The North American elk or wapiti is an indigenous species to Canada. Understanding of the reproductive physiology of elk is limited, as little research has been conducted in this field as compared to domestic farmed species. In order to make available the tools to study reproductive physiology of the elk this thesis describes the cloning and expression of elk pituitary glycoprotein hormone cDNAs. The common gonadotropin a-subunit, and FSH, LH and TSH b-subunit elk cDNAs were amplified by reverse transcription and polymerase chain reaction (RT-PCR). There was a high degree of nucleotide similarity between the elk a and b subunits when compared with reported sequences from other species. The cDNAs for the pituitary glycoprotein hormone genes were used as probes to investigate seasonal expression in the female elk pituitary gland. Steady state levels of the common a-subunit mRNA was observed regardless of the reproductive season, but a significant increase in expression occurred during the breeding season. The FSH and LH b-subunit genes were expressed at low levels in pituitary glands of animals during presumed anestrous and pregnancy, but levels considerably increased during estrus. In contrast, levels of TSH b-subunit mRNA were similar regardless of the reproductive status. The FSH cDNAs were also transfected into a Chinese hamster ovary (CHO) mammalian expression system, aimed at the production of recombinant elk FSH. Transfected CHO cell lines were screened for expression of a- and FSH b-subunit mRNA by Northern blot. Activity of FSH was equivalent to ~100 mIU/ml of recombinant human FSH (Gonal-FTM), identified by FSH receptor signaling in an in vitro cell based assay. In conclusion, this work represents an advance towards understanding the molecular basis of seasonal reproduction in elk. This information and the availability of elk recombinant FSH will be useful for the application of advanced reproductive technologies required for the rapid expansion of healthy, disease resistant, and genetically superior animals, which are important for domestic production and wildlife management.

The use of reduced oxygen-packaging techniques has resulted in anaerobic bacteria emerging as a problem for the brewing industry over the last twenty-five years. The genus Pectinatus, consisting of the species<i>P. cerevisiiphilus</i> and<i> P. frisingensis</i>, is a concern for producers of unpasteurized beer. As a result, there is an ongoing need to both understand this genus and develop rapid detection methodologies to combat its presence in the brewery. The objectives of this study were to sequence and characterize the flagellin genes from both Pectinatus species and evaluate the genes and proteins from a taxonomic and detection-suitability standpoint. <p>A combination of micro-protein sequencing, polymerase chain reaction (PCR) and Bubble-PCR was used to completely sequence one flagellin gene from each Pectinatus species. This knowledge was then utilized to sequence the flagellin gene from four additional Pectinatus isolates, two from each species. To confirm the identity of the flagellin genes, one flagellin gene from each species was cloned, expressed and detected with Pectinatus-specific antibodies. A discrepancy between of the predicted protein size and the actual protein size led to tests for glycosylation, a post-translational modification. Taxonomic analyses, based on the flagellin genes, were conducted at both the superkingdom and genus levels. Finally, genus- and species-specific PCR primer sets were designed and tested for the specific detection of Pectinatus in the brewery. <p>Cloning and expression data confirmed the identity of the sequenced genes as Pectinatus flagellin genes. Glycosylation was positively confirmed to be a post-translational modification for five of the six strains tested. Phylogenetic analysis revealed that both of the Pectinatus species grouped with the phylum Firmicutes (low G+C, Gram-positive bacteria) and that there was more diversity at the species level within the <i>P. frisingensis</i> flagellin gene than the <i>P. cerevisiiphilus</i> flagellin gene. As a final point, the detection of most Pectinatus isolates was achieved with the preliminary PCR primer sets designed, however, some non-Pectinatus beer spoilage organisms, primarily wort spoilage organisms, were also detected. Both the basic science and the applied results generated from this study will aid the brewing industry in its ongoing battle to control Pectinatus contamination.

