Liquidambar L. genus are represented with four species in the world and one of these species, Turkish sweet gum (Liquidambar orientalis Miller) is naturally found only in southwestern Turkey with limited distribution in Mugla Province. The presence of increasing threats to its genetic resources signifies the importance of studying the phylogenetic relationships and genetic diversity in this relict endemic species. In this study, 18 different populations were sampled throughout the species range and noncoding transfer ribonucleic acid (trn) region of chloroplast DNA was studied to asses the phylogenetic relationships and genetic diversity. Experimental studies included the extraction of DNA, amplification and sequencing of the trn region of the chloroplast DNA. Molecular evolutionary analysis was done by using MEGA version 3.1 and Arlequin 2.000 softwares. Sequences from six other species of Liquidambar (L. styraciflua from USA, L. macrophylla from Mexico, L. formosana from Vietnam, L. acalycina from China, L. formosana from China and L. acalycina from USA) in the database were also included in the analysis. Moleculer diversity results show that population located in Mugla-Yatagan district has the highest number of polymorphic sites among the other populations of Turkish sweet gum. Population located in Marmaris-G&uuml / nn&uuml / cek has an average genetic distance value of 0.0032 within population, being the highest within the studied populations of Turkish sweet gum. The average genetic distance within variety orientalis (0.0011) was the greatest among all the varieties, but the most separated or divergent populations were members of variety integriloba. For both varieties and geographic groups, average diversity within was found to be the greatest portion (greater than 80%) of the total sequence diversity. The geographic groups located in Denizli and Mugla-Yatagan showed the highest average genetic distances within location, with a value of 0.0014. The genetic distance between the closest neighbor of Turkish sweet gum, American L. styraciflua was 0.0002, whereas the genetic distance between the most distant neighbors (Vietnamese L. formosana, Chinese L. acalycina and L. formosana) was 0.0051. Based on the molecular diversity analysis, seven populations were found to be important for conservation issues and two of them located in Marmaris have the highest priority. The most variant geographic groups are located in Denizli and Mugla-Yatagan districts. These populations could be considered as good candidates for future in-situ or ex-situ conservation programs

The genus Picea is located from temperate to Taiga (boreal) regions of northern hemisphere from subtropical to high altitude with 34 species. Picea orientalis is endemic to Eastern Black Sea Mountainous region of Turkey and Western Caucasus. To determine the genetic relatedness within Picea orientalis populations, as well as the relationship between other Pinaceae species from database, populations were sampled from 15 different locations within the natural range of species and grouped into 5 depending on several criteria. In order to evaluate the genetic structure of the taxon, non-coding trn and matK regions of choloroplast DNA (cpDNA) were sequenced. According to genetic diversity analysis of 15 Picea orientalis populations with respect to trn and matK regions, there is not much variation among populations. Among 3 non-coding trn and the matK region, there is only one variable site which was parsimony informative. The results indicated that the populations from Artvin had the highest divergence. In this study, the genetic divergence of Picea orientalis from other Pinaceae species were also observed. According to the results obtained from trnV region the studied Picea orientalis observed to display a close relationship with Larix and distinct from other Pinaceae especially Pinus genus. This result is unrepresentative due to the results of other studies. Moreover, as a result of analysis with trncd-ef region, the studied Picea orientalis populations possessed close relationship with species from clade Picea. Moreover, based on molecular clock estimations the studied Picea orientalis populations had close relationships with the species form Asia. Finally, the relationship of Picea orientalis with other Picea species were analyzed with respect to matK region. The result is consistent with the results of trncd-ef region and also with other studies.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

La caractérisation chimique des bois raméaux fragmentés (BRF) et du bois de tronc de Trema orientalis et de Leucaena leucocephala a révélé un plus fort degré de lignification dans les branches de Trema que dans le tronc, alors que le résultat inverse a été observé pour le Leucaena. De même des plus forts taux en cendres ont été trouvés dans les branches que dans le tronc, avec des valeurs globalement plus élevées pour le Trema. Par ailleurs, le ratio C/N déterminé par analyse élémentaire, a révélé des valeurs plus élevées pour le bois de tronc que pour les BRF des deux essences. En revanche, le ratio lignine/N des branches, naturellement plus faible, a conduit à la prédiction selon laquelle la décomposition de la matière ligno-cellulosique des branches serait plus rapide que celle du bois de tronc. De plus, les meilleurs ratios C/N et lignine/N ont été trouvés pour Leucaena. Enfin plusieurs composés identiques, dérivés de la lignine, ont été majoritairement identifiés par pyrolyse-GC-MS dans le bois de tronc comme dans les BRF. / The chemical characterization of ramial chipped wood (RCW) and of the T. orientalis and the L. leucocephala stem woods has shown a higher degree of lignification in the branches of Trema than in the stem woods whereas reverse results have been found for the Leucaena. Besides, higher rates in ashes have been found in the branches than in the trunk, with values globally higher for Trema. The C/N ratio, determined by elementary analysis, has revealed higher values for stem wood than for the RCW of the two species. In contrast, the lignin/N ratio of the branches, naturally low, for the two species, has led to the prediction stating that the decomposition of the lignin-cellulosic matter of the branches would be faster than that of the stem wood. Besides, best ratio C/N and lignin/N have been found for Leucaena. At last, several identical compounds, derivatives of the lignin, have been mostly identified in the stem wood as in the RCW.

