0300-9629/92 $5.00 -t 0.00 Q 1992 Permmon PressLtd
Camp.Eiochem.f’iwsiol.Vol. 102A, No. 4. pp. 759-768, 1992 Printed in Great i&in
~~~PA~ATIVE
NUTR~I~N
PAPERS
DIGESTIVE ENZYME LEVELS AND HISTOPATHOLO~Y OF PANCREAS DISEASE IN FARMED ATLANTIC SALMON ~~~~~~ ~~~~~~ G. M.
* K. R. CALLANAN,*A. I. MITCHELL,*R. S. RAYNARD~ NCHJLIHAN, and G.&i. ~~UGHTON~ ~Department of Zoology, University of Aberdeen, AB9 2TN,U.K. Tel.: (0224) 272000; PRINCXE,*
D. F.
Fax: (0224) 272396; @OAFD, Marine Laboratory, PO Box 101, Aberdeen AB9 8DB, U.K.
Abstract--.~I. The pancreatic digestive enzyme act~vjties of tr~psin and chymotry~sin were assessed in vilro and were found to correlate well with the histological changes characteristic of pancreas disease (PD) in farmed Atlantic salmon (S&m s&r). 2. Pancreatic enzyme activity was assessed in Vito using the chymotr~sin specific substrate N-benzoylL-tyrosyl-~-aminobenzo~c acid. In all cases, signi~~ntly less ~-~rn~~o~nzo~c acid was excreted by fish later found to be suffering from PD. 3. It is concluded that the in uitm digestive enzyme assay was effective in djagnosing PD with the irr t&o method, indicating Further promise for assessing exocrine pancreatic function.
EVTRODUCTION
with measurements at different times after feeding and in animals of different body weight. It was also the aim of this study to investigate the use of an in uivo assessment of pancreatic activity in pancreas-disease fish. This was achieved by adapting a technique normally used to assess human pancreatic function and was based on the orat introduction of ~-benzoyl-L-tyrosyl-~-amino~nzoic acid (Bz-tyPABA) (Dabrowski et al., 1989) which is cleaved by pancreatic ch~otrypsin li~rating ~-amino~nzoic acid (PABA). The amount af PABA absorbed by the intestine and consequently excreted in the urine is used as an index of pancreatic function (Yamato and Kinoshita, 1979; Toskes, 1983). The relationship between the patho~enesis of the disease and enzyme activities were measured in populations of salmon smelts farmed in sea-water and in a transmission experiment carried out in freshwater using salmon parr.
bined
Pancreas disease (PD), a pathogenic condition affecting farmed Atlantic salmon (S&IO s&r), was first recognised in Scotland in 1975 and has since appeared wherever Atlantic salmon have been intensively reared on a significant scale in Europe and North America (~cVjcar and Munro, 1987). The pathological characteristics of the disease include the generalised degeneration of the exocrine pancreas (Munro et al., 1984; McVicar, 1987) and often results in severe ema~iatjon of the affected fish (McVi~ar, 1987). Usually, the condition affects post smelts in their first sea year, aIthougb farmed salmon of alI ages are susceptible to the disease (McVicar and Munro, 1987). Current research suggests an infectious agent is responsible (McVicar and Munro, 1988; MeVicar, I990) and PD has been successfully experimentally transmitted (MeVicar, 1987; McVicar and Munro, 1987; Raynard et ai., 1991). At present, the only reliable pathological diagnosis of PD is by the histopathological examination of the exocrine pancreas, a necessarily slow and laborious tecbn~q~e, but from which it is possible to disti~gujs~ stages in the acinar cell collapse which can be described as normal, pre-acute, acute, chronic and recovery (Munro et al., 1984). The aims of this study were to correlate pancreatic enzyme activities of trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) with the histo~o~~a~ diagnosis of healthy fish and those suffering from PD. Total trypsin and ~hymotrypsin in a fish will depend upon the enzyme activity per gram ofpyloric tissue and the total weight of the pyloric caeca. Pyloric caeca have been found to be variable in mass, and digestive enzyme activity may also vary with time after feeding and composition of the food (Kawai and Ikeda, 1973; Overnell, 1973; Stevens and McLeese, f988). Therefore, studies of digestive enzyme activities were com-