Lygus Hahn (Hemiptera: Miridae) are serious pests of economically important field, fruit, vegetable, and greenhouse crops in Canada. The release of European Peristenus Förster (Hymenoptera: Braconidae) in the USA has resulted in significant suppression of this pest and has renewed interest in the release of European Peristenus spp. in Canada. Prior to the release of exotic Peristenus spp., ecological host range studies need to be conducted to define their habitat and host associations. <p>These associations can be difficult to study using conventional methods. Morphological similarity of related parasitoids prevents species-level identification by dissection. Host rearing is time-consuming and can result in high levels of host and parasitoid mortality. To facilitate identification of immature Peristenus spp. in their hosts, a multiplex PCR assay was developed. This assay provided a specific and sensitive tool to screen individual insects for three parasitoid species simultaneously. <p>To validate the utility of the multiplex PCR assay in ecological host range studies, parasitism and parasitoid species composition obtained using conventional and molecular techniques were compared. Molecular methods compared favorably with conventional methods; however, more complete species composition information was available with the multiplex assay. To improve the quality of risk assessment studies and extract the most accurate ecological host range data, molecular methods were used to evaluate host-parasitoid associations in mirid populations collected in two ecoregions. Several new host-parasitoid associations were recorded for <i>P. digoneutis</i> and <i>P. relictus</i>, but parasitism of non-target mirids was low. <p>Parasitism of the target host collected from different plant species was evaluated to help clarify Peristenus host-plant associations. Despite the investigation of three different host plant species, no difference was observed in the parasitism level or parasitoid species composition in <i>L. rugulipennis</i>. The post-release utility of the multiplex assay was investigated in Canada, where Lygus parasitoids may have dispersed following release in the USA. To confirm establishment, samples were analyzed using the multiplex PCR assay, and P. digoneutis was detected for the first time in southern Ontario.

Mould is a common contamination in cereals. The growth of mould can stimulate mycotoxins production andsome of which at critical concentrations cause health problems in humans and animals. Fusarium is one of thefungus species that has been found in crops and can cause major problems for farmers such as reduced harvestand economic losses. A group of Fusarium species, Fusarium avenaceum, Fusarium poae and Fusariumtricinctum express a mycotoxin, enniatin. The limited information available today about enniatin-forming fungiis that they grow out on fields of wheat in colder climates. This project aims at developing methods for detection,quantification and identification of known and unknown fungi present in Swedish cereals during 2009-2011. Theproject was carried out using two previously published methods, TMAV and MGB, which both use TaqManprobes with realtime-PCR detection. The methods were evaluated for robustness, efficiency, accuracy, inclusionand exclusion. The results showed that both methods, TMAV and MGB, could be used to detect Fusariumavenaceum. The results for the TMAV method were that it could be used with custom annealing temperatureand chemical concentrations for the best detection. The MGB method can be used to detect Fusarium avenaceumwith Fusarium tricinctum in the same analysis. Both methods can be included in future mapping projects when itis of interest to quantify enniatin producing moulds.

An ideal polymerase chain reaction (PCR) system should be capable of rapidly amplifying a wide range of targets in both single and multiplex formats. Unfortunately, the timescales and complexities involved in many existing technologies impose significant limitations on achievable throughput. Buoyancy driven PCR is emerging as a simplified version of thermally driven bio-analysis systems. Here, we demonstrate a simplified convectively driven thermocycler capable of performing single and multiplex PCR for amplicons ranging from 191 bp to 1.3 kb within 10 to 50 minutes using 10 to 25 µL reaction volumes. By positioning two independent thermoelectric heating elements along the perimeter of a flow loop reactor constructed using ordinary plastic tubing, a buoyancy-driven flow is established that continuously circulates reagents through temperature zones associated with the PCR process. Unlike conventional benchtop thermocyclers, this arrangement allows reactions to be performed without the need for dynamic temperature control of inactive hardware components while maintaining comparable product yields and requiring no modifications to standard PCR protocols. We also provide a general correlation that can be applied to design reactor geometries satisfying virtually any combination of reagent volume and cycling time. In addition to offering an attractive combination of cost and performance, this system is readily adaptable for portable battery powered operation, making it feasible to perform PCRbased assays in a broader array of settings.

