Non-resorbable glass fibre-reinforced composite with porous surface as bone substitute material: Experimental studies in vitro and in vivo focused on bone-implant interface

The development of load-bearing osseous implant with desired mechanical and surface properties in order to promote incorporation with bone and to eliminate risk of bone resorption and implant failure is a very challenging task. Bone formation and resoption processes depend on the mechanical environment. Certain stress/strain conditions are required to promote new bone growth and to prevent bone mass loss. Conventional metallic implants with high stiffness carry most of the load and the surrounding bone becomes virtually unloaded and inactive. Fibre-reinforced composites offer an interesting alternative to metallic implants, because their mechanical properties can be tailored to be equal to those of bone, by the careful selection of matrix polymer, type of fibres, fibre volume fraction, orientation and length. Successful load transfer at bone-implant interface requires proper fixation between the bone and implant. One promising method to promote fixation is to prepare implants with porous surface. Bone ingrowth into porous surface structure stabilises the system and improves clinical success of the implant. The experimental part of this work was focused on polymethyl methacrylate (PMMA) -based composites with dense load-bearing core and porous surface. Three-dimensionally randomly orientated chopped glass fibres were used to reinforce the composite. A method to fabricate those composites was developed by a solvent treatment technique and some characterisations concerning the functionality of the surface structure were made in vitro and in vivo. Scanning electron microscope observations revealed that the pore size and interconnective porous architecture of the surface layer of the fibre-reinforced composite (FRC) could be optimal for bone ingrowth. Microhardness measurements showed that the solvent treatment did not have an effect on the mechanical properties of the load-bearing core. A push-out test, using dental stone as a bone model material, revealed that short glass fibre-reinforced porous surface layer is strong enough to carry load. Unreacted monomers can cause the chemical necrosis of the tissue, but the levels of leachable resisidual monomers were considerably lower than those found in chemically cured fibre-reinforced dentures and in modified acrylic bone cements. Animal experiments proved that surface porous FRC implant can enhance fixation between bone and FRC. New bone ingrowth into the pores was detected and strong interlocking between bone and the implant was achieved.

In vivo sensitivity reduction of Puccinia triticina races, causal agent of wheat leaf rust, to DMI and QoI fungicides

Experiments were carried out to determine in vivo the IC50 and the IC90 for demethylation-inhibitor fungicides (DMIs, triazoles) and quinone outside inhibitors (QoIs, strobilurins) to the five most frequent races of Puccinia triticina in 2007 growing season in Southern Brazil. The tests were done in a greenhouse with wheat seedlings. DMI fungicides were tested at the concentrations, in mg/L, 0.0; 0.02; 0.2; 2.0; 20.0; 100.0 and 200.0, and QoIs at the concentrations 0.0; 0.0001; 0.001; 0.01; 0.1; 1 and 10.0 mg of active ingredient/L water. Fungicides were preventively applied at 24 hours before the inoculation of seedlings with the fungal spores. The effect of treatments was assessed based on the number of uredia/cm². The lowest IC50 (inhibitory concentration) for DMI fungicides determined for MCG-MN, sensitive race, ranged from 0.33 to 0.91 mg/L, while the highest values for MDP-MR, MDT-MR, MDK-MR, MFH-HT races, varied from 9.63 to 85.64 mg/L (suspected insensitivity). QoI fungicide presented an IC50 varying from 0.0018 to 0.14 mg/L. The sensitivity reduction factor for DMIs varied from 8.8 to 238.8, and for QoIs from 0.3 to 1.5 mg/L. Sensitivity reduction was confirmed for the races MDP-MR, MDT-MR, MDK-MR, MFH-HT to DMIs, as well as their sensitivity to QoI fungicides.