Fish The freshwater transmission experiment was carried out with Atlantic salmon (S~~~~ solar) parr maintained at the SOAFD fish cuitivation unit, Aultbka, Wester Ross, Scotland. Eighty fish were used in the trial (mean weight 24 a). Head k&&y homogenate of known in&ted fish kas us&l to transmit pancreas disease. Protein con~ntrations of infective and control material were measured with the Bradford technique (19%) and injected at constant amounts (3 fig protein/g fish). Forty fish were injected intraperitoneally in the pelvic region with 5.1 ml of infective material. The control group consisted of 45 fish injected with 5.1 ml of non-infective homogenate (Raynard et al., 1991). Before and after the injection the fish were kept in tanks, 50 cm in depth at 14°C. Fish were fed using automatic feeders which supplied food at a rate of 2.5% body weight/day. Ten animals were removed from the control and injected groups at weekly intervals foliowing the injection for 4 weeks. On
759
760
G. M. PRINGLEet al
the day of sampling the animals were anaesthetized with 10% (w/v) benzocaine and killed bv a sharp blow to the head. ‘Samples of caecal tissue from- each fish were frozen individually in liquid nitrogen and fixed for light microscopy as described below. Salmon smolts in their first year in sea-water, transferred to sea-cages in May, 1990, were used for the field survey. The fish were obtained from a large-scale salmon farming company on the west coast of Scotland (mean weight 261 g). These fish were fed on commercial salmon feeds three times a day at a rate of approximately 2% body weight/day. Variation in trypsin activity with time after feeding was determined with-51 farmed salmon smolts (mean weight 265 n) maintained in 2 m diameter sea-water tanks (125’C) at the Dunstaffnage Marine Laboratory, near Oban, Scot: land. They were all starved, then a group of seven fish was sampled (method described below) and the remaining fish were fed one feed to satiation; these animals were then sampled in groups at various times post feeding. Salmon smolts were obtained from Dunstaffnage Marine Laboratory, which had been maintained in ambient sea-water temperature conditions since May 1990 (mean weight 270 g) in 2 m diameter tanks of 1 m depth. For approximately 6 weeks prior to sampling they had been fed at about “maintenance” ration (approximately 1.0% body weight/day). The fish to be sampled was immediately killed by a sharp blow to the back of the head and the spinal cord was severed just behind the head. Wet weight and length were recorded. The whole pyloric caeca was then removed by cutting immediately at the anterior and posterior ends of the organ (allowing the inclusion of all the caecal fingers) and its wet weight recorded. Two sub-samples of caecal tissue were taken from the same region for histology (light and electron microscopy) before the pyloric caeca was frozen and stored in liquid nitrogen. On return to the laboratory, the frozen caecal samples were stored at -70°C. Histology
Pyloric caeca were fixed in 10% buffered formal saline for light microscopy. Sections (5 pm) of paraffin wax-embedded tissue were stained with haematoxylin and eosin. For electron microscopy, pyloric caeca with pancreas were fixed in 2% paraformaldehyde plus 2.5% gluteraldehyde in 0.1 M Sorenson’s phosphate buffer (pH 7.2), post fixed in 1% osmium tetroxide, dehydrated in graded alcohols and propylene oxide, and embedded in EPON 812 resin, Sections were taken on a Reichert-Jung 0MV3 ultratome, stained with uranyl acetate and lead citrate, and observed with a Philips EM301. Diagonsis of pancreas disease was based on the protocol suggested by Munro er al. (1984) using the wax-embedded sections.
