Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Please refer to fulltext for abstract / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Antimicrobial peptides (AMPs) are currently the most researched group of compounds for new antimicrobial drugs especially with the rise in resistance to almost all available drugs by public health relevant pathogens. In this study we set out to characterise small cyclic AMPs in terms of their activity towards human pathogens Listeria monocytogenes, a food-borne pathogen causing listeriosis and Plasmodium falciparum, a parasite that causes malaria respectively, each a threat to public health. One of the small cyclic peptide libraries examined is the tyrocidines (Trcs) and analogues, which are cyclic decapeptides [cyclo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val- (Orn/Lys)-Leu] produced by the Gram-positive bacteria Bacillus aneurinolyticus as part of the tyrothricin complex which is non-ribosomally synthesised during sporulation. Previous research found that the six major Trcs were active against Listeria monocytogenes and Plasmodium falciparum and it was found that the identity of the aromatic residues in the aromatic dipeptide unit has an important role in activity. We set out to extend the qualitative structure to activity relationship (QSAR) studies using more Trc analogues and small synthetic Arg- and Trp-rich cyclic peptides (RW-peptides) in a bid to establish essential structural motifs and pre-requisites for activity. Eight natural and three synthetic Trc analogues and fifteen RW-peptides were either naturally or by chemical synthesis produced and characterised in terms of chemical character and biological activity. The Trcs were significantly more active than RW peptides, although much more haemolytic and thus toxic. Results indicated the relevance for hydrogen bonding with an aromatic amino acid residue for selective activity towards the leucocin A resistant L. monocytogenes B73-MR1. However, structural properties favouring a tighter membrane interaction hindered the Trc mode of action (MOA). We determined that Gln6 and hydroxyl group of Tyr7 may be involved in interaction with the putative target in L. monocytogenes. There was also need for an amphipathic balance between hydrophobicity and size/steric parameters for optimal activity. From our QSAR studies we predict as lead peptide for a future library of antilisterial Trcs: cyclo(VOMe3LfPWfNQY). Furthermore, the antilisterial activity of the Trcs was found to be predominantly lytic and salt tolerant while RW-peptides were non-lytic and sensitive to Ca2+. We confirmed that Ca2+ enhanced Trc antilisterial activity with Ca2+ increasing the Trc anti-metabolic activity, but conversely inducing a non-lytic mechanism of action. From model membrane studies, we propose that the calcium induced Trc non-lytic MOA could be due to detrimental lipid demixing, presence of a Trc sensitive Ca2+-induced non-membrane target in the prematurely calcium induced intracellular anaerobic form of Listeria monocytogenes, and/or the Trc-Ca2+ complexes may inhibit key components such as membrane bound electron transport system or bacterial dehydrogenases. We confirmed, as previously found, that the Trcs have potent antimalarial activity that is sequence specific and non-lytic. The RW-peptides had very weak activity, but our results again indicated that more hydrophobic and haemolytic peptides tend to be more active, particularly the RW-peptide containing the Trp analogue β-(benzothien-3-yl)-alanine (Bal). A novel finding was that one of the more polar Trc C analogues, namely tryptocidine C (Tpc C), in contrast to Trc C showed potent antimalarial activity indicating the specific sequence and the role of the Trp7 in activity. From these results a proposed lead peptide for future research is cyclo[VOLfP(Bal)fNQ(Bal)]. Furthermore, in our search for the Trc and Tpc C target(s) we employed high resolution fluorescence microscopy. Results show that Trc led to disorganisation of neutral lipid structures and chromatin halting growth in late trophozoite/early schizont stages. This indicated that membrane structures containing neutral lipids, as well as chromatin may