Salmonellae are enteric bacteria infecting animals and humans. Large animal clinics and Veterinary Teaching Hospitals are greatly affected by Salmonella outbreaks and nosocomial infection. The risk of environmental contamination and spread of infection is increased when animals are confined in close contact with each other and subjected to increased stress factors. This study was designed to compare double-enrichment culture techniques with Gel-based and Real-time PCR assays in the quest for improved diagnostic methods for detecting Salmonella in equine fecal samples. 120 fecal samples submitted to the Clinical Microbiology Laboratory of the Veterinary Medical Teaching Hospital at Texas A&M University (CML, VMTH, TAMU) were tested for Salmonella using all three techniques. Double-enrichment bacterial culture detected 29 positive results (24%), Real-time PCR detected 33 positive results (27.5%), and Gel-based PCR detected 73 positives results (60.8%). While culture and real-time PCR methods had similar results, the gel-based PCR method detected many more positive results, indicating probable amplicon contamination. Real-time PCR can be completed as soon as the day after submission while culture techniques may take 2 to 5 days to complete. However, viable bacterial cells are needed for antimicrobial susceptibility testing and serotyping: both important for epidemiological studies. Therefore, double-enrichment bacterial culture performed concurrently with real-time PCR methods could be efficient in clinical settings where both accurate and expedient results are required.

In these studies we evaluated specific environmental stimuli relevant to Salmonella virulence and physiology in the gastrointestinal tract of chickens. Results from Salmonella growth in steady state, glucose-limiting continuous culture (CC) indicated that the optimal growth condition was observed between 0.05 h-1 and 0.27 h-1 dilution rates (D). Cell protein concentrations increased proportionally with an increase in D at each steady state, but after D 0.27 h-1 there was a reduction in the cell protein concentrations as the D increased. Genetic responses generally indicated that the lowest D exhibited highest hilA relative expression. Relatively higher expression of hilA was largely observed at low D (low glucose) (0.0125 h-1, 0.025 h-1, 0.05 h-1). Salmonella incubated in CC at different pH shifts demonstrated that cell protein concentration, glucose utilization, Yield ATP and Acetate:Propionate ratios were influenced by an increase in pH (6.14 to 7.41). These parameters increased and decreased consistently with a corresponding increase and decrease in pH. Polymerase chain reaction-based denaturant gradient gel electrophoresis showed that the overall amplicon band patterns of microbial similarity have demonstrated that hens molted with Alfalfa (ALC+) diet were similar to the Full-Fed (FF+) treatment group. Additional, FF+ and ALC+ treatment groups exhibited a higher percentage similarity coefficient (>90%) than the feed deprived treatment group. Fermentation response from cecal inocula on feed substrates revealed that alfalfa based samples yielded consistently higher short chain fatty acid levels when compared to other feed substrates. Salmonella Enteritidis (SE) colonization in liver, spleen and ovaries was significantly (P < 0.05) higher in FW+ hens compared to ALC+ and FF+ treatments groups. A 4-fold (log10 1.29) reduction in SE colonization for ALC+ hens compared to feed withdrawal hens (FW+) (log10 5.12) SE colonization was observed. Relative expression of hilA in all treatment groups was significantly (P < 0.05) higher in FW+ compared to FF+ and ALC+ groups. hilA expression in FW+ hens was 3.2-, 4.2-, and 1.9-fold higher for Days 6, 11 and 12 respectively, when compared with to ALC+ hens. These results suggest that Salmonella virulence in the gastrointestinal ecology of chickens could be impacted by a combination of low nutrients availability and pH shifts.