Trypsin activity was measured using the synthetic, a-N-benzoylchromogenic trypsin-specific substrate, DL-arginine-p-nitroanilide (BAPNA), which liberates p-nitroaniline upon hydrolysis by trypsin (Preiser et al., 1975). A IO-fold gain in sensitivity for the trypsin determination was achieved by using BAPNA in combination with the Bratton-Marshall (BM) reaction. The released p-nitroaniline gives a purple colour in the presence of N-lnaththylethylenediamine (Bratton and Marshall, 1939). The reaction was employed by the additions of 0.5 ml each of sodium nitrite, ammonium sulphamate and N-l-naphthylenediamine at 3 min intervals, respectively. The optical density (OD) of the resulting purple coloured samples were read on a Pye UNICAM SP-550 UV/VIS spectrophotometer at 550 nm. Weighed amounts of frozen caecal tissue (approximately 500 mg for smelts and 100 mg for Parr) were homogenized with a Polytron homogeniser in 0.05 M Tris buffer pH 8, containing 0.02 M CaCl, and the homogenate was cen-
trifuged at 0°C for 15 min at 13,000g. The supematant was removed and stored on ice. The remaining pellet was resus~nded in fresh buffer, re-homogeni~ and the supernatant added to the first extract. The incubation conditions were 50 p I of extract plus 50 p 1 of buffer added to 0.7 ml of BAPNA at 15°C for 10 min at pH 8; the reaction was stopped by the addition of 0.8 ml of 0.2 N HCI. The substrate used for the chymotrypsin assays was glutaryl-L-phenylalanine-p-nitroanilide (GPNA) (Sigma, Poole, U.K.) using the methods described by Erlanger et ol. (1966) using incubation conditions of 15°C for 30 min at pH 7.6. Throughout this study, enzyme activities are expressed per gram of caecal tissue. The total protein con~n~ation in the supematant containing the enzyme activities prepared from caecal samples was determined using the- method described by Bradford (1976); no significant differences were found between the samples. In vivo measurement qf’pancreatic function Cylindrical pills 5 mm long by 5 mm diameter, each containing 100mg of the specific synthetic substrate of ch~otrypsin, ~-~nzoyl-L-tyrosyl-p-~ino~nzoi~ acid (By-ty-PABA) (Sigma) were formed using a Gall~kamp pill press. Experiments were carried out on healthy farmed salmon smolts (N = 6, mean weight 302 g) and animals suspected as having PD (N = 11, mean weight 215 g). Each animal received a single pill, placed in the stomach by tube and was kept in 10 I of sea-water at approximately l1.5”C with constant aeration. Water samples (lOm1) were collected at various times after pill administration for up to 48 hr. Each fish was then killed and sampled by the methods described above, in order to determine the condition of the pancreas. For the determination of PABA, 0.5 ml of 1.5 N HCI was added to 1 ml of water sample and heated in boiling water for 15 min. The samples were then allowed to cool before carrying out the BM reaction and subsequent reading of optical density at 550 nm (described above). Statistical methods
The results from enzyme measurements are expressed as pmol of substrate released per g fresh caecal tissue weight per min. Mean _frSE are given throughout. Total pyloric caecal weight, trypsin activities per g of caecal tissue and total trypsin activities per fish were related to total ftsh weight using the allometric equation Y = aXb in the logtransformed form. Regression analysis was carried out by linear least squares analysis and slopes and elevations compared by covariance analysis. Means were compared by one-way analysis of variance and, where appropriate, further analysis was by Student’s f-test for both paired and unpaired data. The 5% level of significance was used throughout. RESULTS Histology
The acinar (i.e. exocrine) pancreas sea-year salmon smolts are organ&d
cells of first
in functional units surrounding a lumen; the organelles are distributed with an orientation around this lumen (Fig. I). The basal region of the cells contains the nuclei and the rough-surfaced endoplasmic reticulum while the apical cytoplasm is normally packed with numerous storage or zymogen granules. The mean acinar cell diameter of normal fish was 14.66pm (kO.715 pm, N = 15) with a mean nuclear diameter of 4.88 ,um (kO.263 pm). The presumed centroacinar cells (Weiss, 1988) found next to the lumen were elongate and characterised by relatively large
Salmon
pancreas
761
disease
Fig. 1. Normal exocrine pancreas of salmon (< I sea-year) showing two cell types: acinar cells predominate and are characterised by the presence of vesicular endoplasmic reticulum (ER), large mitochondria (M), and nucleus (N) and numerous zymogen granules (Zm). The orientation of these granules is towards the lumen. The presumed centro-acinar cell (Be) is elongate and is characterised by its large nucleus.