Wood as a model material for medical biomaterials. In vivo and in vitro studies with bone and Betula pubescens Ehrh

Novel biomaterials are needed to fill the demand of tailored bone substitutes required by an ever‐expanding array of surgical procedures and techniques. Wood, a natural fiber composite, modified with heat treatment to alter its composition, may provide a novel approach to the further development of hierarchically structured biomaterials. The suitability of wood as a model biomaterial as well as the effects of heat treatment on the osteoconductivity of wood was studied by placing untreated and heat‐treated (at 220 C , 200 degrees and 140 degrees for 2 h) birch implants (size 4 x 7mm) into drill cavities in the distal femur of rabbits. The follow‐up period was 4, 8 and 20 weeks in all in vivo experiments. The flexural properties of wood as well as dimensional changes and hydroxyl apatite formation on the surface of wood (untreated, 140 degrees C and 200 degrees C heat‐treated wood) were tested using 3‐point bending and compression tests and immersion in simulated body fluid. The effect of premeasurement grinding and the effect of heat treatment on the surface roughness and contour of wood were tested with contact stylus and non‐contact profilometry. The effects of heat treatment of wood on its interactions with biological fluids was assessed using two different test media and real human blood in liquid penetration tests. The results of the in vivo experiments showed implanted wood to be well tolerated, with no implants rejected due to foreign body reactions. Heat treatment had significant effects on the biocompatibility of wood, allowing host bone to grow into tight contact with the implant, with occasional bone ingrowth into the channels of the wood implant. The results of the liquid immersion experiments showed hydroxyl apatite formation only in the most extensively heat‐treated wood specimens, which supported the results of the in vivo experiments. Parallel conclusions could be drawn based on the results of the liquid penetration test where human blood had the most favorable interaction with the most extensively heat‐treated wood of the compared materials (untreated, 140 degrees C and 200 degrees C heat‐treated wood). The increasing biocompatibility was inferred to result mainly from changes in the chemical composition of wood induced by the heat treatment, namely the altered arrangement and concentrations of functional chemical groups. However, the influence of microscopic changes in the cell walls, surface roughness and contour cannot be totally excluded. The heat treatment was hypothesized to produce a functional change in the liquid distribution within wood, which could have biological relevance. It was concluded that the highly evolved hierarchical anatomy of wood could yield information for the future development of bulk bone substitutes according to the ideology of bioinspiration. Furthermore, the results of the biomechanical tests established that heat treatment alters various biologically relevant mechanical properties of wood, thus expanding the possibilities of wood as a model material, which could include e.g. scaffold applications, bulk bone applications and serving as a tool for both mechanical testing and for further development of synthetic fiber reinforced composites.

Regulation of lymphocyte response in vitro and in vivo

CD4+ T helper (Th) cells have an important role in the defence against diverse pathogens. Th cells can differentiate into several functionally distinct subtypes including Th1 and Th2 cells. Th1 cells are important for eradicating intracellular pathogens, whereas Th2 cells pro¬tect our body against extracellular parasites. However if uncontrolled, Th cells can mediate immunopathology such as asthma or allergies, but inappropriate Th response can also lead to autoimmune diseases such as multiple sclerosis or type 1 diabetes. Deeper knowledge of the regulation of the lymphocyte response both in vitro and in vivo is important for un¬derstanding the pathogenesis of immune-mediated diseases and microbe-host interactions. In the work presented in this thesis, the first goal was to elucidate the role of novel factors, PIM kinases and c-FLIP in the regulation of human Th cell differentiation. The oncogenic serine-threonine kinases of the PIM family were shown to be preferentially expressed in Th1 cells and in addition, by using RNA interference, they were also shown to be positive regulators of Th1 differentiation. The PIM depletion experiments suggest that PIM kinases promote the expression of the hallmark cytokine of Th1 cells, IFNγ, and influence the IL12/STAT4 pathway during the early Th1 cell differentiation. In addition to cytokine and T cell receptor (TCR) induced pathways, caspase activity has been shown to regulate Th cell proliferation. In the work presented in this thesis, the two isoforms of the caspase regulator protein, c-FLIP, were shown to be differentially ex¬pressed in Th1 and Th2 cells. Both of the isoforms were up-regulated in response to TCR activation, but the expression of the short isoform was selectively induced by IL4, the Th2 inducing cytokine. Furthermore, the c-FLIP isoforms had distinct and opposite roles during the early differentiation of Th1 and Th2 cells. The knockdown of the long isoform of c-FLIP led to the induction of Th1 marker genes, such as IFNγ and TBET, whereas the depletion of c-FLIP short down-regulated Th2 marker genes IL-4 and GATA3. The third goal was to elucidate the gene expression profiles characterizing the T- and B-lymphocyte responses in vivo during experimental infection by intracellular bacte¬rium Chlamydia pneumoniae. Previously, it has been shown that CD8+ and CD4+ T cells are important for the protection against Chlamydia pneumoniae. In this study, the analysis revealed up-regulation of interferon induced genes during recurrent infection underlining the importance of IFNγ secreted by Th1 and CD8+ T cells in the protection against this pathogen. Taken together, in this study novel regulators of Th cell differ¬entiation were discovered and in addition the gene expression profiles of lymphocytes induced by Chlamydia pneumoniae infection were characterized.

