Development of a multiplex real-time PCR assay using two thermocycling platforms for detection of major bacterial pathogens associated with bovine respiratory disease complex from clinical samples

Bovine respiratory disease complex (BRDC) is one of the most significant diseases of cattle. Bacterial pathogens involved in BRDC include Mannheimia haemolytica, Mycoplasma bovis, Histophilus somni, and Pasteurella multocida. We developed and evaluated a multiplexed real-time hydrolysis probe (rtPCR) assay using block-based Peltier and rotary-based thermocycling on lung tissue, nasal swabs, and deep nasopharyngeal swabs. The rtPCR results were compared to culture or a gel-based M. bovis PCR using statistical analysis to determine optimum quantification cycle (Cq) cutoffs to maximize agreement. The limits of detection were 1.2–12 CFU/reaction for each pathogen. M. haemolytica was the most prevalent organism detected by rtPCR, and was most frequently found with P. multocida. The rtPCR assay enabled enhanced levels of detection over culture for all pathogens on both thermocycling platforms. The rotary-based thermocycler had significantly lower Cq cutoffs (35.2 vs. 39.7), which maximized agreement with gold standard culture or gel-based PCR results following receiver operating characteristic analysis to maximize sensitivity (Se) and specificity (Sp). However, overall assay Se and Sp were similar on both platforms (80.5% Se, 88.8% Sp vs. 80.1% Se, 88.3% Sp). Implementation of these tests could enhance the detection of these pathogens, and with high-throughput workflows could reduce assay time and provide more rapid results. The assays may be especially valuable in identifying coinfections, given that many more antemortem samples tested in our study were positive for 2 or more pathogens by rtPCR (n = 125) than were detected using culture alone (n = 25)

Gamma rays induced enhancement in the phytonutrient capacities of tomato (Solanum Lycopersicum L.)

One of the most important problems for the field-grown Ayaş tomato (Solanum lycopersicum Ayas population), which is preferred for consumption in Central Anatolia because of its aromatic taste, is that the shelf life is short because of insufficient fruit firmness. Therefore, a study was initiated to develop high-quality lines and variety candidates through mutation breeding in the current Ayaş population. In this study, the effective mutation dose (EMD50) was found to be 150 Gy for seeds using a Cobalt-60 gamma ray source. The main mutant population was generated by applying EMD50. During the study, mutant lines selected by following the classical mutation breeding stages were evaluated in terms of yield and quality traits (antioxidant content, total soluble solid amount, fruit firmness, Brix, etc.) starting from the M4 stage. One of our aims was to determine and improve the phytonutrient content of field-grown tomatoes. The fruits of selected lines at the M4 stage were extracted by liquid-liquid partition and accelerated solvent extraction techniques and analyzed by High Performance Liquid Chromatography (HPLC) in this study. It was found that 28 of the 29 mutant lines were significantly different from the control and mutant lines. Mutant lines 9-22, 8-90, 8-135, and 8-127 were determined to be the most promising for commercialization

Controlling Public Health Risks Posed by Zoonotic Pathogens in Beef Cattle Production Through Consumption of Meat Products and Exposure to Environmental Pathways

The contribution of meat animal production (including beef) to the occurrence of human bacterial infections remains a prominent and contentious issue. Currently, the challenge has centered on the accurate identification of the contamination pathways from food animal production to human populations. Evidence shows that the pathogens can be transferred from food animals to human populations through the consumption of contaminated meat products; or via the exposure to livestock and livestock production associated environmental pathways. Therefore, the goal of this research was to determine and evaluate the pathways of pathogen transmission linked to livestock production and to translate the research findings into safety practices for the reduction of human illnesses. In this dissertation, bacterial contamination pathways for beef production chain was first investigated in a farm-to-processing continuum. Then, the fate and transfer of antimicrobial resistant bacteria and genes in the environment associated with beef cattle primary production and manure storage and land application were studied. Finally, public health risks associated with consumption of lettuce that can be potentially contaminated by fertilization manure were estimated. Results showed that the lairage pen environment at the processing plant is of more importance as a contamination source for beef cattle carcasses, compared to animal feces, and pen surface, feed and water sources on the feedlot. The results indicate that cleaning and disinfection of lairage pen environment may potentially reduce the bacterial contamination in beef products. There is no significant evidence demonstrating the linkage between antimicrobial administration in beef cattle and the increase in resistant bacteria and genes in feedlot pen surface materials, manure or in the air/dust nearby. However, beef cattle manure can be a contamination source of antimicrobial resistance in lettuce as soil amendment. To mitigate the contamination originating from manure, our studies showed manure storage as static piles can be considered as candidate intervention strategy, as both antimicrobial resistant bacteria and genes were effectively reduced in manure after a 3-month storage

Controlling Public Health Risks Posed by Zoonotic Pathogens in Beef Cattle Production Through Consumption of Meat Products and Exposure to Environmental Pathways