Campylobacter jejuni is a commensal microorganism of the poultry gastrointestinal tract. Broilers, layers, ducks, turkeys, and quails can be colonized by Campylobacter without illness occurring. The vast majority of human Campylobacter infections are recognized as being foodborne. For 2008, preliminary FoodNet data showed that the Campylobacter incidence of infection, 12.68 per 100,000 of the U.S. population, is the second highest, only behind Salmonella at 16.20 per 100,000. To further understand Campylobacter’s role as a foodborne pathogen, analysis at the molecular level is needed. Microbial molecular typing allows for identification and differentiation of bacterial strains beneath the species level. In this study, the “gold standard” method for molecular subtyping, Pulsed Field Gel Electrophoresis (PFGE), along with Diversilab® repetitive element Polymerase Chain Reaction (rep-PCR) and 16S-23S Internal Spacer Region Denaturing Gradient Gel Electrophoresis (ISR DGGE) were used for the molecular typing of Campylobacter jejuni isolates obtained during different stages of commercial broiler production and processing. In addition, the C. jejuni isolates were tested for resistance to antimicrobials commonly used in both veterinary and human medicine. Antimicrobial resistance testing was carried out using a broth dilution system. The majority of recovered isolates came from post-harvest carcass rinsates. Carcass rinses were obtained at post-evisceration, post-chill stages. All isolates (n = 46) were identified by the Polymerase Chain Reaction as Campylobacter jejuni. Three genotypes (n = 44, n = 1, n = 1) were identified by PFGE. The 46 rep-PCR products grouped into seven clusters and two outliers. Clustering of rep-PCR products by sample source was not observed. No relatedness trends were observed for isolates recovered from the same source. The combination of PFGE and Diversilab rep-PCR methods provides highly discriminatory molecular typing results. These results provide practical epidemiological information that shows postevisceration and post-chill stages are still important targets for intervention studies. The very high occurrence of C. jejuni isolates exhibiting genotype A suggests it may differentially express certain gene(s) that enable this strain to more favorably survive under the different harsh environmental conditions encountered during production and processing. In addition, phenotypic testing revealed all of the isolates were not resistant to the antimicrobials azithromycin, ciprofloxacin, erythromycin, gentamycin, tetracycline, florfenicol, nalidixic acid, telithromycin, and clindamycin at any of the concentrations tested. All the C. jejuni isolates exhibited an indistinguishable two-band 16S-23S ISR DGGE profile. Overall, these C. jejuni commercial broiler pre- and post-harvest isolates exhibited an extremely low degree of molecular and phenotypic variability.

Iridovirus infections have led to serious economic loss in the aquaculture industry in Kaohsiung County as well as the whole Southern Taiwan region. Identified susceptible host species in this region includes hybrid grouper (Epinephelus hybrid), giant seaperch (Lates calcarifer), largemouth bass (Micropterum salmoides), king grouper (Epinephelus lanceolatus), spotted butter fish (Scatophagus argus), yellow-wax pomfrat (Trachinotus blochii), goldlined seabream (Sparus sarba), humpback grouper (Chromileptes altivelis), Mangrove red snapper (Lutjanus argentimaculatus). In this study, a diagnostic PCR primer pair CY15n-F/CY15nR, and its nested primer pair RY16-F/RY16-R, were designed and applied to amplify virus-specific products of 1339 bp and 305 bp, respectively. This primer set did not amplify products from lymphocystis disease virus, largemouth bass virus, or healthy control fish DNA. This sensitive technique can detect the presence of 50 fg plasmid with viral DNA insert in the presence of 100 ng/£gl host DNA, or 0.05 fg DNA from infected fish. Comparing sequences of CY15 fragment, ATPase gene, predicted major capsid protein and partial DNA polymerase genes among iridoviruses, it is suggested that the viruses found in this area should be classified as the Megalocytivirus of Iridoviridae. These viruses are clearly different from the Ranavirus, another fish-pathogenic iridovirus. Those iridoviruses can be classified into two genotypes: the CY630 type, which is the Taiwan grouper iridovirus; and the CY113 type, which is similar to red seabream iridovirus (RSIV). The identity in CY15 fragment sequences is about 91%. Microscopically, enlarged cells can be found in organs of infected fishes. They appear in the spleen, head kidney, and trunk kidney in infected groupers. The enlarged cells may be relocated from other organs. In giant seaperch, the enlarged cells appeared in the above mentioned organs, and also in the digestive tract and the heart. Two kinds of the enlarged cells in grouper can be distinguished by their H&E staining properties: the basophilic and the eosinophilic enlarged cells. The result from in situ hybridization and electron microscopy suggest that the viruses only appear in the basophilic enlarged cells. Both nucleus and cell volume increase in basophilic enlarged cells, while only the cell volume increases in the eosinophilic enlarged cells. The viruses appeared first in the nuclei of the basophilic enlarged cells, after the mid-phase of the infection they distributed into the whole cells. Judging from the results of phagocytosis, acid phosphatase activity and the ultrastructure of infected cells, it is suggested that this target cell is macrophage or monocyte. The viral capsid is assembled in the viromatrix, and the virogenic stroma can be either ring-shped or disc-shaped. The diameter of mature virus is 120-130 nm from side to side, or 160-170 nm from apex to apex. The electron-lucent space between the capsid and the envelope is about 20-50 nm. The virus particles can be found in (1) lysosome-like vesicles in the cytoplasm, if the host cell still has its nucleus; or (2) the viromatrix, When the host nucleus is dissolved or only has some vestige nuclear membrane left.

