Identification of Lawsonia intracellularis proteins recognized by neutralizing antibodies and use of these proteins to design a subunit vaccine

Lawsonia intracellularis is an obligate intracellular microorganism which causes diseases known as porcine ileitis or porcine proliferative enteropathy. Due to its obligate intracellular nature, characterization of L. intracellularis antigens and proteins involved in host-pathogen interaction and immune recognition have been difficult to accomplish using conventional microbiological techniques. The overall goal of this thesis is to identify, characterize and evaluate L. intracellularis bacterial proteins as neutralizing antibody targets which may then be suitable candidates for subunit vaccine development. In this thesis, 2-dimensional (2-D) gel electrophoresis coupled with Western-immunoblotting, mass spectrometry, and bioinformatics were used to identify proteins on the surface of the bacteria that interact in vitro with pig intestinal cells (IPEC-1) and have immunogenic properties. Eleven immunogenic bacterial proteins were detected of which LI0710 (annotated as fliC), LI1153 (annotated as Putative protein N), LI0649 (annotated as autotransporter), and LI0169 (OppA; annotated as ABC dipeptide transport system) were predicted to be expressed on the outer membrane. The genes coding for these four proteins were cloned and expressed in Escherichia coli and the corresponding recombinant proteins were purified using affinity chromatography. Porcine hyperimmune serum against whole L. intracellularis lysate established that all four recombinant proteins were immunogenic. To be able to quantify invasion of the vaccine strain of L. intracellularis in McCoy and IPEC-1 cells and to determine whether antibodies specific for the recombinant L. intracellularis proteins inhibit the bacterial attachment and penetration into eukaryotic cells, the bacteria were labeled with cell-permeable fluorescent dye 5’-carboxyfluoroscein succidyl ester (CFSE) prior to cell infection. Flow cytometry was applied to determine the percentage of eukaryotic cells which were infected with fluorescent bacteria. As obligate intracellular bacteria, their replication is dependant on eukaryotic cells and thus qPCR analysis was applied to quantify bacterial growth. qPCR analysis showed increase of bacterial DNA over the course of five days, indirectly showing that bacterial invasion and growth took place. The CFSE+ (i.e. infected) McCoy cells were sorted from the CFSE- (i.e. non-infected) McCoy cells using fluorescence-activated cell sorting (FACS) while confocal microscopy was performed to visually confirm bacterial invasion and cytosolic localization of CFSE-L. intracellularis. Rabbit hyperimmune serum was generated against each recombinant protein and we investigated the effect that hyperimmune sera had on invasion of IPEC-1 cells. Serum antibodies significantly inhibited invasion and replication of CFSE-bacteria, thus indicating that each of the recombinant proteins is a potential neutralizing antibody target and a candidate for subunit vaccine formulation. In conclusion, we used 2-D gel electrophoresis coupled with mass spectrometry to identify four antigens that, when formulated in a vaccine, may lead to production of neutralizing antibodies and disease protection

Wetterprognoseunterstützte Regelung der Gebäudeklimatisierung : Analyse der Einspa-rungsmöglichkeiten bezüglich Kosten und Umwelt

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Review of the speculative role of co-infections in Streptococcus suis-associated diseases in pigs

Streptococcus suis is one of the most important bacterial swine pathogens affecting post-weaned piglets, causing mainly meningitis, arthritis and sudden death. It not only results in severe economic losses but also raises concerns over animal welfare and antimicrobial resistance and remains an important zoonotic agent in some countries. The definition and diagnosis of S. suis-associated diseases can be complex. Should S. suis be considered a primary or secondary pathogen? The situation is further complicated when referring to respiratory disease, since the pathogen has historically been considered as a secondary pathogen within the porcine respiratory disease complex (PRDC). Is S. suis a respiratory or strictly systemic pathogen? S. suis is a normal inhabitant of the upper respiratory tract, and the presence of potentially virulent strains alone does not guarantee the appearance of clinical signs. Within this unclear context, it has been largely proposed that co-infection with some viral and bacterial pathogens can significantly influence the severity of S. suis-associated diseases and may be the key to understanding how the infection behaves in the field. In this review, we critically addressed studies reporting an epidemiological link (mixed infections or presence of more than one pathogen at the same time), as well as in vitro and in vivo studies of co-infection of S. suis with other pathogens and discussed their limitations and possibilities for improvement and proposed recommendations for future studies.info:eu-repo/semantics/publishedVersio

Antioxidant Capacities and Phenolic Levels of Different Varieties of Serbian White Wines

