Sequential study of antigen persistence and concomitant inflammatory reactions relative to side-effects and growth of Atlantic salmon (Salmo salar L.) following intraperitoneal injection with oil-adjuvanted vaccines

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Abstract

The persistence of antigens at the injection site (area around pyloric caeca and spleen), concomitant inflammatory reaction and granuloma development were monitored at 3, 6 and 12 months following intraperitoneal injection with multivalent, oil-adjuvanted vaccines in Atlantic salmon. Parallel assessment of side-effect profiles and growth rate were also performed. Antigen persistence was examined by use of a monoclonal antibody that recognises Aeromonas salmonicida lipopolysaccharide in an immunohistochemical method for in situ identification of bacteria or bacterial fragments. The inflammatory reaction was monitored using standard histological techniques. The amount of persistent antigens and size 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. Antigens were consistently found in inflamed tissues located in the pancreatic region. The size of inflammation increased during the first 6 months but declined thereafter. These findings suggest that persistent antigens at the injection site may act as inflammatory stimulants that induce and perpetuate the inflammatory reaction, eventually leading to adverse side-effects.

Introduction

Vaccination of Atlantic salmon (Salmo salar L.) against furunculosis and cold-water vibriosis with oil-adjuvanted vaccines induces long-lasting protective immunity [1], [2]. Intraperitoneal (i.p.) injection of oil-based vaccines, however, results in the formation of visible injection-site lesions that may persist up to harvest size. This may on some occasions result in downgrading of fish at slaughter or after processing. The intra-abdominal lesions are recognised grossly as melaninisation and adhesions between internal organs or between the organs and the peritoneal wall [2], [3], [4].

Side-effects should be kept at a minimum and are in principle not acceptable because they can lead to reduced growth rate, downgrading of fish and may also be associated with fish suffering. Although the mechanisms are not known, it is not unlikely that specific components in the vaccines, the vaccine formulation and the inflammatory cell profile or any combination thereof, may contribute to the development and maintenance of side-effects.

In Atlantic salmon, side-effects following i.p. injection of oil-adjuvanted vaccines have been characterised by several authors [2], [5] who focused on the duration and severity of lesions. However, there are very few reports that address the effects of persistent/retained antigens at the injection site besides Press et al. [6] whose emphasis was on protection conferred by vaccines administered through different routes. Persistent antigens at the injection site, while important in the maintenance of immunity, can act as inflammatory stimulants that may induce/maintain the inflammatory reaction leading to severe side-effects.

As part of a larger study evaluating the efficacy of various mono- and multi-component furunculosis vaccines, this study was undertaken to examine sequential gross and histomorphological changes at the injection site of Atlantic salmon following i.p. injection with oil adjuvanted vaccines. The pyloric caeca, head kidney and spleen were examined at 3, 6 and 12 months following vaccination. The side-effect profile and the specific growth rate of individual fish were also monitored at the same time points.

Section snippets

Vaccination trial

The materials used in this study were collected as part of a vaccination trial conducted by Alpharma AS at EWOS Innovation facilities in Norway from January 2001 to January 2002. Processing and examination of the samples were done at the Norwegian School of Veterinary Science in Oslo, Norway.

Fish

About 4000 Atlantic salmon pre-smolts, weighing about 70 g each and reared in fresh water were transferred to EWOS Innovation experimental facility in Dirdal. The fish were a Norwegian Salmon Breeders'

Intra-abdominal lesions

All 180 fish sampled from groups A to F exhibited macroscopical lesions. No lesions were observed in the control fish (group G). Of the lesions observed, 98.9% were located around the pyloric caeca and spleen (injection site), 78.3% caudally, along the descending intestine near the urinary bladder, while 56.9% exhibited dorso-anterior lesions near the septum transversum and oesophagus. The lesions comprised of adhesions in the form of soft strands of fibrous tissue between adjacent organs

Discussion

The results presented in this paper suggest that persistent antigens of oil-based vaccines are associated with stimulation and maintenance of inflammatory reaction at the injection site of Atlantic salmon leading to adverse side-effects. In all vaccinated groups, persistent antigens were consistently observed with active inflammation that was characterised by the predominant presence of macrophages and lymphocytes as well as varying numbers of polymorphonuclear cells throughout the observation

Acknowledgements

Financial support for this study was obtained from the European Commission (CT-1999-00799).

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