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Intraperitoneal immunopathological reactions following vaccination of farmed fish. studies of basic immune mechanisms

Deliverables

We studied the in vitro effects of the exposure of explants from EGCs rich tissues to antigen preparations and to the recombinant cytokine TNF2. The antigen preparations consisted of bacterial culture supernatants or of bacterins (bacterial cells) obtained from the bacterial pathogens Aeromonas salmonicida and Vibrio anguillarum. The studies have comprised a quantitative analysis of the changes in the number of EGCs in tissue sections of the explants collected at different times in culture after the exposure to the antigen preparations and to TNF2. These studies allowed us to show that: - The exposure to the antigen preparations produced an increase in the EGC number in the tissues, and their degranulation, which was more evident in the intestine and gills. - The exposure to the recombinant TNF2, either alone or combined with antigen preparations, caused miscellaneous results on the EGC numbers, depending of the tissue and of the antigen formulations. At this moment, it is apparent that the TNF2 suppress the activity in the lymphoid organs, while increasing the responses in the intestine and gill tissues. Such results demonstrated that the antigen preparations have mitogenic properties for the EGCs, or elicit maturation of such cells from immature non-granulated precursors, which are resident in the tissues. The studies also indicate that the trout EGCs contain antimicrobial peptides.
Kinetics of peritoneal leucocyte population after i.p. vaccination. Peritoneal leucocytes did not show any significant differences until 60 days in fish injected with PBS. Water formulated vaccines (23.01 and 37.01) shown a lower response in all peritoneal leucocytes after vaccination; water in oil, alginate and liposome vaccines induced a strong inflammatory response. Oil in water vaccine induced a strong mobilisation of PMN but small mobilisation of Mo. Liposome vaccine induced 16% mortalities. EGC variations after vaccination were as follows: Samples from fish injected with water based (23.01 and 37.01) and liposome (62.01) did not show significant variations from resting values and from PBS values in all times, except 37.01 that showed values of EGC-II at day 30 significantly higher than all other vaccines assayed. Samples from fish injected with water in oil (22.01 and 35.01) and alginate vaccine (63.01) showed a significant increase of the EGC-I, comparing with resting values, at days 30 and 60 and EGC-II at day 60. Oil in water vaccine (61.01) induced higher values of EGC-I comparing with resting population, after 60 days.
The effects of different vaccine formulations on gene expression of pro-inflammation mediators including IL1a, COX2 and IL8 in elicited peritoneal cells are being investigated. Water or water-in-oil based Aeromonas salmonicida vaccines were used to vaccinate fish and expression of IL1a, COX2 and IL8 was monitored in elicited peritoneal cells, head kidney cells and splenocytes weekly up to for 4 weeks. Sample collection has been completed and the preliminary results revealed that a week after injection, IL1a expression was up-regulated in fish vaccinated with water in oil based vaccines but not in control fish or fish injected with water based vaccine.
Some key cells orchestrate the responses to vaccination and they produce different signal molecules that activate or down-regulate the responses of cells. As part of this project it has been shown that interleukin 8 and interleukin 1b (both signal molecules) are produced by cells in the peritoneal cavity following vaccination. These signal molecules attract cells to the site of injection and also give a message to the cell to be activated, e.g. up-regulate their functional capabilities. IL8 will act as a chemotactic signal to neutrophils and IL1a is a strong pro-inflammatory cytokine that will act on macrophages through attraction and activation. Studies of some of the key cells involved in the early responses to vaccination, the eosinophilic granular cells (EGCs) being one of them, have shown that defined components in the vaccine will activate the EGCs. The same cells also respond such that they increase in numbers. The project has shed light on the underlying mechanisms of granuloma formation in vaccinated farmed fish and it has been documented that the characteristics of the vaccine formulation, e.g. extracellular products, antigen content and stability has a bearing on the extent of injection site reactions. It has been shown that different cytokines are produced at the site of injection and that it is also up-regulated in immunocompetent organs like the spleen a few days after vaccination.
Studies during the project period have allowed for characterization of the dynamics of the cell responses in the peritoneal cavity over time showing that the initial response to injection of a vaccine preparation is rapid. In salmonids the response is typically biphasic but with differences even between different salmonid species like Atlantic salmon and rainbow trout. - The responses in Atlantic salmon persist over extended time periods (increasing up to 4-6 months) after which the responses level off and start to decline. The reaction at the injection site is associated with the presence of antigens retained at the injection site through an oil-in-water formulation. - In rainbow trout the responses have waned already at 4 months. Cell responses are diffuse and developed into immune granulomas over time. In the marine fish species sea bass, there is an initial inflammation that develops into a different type of granuloma, with less immune involvement and with resemblance to a foreign-body granuloma at the injection (area around the pyloric caeca). The responses in general are in general milder in sea bass than what is found in Atlantic salmon and rainbow trout.