be targeted by the Trcs. Another novel finding in our studies was that chloroquine (CQ) resistance not only correlated with resistance to Trcs, but the Trcs and CQ were found to be antagonistic towards each other’s activity. This indicated a shared target and we propose the food vacuole as another of the Trc targets in P. falciparum. / AFRIKAANSE OPSOMMING: Antimikrobiese peptiede (AMPe) is tans die mees nagevorsde groep verbindings in die soeke na nuwe antimikrobiese middels, veral weens 'n toenemende weerstandigheid van patogene in die openbare gesondheidsektor teen alle beskikbare middels. Die doel van hierdie studie was om klein, sikliese AMPe in terme van hul aktiwiteit teenoor twee menslike patogene wat 'n bedreiging vir openbare gesondheid is, Listeria monocytogenes, 'n voedsel-oordraagbare patogeen wat listeriose veroorsaak, asook Plasmodium falciparum, die parasiet verantwoordelik vir malaria, te karakteriseer. Een van die klein, sikliese peptiedbiblioteke wat ondersoek is, is die tyrocidines (Trcs) en analoë (sikliese dekapeptiede [siklo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val- (Orn/Lys)-Leu]). Hierdie peptiede deur die Gram-positiewe bakterie Bacillus aneurinolyticus word wat nie-ribosomaal gesintetiseer as deel van die tirotrisien kompleks word tydens sporulasie. Vorige navorsing het gewys dat die ses hoof Trcs teen Listeria monocytogenes en Plasmodium falciparum aktief is en dat die identiteit van die aromatiese residue in die aromatiese dipeptiedeenheid 'n belangrike rol speel in die Trc-aktiwiteit. Ons het gepoog om die kwalitatiewe struktuur-aktiwiteit-verwantskap (QSAR) studies uit te brei deur meer Trc analoë en klein sintetiese Arg- en Trp-ryke sikliese peptiede (RW-peptiede) te gebruik en sodoende essensiële struktuur-motiewe en voorvereistes vir aktiwiteit vas te stel. Agt natuurlike en drie sintetiese Trc analoë, asook vyftien RW-peptiede is of deur natuurlike of chemiese sintese geproduseer en gekarakteriseer in terme van chemiese karakter en biologiese aktiwiteit. Die Trcs het beduidend meer aktiwiteit as RW-peptiede getoon, maar is ook meer hemolities en dus meer toksies. Die resultate dui op die belang van waterstofbinding met 'n aromatiese aminosuurresidu vir die selektiewe aktiwiteit teenoor die leucocin A weerstandige L. monocytogenes B73-MR1. Strukturele eienskappe wat tot 'n sterker membraan-interaksie lei, verhinder egter die werkingsmeganisme. Ons het vasgestel dat Gln en die hidroksielgroep van Tyr betrokke kan wees in die interaksie met die vermeende teenmiddelteiken in L. monocytogenes. 'n Balans tussen amfipatiese/hidrofobiese en grootte/steriese parameters is ook noodsaaklik vir optimale aktiwiteit. Vanuit ons QSAR studies word die peptied siklo-(VOMe3LfPWfNQY) as die voorloper vir 'n toekomstige peptiedbiblioteek van antilisteriale Trcs voorgestel. Verder is daar gevind dat die antilisteriese aktiwiteit van die Trcs oorwegend lities en sout-verdraagsaam is, terwyl die RW-peptiede nie-lities en Ca2+ sensitief is. Ons het bevestig dat Ca2+ die Trc antilisteriese aktiwiteit verbeter, deur die Trc se antimetaboliese aktiwiteit verhoog, maar terselfdertyd 'n nie-litiese werkingsmeganisme induseer. Vanuit model-membraan studies word voorgestel dat Trc se nie-litiese werkingsmeganisme, soos teweeggebring deur Ca2+, die gevolg kan wees van nadelige lipied vermenging, die teenwoordigheid van 'n kalsium geïnduseerde Trcsensitiewe nie-membraan teiken in 'n vervroegde kalsium geïnduseerde intrasellulêre anaerobiese vorm van Listeria monocytogenes, en/of dat die Trc-Ca2+ komplekse belangrike komponente soos ’n membraan-gebonde elektron transport sisteem of bakteriële dehidrogenases inhibeer. Daar is ook bevestig, soos voorheen gevind, dat die Trcs kragtige, antimalaria aktiwiteit besit wat volgorde-spesifiek en nie-lities is. Die RW-peptiede het swak aktiwiteit getoon, maar ons resultate het weereens bewys dat peptiede wat meer hidrofobies en hemolities is, meer aktief is, veral die RW-peptiede wat die Trp analoog β-(bensoteïen-3-iel)-alanien (Bal) bevat. 