The biologically active compounds in wine, especially phenolics, are responsible for reduced risk of developing chronic diseases (cardiovascular disrease, cancer, diabetes, etc.), due to their antioxidant activities. We determined the contents of total phenolics (TP) and total flavonoids (TF) in selected Serbian white wines by colorimetric methods. Total antioxidant activity (TAA) of the white wines was analyzed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity assay. Međaš beli had the highest content of TP, TF and TAA. The radical scavenging capacity (RSC) and total antioxidant activity (TAA) of white wines were 15.30% and 1.055 mM Trolox equivalent, respectively. Total phenolic (TP) and total flavonoid (TF) contents in white wines ranged from 238.3 to 420.6 mg gallic acid equivalent per L of wines and 42.64 to 81.32 mg catechin equivalent per L of wines, respectively. A high and significant correlation between antioxidant activity and total phenolic content was determined in wines (R2 = 0.968, p < 0.01). For the individual polyphenols determination we used a high performance liquid chromatography (HPLC)-diode array detection (DAD) technique. The majority of white wine polyphenols was represent by four hydroxycinnamic acids (HCAs)

Pectobacterium carotovorum subsp. carotovorum – prouzrokovač vlažne truleži biljaka kale u Srbiji i Crnoj Gori

Bacterial strains were isolated from above- and underground parts of diseased calla plants originating from different localities in Serbia and one locality in Montenegro. They were characterized by studying their pathogenic, cultural, biochemical and physiological characteristics. All investigated strains caused soft rot of calla leaf stalks, potato slices and aloe leaves, and induced hypersensitive reaction on tobacco. Bacteriological properties of the strains indicated that symptoms on calla plants were caused by Gram-negative, nonfluorescent, oxidase negative, catalase positive and facultatively anaerobic bacterium belonging to the genus Pectobacterium. The investigated strains grew at 37ºC and in 5% NaCl, utilised lactose and trechalose, and produced neither indol nor lecitinase. These results, as well as the characteristic growth on Logan’s differential medium indicated that soft rot of tuber and stem base of calla plants was caused by Pectobacterium carotovorum subsp. carotovorum. This is the first report of this pathogen affecting calla plants in Serbia.Proučene su patogene, odgajivačke i biohemijsko-fiziološke odlike sojeva izolovanih iz nadzemnih i podzemnih delova obolelih biljaka kale gajene u različitim lokalitetima u Srbiji i jednom lokalitetu u Crnoj Gori. Svi proučavani sojevi ispoljili su izraženu pektolitičku aktivnost prouzrokujući vlažnu trulež lisnih drški kale, kriški krompira, liski aloje ili sanseverije, a prouzrokovali su i hipersenzitivnu reakciju duvana. Proučavanjem bakterioloških karakteristika utvrđeno je da promene na biljkama kale prouzrokuju Gram-negativni, nefluorescentni, oksidaza-negativni, katalaza-pozitivni i fakultativno-anaerobni sojevi bakterije, koja prema navedenim karakteristikama pripada rodu Pectobacterium. Proučavani sojevi se razvijaju pri 37ºC i u prisustvu 5% NaCl, razlažu laktozu i trehalozu, ne stvaraju indol i lecitinazu. Navedeni rezultati, kao i karakterističan razvoj na Loganovoj diferencijalnoj podlozi ukazuju da je vlažnu trulež korena i prizemnog dela biljaka kale prouzrokovala bakterija Pectobacterium carotovorum subsp. carotovorum. Ovo je prvo saopštenje o pojavi P. c. ssp. carotovorum kao patogena kale u Srbiji