Of the four most economically important tickborne diseases of cattle in the world, two have been identified in Virginia, occasionally as co-infections: anaplasmosis and theileriosis. The latter is caused by the emerging infectious agent, the Theileria orientalis complex, in particular the Ikeda and Chitose genotypes. These organisms are carried by the ixodid tick, Haemaphysalis longicornis, recently identified in the United States. Our work has been focused on initially identifying the protozoal organisms, crafting assays to aid in the identification of these organisms in clinically affected animals, and briefly examining the rate of co-occurrence of theileriosis and anaplasmosis. This is important, as Anaplasma marginale - the most common etiologic agent of anaplasmosis in cattle in Virginia - is treatable with a safe, effective, FDA-approved compound, whereas there is no currently approved treatment for theileriosis. Finally, we seek to contextualize theilerosis as a cause of infectious bovine anemia (IBA) and its expected economic impact on the cattle industry in Virginia. / Doctor of Philosophy / Theileriosis is a disease that infects cattle, caused by the blood parasite, Theileria orientalis, specifically two distinct genotypes of T. orientalis, Ikeda and Chitose. Theileriosis is transmitted to cattle by the Asian longhorned tick, Haemaphysalis longicornis, which was recently identified in the United States. Globally, theileriosis is one of four major tickborne diseases of cattle with significant economic importance, so the discovery of this parasite in the state of Virginia is of special importance to the cattle industry in Virginia. My work has revolved around making the initial discovery of T. orientalis Ikeda in the United States, and developing tests for cattle producers and veterinarians to use to help diagnose theileriosis in sick animals. Another tickborne disease of cattle, anaplasmosis, is caused by a bacterium, A. marginale. These two organisms have different biology, are transmitted by different ticks, and are treated differently, but cause identical clinical disease in cattle. In addition to identifying T. orientalis, we have developed a single test that can determine if sick cattle have T. orientalis or A. marginale – this is important, because the antibiotic used to treat A. marginale does not work to treat T. orientalis. In fact, there is no treatment for T. orientalis available in the United States. In addition to developing diagnostic assays, I seek to put into pathobiological, ecological, and economic context the importance of theileriosis on the cattle industry in Virginia.

