The w~desp read loss of exocnne pancreas secretory aclnai cells, slmllai to the pathology observed In Atlantic salmon Salmo salaraffected by pancreas dlsease, was reproduclbly transmitted to salmon parr in fresh water by intraperltoneal injections of homogenised k ~ d n e y and spleen removed from affected f ~ s h . In fresh water the prevalence of pancreas pathology in experimental populat~ons increased to a maximum of 40 to 70 U/;) over 2 to 4 wk followed by an apparent recovery w ~ t h l n a further 4 w k . In sea water fish the incidence of affected salmon was lower and the temporal pattern of progresslon more varied than was the case In fresh water Infectious pancreatic necrosis virus was not detected in experimental fish or homogenised fish t~ssues . In both fresh and sea water the lncldence of the pancreas pathology was dependent upon the dose of homogenate injected. Preliminary results indicate that experimentally affected fish in sea water are less likely to feed than non-affected fish. Evldence supporting a n infectious cause of the pancreas pathology ~nc luded : f lnd~ngs consistent with the I-epllcation of the causative agent in fish tissue, transmission to salmon cohabited with affected animals and no dim~nution in the effectiveness of homogenates in transmitting the pancreas pathology after 0.22 pm filtration INTRODUCTION tal transmission described by McVicar (1990) resulted in variable success particularly for salmon in sea water Pancreas disease is a condition of farmed Atlantic when it was often not possible to transmit the disease. salmon which was first recorded in 1976 (Munro et al. Since other workers have con~n~unicated to us their 1984). The major pathological characteristic of the disdifficulties in transmitting the disease the primary purease is the degeneration and widespread loss of acinar pose of the present paper is to describe our attempts to cells of the exocrine pancreas (Munro et al. 1984, investigate possible causes of this variability. McVicar 1987). Epidemiological evidence obtained from salmon farms in Scotland was judged by McVicar (1987) to MATERIALS AND METHODS support the idea that pancreas disease had an infectious cause. However, a pathogen which can cause Terminology. For convenience, and in the absence pancreas disease has not yet been identified or isoof proof of an infectious agent causing pancreas dislated. The first experimental evidence that pancreas ease, the term infective is used to describe putatively disease was transmittable and, therefore, might be ininfectious material derived from previously affected fectious was presented by McVicar (1987, 1990). The populations of fish. exocrine pathology which was typical of pancreas disPreparation of homogenates. Large batches of conease was reproduced in previously healthy salmon by trol and putatively infective material were made by the injecting homogenised kidney dissected from salmon following procedure. A total of 650 parr in fresh water in the early stages of the disease. However, in our own (temperature 13 C, mean weight 18 g ) were injected studies of pancreas disease, the method for experimenwith 0.1 m1 of putatively infective or control material O Inter-Research 1993 124 Dis. aquat. Org. (McVicar 1990) diluted 1:lO (v/v) with PBS pH 7.2, without magnesium or calcium (Gibco). Five days after injection, kidneys were asceptically removed from 600 fish, from each group, and stored on ice until they were homogenised in an equal volume of PBS and filtered (120 pm) before storage in liquid nitrogen. The remaining 50 fish in control and experimental populations were maintained and monitored by histological examination at intervals to confirm subsequent absence or presence of acinar cell loss respectively. Further ba.tches of kidney homogenates were made by injecting parr with aliquots of the above kidney homogenates. Additionally, homogenates of spleen were prepared as above. Homogenates were filtered using a 0.22 Fm (Millipore) membrane filter. Pools of infective matenal generated at diiierent times were ~idl lddl dised on the empirical basis of their protein concentration (Sigrna Chemical Co. Ltd, procedure 690). Injection of fish. Control and experimental fish in each trial received standardised, body weight corrected (pg control or infective hornogenate protein g ' body weight) intraperitoneal injections of 0.1 ml. Fish. Atlantic salmon were reared at the fish cultivation unit of the Scottish Office Agriculture and Fisheries Department (SOAFD), Aultbea, Wester Ross, Scotland. The absence of pancreas disease and infectious pancreatic necrosis (IPN) was established for all stocks prior to their use (Smail & Munro 1989). Unless otherwise stated, fish which were injected with control homogenates were always held in different tanks from fish which were injected with infective homogenates. Salmon parr were maintained in 1 m diameter tanks containing 350 1 of water, supplied at ca l 0 1 min-' tank-'. Salmon post-smolts were maintained in 1 m diameter tanks containing sea water, supplied at a rate of ca 10 l min-I Fish were fed (Mainstream diets, BP Nutrition) to satiation. Since the water temperature was seasonally variable temperatures for specific experiments are Indicated in the results. Prior to all experimental procedures fish were anaesthetised using ethyl-4-aminobenzoate (benzocaine, BDH Chemicals, Poole, Dorset, UK) . Diagnosis of pancreas disease. Fish were subjected to histological examination. (Munro et al. 1984) at various intervals after injection. Fish were classified as affected when the normal acinar arrangement of the exocrine pancreas secretory cells had become transformed Into one of total apparent necrosis and when no zymogen containing cells (eosinophilia) could be observed. In practice, fish with altered pancreas histology but with a few intact acinar cells were encountered. These fish were classified as non-affected, along with fish having normal pancreas appearance. They probably represent salmon in an intermediate stage of the disease. It was considered impractical to establish a satisfactory, unbiased scoring system for these intermediate fish. Transmission by cohabitation. Forty salmon postsmolts were injected with infective spleen homogenate and maintained in the same tank as 40 non-injected fish. The mean weight of fish was 130.2 + 14.6 g and each fish received a spleen hornogenate dose of 15 pg protein g' body wt. Water temperature was 14 C and samples of 10 fish from each group were assessed for pancreas histology at weekly intervals. 0 10 20 30 40 50 60 70 80 90 Time after injection (days) Fig. 1 Sa ln~o salar. Development of complete acinar cell loss in potential S2 salmon parr in fresh water following injection with kidney homogenates. (a) Results of 4 experiments. Fish were 13 to 31 g and water temperature varied from 10 to 20 'C between experiments. All flsh were Injected with kidney homogenates at a dose of 3 pg protein g ' body wt. Expts 1 to 3 (W, A and 0) used the same batch of kidney material. Expt 4 (L.) used a different batch of kidney material. ( ) controls for Expt 3. All experiments used control fish, none of which showed altered pancreas histology (results only shown for 1 experiment). Each point represents a subsample of 10 fish from an lnitial population of 70. (b) Results of 2 experiments. Fish weighed 14 to 18 g and received 30 pg protein g ' body weight. Expt 1 (water temperature 13 C): (0) experimental fish; (C) control fish. Expt 2 (water temperature was 2 C (A) experimental fish; (A) control fish Each point represents a sample of 25 fish Raynard & Houghton: T ransm~ss~on f pancreas d ~ s e a s e of Atldntic salmon 125