk diversitet under prekambrium. Det är därför viktigt att studera diversiteten för att förstå utvecklingen av biosfären under denna period i samband med utvecklingen av miljöer. Den studerade samlingen härrör från ett borrhål på Visingsö i Vättern, och visar på större diversitet än från tidigare studier.Denna nya studie syftar till att bestämma biodiversiteten i den övre formationen av Visingsögruppen och att känna igen affiniteten av mikrofossilen med organiska väggar och deras ekologi. Vissa av de undersökta mikrofossilen hör sannolikt till grönalgerna. Kluster och fiber av cyanobakterier är rikligt förekommande, och några prover är ej biologiskt igenkännbara. Med hjälp av moderna analoger och sedimentologiska data är ekologin hos dessa mikrofossil utläs

Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are economically important pests of potato (Solanum tuberosum) crops in potato growing regions worldwide. Integrated management is under threat, with effective nematicides increasingly being withdrawn on environmental and health grounds. Alternative strategies are urgently needed and trap cropping could be one of them. The non-tuber-bearing Solanum sisymbriifolium is regarded as an effective trap crop for PCN with strong hatching ability and immunity to PCN infection and has been used in the UK and The Netherlands. However, its mode of action is unknown. In order to shed light on the mode of action so that a novel control strategy could be identified, the interactions between G. pallida and S. sisymbriifolium were investigated using in vitro bioassays. In choice assays, G. pallida J2s were equally attracted to the roots of S. sisymbriifolium and to those of S. tuberosum. However, potato root diffusate (PRD), which is routinely used to induce PCN hatch, failed to attract G. pallida J2s in chemotaxis bioassays, indicating hatching factors (HFs) and soluble compounds present in PRD are not involved in attraction of G. pallida J2s to potato roots. The J2s invaded the roots of S. sisymbriifolium in large numbers but failed to develop further. To facilitate continuous observation of nematode development, a novel in vitro method was devised with the use of Pluronic F-127, which requires no sterilisation, and the life cycle of G. pallida was successfully observed in S. tuberosum roots. Quantitative real-time polymerase chain reaction analyses of defence related genes of S. tuberosum and S. sisymbriifolium infected with G. pallida revealed up-regulation of the chitinase gene (ChtC 2.1) at 3 days post inoculation in S. sisymbriifolium but not in S. tuberosum. Electrospray ionisation-mass spectrometry analyses of root exudate extracts of the two Solanum species and subsequent bioassay-guided fractionation showed that the HF of S. sisymbriifolium differs from that of S. tuberosum. Previously, attention had been solely paid to the hatching ability of the root exudate of S. sisymbriifolium, but this study revealed for the first time that the aerial part extract possesses a significant hatching ability.