'n Nuwe bevinding is dat een van die meer polêre Trc C analoë, genaamd triptosidien C (Tpc C), in teenstelling met Trc C, sterk antimalaria aktiwiteit het, wat 'n aanduiding is van die spesifieke volgorde en die rol van die Trp7 in aktiwiteit. Vanuit hierdie bevindinge word die peptied siklo- (VOLfP(Bal)fNQ(Bal)) as 'n voorloper vir toekomstige navorsing aangedui. Vir ons soeke na die Trc en Tpc C teiken(s), het ons hoë resolusie fluoressensie mikroskopie aangewend. Resultate toon dat Trc tot die ontwrigting van 'n neutrale lipied strukture en chromatien lei en sodoende groei beperk in die laat trofosoïet/vroeë skisont fases. Dit het aangedui dat die membraanstrukture wat neutrale lipiede bevat, sowel as chromatien, deur die Trcs geteiken word. 'n Verdere nuwe bevinding in hierdie studie was dat chloroquine (CQ) weerstandigheid nie net korreleer met weerstandigheid teen Trcs nie, maar dat die Trcs en CQ antagonisties optree teenoor mekaar se aktiwiteite. Dit dui op 'n gemeenskaplike teiken en die kosvakuool as 'n addisionele Trc teiken in P. falciparum word voorgestel.

No description available.

Pathogenic Listeria are the causative agents of listeriosis, a severe food-borne infection. They are able to invade various non-phagocytic cell types including epithelial cells. The life cycle of these intracellular parasites involves penetrating into host cells, rupturing of the phagocytic vacuole, rapidly proliferating in the cytosol, and directly spreading cell to cell. Each step of the listerial intracellular infection involves activation of virulence factors dependent on PrfA, the master regulator of Listeria virulence. PrfAmediated virulence gene activation occurs within host cells by mechanisms that remain unknown. This thesis explores several aspects of PrfA regulation and its impact in the host-pathogen interaction. Methods for assessing PrfA-dependent gene expression were first developed and standardized, including a highly sensitive and accurate quantitative reverse-transcription real-time PCR (RT-QPCR), as well as procedures to investigate the correlation with virulence using cell culture-based assays. These techniques were applied in an investigation into the structure-function of PrfA. We studied the role of a solvent-accessible pocket identified in the N-terminal domain of PrfA, homologous to the cyclic nucleotide-binding (CNB) domain of Crp and other cAMP-regulated proteins, in intracellular virulence gene activation. Site-directed PrfA mutants were constructed. Our data support the notion that PrfA activity is allosterically regulated and are consistent with a role for the pocket as putative binding site for the PrfA-activating allosteric effector. The characterization of spontaneously occurring PrfA mutations that identified in our laboratory as PrfA*- suppressor or attenuator mutations, A129T, E173G and C229Y, allowed us to gain additional insight into PrfA structure-function. The role of the C229Y in sugar-mediated repression was investigated and found to explain the anomalous phenotype of strain NCTC 7973, a prfA* (G145S) mutant that carries this second mutation and is repressed by cellobiose but not glucose. We also carried out experiments to address the intriguing activation of PrfAdependent virulence genes upon addition of an adsorbent to the culture medium, the socalled "charcoal effect". Using a chemically defined culture medium and resin, Amberlite™ XAD-4, we provided evidence that the virulence gene activation may involve the sequestration of a medium component rather than a bacteria-derived autorepressor, as initially thought. We also explored the role of PrfA and the sigma factor σB in L. monocytogenes entry into host cells. ΔsigB mutants in different prfA regulation backgrounds were constructed. We showed that σB has no major effect on host cell invasion, and that L. monocytogenes invasiveness is a strictly PrfA-dependent trait. Our results also demonstrate a differential role of σB in L. monocytogenes serotypes. σB apparently plays no role in stress tolerance in serotype 4b, whereas it is important in serotype 1/2a for maintenance of bacterial fitness in stress conditions. Finally, we investigated the occurrence of apoptosis in Listeria-infected cells and developed normalized methods to accurately determine and quantify this cellular response in infected cell monolayers.