Mitochondrial DNA synthesis studied autoradiographically in various cell types in vivo

It is generally accepted that mitochondria are able to proliferate even in postmitotic cells due to their natural turnover and also to satisfy increased cell energy requirements. However, no detailed studies are available, particularly with respect to specific cell types. Since [3H]-thymidine is incorporated not only into nuclear (n) DNA but also into the DNA of cytoplasmic mitochondria, an autoradiographic approach was developed at the light microscopy level in order to study basic questions of mitochondrial (mt) proliferation in organs of rodents in situ via the cytoplasmic incorporation of [3H]-thymidine injected into the animals 1 h before sacrifice. Experiments carried out on mice after X-irradiation showed that cytoplasmic labeling was not due to a process such as unscheduled nuclear DNA synthesis (nUDS). Furthermore, half-lives of mitochondria between 8-23 days were deduced specifically in relation to cell types. The phase of mtDNA synthesis was about 75 min. Finally, mt proliferation was measured in brain cells of mice as a function of age. While all neurons showed a decreasing extent of mtDNA synthesis during old age, nUDS decreased only in distinct cell types of the cortex and hippocampus. We conclude that the leading theories explaining the phenomenon of aging are closely related, i.e., aging is due to a decreasing capacity of nDNA repair, which leads to unrepaired nDNA damage, or to an accumulation of mitochondria with damaged mtDNA, which leads to a deficit of cellular energy production

Food restriction induces in vivo ventricular dysfunction in spontaneously hypertensive rats without impairment of in vitro myocardial contractility

Cardiac structures, function, and myocardial contractility are affected by food restriction (FR). There are few experiments associating undernutrition with hypertension. The aim of the present study was to analyze the effects of FR on the cardiac response to hypertension in a genetic model of hypertension, the spontaneously hypertensive rat (SHR). Five-month-old SHR were fed a control or a calorie-restricted diet for 90 days. Global left ventricle (LV) systolic function was evaluated in vivo by transthoracic echocardiogram and myocardial contractility and diastolic function were assessed in vitro in an isovolumetrically beating isolated heart (Langendorff preparation). FR reduced LV systolic function (control (mean ± SD): 58.9 ± 8.2; FR: 50.8 ± 4.8%, N = 14, P < 0.05). Myocardial contractility was preserved when assessed by the +dP/dt (control: 3493 ± 379; FR: 3555 ± 211 mmHg/s, P > 0.05), and developed pressure (in vitro) at diastolic pressure of zero (control: 152 ± 16; FR: 149 ± 15 mmHg, N = 9, P > 0.05) and 25 mmHg (control: 155 ± 9; FR: 150 ± 10 mmHg, N = 9, P > 0.05). FR also induced eccentric ventricular remodeling, and reduced myocardial elasticity (control: 10.9 ± 1.6; FR: 9.2 ± 0.9%, N = 9, P < 0.05) and LV compliance (control: 82.6 ± 16.5; FR: 68.2 ± 9.1%, N = 9, P < 0.05). We conclude that FR causes systolic ventricular dysfunction without in vitro change in myocardial contractility and diastolic dysfunction probably due to a reduction in myocardial elasticity.

Comparative effects of organic and inorganic mercury on in vivo dopamine release in freely moving rats