In der molekularen Diagnostik besteht ein Bedarf an schnellen und spezifischen Testsystemen, die entweder für die Labordiagnostik oder in Point of Care-Umgebungen eingesetzt werden können. Um dieses Ziel zu erreichen, stehen die Miniaturisierung und Parallelisierung im Mittelpunkt des Forschungsinteresses. Die führende Methode im Bereich der DNA-Analytik ist derzeit die Realtime-PCR. Dieser Technologie sind hinsichtlich der Multiplexfähigkeit technologischen Hürden gesetzt, da derzeit nur eine Analyse von maximal vier Parametern parallel in einem Versuchsansatz erfolgen kann. Microarrays stellen hingegen die benötigten Voraussetzungen zur Verfügung, um als Werkzeuge für die Multiparameteranalyse in verschiedensten Anwendungsbereichen zu dienen. Ein Schwerpunkt dieser Arbeit war es, Multiplex-PCRs und diagnostische Microarrays zu entwickeln, die für analytische Fragestellungen eine schnelle und zuverlässige Multiparameteranalytik ermöglichen, um die bisherigen Einschränkungen aktueller Nachweisverfahren zu vermeiden. Als Anwendungen wurden zum einen ein Nachweissystem für acht relevante Geflügelpathogene zur Überwachung in der Geflügelzucht, zum anderen ein Nachweissystem zur Identifikation potentiell allergener Lebensmittelinhaltstoffe entwickelt. Neben der Entwicklung geeigneter PCR und Multiplex-PCR-Verfahren sowie spezifischer Microarrays für die Detektion der gesuchten Zielsequenzen stand auch die weiterführende Integration von DNA-Amplifikation und Microarray-Technologie im Fokus dieser Arbeit. Die OnChip-Amplifikation stellt eine Möglichkeit dar, um DNA-Analytik und Detektion in einem Reaktionsschritt zu integrieren. Entsprechend wurden die in der Arbeit entwickelten PCR- und Multiplex-PCR-Verfahren zum Nachweis potentieller allergener Lebensmittelinhaltsstoffe für die OnChip-Amplifikation adaptiert und Reaktionsbedingungen getestet, die eine Multiparameteranalyse auf dem Chip ermöglichen. Die entwickelten OnChip-PCR-Verfahren zeigten eine hohe Spezifität sowohl in Single- als auch in der Multiplex-OnChip-PCR. Eine Sensitivität von 10 Kopien bzw. <10ppm konnte in Single-OnChip-PCRs für den Nachweis allergener Lebensmittelinhaltsstoffe gezeigt werden. In Multiplex-OnChip-PCRs konnten 10-100ppm allergene Verunreinigungen spezifisch in unterschiedlichen Lebensmitteln nachgewiesen werden. Ein weiterer Schritt in Richtung einer möglichen Verwendung im Point of Care-Bereich stellt der Einsatz eines isothermalen Amplifikationsverfahrens dar. Vorteil eines solchen Verfahrens ist die Möglichkeit, auf das ansonsten benötigte Thermocycling zu verzichten. Dies vereinfacht eine Integration der OnChip-Amplifikation in mobile Analysegeräte oder Lab on Chip-Systeme und qualifiziert das Verfahren für den Einsatz in Point of Care-Umgebungen. In dieser Arbeit wurde eine noch junge isothermale Amplifikationsmethode, die helikase-abhängige Amplifikation (HDA), hinsichtlich ihrer Eignung für die Integration auf einem Microarray getestet. Hierfür konnte die bislang erste OnChip-HDA für Einzel- und Duplex-Nachweise von Pathogenen entwickelt werden. / In molecular diagnostics there is a need for fast and specific assay systems that could be used in the clinics and in point of care settings alike. Therefore miniaturisation and parallelisation are in the main focus of current assay development researches. The current gold standard for DNA analytics is the realtime PCR. However, this technology has its restraints in context to multiplex analysis. With the currently available technology an efficient multiplexing is only possible for four different targets per analysed sample. Microarrays in contrast offer the needed multiplex capabilities and have advanced to capable tools used in multiple fields of application. One focus of this work was the integration of Multiplex PCR and microarray technology, developing a microarray capable of analysing multiple parameters in one given sample, circumventing the problems and restraints of the exsisting technologies. As an example microarray assays for two different application fields were developed. One microarray assay for the detection of pathogens in poultry and another microarray assay for the detection of potentially allergenic food ingredients. Single- and Multiplex OnChip-PCR assays for both applications were developed and tested. OnChip-PCRs developed in this work showed high specificity in Single- and Multiplex-OnChip amplifications. The sensitivity was in the range of 10 DNA copies or 10ppm respectively for Single-OnChip-PCR in experiments for the detection of allergenic food contaminations. In Multiplex-OnChip-PCR experiments 100 DNA copies or 100ppm of food contaminents could be detected in different food matrices. A further focus of this work was the adaption of the OnChip amplification for the use in Point of Care settings. Isothermal amplification is a promising approach having the advantage of avoiding the thermocycling needed in the PCR. This opens up certain opportunities for the development of smaller, more flexible mobile diagnostic analysis devices. In this work we have evaluated the helicase dependent amplification (HDA) in terms of usability in OnChip amplification. In this work it was shown for the first time that HDA could be used for the detection of different pathogens in an Duplex-OnChip-PCR, showing the potential of this technology for integration in Point of Care settings.