La surveillance de la listériose en France s’est construite par étapes depuis les années 1980 sur deux piliers, la microbiologie et l’épidémiologie. Grâce à la création du Centre National de Référence des Listeria et à la mise au point de techniques de typage performantes, l’Institut Pasteur assure une surveillance microbiologique depuis 1987. Une surveillance épidémiologique initiée entre 1984 et 1992 par le Laboratoire National de la Santé, a été développée par le Réseau National de Santé Publique de 1993 à 1999, puis amplifiée par l’Institut de Veille Sanitaire à partir de 2000. Le premier objectif de cette thèse est de décrire les différentes phases de la construction de cette surveillance afin d’analyser leurs contributions respectives au cours de ces 30 dernières années. Cette construction s’est faite en 4 phases : 1. L’étape fondatrice de 1982 à 1992 a été la reconnaissance et la prise en compte du rôle des aliments dans la transmission de la maladie et dans la survenue d’épidémies. 2. La deuxième phase de 1993 à 2000 a été l’édification d’un système de surveillance opérationnel pour détecter et investiguer les épidémies en France. 3. La troisième phase de 2000 à 2005 a permis de consolider le système de surveillance et de le perfectionner en ajoutant un volet complémentaire avec des prélèvements alimentaires.4. Depuis 2005, nous sommes dans la quatrième phase avec comme objectif l’optimisation du système. Cette optimisation repose sur l’adaptation des outils de surveillance et d’alerte aux connaissances. Ainsi, après avoir montré que la durée d’incubation de la maladie varie selon la forme clinique de la maladie, nous avons proposé d’intégrer cette variation pour déterminer la période d’évaluation des expositions alimentaires à risque. L’analyse des performances du système a permis à deux reprises de proposer de nouveaux seuils de signalement plus spécifiques afin d’optimiser cette surveillance tout en réduisant son coût. Le deuxième objectif de cette thèse est de montrer la contribution des données de surveillance à une politique de santé publique. Un premier travail a consisté à mettre en perspective les variations temporelles d’incidence observées avec les différentes sources de données disponibles afin d’en analyser les déterminants. La phase de décroissance de 1987 à 1997 a été concomitante des mesures de contrôles prises par l’industrie agro-alimentaire et de la réduction de la contamination des aliments. La phase d’augmentation en 2006-2007 semble multifactorielle. L’augmentation de la prescription de traitements de réduction de l’acidité gastrique par des inhibiteurs de la pompe à protons pourrait être l’un des déterminants majeurs de cette augmentation.Dans une deuxième analyse, nous avons hiérarchisé les groupes à risque de listériose sur la base de l’estimation du taux d’incidence de listériose et de sa mortalité dans ces groupes. Cela a permis d’identifier les groupes les plus vulnérables : hémopathies, certains cancers (digestifs, cérébral et pulmonaire), maladie de Horton, cirrhose hépatique, les dialysés rénaux, les greffés, et les femmes enceintes. Une analyse épidémiologique des listérioses materno-néonatales (MN) a montré une association entre les régions avec une incidence plus faible de listérioses materno-néonatales et les régions où la séroprévalence toxoplasmique des femmes enceintes est la plus faible, ce qui suggère un effet positif des recommandations contre la toxoplasmose pour la prévention de la listériose MN. / Listeriosis surveillance was built up stage by stage in France since the 1980s on a twofold basis: microbiology and epidemiology. Thanks to the creation of the Listeria National Reference Centre (Centre National de Référence des Listeria), the Pasteur Institute has been doing microbiological surveillance since 1987. Epidemiological surveillance was initiated by the National Health Laboratory, then conducted by the National Health Network and further developed by the National Institute of Health Surveillance. This thesis aims first of all to describe the different stages in the setting up of this surveillance system in order to analyze their respective inputs during these last thirty years. The four stages are:1. From 1982 to 1992: awareness and recognition of the role of food in the transmission of listeriosis and as the source of outbreaks. 2. From 1993 to 2000: building a reliable surveillance system in order to detect and investigate outbreaks in France. 