These cells may function in the production of bicarbonate solution which flushes the trypsinogen out of the ducts (Weiss, 1988). Each acinus was surrounded by a basal lamina and a loose connective tissue matrix. Using the diagnostic protocol for the assessment of histological samples (Table 1) the corresponding electron microscopic appearance of acinar cells in the acute phase of PD was characterised by the endoplasmic reticulum (ER) being condensed into sheets and whorls (Fig. 2). Vacuolation (V) of the cytoplasm was evident. The nuclei (N) were irregular in shape and stained darkly due to condensation of the nuclear components. No zymogen granules were present. In the chronic phase of PD the acinar cells were characterised by the presence of sheets and whorls of collagen (C) (Fig. 3). Large vacuoles were also present. There was an absence of any characteristics typicai of normal acinar cells and it was impossible to differentiate between any cell types. In the acute phase the mean acinar cell diameter was 4.76.um (20.257, N = 15) (significantly smaller than that of normal cells, P < 0.05) although the mean nuclear diameter (3.68 him + 0.245) was similar to those of normal cells. This suggests that the nuclei of these diseased cells remained morphologically intact despite the degenerative condition of the acinar cells. Fish diagnosed as being histologically normal by light microscopy but which did not have any nuclei.
detectable trypsin or chymot~psin activities had two acinar cell types (Fig. 4). The first type, (A), exhibited no signs of necrosis and had the normal acinar cell characteristics. The second cell type, (B), had dark condensation of the endoplasmic reticulum (ER), the mitochondria (M) were enlarged and many were crescent-shaped. The cell membrane, nucleus and zymogen granules appeared to remain intact.
Table 1. Summary of the modified characteristic histopathology (light) used to distinguish the stages of acinar cell collapse during pancreas disease, based on the original protocol suggested by Munro rt crl. (1984) Condition NCXltlal
Degenerative Pre-acute Acute Chronic
Recovery
Histooatholoav
(linht)
None. Dense sheets of acinar cells with zymogen granules, fat and endocrine cells, i.e. islets of Lange&am, ducts, veins, arterioies and bicarbonate cells. Some acinar cells exhibit degenerative changes, but without vacuolatians or signs of necrosis. Some acinar cells develop vacuolatinns along with focal areas of necrosa. Acute and generalised haemorrhagic pancreatic necrosis, deposition of collagen. Total absence of acinar tissue replaced by fibrotic tissue, infiltration with fibrocytes and/or leucocytes. Remaining endocrine pancreatic components remain intact. Small groups of presumed acinar cells can be seen amongst the necrotic tissue, particulariy in assocmtion with islet tissue and/or ducts.
762
6. M.
PRINCLE et it/.
FEg.2. Acute pancreas disease: the endoplasmic reticulum (ER) is condensed into sheets and whorls. vacuolation (V” of the cytoplasm is evident. The nuclei (PI) are irregular in shape and stain darkly due to condensation of the nuclear components. No zymogen granules are present.
The mntral group showed no bistolo~i~~ signs of pancreatic degeneration during the course of the trial (Table 2). The mean try&n and chym~tr~~s~n activities of the control animals declined during the course of the experiment and were signi~cant~y lower by week 4 compared with week 1 in the case of trypsin and by weeks 3 and 4 in the case of chymotryps~n (Fig. 5a and Sb). In animals injected with infective material there were no signs of PD in weeks 1 and 2 but it was diagnosed in some fish in weeks 3 and 4 (Table 2). Mean enzyme activities for trypsin and c~~~~~ry~§i~ were ~igni~~a~~iy lower from the controls in weeks 3 and 4. The majority of the PD diagnosed fish were in the chrome phase with significantly tower trypsin activities compared with the controls in week 3. No trypsin was detected in week 4 (Table 2). Chymotrypsin activities were found to be consistent& lower than trypsin activities but chym~tryps~n activities were measurable in ah the PD fish, although the vatues were s~~n~~~nt~y lower than those of histologically normal animals, There was a significant correlation between the activities of trypsin (X) and chymotrypsin (Y) which can be described by the equation: Y = 0.0085 -I-0.034x (N = 80, Y2= 0.034, P -c 0.05). This means that overall the amounts of the two enzymes are linked afthough chymotrypsin ao tivities were still detestable when trypsin activities were absent in the PD fish.