Piscine francisellosis in an infectious emerging bacterial disease that affects several marine and fresh water fish species worldwide, including farmed salmon, wild and farmed cod, farmed tilapia and several ornamental species, for which no commercial treatment or vaccine exists. During 2011 and the first semester of 2012, chronic episodes of moderate to high levels of mortality with nonspecific clinical signs, and widespread multifocal white nodules as the most consistent gross pathological lesion were experienced by farmed tilapia fingerlings at two different locations in Northern Europe. In this study such outbreaks of granulomatous disease were diagnosed as francisellosis with a genus-specific PCR, and 10 new isolates of the bacterium including the one named STIR-GUS-F2f7, were recovered on a new selective “cysteine blood-tilapia” agar and cysteine heart agar with bovine haemoglobin. Ultrastructural observations of the pathogen in Nile tilapia (O. niloticus) tissues suggested the secretion of outer membrane vesicles (OMVs) by the bacterial cells during infection in these fish. This represented the first documented report of isolation of pathogenic Francisella strains from tilapia in Europe. The phenotypic characterisation indicated that isolates recovered were able to metabolise dextrin, N-acetyl-D glucosamine, D-fructose, α-D-glucose, D-mannose, methyl pyruvate, acetic acid, α-keto butyric acid, L-alaninamide, L-alanine, L-alanylglycine, L-asparagine, L-glutamic acid, L-proline, L-serine, L-threonine, inosine, uridine, glycerol, D L-α-glycerol phosphate, glucose-1-phosphate and glucose-6-phosphate. The predominant structural fatty acids of the isolates were 24:1 (20.3%), 18:1n-9 (16.9%), 24:0 (13.1%) 14:0 (10.9%), 22:0 (7.8%), 16:0 (7.6%) and 18:0 (5.5%). Anti-microbial resistance analyses indicated that STIR-GUS-F2f7 was susceptible to neomycin, novobiocin, amikacin, ciprofloxacin, imipenem, gatifloxacin, meropenem, tobramycin, nitrofurantoin, and levofloxacin using the quantitative broth micro-dilution method, while the qualitative disc diffusion method indicated susceptibility to enrofloxacin, kanamycin, gentamicin, tetracycline, oxytetracycline, florfenicol, oxolinic acid and streptomycin. The use of the following housekeeping genes: mdh, dnaA, mutS, 16SrRNA-ITS-23SrRNA, prfB putA rpoA, rpoB and tpiA indicated 100% similarity with other isolates belonging to the subspecies F. noatunensis orientalis (Fno). Koch’s postulates were successfully fulfilled by establishing an intraperitoneal injection (IP) challenge model with STIR-GUS-F2f7 in Nile tilapia. Moreover, the challenge model was used to investigate the susceptibility of 3 genetic groups of tilapia to STIR-GUS-F2f7. The lowest amount of bacteria required to cause mortality was 12 CFU/ml and this was seen as early as only 24 hours post infection in the red Nile tilapia and in the wild type after 26 days, no mortalities were seen in the species O. mossambicus with this dose. The mortality in red O. niloticus was significantly higher than that of the other two tilapia groups when 12 and 120 CFU/fish were injected. It was also observed that when a dose of 1200 CFU/ml was used, the mortality in O. niloticus wild type was significantly lower than that of the other two tilapia groups and no differences were seen among the 3 groups when the highest dose (1.2 x105 CFU/fish) was used. The median lethal dose (LD50) of O. niloticus wild type was the most stable during the experiment (values around 104 CFU/ml) and the highest of the three groups after day 25 post infection. At the end of the experiment (day 45) the LD50 was 30 CFU/ml in the red Nile tilapia, 2.3x104 CFU/ml for the wild type and 3.3x102 CFU/ml for O. mossambicus. This pattern, where the LD50 of the red tilapia was lower than that of the other two groups, was observed during the whole experiment. The outcomes of these experiments suggested that the red Nile tilapia family appeared to be the most susceptible while the wild type Nile tilapia family the most resistant. The complete genome of STIR-GUS-F2f7 was sequenced using next generation sequencing (NGS) Illumina Hi-Seq platform™, and the annotation of the assembled genome predicted 1970 protein coding sequences and 63 non-coding rRNA sequences distributed in 328 sub-systems. The taxonomy of the species Francisella noatunensis was revised using genomic-derived parameters form STIR-GUS-F2f7 and other strains in combination with a polyphasic approach that included ecologic, chemotaxonomic and phenotypic analyses. The results indicated that STIR-GUS-F2f7 and all the other strains from warm water fish represent a new bacterial species for which the name Francisella orientalis was assigned. Moreover the description of F. noatunensis was emended and the creation of a new subspecies within this taxon i.e. Francisella noatunensis subsp. chilense was proposed. The results of this study led to the development of a highly efficacious vaccine to protect tilapia against francisellosis.