3. From 2000 to 2005: strengthening and perfecting the surveillance system by taking additional measures, such as food sampling.4. Since 2005, we have reached the fourth stage, designed to optimize the surveillance system. This optimization involves adapting surveillance and early warning tools to new knowledge and information. For instance, having established that listeriosis incubation periods vary according to the clinical form of the illness, we suggested the integration of the variation of exposure period factor when interviewing patients with the food questionnaire. On two separate occasions, analysis of the surveillance system performance results made it possible to modify the criteria for early warning so as to optimize surveillance by increasing its specificity whilst reducing costs.The second aim of this thesis is to illustrate how surveillance data can contribute to public health policies. A first study analyzed temporal trends, using all available data in order to give some explanation as to major trends. The first trend was a reduction of incidence from 1987 to 1997 that was concomitant with control measures by the food industry and a drop in food contamination. The increased trend observed in 2006-2007 appears to be due to several factors. The increased rate of sales of proton pump inhibitors medication could be the major factor in this increase. In a second study, we ranked groups at risk of acquiring listeriosis based on the incidence of listeriosis and its lethality in each group. This enabled us to identify the most vulnerable groups : hematological malignancy, some cancers (digestive, lung, and brain cancer), dialysis, cirrhosis, organ transplantation and pregnancy. Epidemiological analysis of listeriosis cases associated with pregnancy indicated an association between regions with low rate listeriosis associated with pregnancy and regions where toxoplasmosis prevalence of pregnant women is low. This suggests that recommendations for avoiding toxoplasmosis have a positive effect on preventing listeriosis during pregnancy.

The bacterium Listeria monocytogenes can cause the disease listeriosis in both humans and animals. For the epidemiological investigation of listeriosis detection and characterisation of the organism are important steps. Paper I. There are few reports on the incidence of L. monocytogenes in clinical samples from humans in India. Therefore, we investigated 144 samples from immunocompromised patients. L. monocytogenes was isolated from two placental bits from women with poor obstetric history, one patient with renal failure and three other patients. Five isolates were positive for the virulence genes hlyA, actA and iap. The sixth isolate was positive for hlyA and actA genes. Paper II. Characterisation of 601 human L. monocytogenes isolates causing invasive listeriosis during the period 1986 to 2007 in Sweden reveals a decrease in serovar 4b strains. Since 1996, serovar 1/2a has become the predominant serovar causing human listeriosis: PFGE analysis revealed two clusters including different serotypes suggesting that we need more studies on genetic relatedness among clinical isolates. Paper III. The incidence of Listeria species in seafood from markets in Goa was studied. One hundred and fifteen raw/fresh seafoods bought at the fish markets were sampled and tested for presence of Listeria spp. L. monocytogenes was detected in 10 samples. L. monocytogenes in raw seafood may pose a health risk in kitchen if contaminating ready-to-eat food. Paper IV. Gravad and cold-smoked salmon are associated with human listeriosis in Sweden. L. monocytogenes was isolated from 11 of 56 products. Serovar 1/2a was predominant, followed by 4b. REA/PFGE typing of the isolates identified five types of L. monocytogenes. One type was identical to a human type, two other were closely related.These findings suggest that gravad and cold-smoked salmon are still possible sources of listeriosis in Sweden. Paper V. Many outbreaks of listeriosis have been related to consumption of dairy-associated products. Therefore, 123 farm bulk milk samples in India and 20 cervico-vaginal samples from dairy cows with reproductive disorders were investigated for L. monocytogenes. L. monocytogenes was isolated from 17.9% of bulk milk samples and from 10% of cervico-vaginal swabs. The virulence gene hlyA was detected in all isolates. These