The present study was carried out in order to compare the effects of administration of organic (methylmercury, MeHg) and inorganic (mercury chloride, HgCl 2 ) forms of mercury on in vivo dopamine (DA) release from rat striatum. Experiments were performed in conscious and freely moving female adult Sprague-Dawley (230-280 g) rats using brain microdialysis coupled to HPLC with electrochemical detection. Perfusion of different concentrations of MeHg or HgCl 2 (2 µL/min for 1 h, N = 5-7/group) into the striatum produced significant increases in the levels of DA. Infusion of 40 µM, 400 µM, or 4 mM MeHg increased DA levels to 907 ± 31, 2324 ± 156, and 9032 ± 70% of basal levels, respectively. The same concentrations of HgCl 2 increased DA levels to 1240 ± 66, 2500 ± 424, and 2658 ± 337% of basal levels, respectively. These increases were associated with significant decreases in levels of dihydroxyphenylacetic acid and homovallinic acid. Intrastriatal administration of MeHg induced a sharp concentration-dependent increase in DA levels with a peak 30 min after injection, whereas HgCl 2 induced a gradual, lower (for 4 mM) and delayed increase in DA levels (75 min after the beginning of perfusion). Comparing the neurochemical profile of the two mercury derivatives to induce increases in DA levels, we observed that the time-course of these increases induced by both mercurials was different and the effect produced by HgCl 2 was not concentration-dependent (the effect was the same for the concentrations of 400 µM and 4 mM HgCl 2 ). These results indicate that HgCl 2 produces increases in extracellular DA levels by a mechanism differing from that of MeHg.

Ultrasound method applied to characterize healthy femoral diaphysis of Wistar rats in vivo

A simple experimental protocol applying a quantitative ultrasound (QUS) pulse-echo technique was used to measure the acoustic parameters of healthy femoral diaphyses of Wistar rats in vivo. Five quantitative parameters [apparent integrated backscatter (AIB), frequency slope of apparent backscatter (FSAB), time slope of apparent backscatter (TSAB), integrated reflection coefficient (IRC), and frequency slope of integrated reflection (FSIR)] were calculated using the echoes from cortical and trabecular bone in the femurs of 14 Wistar rats. Signal acquisition was performed three times in each rat, with the ultrasound signal acquired along the femur's central region from three positions 1 mm apart from each other. The parameters estimated for the three positions were averaged to represent the femur diaphysis. The results showed that AIB, FSAB, TSAB, and IRC values were statistically similar, but the FSIR values from Experiments 1 and 3 were different. Furthermore, Pearson's correlation coefficient showed, in general, strong correlations among the parameters. The proposed protocol and calculated parameters demonstrated the potential to characterize the femur diaphysis of rats in vivo. The results are relevant because rats have a bone structure very similar to humans, and thus are an important step toward preclinical trials and subsequent application of QUS in humans.

Developing correlative Optical-Photoacustic microscopy for high-resolution in vivo imaging

PhotoAcoustic Imaging (PAI) is a branch in clinical and pre-clinical imaging, that refers to the techniques mapping acoustic signals caused by the absorption of the short laser pulse. This conversion of electromagnetic energy of the light to the mechanical (acoustic) energy is usually called photoacoustic effect. PAI, by combining optical excitation with acoustical detection, is able to preserve the diffraction limited spatial resolution. At the same time, the penetration depth is extended beyond the diffusive limit. The Laser-Scanning PhotoAcoustic Microscope system (LS-PAM) has been developed, that offers the axial resolution of 7.75 µm with the lateral resolution better than 10 µm. The first in vivo imaging experiments were carried out. Thus, in vivo label-free imaging of the mouse ear was performed. The principle possibility to image vessels located in deep layers of the mouse skin was shown. As well as that, a gold printing sample, vasculature of the Chick Chorioallantoic Membrane Assay, Drosophila larvae were imaged by PAI. During the experimental work, a totally new application of PAM was found, in which the acoustic waves, generated by incident light can be used for further imaging of another sample. In order to enhance the performance of the presented system two main recommendation can be offered. First, the current system should be transformed into reflection-mode setup system. Second, a more powerful source of light with the sufficient repetition rate should be introduced into the system.

Comparison of epicardial mapping and noncontact endocardial mapping in dog experiments and computer simulations