Arbuscular mycorrhizal fungi (AMF) are important components of agro-ecosystems and are especially significant for productive low-input agriculture. Traditional spore morphology-based identification of AMF in biodiversity studies is subjective and requires expertise and time. Researchers have used molecular techniques to investigate community composition of AMF in uncultivated, disturbed, or contaminated soils, but this approach to community analysis of AMF in agricultural soils has not been reported. In this study, a polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) procedure for the detection of fungal 18S ribosomal RNA gene was developed with reference cultures. Five AMF species were procured from the International Culture Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi (INVAM). These reference cultures were chosen because isolates of their species were putatively identified in a previous survey of farm field soils in Saskatchewan, Canada. A reference PCR-DGGE profile was generated using DNA extracted and amplified from the spores of these INVAM cultures. The methods technical limitations were investigated. The optimized procedures effectiveness was tested by its application to soil samples from 38 farms. Bands from the PCR-DGGE profiles of these samples were excised for sequence analysis. The total number of species recovered was low in comparison to other AMF community surveys of temperate climate locations. The majority of the sequences recovered were Glomus species. Scutellospora calospora, a previously undetected AM fungus in Saskatchewan was found. A trend in AMF distribution in Saskatchewan was observed and it was relatable to their phylogenetic taxonomy. Though not without its drawbacks, this approach to community composition analysis of AMF was faster than conventional trap cultivation methods.

Arbuscular mycorrhizal fungi (AMF) are important components of agro-ecosystems and are especially significant for productive low-input agriculture. Traditional spore morphology-based identification of AMF in biodiversity studies is subjective and requires expertise and time. Researchers have used molecular techniques to investigate community composition of AMF in uncultivated, disturbed, or contaminated soils, but this approach to community analysis of AMF in agricultural soils has not been reported. In this study, a polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) procedure for the detection of fungal 18S ribosomal RNA gene was developed with reference cultures. Five AMF species were procured from the International Culture Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi (INVAM). These reference cultures were chosen because isolates of their species were putatively identified in a previous survey of farm field soils in Saskatchewan, Canada. A reference PCR-DGGE profile was generated using DNA extracted and amplified from the spores of these INVAM cultures. The methods technical limitations were investigated. The optimized procedures effectiveness was tested by its application to soil samples from 38 farms. Bands from the PCR-DGGE profiles of these samples were excised for sequence analysis. The total number of species recovered was low in comparison to other AMF community surveys of temperate climate locations. The majority of the sequences recovered were Glomus species. Scutellospora calospora, a previously undetected AM fungus in Saskatchewan was found. A trend in AMF distribution in Saskatchewan was observed and it was relatable to their phylogenetic taxonomy. Though not without its drawbacks, this approach to community composition analysis of AMF was faster than conventional trap cultivation methods.