In f&h sampled from a he&by ~~~~~~~~~~ and having histo~og~ca~~y normal pancreas adnar cells, a greater than IO-fold variation in trypsin activity was found (Fig. Ba): however. most fish had activities close to the mean (0.135 +- 0.0086 ~moles pNA/g/min, N = 68). Thirty-eight fish that were sampted on the same date from a population apparently suffering from PD were found, after histologicat examination using the criteria described in Table I. to have PD. These animals had no detestable Ievefs of trypsin activity. A comparison between the trypsin assay and the conventional histopathoRogica~ method of PD diag nosis from 185samples is summar~sed in Fig.. 6b. The results clearly demonstrate that trypsin actrvtties and histology agree almost completely; pancreas acinar ceils classified as histologically normal almost always exhibited trypsin activities whereas acinar cells &ass&d at various stages of PD bad generally no trypsin activity even when classified to be in the recovery stage {nine out of 80). Of the IO5 samples h~stopaYho~o~ically classified as being normal, 99 (94.3%) had detectable levels of trypsin activity and six (5.7%) had no detectable levels of tryptic activity. Of the 80 samples b~stopathologica~~y classified as having PD, 77 (96.3%) had no detectable levels of tryptic activity and three (3.7%) did have detectable fevels. 1~was found that the three sampIes which had PD with detectable levels of tryptic activity came
Salmon pancreas disease
163
Fig. 3. Chronic pancreas disease: the pancreas tissue is characterised by the presence of sheets and whorls of collagenous fibrotic tissue (F). Large vacuoles are also present. There is an absence of any characteristics typical of normal acinar cells.
from a “degenerative” normal and pre-acute. Variation
category,
in trypsin activity
diagnosed
between
in normalJish
In view of the variation in trypsin activity found in histologically normal animals, experiments were carried out to determine whether this variation was due to the site of caecal sampling or time since feeding. The caecal mass consisted of a clearly distinguishable anterior section which has caeca both dorsally and ventrally and a posterior section with only dorsal caeca. The caeca of seven animals from the same population were divided up into six sections and trypsin activity determined. The mean levels of activity in each section were found to be similar (Table 3) and no significant difference was found between any section (paired r-test). In feeding animals, the mean trypsin activities declined significantly by 5 hr post feeding. Mean activities returned to close to pre-feeding levels by 47 hr after the last meal (Fig. 7). Eflects of body size The weight of the pyloric caeca of histologically normal, fed fish were positively correlated with total fish weight (Fig. 8) (Table 4); combining the results from parr and sea-water smolts did not significantly alter the slope or intercept of the regression equations. The results from sea-water animals that were histologically normal but which had been main-
tained on minimal diets or without food gave a regression equation which was significantly lower than that of the fed fish (Fig. 8). The pyloric caecal weights of the PD fish were almost all within the range found for the minimal ration or starved fish. Regression analysis of all the low ration, starved and PD fish had a similar slope to that from the fed fish but had a significantly lower elevation (Table 4). Trypsin activity per g was also correlated with fish weight in normal, fed fish (Table 4). Animals which had been starved for 12 days had trypsin activities which were within the range found from normal feeding animals (Fig. 9). Total trypsin activity (from the product of total caecal weight and weightspecific trypsin activity) was correlated with total fish weight (Fig. 10); in fed sea-water smelts the slope was greater than one but when the parr were included, the slope was not significantly different from one (Table 4). Bz-ty-PABA function
and in vivo measurement
of‘pancreatic
In salmon smolts which were later found to have histologically normal pancreas cells and normal in vitro chymotrypsin activities (0.0052 pmoles pNA/g/min) (Fig. ll), the appearance of the breakdown product of the chymotrypsin-sensitive substrate followed a sigmoidal curve with time. After 44 hr 67 + 2.6% of the calculated dose had been recovered. Control experiments where the pill was left in tank water gave 6.22% recovery after 48 hr. Animals
763
G.