Sand flies (Diptera, Phlebotominae) are small biting insects and vectors of Leishmania spp. which cause medically and veterinary important disease - leishmaniasis. During the piercing of the host skin, sand fly females inject saliva to facilitate the blood feeding. The sand fly saliva is composed of many bioactive molecules which were shown to possess anti-inflammatory and anti-haemostatic functions. The saliva affects host's immunity in the bite site and consequently enhances the survival and development of transmitted pathogens. Most of the studies focus on salivary proteins and enzymes of sand flies belonging to Phlebotomus and Lutzomyia genera, while salivary proteins from sand flies of the third genus Sergentomyia were neglected so far. In this thesis we focused on comparison of salivary proteins from two Phlebotomus species, namely Phlebotomus perniciosus and Phlebotomus orientalis, and Sergentomyia schwetzi. These sand fly species differ not only by the ecology and geographical distribution but also by host preferences. Both Phlebotomus species prefer large or medium-size mammals as the bloodmeal source, particularly rabbits, hares and dogs for P. perniciosus and cattle, goats, sheep and humans for P. orientalis. Contrarily, Sergentomyia sand flies are known for preferred feeding on reptiles...

No / (137)Cs concentrations in ten species of foliose lichens collected within Tsukuba-city in August 2013 ranged from 1.7 to 35 kBq/kg. The relationships between (137)Cs in two dominant species, Dirinaria applanata and Physcia orientalis, and the air dose rate (muSv/h) at the sampling sites were investigated. (137)Cs in P. orientalis measured about 1 year after the Fukushima nuclear accident was correlated (r(2) = 0.80) more closely with the air dose rate than those measured after about 2 years (r(2) = 0.65), possibly demonstrating its continued value as a biomonitor to reflect ambient fall-out levels. In contrast, those of Dirinaria applanata were not correlated with the air dose rate in either year.

Nile tilapia, Oreochromis niloticus, is one of the most important farmed fish globally. One of the most serious bacterial diseases constraining global tilapia production is Francisellosis caused by Francisella noatunensis subsp. orientalis (Fno). Although outbreaks of Fno are increasing worldwide, there are no licenced commercial vaccines to prevent the disease for use on tilapia farms. Thus, the current treatment of choice is the use of antibiotics combined with increasing water temperature up to 30°C. Studies investigating the diversity of circulating Fno isolates and the immune response of tilapia elicited by vaccination against piscine francisellosis are lacking. In addition, the current conventional and molecular tools used for detection of Fno have many drawbacks, making detection of Fno a challenging process. In this study, five clinical isolates of Fno from diverse geographical locations (UK, Costa Rica, Mexico, Japan and Austria), previously characterised by morphology, genotype, antimicrobial susceptibility and virulence, were used in a proteomic study. The whole proteomic cell profile of the five isolates were homogenous by one-dimension sodium dodecyl polyacrylamide gel electrophoresis (1D-SDS-PAGE), while minor differences in the intensity of 15 proteins between the strains were observed by two-dimension SDS-PAGE (2DE), including some important virulence related proteins. The UK isolate was the most significantly different isolate when compared to the other Fno isolates in the current study. The Fno UK isolate had significantly higher abundance of 10/15 of the significantly expressed proteins including four of the essential pathogenicity and virulence related proteins (IglC, GroEL, DnaK, ClpB) compared to the other used Fno isolates. The antigenic profiles of the five Fno isolates were studied by 1D western blotting using tilapia hyper immune sera which recognised an immunodominant band of a molecular weight of ~ 17-28 kDa in all tested Fno isolates. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI/MS/MS) identified 47 proteins in this antigenic band. Some of the identified proteins are associated with Fno pathogenicity. 2D western blot analysis of the vaccine isolate (Fno UK) revealed differential antigen recognition between sera from vaccinated and non-vaccinated fish following experimental challenge (26 antigenic spots recognised by sera from vaccinated fish; 31 antigenic spots recognised by sera from vaccinated and challenged fish and 30 antigenic spots recognised by non-vaccinated and challenged fish). The identity of these proteins was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and some of them are known Francisella virulence related proteins. Bioinformatics analyses revealed diverse categories of proteins with high biological functions, however the vast majority of these proteins are involved in energy production and metabolic pathways of the bacteria. This