Nuvarande metoder för att diagnostisera svampinfektioner i nagel är långsamma och troligen ganska okänsliga. Diagnostik görs vanligen genom direktmikroskopi och odling på svampsubstrat. I ett försök att minska analystiden och att öka känsligheten användes i denna studie realtids-PCR (Polymerase Chain Reaction) för att påvisa dermatofyter. Optimering och validering gjordes av en PCR som var generell för dermatofyter och en PCR som var specifik för Trichophyton rubrum. Optimeringen innefattade annealingtemperatur, Mg2+-koncentration och primerkoncentration. Valideringen gjordes genom analys av ett templat som spätts seriellt och av isolerade svampstammar. PCR-produkter identifierades med smältpunktsanalys. 241 konsekutiva nagelprover analyserades med PCR och resultaten jämfördes med de från odling/mikroskopi. PCR-effektiviteten bestämdes till 102 % resp. 104,5 % och det dynamiska området täckte ett koncentrationsområde på minst 104. Mellan-assay-variationen bestämdes till 1,6 % resp. 1,1 % för Ct-värdet (Threshold cycle) och 0,3 % resp. 0,2 % för smältpunktsbestämningen. Smältpunktsanalysen av stammarna visade att T. rubrum kunde identifieras med hjälp av PCR och att övriga dermatofyter kunde påvisas men inte artbestämmas. Analysen av nagelproverna visade att känsligheten hos PCR var bättre än känsligheten hos både odling (54 % i odlingen mot 99 % i PCR) och mikroskopi (83 % i mikroskopi mot 90 % i PCR). Metoden går att använda för att påvisa dermatofyter och för att identifiera T. rubrum.

In these studies we evaluated specific environmental stimuli relevant to Salmonella virulence and physiology in the gastrointestinal tract of chickens. Results from Salmonella growth in steady state, glucose-limiting continuous culture (CC) indicated that the optimal growth condition was observed between 0.05 h-1 and 0.27 h-1 dilution rates (D). Cell protein concentrations increased proportionally with an increase in D at each steady state, but after D 0.27 h-1 there was a reduction in the cell protein concentrations as the D increased. Genetic responses generally indicated that the lowest D exhibited highest hilA relative expression. Relatively higher expression of hilA was largely observed at low D (low glucose) (0.0125 h-1, 0.025 h-1, 0.05 h-1). Salmonella incubated in CC at different pH shifts demonstrated that cell protein concentration, glucose utilization, Yield ATP and Acetate:Propionate ratios were influenced by an increase in pH (6.14 to 7.41). These parameters increased and decreased consistently with a corresponding increase and decrease in pH. Polymerase chain reaction-based denaturant gradient gel electrophoresis showed that the overall amplicon band patterns of microbial similarity have demonstrated that hens molted with Alfalfa (ALC+) diet were similar to the Full-Fed (FF+) treatment group. Additional, FF+ and ALC+ treatment groups exhibited a higher percentage similarity coefficient (>90%) than the feed deprived treatment group. Fermentation response from cecal inocula on feed substrates revealed that alfalfa based samples yielded consistently higher short chain fatty acid levels when compared to other feed substrates. Salmonella Enteritidis (SE) colonization in liver, spleen and ovaries was significantly (P < 0.05) higher in FW+ hens compared to ALC+ and FF+ treatments groups. A 4-fold (log10 1.29) reduction in SE colonization for ALC+ hens compared to feed withdrawal hens (FW+) (log10 5.12) SE colonization was observed. Relative expression of hilA in all treatment groups was significantly (P < 0.05) higher in FW+ compared to FF+ and ALC+ groups. hilA expression in FW+ hens was 3.2-, 4.2-, and 1.9-fold higher for Days 6, 11 and 12 respectively, when compared with to ALC+ hens. These results suggest that Salmonella virulence in the gastrointestinal ecology of chickens could be impacted by a combination of low nutrients availability and pH shifts.