M.
F'RINGLE~I al
Fig. 4. Fish diagnosed as being hist~logicaily normal by light microscopy but having trypsin activities of zero, show two acinar cell types. The first type, (A), exhibits no signs of necrosis and has the normal acinar cell characteristics. The second cell type, (B), shows dark condensation of the endoplasmic reticulum (ER), the mitochondria (M) are enlarged and many are crescent shaped. The cell membrane, nucleus and zymogen granules appear to remain intact.
which were later determined to have PD from histological ex~~mination of their pancreas cells and had zero in vitrochymotrypsin activities had significantly lower levels of breakdown products at all time points and the excretion rate appeared to be linear; percentage recovery was only 36 + 2.8% after 44 hr. Taking the 24-hr time point, the normal fish had excreted 47.1 rt 6.6% of the calculated dose whereas the PD fish had excreted 17.4 i: 3.4% (P < 0.0001~.
Previous, unpublished studies into the pathology of PD have also investigated the levels of trypsin activity, but a non-specific protease assay method was used. It is possible, therefore, that some of the results obtained may have been erroneous due to contamination by other proteolytic enzymes. The trypsin assay used in this study was found to be highly specific and repeatable; it does not
Table 2. Percentage of fish in different histological categories of the 40 infected fish from the ~~ansrnissiantrials with salmon parr in fresh water Enzyme Week (E) ____ -._..---
i
% _--.. ~.
Normal .._..E:_
Acute
% ~~_____
Chronic E
oio
E .____..._..I^
Recovery 91. ..___.. _. .,_..-. E
100 :
2
0.232 5 0.024 D.Ol52 ri:o.w9 100
7 e 3
o.zau & 0.040 0.0113 + 0.0014 60
4
10 0.140 & 0,025 0.008 i: 0.0015
T c so T c
30
0.058
0.007 +- 0.0004* 0.0001 1.0.0001*
0.0046 40
0.062 4 0.01 L 0.0067 f 0.0009
IO 0.000 0.00009 4 o.O0005*
Ten animals were sampled at each week. The mean + SE trypsin CT) and c~ymot~~jn (C) levels fpmoles ~~gh~~rni~~ in the different categories of pancreas-diseased animals are indicated. *P < O.Oal compared with mean confrol values.
0.000 0.0005 @A!$
fresh
765
Salmon pancreas disease
a very beneficial effect in greatly reducing the impact
of PD. Lowered ration levels or starvation also resulted in the same wide variations in trypsin levels but there was a marked reduction in caecal mass compared with feeding animals. Nevertheless, the total trypsin activity per fish of animals starved for 12 days appears to be within the normal range of total trypsin activities for feeding animals (Fig. IO). Reduction in
week 1
Week 2
weak 9
week4
Week number
.,. .c E
0.018 ,
b)
pyloric caecal size has been reported previously and found to be associated with reduced digestibility (Windell, 1967). The small differences in the levels of tryptic activity from the different areas of the pyloric caeca were not found to he significant (Table 3), suggesting that dispersed pancreatic tissue is homogeneously
0.016
‘;gJ) 0.014 2
0.012
1
0.010
8 1
0.006
‘;
0.006
6
0.004
E 3 0
0.(x12 0.060 week 1
w&k 2
week 3
Week number
Fig. 5 (a) Mean (&SE) trypsin activities in the caeca of the control and infected groups of salmon parr in the transmission trial with time. (b) Mean (&SE) chymotrypsin activities of the same animals. Ten animals from each group were analysed for each time point.