findings represent a public health risk where unpasteurised milk and milk products are largely consumed. Paper VI. Isolates of L. monocytogenes (n=177) from 22 animal species were characterized and compared with human strains isolated between 1986-2006 in Sweden. Although many animals and humans shared pulsovars, they did not appear at the same time or with the same proportion of strains. The pulsovars shared by both animals and humans may indicate that there is an exchange of L. monocytogenes strains between these two groups due to either direct or indirect transmission. / <p>The work is done in cooperation with the School of Hospitality, Culinary Arts &amp; Meal Science, Örebro UniversityVishal Singh Parihar, Örebro University, Department of Restaurant and Culinary Arts, Sörälgsvägen 2, SE-712 60 Grythyttan, Sweden or ICAR Research Complex for Goa, Ela, Old Goa – 403402, Goa, India. Phone 0832-2284678/79; Fa:0832-2285649. E-mail: drvishu@yahoo.co.in</p>

La listeriosis murina experimental se caracteriza por la rápida llegada de leucocitos, especialmente neutrófilos (PMNNs) y macrófagos a los órganos diana de la infección, hígado y bazo. El rápido reclutamiento de estas células a los focos de infección es vital para el control inicial de la infección. Las lesiones nerviosas en la listeriosis murina experimental se caracterizan por la afluencia masiva de PMNNs hacia las meninges y al sistema ventricular. Sin embargo, de la misma manera que el rápido reclutamiento de estas células a los focos de infección es vital para el control inicial de la infección, no se puede excluir la posibilidad de que el reclutamiento de PMNNs en el sistema nervioso central contribuya al desarrollo de las lesiones nerviosas mediante la liberación de mediadores inflamatorios y/o participando a la vehiculación y diseminación de la listerias. Uno de los objetivos principales de este trabajo doctoral fue el estudio de la expresión de las moléculas de adhesión endotelial E-selectina, P-selectina e ICAM-1 en el transcurso de la listeriosis murina experimental y determinar la correlación entre la expresión de dichas moléculas y las lesiones inflamatorias observadas. Asimismo, se intentó elucidar la importancia de los PMNNs en la patogenia de las lesiones nerviosas mediante la utilización del anticuerpo monoclonal RB6-8C5, el cual depleciona específicamente la población de PMNNs in vivo .La estandarización de las técnicas inmunohistoquímicas para la detección de las moléculas de adhesión endotelial E-selectina, P-selectina e ICAM-1 en tejidos de ratón fijados en formol e incluidos en parafina fue llevada a cabo mediante la caracterización de su expresión en ratones normales y en un modelo de activación endotelial por lipopolisacárido bacteriano. E-selectina fue únicamente detectada tras activación endotelial en la mayoría de los órganos. ICAM-1fue detectada de forma constitutiva en células endoteliales de todos los órganos estudiados y su expresión fue sobre-regulada tras activación con lipopolisacárido. Estos resultados están en concordancia con los resultados obtenidos previamente por otros autores en tejidos congelados.En el modelo murino de listeriosis, tanto P-selectina como ICAM-1 fueron inducidas y/o sobre-expresadas rápidamente tras la infección. E-selectina fue detectada únicamente de forma espóradica en este modelo. Tras esta primera fase de activación endotelial inespecífica, la sobre-expresión de estas moléculas estuvo asociada a la presencia de lesiones inflamatorias en los órganos diana de la infección, especialmente hígado, órganos linfoides secundarios y SNC. En el hígado, la aparición de las lesiones inflamatorias está correlacionada con la expresión de ICAM-1 en los sinusoides hepáticos y de P-selectina e ICAM-1 en las venas centrolobulillares y en los vasos situados en el espacio porta. La lesiones esplénicas estuvieron asociadas a la expresión de ICAM-1 en la zona marginal de la pulpa blanca. En los linfonodos inflamados, P-selectina e ICAM-1 fueron detectadas en las venas de endotelio alto. En el SNC, las lesiones iniciales consistieron en una leptomeningitis purulenta asociada a una fuerte expresión endotelial de P-selectina e ICAM-1 en vénulas del espacio subaracnoideo, especialmente en aquellas situadas en la fisura del hipocampo. La aparición de ventriculitis y/o coroiditis purulentas en nuestro modelo fue un fenómeno posterior a la presencia de meningitis y está asociado