La fibrillation auriculaire, l'arythmie la plus fréquente en clinique, affecte 2.3 millions de patients en Amérique du Nord. Pour en étudier les mécanismes et les thérapies potentielles, des modèles animaux de fibrillation auriculaire ont été développés. La cartographie électrique épicardique à haute densité est une technique expérimentale bien établie pour suivre in vivo l'activité des oreillettes en réponse à une stimulation électrique, à du remodelage, à des arythmies ou à une modulation du système nerveux autonome. Dans les régions qui ne sont pas accessibles par cartographie épicardique, la cartographie endocardique sans contact réalisée à l'aide d'un cathéter en forme de ballon pourrait apporter une description plus complète de l'activité auriculaire. Dans cette étude, une expérience chez le chien a été conçue et analysée. Une reconstruction électro-anatomique, une cartographie épicardique (103 électrodes), une cartographie endocardique sans contact (2048 électrodes virtuelles calculées à partir un cathéter en forme de ballon avec 64 canaux) et des enregistrements endocardiques avec contact direct ont été réalisés simultanément. Les systèmes d'enregistrement ont été également simulés dans un modèle mathématique d'une oreillette droite de chien. Dans les simulations et les expériences (après la suppression du nœud atrio-ventriculaire), des cartes d'activation ont été calculées pendant le rythme sinusal. La repolarisation a été évaluée en mesurant l'aire sous l'onde T auriculaire (ATa) qui est un marqueur de gradient de repolarisation. Les résultats montrent un coefficient de corrélation épicardique-endocardique de 0.8 (expérience) and 0.96 (simulation) entre les cartes d'activation, et un coefficient de corrélation de 0.57 (expérience) and 0.92 (simulation) entre les valeurs de ATa. La cartographie endocardique sans contact apparait comme un instrument expérimental utile pour extraire de l'information en dehors des régions couvertes par les plaques d'enregistrement épicardique.

Δ9-tetrahydrocannabivarin suppresses in vitro epileptiform and in vivo seizure activity in adult rat

Purpose: We assessed the anticonvulsant potential of the phytocannabinoid Δ9-tetrahydrocannabivarin (Δ9-THCV) by investigating its effects in an in vitro piriform cortex (PC) brain slice model of epileptiform activity, on cannabinoid CB1 receptor radioligand-binding assays and in a generalized seizure model in rats. Methods: Δ9-THCV was applied before (10 μmΔ9-THCV) or during (10–50 μmΔ9-THCV) epileptiform activity induced by Mg2+-free extracellular media in adult rat PC slices and measured using multielectrode array (MEA) extracellular electrophysiologic techniques. The actions of Δ9-THCV on CB1 receptors were examined using [3H]SR141716A competition binding and [35S]GTPS assays in rat cortical membranes. Effects of Δ9-THCV (0.025–2.5 mg/kg) on pentylenetetrazole (PTZ)–induced seizures in adult rats were also assessed. Results: After induction of stable spontaneous epileptiform activity, acute Δ9-THCV application (≥20 μm) significantly reduced burst complex incidence and the amplitude and frequency of paroxysmal depolarizing shifts (PDSs). Furthermore, slices pretreated with 10 μmΔ9-THCV prior to induction of epileptiform activity exhibited significantly reduced burst complex incidence and PDS peak amplitude. In radioligand-binding experiments, Δ9-THCV acted as a CB1 receptor ligand, displacing 0.5 nm [3H]SR141716A with a Ki∼290 nm, but exerted no agonist stimulation of [35S]GTPS binding. In PTZ-induced seizures in vivo, 0.25 mg/kg Δ9-THCV significantly reduced seizure incidence. Discussion: These data demonstrate that Δ9-THCV exerts antiepileptiform and anticonvulsant properties, actions that are consistent with a CB1 receptor–mediated mechanism and suggest possible therapeutic application in the treatment of pathophysiologic hyperexcitability states.

Modulation of detoxification enzymes by watercress: in vitro and in vivo investigations in human peripheral blood cells