The contribution of meat animal production (including beef) to the occurrence of human bacterial infections remains a prominent and contentious issue. Currently, the challenge has centered on the accurate identification of the contamination pathways from food animal production to human populations. Evidence shows that the pathogens can be transferred from food animals to human populations through the consumption of contaminated meat products; or via the exposure to livestock and livestock production associated environmental pathways. Therefore, the goal of this research was to determine and evaluate the pathways of pathogen transmission linked to livestock production and to translate the research findings into safety practices for the reduction of human illnesses. In this dissertation, bacterial contamination pathways for beef production chain was first investigated in a farm-to-processing continuum. Then, the fate and transfer of antimicrobial resistant bacteria and genes in the environment associated with beef cattle primary production and manure storage and land application were studied. Finally, public health risks associated with consumption of lettuce that can be potentially contaminated by fertilization manure were estimated. Results showed that the lairage pen environment at the processing plant is of more importance as a contamination source for beef cattle carcasses, compared to animal feces, and pen surface, feed and water sources on the feedlot. The results indicate that cleaning and disinfection of lairage pen environment may potentially reduce the bacterial contamination in beef products. There is no significant evidence demonstrating the linkage between antimicrobial administration in beef cattle and the increase in resistant bacteria and genes in feedlot pen surface materials, manure or in the air/dust nearby. However, beef cattle manure can be a contamination source of antimicrobial resistance in lettuce as soil amendment. To mitigate the contamination originating from manure, our studies showed manure storage as static piles can be considered as candidate intervention strategy, as both antimicrobial resistant bacteria and genes were effectively reduced in manure after a 3-month storage

Klinik insan vakaları, klinik olmayan hayvan vakaları ve farklı gıdalardaki salmonella çeşitliliğinin vuruşlu alan jel elektroforezi (PFGE) kullanılarak moleküler düzeyde belirlenmesi.

Salmonella is one of the most reported pathogens in foodborne outbreaks worldwide. In order to ensure safety of foods, farm-to-fork surveillance and control systems must be utilized. Development of numerous typing methods have improved the ability to detect salmonellosis outbreaks, enabling to trace the contaminated source from farm to fork. Facilitating prevention and regulation of techniques, knowledge of the epidemiology, genetics and ecology of Salmonella infections depend on the information provided by typing methods. In this study a phenotypic subtyping method, serotyping, and the current “gold standard” molecular subtyping method for bacterial pathogens, pulsed-field gel electrophoresis (PFGE) was used for characterization of 59 isolates of food origin, 53 isolates of animal origin and 50 clinical human isolates collected from Şanlıurfa region. Salmonella was prevalent mostly in chicken and offal samples (58.3 %), while Salmonella isolates from positive samples were much diverse in animal feces. Paratyphi B was the main serovar recorded for clinical human isolates; serovars Enteritidis, Typhimurium and Kentucky were recovered from all three sources. Having the most diversity from both typing methods, 53 animal isolates were represented by 13 subsp. enterica serovars and subspecies diarizonae and salamae; moreover, 28 PFGE types were observed. For all isolates, PFGE (SID: 0.975) represented a higher discriminatory power with respect to serotyping (SID: 0.915); serovar Montevideo had the most variation with 8 different PFGE types. The results were uploaded to a publicly available databanks (Food Microbe Tracker: http://www.foodmicrobetracker.com/login/login.aspx and Pathogen Detector: pathogendetector-metu.rhcloud.com); therefore, the information could be useful for investigations on farm-to fork chain, as well as for evolution, ecology and transmission of Salmonella in Turkey. From our knowledge, this is the first study reporting Salmonella diversity through farm to fork chain in the same period of time and at the same location in Turkey.M.S. - Master of Scienc

Children, monsters and mothers where the wild things are and beasts of the southern wild

64 page

FEN BİLİMLERİ ENSTİTÜSÜ/LİSANSÜSTÜ TEZ PROJESİ

ÇOKLU İLAÇ DİRENÇLİ SALMONELLA İZOLATLARINDAKİ PLAZMİDLERİN İLETİŞİM KABİLİYETLER

KLİNİK İNSAN VE HAYVAN VAKALARI İLE GIDALARDAKİ SALMONELLA ÇEŞİTLİLİĞİNİN VURUŞLU ALAN JEL ELEKTROFOREZİ ( PFGE) KULLANILARAK MOLEKÜLER DÜZEYDE BELİRLENMESİ

KLİNİK İNSAN VE HAYVAN VAKALARI İLE GIDALARDAKİ SALMONELLA ÇEŞİTLİLİĞİNİN VURUŞLU ALAN JEL ELEKTROFOREZİ ( PFGE) KULLANILARAK MOLEKÜLER DÜZEYDE BELİRLENMES

FEN BİLİMLERİ ENSTİTÜSÜ/LİSANSÜSTÜ TEZ PROJESİ

KLİNİK İNSAN VE HAYVAN VAKALARI İLE GIDALARDAKİ SALMONELLA ÇEŞİTLİLİĞİNİN VURUŞLU ALAN JEL ELEKTROFOREZİ ( PFGE) KULLANILARAK MOLEKÜLER DÜZEYDE BELİRLENMES