CORDIS provides links to public deliverables and publications of HORIZON projects.
Links to deliverables and publications from FP7 projects, as well as links to some specific result types such as dataset and software, are dynamically retrieved from OpenAIRE .
Deliverables
The fundamental aspects of host-pathogen interaction were elucidated through the construction of in vitro and in vivo models, which provided data on the agents pathogenetic mechanisms.
We have demonstrated that cell-line models represent an attractive and reproducible method for the study of host-pathogen interactions, allowing the screening of a large number of strains and the investigation of the different aspects of virulence (e.g. adhesion, colonization, penetration, intracellular survival, trafficking inside cells and cytotoxicity). Fish cell primary cultures, generated during the project, and established cell-lines were used to investigate basic aspects of host-pathogen interactions, with special emphasis on apoptotic/necrotic outcomes, invasion and intracellular survival.
Apoptosis/necrosis was investigated by three methods
- DNA laddering,
- Detection of apoptotic nuclei and analysis of cell cycle, and (c) morphological changes that are markers of apoptosis (i.e. detection of cell surface phosphatyl serine within apoptotic cells). We have shown that cell lines are suitable in-vitro techniques for the replacement of in-vivo animal experimentation. Results demonstrate that a specific apoptotic process plays a crucial role in the establishment of Streptococcus iniae infection.
In order to investigate early events that take place during the initial stages of the infection (adhesion, colonization and penetration) and to explore the role of immune cells, we used a macrophagic cell-line. Data were then confirmed by ex-vivo pronephros model. Results show that S. iniae specifically invade macrophages, survive within these immune cells and exploit them as part of their pathogenic mechanisms (Trojan horse effect). In order to explore the role of the skin as primary site of infection, we used an epithelial primary cell-line. Its establishment was performed in an innovative fashion. Briefly, trout were sacrificed, dipped in 5% chlorex for 5 min and then wiped in ethanol 70%. Scales were washed three times in a penicillin-streptomycin-nystatin solution, minced with scissors and agitated in a trypsine-EDTA solution. Released cells were collected by centrifugation and transferred into 25-ml tissue culture flasks in DMEM medium supplemented with 25% FCS. Results, based on transepithelial voltage measurements and EM analysis, showed that epithelial cells are invaded by S. iniae, allowing its invasion into the organism.
Fulfillment of Kochs postulates, through the establishment of methodologies to induce an experimental disease, enabled integration with the in-vitro data. In-vivo models determined: - the time post-infection at which bacteria are first identified in the various tissues; - the onset and the character of the inflammatory response; - the extend of phagocytized bacteria killed within macrophages; - the similarity between the natural and the experimental infections. The availability of reproducible in-vivo models for infection is of primary significance, as it allow correlation between various research groups and sets up a basic standard for the estimation of pathogenicity.
What generated within workpackage 1 provided new insights of the immunopathological machinery of fish versus infections sustained by pathogenic Gram-positive cocci. In additions, it provided novel scientific data regarding the virulence mechanisms sustained by Streptococcus iniae.
Work was oriented to explore potential remedies to L. garvieae and S. iniae losses.
2 L. garvieae and S. iniae preparations were tested: a bacterin (5x109cfu/ml) and a W/O (30%-70%) vaccine, consisting of IFA (1x109cfu/ml). Fishes were vaccinated by i-p (200 l) injection. Adjuvated vaccines elicited titres of 1:10/20 at 60 and 90 days, while bacterins elicited titres of 1: 10/20 at 60 days and 1: 1/2 at 90 days (L. g./S. i., respectively).
Phagocytosis, oxidative burst and lymphoproliferation were assessed.
Phagocytosis was assayed in-vitro after 1, 3,18 hours contact between bacteria and pronephros (PN) leukocytes. An obvious enhancement of phagocytosis was observed at 1 and 3 hours in L. g. vaccinated fish.
Oxidative activity was assayed by flow cytometry, 4 hours post-activation with bacteria. Identical levels of activation were observed in vaccinated and non-vaccinated fish.
Specific blastogenic response of lymphocytes from blood and PN was assayed by tritiated thymidine incorporation. Lymphoproliferation was induced with killed L. g. and S. i. or mitogens. Blood lymphocytes proliferative response differed from that of PN lymphocytes. In PN cells the magnitude of the proliferative responses to mitogens were similar to the responses induced with streptococcal antigens.
L. garvieae and S. iniae infections represent the major cause of rainbow trout losses in several Mediterranean countries.
During the infection first events are very important either in the establishment of the disease or in the elimination of the pathogen. At an early step of the infection, the non-specific mechanisms play a major role in the fish defence system.
Pronephros invasion and opsonin-dependent phagocytosis of S. iniae was assessed: higher phagocytic indexes were observed among vaccinated fish.
Apoptosis of leukocytes was monitored by DNA fragmentation analysis and quantified through flow cytometry (FCM). Results demonstrated that apoptosis induced by S. iniae serotype II is stronger than the one induced by serotype I. In-vitro, similar levels of L. garvieae induced apoptosis were observed in leukocytes, while in-vivo blood leukocytes displayed higher susceptibility to apoptosis.
The gut-associated lymphoid tissue-GALT is a component of the immune system that ensures protection against pathogens. The intraepithelial lymphocytes-IELs are postulated to play an important role in the mucosal defence system. In this study, we determined the morphological and immunocytochemical parameters of rainbow trout intestine leukocytes populations, isolated intraepithelial lymphocytes and performed in vitro assays. For the purpose, an essentially mechanical method allowing the rapid harvesting of viable IELs was developed.
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