Epidemiological studies indicate that consumption of cruciferous vegetables (CV) can reduce the risk of cancer. Supposed mechanisms are partly the inhibition of phase I and the induction of phase II enzymes. The aim of this study was to investigate in vitro and in vivo effects of watercress (WC), a member of the CV family, on chemopreventive parameters using human peripheral blood mononuclear cells (PBMC) as surrogate cells. We investigated the hypothesis that WC reduces cancer risk by inducing detoxification enzymes in a genotype-dependent manner. In vitro gene expression and enzyme activity experiments used PBMC incubated with a crude extract from fresh watercress (WCE, 0.1-10 mu L/mL with 8.2 g WC per 1 mL extract) or with one main key compound phenethyl isothiocyanate (PEITC, 1-10 mu M). From an in vivo perspective, gene expression and glutathione S-transferase (GST) polymorphisms were determined in PBMC obtained from a human intervention study in which subjects consumed 85 g WC per day for 8 weeks. The influence of WC consumption on gene expression was determined for detoxification enzymes such as superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1), whilst the SOD and GPX activities in red blood cells were also analysed with respect to GST genotypes. In vitro exposure of PBMC to WCE or PEITC (24 h) increased gene expression for both detoxification enzymes GPX1 (5.5-fold, 1 mu L/mL WCE, 3.7-fold 1 mu M PEITC) and SOD2 (12.1-fold, 10 mu L/mL WCE, 7.3-fold, 10 mu M PEITC), and increased SOD2 activity (1.9-fold, 10 mu L/mL WCE). The WC intervention had no significant effect on in vivo PBMC gene expression, as high individual variations were observed. However, a small but significant increase in GPX (p = 0.025) and SOD enzyme activity (p = 0.054) in red blood cells was observed in GSTM1*0, but not in GSTM1*1 individuals, whilst the GSTT1 genotype had no impact. The results indicate that WC is able to modulate the enzymes SOD and GPX in blood cells in vitro and in vivo, and suggest that the capacity of moderate intake of CV to induce detoxification is dependent in part on the GSTM1 genotype.

Intracellular metabolism and bioactivity of quercetin and its in vivo metabolites

Understanding the cellular effects of flavonoid metabolites is important for predicting which dietary flavonoids might be most beneficial in vivo. Here we investigate the bioactivity in dermal fibroblasts of the major reported in vivo metabolites of quercetin, i.e. 3'-O-methyl quercetin, 4'-O-methyl quercetin and quercetin 7-O-beta-D-glucuronide, relative to that of quercetin, in terms of their further metabolism and their resulting cytotoxic and/or cytoprotective effects in the absence and presence of oxidative stress. Uptake experiments indicate that exposure to quercetin led to the generation of two novel cellular metabolites, one characterized as a 2'-glutathionyl quercetin conjugate and another product with similar spectral characteristics but 1 mass unit lower, putatively a quinone/quinone methide. A similar product was identified in cells exposed to 3'-O-methyl quercetin, but not in the lysates of those exposed to its 4'-O-methyl counterpart, suggesting that its formation is related to oxidative metabolism. There was no uptake or metabolism of quercetin 7-O-beta-D-glucuronide by fibroblasts. Formation of oxidative metabolites may explain the observed concentration-dependent toxicity of quercetin and 3'-O-methyl quercetin, whereas the formation of a 2'-glutathionyl quercetin conjugate is interpreted as a detoxification step. Both O -methylated metabolites conferred less protection than quercetin against peroxide-induced damage, and quercetin glucuronide was ineffective. The ability to modulate cellular toxicity paralleled the ability of the compounds to decrease the level of peroxide-induced caspase-3 activation. Our data suggest that the actions of quercetin and its metabolites in vivo are mediated by intracellular metabolites.

Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.

In vivo characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry

Aims: To test the efficacy of Lactobacillus johnsonii FI9785 in reducing the colonization and shedding of Salmonella enterica serotype Enteritidis, Escherichia coli O78:K80 and Clostridium perfringens in poultry. Methods and Results: Specific pathogen-free chicks (1 day old) were dosed with a single oral inoculum of 1 x 10(9) CFU. Lactobacillus johnsonii FI9785 and 24 h later were challenged in separate experiments with S. Enteritidis (S1400, nal(r)) and E. coli O78:K80 (EC34195, nal(r)). There were no significant effects against S. Enteritidis whereas colonization of the small intestine by E. coli O78:K80 was reduced significantly. Both S. Enteritidis and E. coli colonized the caeca and colon to levels equivalent to control birds and there was no reduction in shedding as assessed by a semi-quantitative cloacal swabbing technique. Specific pathogen-free chicks (20 day old) were dosed with a single oral inoculum of 1 x 10(9) CFU L. johnsonii FI9785 and 24 h later were challenged with C. perfringens. A single oral dose of L. johnsonii FI9785 was sufficient to suppress all aspects of colonization and persistence of C. perfringens. Conclusions: Lactobacillus johnsonii FI9785 may be given to poultry for use as a competitive exclusion agent to control C. perfringens. Significance and Impact of the Study: Lactobacillus johnsonii FI9785 may be a valuable tool to control the endemic disease of necrotic enteritis, thereby reducing economic losses associated with reduced use of antimicrobials in the poultry industry.