The persistence of antigens at the injection site (area around pyloric caeca and spleen) and the concomitant inflammatory reaction and granuloma development of Atlantic salmon was monitored at 3, 6 and 12 months following intraperitoneal injection with multivalent, oil-adjuvanted vaccines. Parallel assessment of side effect profiles and growth rates of the same fish were also performed. Antigen persistence was examined by use of a monoclonal antibody that recognises Aeromonas salmonicida lipopolysaccharide was used in combination with a standard Steptavidin alkaline phosphatase immunohistochemical method for in situ identification of bacterium/ bacterial fragments that form part of these vaccines. The inflammatory reaction was monitored using standard histological techniques. The amount of persistent antigens and degree of inflammation/adhesions were estimated semi-quantitatively. A steady decrease in the quantity of antigens at the injection site was observed from 3 to 12 months. These antigens were consistently found in inflamed tissues located in the pancreatic region. A delay between the onset of the decline in antigen concentration and inflammatory reaction was demonstrated. The degree of inflammation increased during the first 6 months but declined thereafter. Persistent antigens at the injection site act as inflammatory stimulants that induce and perpetuate inflammatory reaction that can lead to adverse side effects. Understanding the reaction to persistent antigens in the vaccine is important for an optimised formulation of adjuvant, oil-based vaccines, the ultimate goal being to develop vaccines with no or reduced side effects.
Extracellular products (ECP) are part of the oil-based vaccines against furunculosis that induce side effects following intraperitoneal injection in Atlantic salmon. In this study, we evaluated the contribution of whole ECP and two of its major components, serine protease and glycerophospholipid:cholestrol acyltransferase (GCAT) in the induction inflammatory reactions. Six groups of Atlantic salmon pre-smolts were intraperitoneally injected with antigen preparation containing either: - Standard amounts of ECP, - ECP concentrated 5 times, - Washed antigens, and - ECP lacking serine protease. Two controls, water-in-oil as well as physiological saline were also included. Tissue samples of the injection site (pyloric caeca and surrounding area) were collected in phosphate buffered formal in and processed for light microscopy. Computer-assisted microscopy with the help of Image Pro® analysis program was used to measure the area of inflammation on H&E stained sections. Antigen preparations containing ECP induced early, biphasic reactions associated with more intense inflammation compared to slower, monophasic inflammations caused by washed preparations.
The development of the chronic inflammation and in granuloma formation was studied in sea bass. Samples were collected from one to eleven months after vaccination (mainly from fish injected with water in oil vaccines). The granulomata have different organisation from what is seen in Atlantic salmon. While in salmon loosely to well organised classical granulomas are typical, in sea bass the responses consist of a granulomas structure that is comprised of a capsule organised in two different layers that surrounds a central space containing liquid substance mainly composed of oil (onion-like). The external layer is mainly composed by collagen fibres. The inner layer presents a lamellar structure composed by cell debris and oil, and together these structures are organised with an onion-like appearance. By immunohistochemistry using an antibody to V. anguillarum it was shown that antigens are present in the inner layer and central space of granulomas. The reactions are more of a typical foreign-body type of granulomatous reaction with less immune involvement compared to what is seen in salmon.
Intraperitoneal injection of Atlantic salmon with compound 48/80 or cortisol prior to vaccination resulted into marginally higher inflammatory reactions than the control group (PBS prior to vaccination) 4 weeks following vaccination. At week 8, the group injected with compound 48/80 had significantly lower inflammatory reaction than the control. No differences were observed between groups thereafter. There were no significant differences in the relative participation of EGCs in the inflammatory reaction following treatment with degranulation agents. These studies are difficult to carry out as the treatment with 48/80 and particularly cortisol result in severe immunosuppresion of the fish with increased risk of secondary infection.
Studies during the project have allowed for characterization of the kinetics of the cell responses in the peritoneal cavity over time showing that the initial response to injection of a vaccine preparation is rapid also in the cold-water species. In salmonids the response is typically biphasic. The responses in Atlantic salmon persist over extended time periods (increasing up to 6 months) while in rainbow trout the responses have waned already at 4 months. The cell responses are diffuse initially and develop at later stage into a granulomatous response with formation of granulomas. It was also an interesting finding that water-in-oil emulsion (including the antigen) induced a response that was immune mediated, observed by the presence of lymphocytes at the site of injection. In water-in-oil emulsions only (without antigens included) there was little to no immune involvement; lymphocytes being absent at the injection site. This is the first documentation of vaccine related granulomas at the injection site being immune mediated.