detailed analysis will facilitate the development of cross-strain protective subunit Fno vaccines and antigen-targeted Fno diagnostics. The outer membrane proteins (OMPs) of the same five Fno isolates were extracted using the ionic detergent sarkosyl. The OMP fraction of the different isolates were separated via 1D-SDS PAGE and the digested peptides of the UK isolate were analysed by LC/ESI/MS/MS. High degree of similarity was observed in the OMP profile of the five Fno isolates with an abundant protein band at 17-28 kDa, which was found to be antigenic by 1D western blot using convalescent tilapia sera. LC/ESI/MS/MS analysis of the OMPs of the Fno UK isolate identified 239 proteins, including 44 proteins in the antigenic band (17-28 kDa). Comparison between the proteins identified in the immunogenic band of whole cell lysate and OMP fraction of the Fno UK isolate showed 30 common proteins between the two preparations, 17 proteins were identified only in the whole cell extract and 14 were identified only in OMP fraction. Outer membrane proteins (e.g. Omp-A), virulence related proteins such (e.g. IglC) and other stress related proteins (e.g. AhpC/TSA family peroxiredoxin) were more abundant in the OMP fraction than the whole cell lysate. In silico analysis enabled prediction of the function and location of the OMPs identified by Mass-spectrometry. The findings of this study provide preliminary data on bacterial surface proteins that exist in direct contact with the host immune defence during infection and offering an insight into their potential role as novel targets for Fno diagnostics and vaccine development. The efficacy of an injectable whole cell oil-adjuvanted vaccine was evaluated against challenge with heterologous Fno isolates in Nile tilapia, Oreochromis niloticus. Three duplicate groups of 130 healthy Nile tilapia (~15 g) were intraperitoneally (i.p.) injected with the vaccine, adjuvant-alone or PBS followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection to all immunised tilapia groups with a significantly higher relative percent survival (RPS) of 82.3% against homologous challenge, compared to 69.8% and 65.9% after heterologous challenge. Protection correlated with significantly elevated specific antibody responses and western blot analysis demonstrated cross-isolate antigenicity with sera from fish post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by quantitative real-time polymerase chain reaction (qPCR) in conjunction with significantly greater expression of IgM, IL-1β, TNF-a and MHCII 72 hours post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to those of adjuvant-alone and control fish. The latter results suggested stimulation of protective immune responses following vaccination. In addition, a whole cell formalin killed autogenous immersion vaccine against Fno was developed using the same isolate used for the injectable vaccine. Duplicate tanks of 35 tilapia fry were immersed in the vaccine or in sterile Modified Muller Hinton broth (MMHB) diluted in tank water (1:10 dilution) for 30 s and at 30 days post-vaccination (dpv), all fish groups were immersion challenged with the homologous Fno isolate and monitored for 21 days. A moderate RPS of 43.7% was provided by the vaccine. Serum IgM levels were below the threshold in 30 % of the vaccinated fry 30 dpv. Also, the IgM levels of the vaccinated fry were not significantly different from control fry 21 days-post challenge. A recombinase polymerase amplification (RPA) assay was developed and validated for rapid detection of Fno. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in fish farms, but also by screening 78 Nile tilapia and 5 water samples collected from UK and Thailand. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other species of bacteria tested. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The Fno-RPA was rapid, giving results in approximately 6 min in contrast to the qPCR that required approximately 90 min to reach the same detection limits. Moreover, the RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to control the infection through prompt on-site detection of Fno. The overall results of this study indicated that Fno isolates from different origins share a high degree of homology in their proteomic and antigenic profile. Proteomic characterisation data of Fno isolates has contributed to understanding the diversity of Fno isolates and assisted in identifying suitable candidates for developing an effective Fno vaccine. / Moreover, this study has proven the efficacy of a cross protective Fno injection vaccine in tilapia fingerlings, with further optimisation needed for immersion vaccination of fry, and given insights into the immune response of tilapia to vaccination against francisellosis. In addition, it provided a rapid, sensitive, specific and robust molecular tool for detection of Fno that can assist surveillance and control of piscine francisellosis on tilapia farms.