Karakterizacija sojeva Xanthomonas euvesicatoria, patogena paprike u Srbiji

During spring and summer of 2008, 101 bacterial strains was isolated from the diseased pepper leaves collected from different pepper growing areas in the Republic of Serbia. The aim of this research was to characterize the isolated strains and determine their taxonomic position according to the most recent nomenclature. Pathogenic, biochemical and physiological characteristics of isolated bacteria were tested using standard bacteriological tests. The pathogen races were determined according to the reaction of differential varieties of Early Calwonder (ECW), their isogenic lines (ECW-10R, ECW-20R, ECW-30R) and Capsicum pubescens. The sensitivity of strains to bactericides was studied in vitro by culturing bacteria on sucrose pepton agar (SPA) plates, amended with filter-sterilized aqueous solution of streptomycin and kasugamycin (50, 100, 200 ppm) or copper-sulphate (100, 200 ppm). Based on pathogenic, biochemical and physiological characteristics, the investigated strains belonged to Xanthomonas euvesicatoria. The reaction of pepper differential varieties indicated that these strains belonged to pepper races P1, P3, P7, P8. Streptomycin resistant strains were not detected, but 6 strains were resistant to kasugamycin (50 ppm) and 13 strains to copper-sulphate (200 ppm), indicating bacterial resistance development.Tokom 2008. godine prikupljeni su uzorci obolelog lišća paprike sa simptomima bakteriozne pegavosti iz različitih lokaliteta Republike Srbije. Izolacijom iz zaraženih listova dobijen je 101 soj bakterija. Cilj ovog rada je proučavanje odlika i identifikacija izolovanih sojeva bakterije u skladu sa najnovijom nomenklaturom. Patogene i biohemijsko-fiziološke karakteristike sojeva proučene su korišćenjem standardnih bakterioloških testova. Određivanje fizioloških rasa bakterijske populacije izvršeno je na osnovu reakcije diferencijalnih sorti paprike Early Calwonder (ECW), njenih izogenih linija (ECW-10, ECW-20 ECW-30) i reakcijom sorte Capsicum pubescens. Proučena je osetljivost sojeva u in vitro uslovima na streptomicin, kasugamicin i bakar-sulfat. Rezultati proučavanja biohemijsko-fizioloških odlika sojeva ukazuju da naši sojevi pripadaju genetičkoj grupi „A“, odnosno vrsti Xanthomonas euvesicatoria. Proučavani sojevi, takođe, predstavljaju heterogenu populaciju u kojoj su zastupljene četiri fiziološke rase bakterije X. euvesicatoria (P1, P3, P7, P8). Sojevi rezistentni na streptomicin nisu detektovani ovim istraživanjima. Otpornost na 50 ppm kasugamicina utvrđena je kod 6 sojeva, a 13 sojeva je bilo otporno na 200 ppm bakar-sulfata. Prikazani rezultati ukazuju na opasnost od razvoja rezistentnosti bakterija na ova jedinjenja

Efikasnost organskih kiselina u očuvanju kvaliteta obroka za ishranu goveda

This paper presents the efficiency of organic acids (sorbic acid, benzoic acid and palm and coconut oil acids, ammonium propionate, etc.) in prevention of microorganisms activities and undesirable spoilage process of total mixed ration (TMR) for cattle feeding during the summer months. Meals treated with these acids have a lower total number of bacteria and yeasts (cfu/g) for 25 to 78%, are less heated up, and are eaten better. Usage of these acid (1-2 L/t of food) does not require the use of expensive equipment. Purchase costs are compensated by the nutrients less loss and more milk production. Protection of TMR meals by organic acids is justified in the cattle diet once a day and at a time of high summer temperatures.U radu je prikazana efikasnost organskih kiselina (sorbinska, benzijeva, kiseline palminog i koksovog ulja, amonijum propionat i dr.) u sprečavanju aktivnosti mikroorganizama i nepoželjnih procesa kvarenja potpunih mešanih obroka (TMR) za ishranu goveda. Obroci tretirani ovim kiselinama imaju manji ukupan broj bakterija i kvasaca, slabije se greju i bolje konzumiraju. Korišćenje kiselina (1-2 L/t hrane) ne zahteva upotrebu skupe opreme. Troškovi nabavke se nadoknađuju manjim gubitkom hranljivih materija i većom proizvodnjom mleka. Zaštita TMR obroka organskim kiselinama ima opravdanje u ishrani goveda jednom dnevno, posebno u vreme visokih letnjih temperatura

Polyphenol Content and Antioxidant Activity of Sour Cherries From Serbia

The aim of this study was to evaluate the content of phenolics: the total phenols (TP), flavonoids (TF), anthocyanins (TA), as well as the total antioxidant\ud capacity (TAC) in three sour cherry cultivars (Prunus cerasus L.) introduced to the southeast Serbia climate conditions. Among the investigated sour cherries,\ud „Oblačinska“ cultivar contained the highest amounts of all groups of phenolics, followed by „Cigančica“ > „Marela“. A significant difference were observed in the phenolic content among different cultivars and growing seasons (p  0.05), and the phenolic compounds were significantly higher in the growing season 2009. The examined cultivars possess a high antioxidant capacity, and all phenolics of highy correlation with TAC. The following compounds were identified and quantified using HPLC-DAD: 4 anthocyanins, the most abundant of which was cyanidin-3-glucoside in “Marela” and “Oblačinska”, and cyanidin-3-glucosylrutinoside in „Cigančica“, and 4 hydroxycinnamic acids, the most abundant of which was neochlorogenic acid in all sour cherry cultivars. The growing and ripening process on the tree of sour cherry cv. „Oblačinska“ was evaluated also. The results showed significant increases in total phenols during the ripening, the total anthocyanins and total antioxidant capacity and 4 quantified anthocyanins, however the neochlorogenic acid decreased during the ripening. The study indicated that the growing and climate conditions in southeast Serbia are convenient for introducing sour cherry cultivars.\u