Three studies were conducted to investigate the use of molecular techniques to identify Salmonella serotypes in poultry. In the first experiment, two polymerase chain reaction (PCR)-based techniques: denaturing gradient gel electrophoresis (DGGE) and polyacrylamide gel electrophoresis (PAGE) were used to analyze Salmonella serotype isolates from two turkey processing plants (A and B). Genotypic patterns of each isolate were compared with those of known serotypes identified by traditional antibody precipitation methods. In Plant A, four different Salmonella serotypes were identified: Derby, Hadar, Montevideo, and Senftenberg. In plant B, ten serotypes were identified: Agona, Anatum, Brandenburg, Derby, Hadar, Meleagridis, Montevideo, Reading, Senftenberg, and Typhimurium. S. Derby was predominant in Plant A (83%) while S. Typhimurium was the most common serotype recovered in Plant B (39%). Overall, DGGE was more sensitive than PAGE. Isolates of the same serotypes were all grouped together by DGGE, while PAGE failed to group all like serotypes. Next, DGGE and REP-PCR were used as genotyping tools for identifying Salmonella. Fifty-four Salmonella isolates from two turkey processing plants (A and B) were evaluated. The isolates were comprised of the following serotypes: Brandenburg, Derby, Hadar, and Typhimurium (n = 6, 21, 12, and 15, respectively). Both methods were very sensitive and detected diverse fingerprint profiles among the isolates. The data suggested that REP-PCR and DGGE are useful tools for identifying Salmonella serotypes in research trials of this type. The final trial was carried out to track Salmonella serotypes throughout an integrated poultry operation using DGGE. Four flocks were sampled from grow-out through processing. The data showed that there was correlation between Salmonella serotypes found on processed carcasses and during grow-out. In addition, the isolates were compared against 15 known serotypes in our data base and only S. Hadar from the data base matched the unknown Salmonella isolates. Overall, these studies demonstrate that PCR-based methods could be considered as an alternative to conventional methods of antibody-based serotyping. Molecular methods were found to be reliable, sensitive, inexpensive, reproducible, and less labor intensive than conventional methods.

The annexin-7 (ANX7) gene is located on human chromosome 10q21, a site long been hypothesized to harbor a tumor suppressor gene (TSG) associated with brain, prostate, breast and other cancers. To test whether ANX7 might be a candidate TSG, and its role in the molecular pathogenesis of malignant glioma. We examined the ANX7 expression levels in several kinds of cell lines, 139 glioma specimens and 84 gastric cancer specimences. Consistently, analysis of ANX7 protein expression in human glioma tumor a significantly higher rate (2.14 times) of loss of ANX7 expression in glioblastoma multiformes (GBM) as compared with low grade astrocytoma (p=0.04). The striking correlation ANX7 expression and the differentiation of gastric carcinoma (p = 0.001). ANX7 may play an important role in the tumor progression. Loss or reduced expression of ANX7 is possibly a biomarker for tumor cell progression

Abstract This research used molecular biological techniques such as polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to analyze the dynamic microbial community and biodiversity in the groundwater contaminated with petroleum-hydrocarbons. The 16S rDNA sequences from all water samples were compared with the sequences of relative bacteria in the Ribosomal Database Project Bank to construct a phylogenetic tree. The results allowed us to understand the composition of the microbial communities in the petroleum-hydrocarbon contaminated groundwater. In this study, groundwater samples taken from the Chinese Petroleum Corporation Kaohsiung Refinery (CPCKR), Chinese Petroleum Corporation at Ciaotou fuel Tank Farm (CPCCTF) and China Petrochemical Development Corporation at Kaohsiung Factory (CPDCKF) were analyzed. The contaminated sites at CPDCKR and CPCCTF are remeated by natural attenuation. While the CPDCKF site is remeated by an enhanced air sparging bioremediation. In CPDCKR, we found that the low polluted area contained the richest microbial community, followed by the non-polluted area, and the high polluted area. At the CPCCTF site, the microbial community in the non-polluted area was richer than the high-polluted area. Increased microbial populations and variation in microbial community have beenobserved in non-polluted, less polluted, and highly polluted areas. The microbial community showed a dynamic succession of complexity during the bioremediation process at the CPDCKF site. From the 16S rDNA sequence analysis, it is possible that all samples contained petroleum-hydrocarbon degrading bacteria. These petroleum-hydrocarbon degrading bacteria include Methylobacterium, Xanthobacter, Xanthomonas and Pseudomonas at CPCKR site, Flavobacterium at CPCCTF site, Nocardia, Pseudomonas, Rubrivivax, Methylobacterium, and Candida at CPDCKF site. This study also demonstrates that it is more economic and reliable of using molecular techuiques to analyze the groundwater. Thus, groundwater samples can be used to replace soil samples for future work.