a una intensa expresión de ICAM-1 las células epiteliales coroideas y en las células ventriculares así como en el endotelio de vasos periventriculares. La depleción de PMNNs mediante la administración del anticuerpo monoclonal RB6-8C5 durante la listeriosis murina experimental facilitó la multiplicación de Listeria monocytogenes en el SNC pero aparentemente no previno o interfirió su acceso al encéfalo. Estas observaciones sugieren que los PMNN tienen una función esencial en la prevención del acceso y/o la multiplicación de L. monocytogenes en el SNC. / Systemic murine listeriosis is characterized by the rapid influx of leukocytes, especially neutrophils but also macrophages, into the site of initial bacterial replication, especially the liver and spleen. Neutrophils play a critical role in reducing the bacterial burden in these organs. Neutrophils are also the main inflammatory cell present in meningitis, ventriculitis/choroiditis and rombencephalitis that develop during the course of human and experimental murine listeriosis. Recruited neutrophils are thought to contribute to the elimination of L. monocytogenes, although they could also be involved in the development of progressive brain injury via the release of harmful mediators and/or participating in the dissemination of Listeria . The aim of this study was to detect by immunohistochemistry the expression of the endothelial adhesion molecules E-selectin, P-selectin and ICAM-1 during the course of experimental murine listeriosis and to determine if correlation existed between the expression of these adhesion molecules and the inflammatory infiltrate in the target organs. In addition, the role of neutrophils in the development of the lesions in the central nervous system (CNS) was studied by the in vivo administration of the neutrophil-depleting antibody RB6-8C5. Immunohistochemical techniques to detect ICAM-1, P-selectin and E-selectin in formalin-fixed, paraffin embedded tissues were developed using standard antigen unmasking protocols. The distribution and pattern of expression of E-selectin and ICAM-1 in normal and lipopolysaccharide-stimulated mice was described. No constitutive expression of E-selectin was found in any of the normal tissues investigated. In stimulated animals, E-selectin expression was detected on endothelial cells in most evaluated organs. ICAM-1 was constitutively expressed, and was upregulated in all the organs after LPS inoculation. The results paralleled previous studies where immunohistochemistry on frozen sections was used. During the course of experimental murine listeriosis, a strong up-regulation of P-selectin and ICAM-1 occurred rapidly after the infection. E-selectin was faint and inconstantly detected in all the studied organs. The expression of these adhesion molecules was correlated with the recruitment of leukocytes, especially to the liver, lymphoid organs and CNS. In the liver, typical lesions of murine listeriosis were related to the expression of ICAM-1 on sinusoidal endothelial cells, and to the de novo expression of P-selectin in hepatic portal vessels. Inflammation in the spleen was related to the expression of ICAM-1 on red pulp sinusoidal cells, especially in the marginal sinus. High endothelial venules of inflamed lymph nodes also expressed P-selectin and ICAM-1. In the CNS, the expression of P-selectin and up-regulation of ICAM-1 in meningeal vessels, especially in those located in the hippocampal sulcus, was associated with the initial meningeal inflammation and suggest that neutrophils probably reach the CNS through these vessels during experimental murine listeriosis. Leptomeningitis was followed by the presence of ventriculitis, which was related to the up-regulation of ICAM-1 on choroid plexus epithelial cells, periventricular vessels and ependimal cells.Administration of the neutrophil-depleting antibody RB6-8C5 during experimental murine listeriosis enhanced or facilitated multiplication of L. monocytogenes as well as lesion formation in the CNS of infected mice. Thus, neutrophils not only play a key role in host defense against L. monocytogenes infection in the limiting early bacterial multiplication in the liver and the spleen, but they are also crucial in eliminating bacteria that gain access to the CNS compartment.

Thesis (doctoral)--Helsinki University of Technology, 2007. / Includes bibliographical references. Also available on the World Wide Web.