In this study we used the phytoremediation techniques to treatment diesel contaminated wetland soil. At first, we compared the four common wetland plants, Typha orientalis Presl, Cyperus malaccensis, Bolbos choenus planieulmis and Phragmites communis, on the treatment efficiency of the diesel contaminated wetland soils. From the results, we find out that the Typha orientalis Presl has highest growth rate and activity on rhizosphere among the four species. The Typha orientalis Presl was planted on artifical diesel contaminated wetland soil and incubated inside a greenhouse, while a control system without vegetation is compared. After 240 days, the result shows that soil planted with Typha orientalis Presl can enhance the microbial and dehydrogenase activity. And adding with nutrients can help plants to prevent the diesel stress. Finally, we utilized the PCR/DGGE methods to analyze soil microbial diversity. According to the DGGE profiles, presence of Typha orientalis Presl can augment microbial diversity . So far as degradation of TPH-d to be concerned, because of the period was too short, it doesn¡¦t have significant difference between treatments. However, presence of Typha orientalis Presl and addition of nutrients, the TPH-D degradation rate was measured to be approximately 80 % and concentration of TPH-D could degrade from 16000 mg kg-1 to 3500 mg kg-1 after 240 days.

The multiplex PCR experiment is to amplify multiple regions of a DNA sequence at the same time by using different primer pairs. Although, in recent years, there are lots of methods for PCR primer design, only a few of them focus on the multiplex PCR primer design. The multiplex PCR primer design is a tedious task since there are too many constraints to be satisfied. A new method for multiplex PCR primer design strategy using genetic algorithm is proposed. The proposed algorithm is able to find a set of suitable primer pairs more efficient and uses a MAP model to speed up the examination of the specificity constraint. The dry-dock experiment shows that the proposed algorithm finds several sets of primer pairs for multiplex PCR that not only obey the design properties, but also have specificity.

This research investigated the existence of <10 £gm nitrogen-fixing unicellular cyanobacteria in the South China Sea. The surveys covered the period from February 2004 to January 2005 and a total of seven cruises. The unicellular cyanobacteria that express orange-yellow from cellular phycoerythrin were observed under a fluorescence microscope. Their expressions in nitrogen-fixation were confirmed by the results from reverse transcription polymerase chain reaction (RT-PCR) and whole cell fluorescence immunolocalization of nitrogenase. The nifH gene sequences of the unicellular cyanobacteria collected from the South China Sea was with >90% identities of their nucleotides similar to the nifH gene sequences of unicellular diazotrophs from ALOHA (Hawaii) as well as Synechocystis sp. WH 8501, Cyanothece sp. ATCC 51142, Cyanothece sp. WH 8902, Cyanothece sp. WH 8904 and Synechococcus sp. RF-1. Positive reactions of fluorescence immunolocalization of nitrogenase were only observed in some, not all, of <10 £gm unicellular cyanobacteria, suggesting that cell counting alone can not be used to estimate nitrogen fixation rate. There was great seasonal and spatial variation in the unicellular cyanobacteria cell density. There was, however, no significant relationship between cell density and the investigated environmental factors. Cell density was high when temperature was high or where stratification index of water column was high, such as in summer or in basin in contrast to other seasons or the shelf-slope regions.