Meat and poultry slaughtering and processing practices have been associated with the microbial contamination with Listeria spp. Ready-to-eat poultry products have been considered as a primary agent associated with Listeria monocytogenes illness outbreaks. Developing environmental monitoring programs (EMPs) that are based on product and/or process risk level analysis is a useful approach to reduce contamination in poultry processing plants and enhance food safety. Sampling criteria that is based on product risk levels and process control in ready-to-eat poultry processing facilities was developed to allow users to design and conduct appropriate sampling plans to target Listeria spp. After developing the criteria, an internet-based environmental monitoring program ("EZSafety") was developed to allow poultry producers to enhance their sample collection and analysis of test results over time and conduct appropriate sampling plans for Listeria spp. and other microbiological indicators. The frontend of the program website was built using React Native (an open-source JavaScript library for building user interfaces). The backend of the program website was built using Node.js which executes JavaScript code outside a web browser. MongoDB was used as a document-oriented database for the website. The program was evaluated by 20 food safety professionals to assess its ability to develop appropriate sampling plans to target Listeria spp. The majority of these participants believed that EZSafety has several tools that are effective for targeting Listeria spp. and other indicators and enhancing environmental monitoring. Additionally, most participants agreed that EZSafety is organized and user-friendly. EMPs can play a significant role in improving the detection rate and the prevention of Listeria spp. and other indicators in poultry processing plants. / Master of Science in Life Sciences / Meat and poultry slaughtering and processing practices have been associated with the microbial contamination with a bacterium known as Listeria. Cooked poultry products during the manufacturing process have been considered as a primary agent associated with Listeria monocytogenes (disease causing type of bacteria) sickness outbreaks. Developing environmental monitoring plans to detect and prevent this bacterium in poultry processing establishments is a useful approach to reduce contamination and enhance food safety. Several guidelines and baselines were developed to allow users to design and conduct appropriate environmental monitoring plans to target this bacterium. After developing these guidelines and baselines, an internet-based environmental monitoring program ("EZSafety") was developed to allow poultry processors to enhance their sample collection and analysis of test results over time. The program was developed using several kinds of computer platforms (JavaScript, React Native, and MongoDB) . These open-source platforms were used to design, develop, and store the program over the internet. In order to validate its usefulness, the program was evaluated by 20 users who are majored in food safety and familiar with poultry processing plants hygiene to assess its ability to suggest appropriate monitoring plans. Most of the participants believed that EZSafety has several tools that are effective for targeting Listeria and other kinds of bacteria and enhancing environmental monitoring plans. Additionally, most participants agreed that EZSafety is organized and user-friendly. Such automated monitoring programs can play a significant role in enhancing the detection rate and the prevention of Listeria and other organisms in poultry processing facilities.

Listeriosis caused by Listeria monocytogenes (L. monocytogenes) has been of public concern since the 1980s. Among all the RTE food, deli meats are the major carrier for this pathogen. Eliminating or lowering the initial level of L. monocytogenes in RTE meat and poultry product in the plants is an important practice in reducing the risk of L. monocytogenes to the public due to the growth potential of L. monocytogenes in the RTE food product during storage. Research identifying the contamination at plants provided information for the Food Safety and Inspection Service (FSIS) to establish the Interim Final Rule, requiring the food processing plants that produce post-lethality exposed RTE meat and poultry product choose one of the three alternative plans to ensure good sanitation conditions during food processing or suppress the growth of L. monocytogenes during storage: post-processing treatment and use of growth inhibitor (Alternative1), post-processing alone (Alternative 2a) or use of growth inhibitor and sanitation program (Alternative 2b), and sanitation program alone (Alternative 3). This research developed a comprehensive model that simulated the entire processes of RTE food production, taking into account potential transfer and growth of L. monocytogenes in RTE meat and poultry products. This plant-to-consumption model analyzed the effectiveness of the three alternative processes on reducing the L. monocytogenes in the RTE food products and also investigated the optimal sampling and sanitizing program. Results showed that formulation of food products with growth inhibitor has the greatest impact on reducing the risk of L. monocytogenes, followed by the post-processing treatment and sanitation intervention. Risk can also be reduced depending on alternatives. For example, 70% reduction if all are switched to alternative 2b and 91% reduction if all are switched to Alternative 1, compared with the current alternative selection by food establishments. This study investigated several important factors in the sanitation program, analyzed the sensitivities of these factors, and proposed the reasonable improvement of the hold-and-test strategies by the plant-to-consumer mathematic model. Holding all the lots during the food contact surface (FCS) testing period instead of holding lots after finding the positive FCS would increase the detection rate of positive lots by three "times. These results may help the food establishments under Alternative 3 choosing the proper sampling and sanitation program or switching to Alternative 1 or 2. / Ph. D.