0.e.n
@xl. O.oal. O.?Ol- 0.151-OPl- 0.251. om- om- 0.4w o.oso o.tw O.,JO 0.200 0.250 0.300 0.350 0.400 0.450
n
PDFish
n
Healthy Fish
differentiate between trypsin and its inactive precursor trypsinngen. The assay conditions ensure that all the stored trypsinogen was converted to trypsin, thus achieving a measure of the total potential tryptic activity. Trypsin actim2ie.s
There is clearly a great deal of variation in trypsin activities in normaily feeding salmon smolts. Part of this variation has been found to be due to a decrease in trypsin activities after feeding (Fig. 7). As the trypsin method detected total trypsin it can be inferred that the pancreas had been stimulated by the arrival of the meal to secrete trypsinogen/trypsin from the pancreas into the gut and therefore the enzyme activity was reduced in the region of caecal tissue that was sampled (Love& 1989). What effect repeated meals have on trypsin activities is not known, although if combined with the indicated resynthesis time for the enzyme of around 48 hr (Fig. 7), it can be speculated that the pancreas is being placed under increased physiological pressure {stress) to produce sufficient enzyme to be able to fully digest each meal. This stress may increase the pancreas’s susceptibility to illness. Such speculation is further reinforced by findings from within the salmon farming industry itself, where lowered feeding rations and even starvation at the first signs of PD appear to have
Pancrease disease
HietopethoiogicdClassification TtypeinAssay
Fig. 6 (a) The distribution in trypsin activities from 68 histologically normal farmed Atlantic salmon smelts (mean weight 283 g) randomly sampled in early October 1990. Also included is the data from 38smolts his~oiogic~ly confirmed to be suffering from PD sampled from a different nooulation on the same date. (b) Comp&son of the pancreatic trypsin activities with the histological assessment for PD. The data for histologically normal farmed salmon smelts were taken from 105 animals sampled between late August and November 1990. The 80 farmed salmon smolts diagnosed as having PD were sampled over the same period.
166
G.
Table 3. Distribution
of trypsin activities Anterior
M.
et al.
PRINGLE
@moles pNA/g fresh weight caecal tissue/min) caeca of salmon smelts
pyloric region
in various regions of the pyloric Posterior
Dorsal
Ventral
Anterior
Posterior
Anterior
0.0883 k 0.0119
0.0883 f 0.0135
0.0746 i 0.0082
region
Anterior
Posterior
0.0831 k 0.0114
0.0770 * 0.010
Posterior 0.0794 f 0.01 I4
pyloric Dorsal
The caeca were divided into an anterior region which has both dorsal and ventral caeca and a posterior region which only has dorsal caeca. Each region was in turn divided into anterior and posterior subsections. Each mean is from seven animals sampled from the same population
throughout the pyloric caeca. This agrees with the findings of Stevens and McLeese (1988) for rainbow trout (Onchovhynchus mykiss). Pancreatic tissue is also found around the stomach, bile duct, anterior spleen and upper intestine (Munro er al., 1984), although it is not yet clear whether these cells add significantly to the total trypsin activity determined from the caecal tissue. The amount and activity of the digestive enzymes has previously been related to the time of feeding (Steffens, 1989). In carp, the level of protease activity in the intestine reaches a maximum 5 hr after feeding (Onishi et al., 1976) with the largest rate of enzyme secretion per unit of time being observed during the first 5 hr post feeding (Steffens, 1989). In the European eel (Anguilla anguillu), the highest enzyme secretion and activity levels were recorded 12 hr after feeding; at 46 hours after feeding the rate of rise of total activity was found to be at its highest (Steffens, 1989). There are clearly two separate aspects to digestive enzyme activities in salmon; trypsin and chymotrypsin activities per g and the total weight of the caecal tissue. Although it is recognised that the pyloric caeca increase the intestinal surface area (Buddington and Diamond, !987) most studies have concentrated on the number of caeca. The number of caeca has been found to be correlated with size of fish in rainbow trout (Babushkin, 1974; Bergot et al., 1981). Results from the present measurements clearly demonstrate that caecal mass is related to body weight in an isometric fashion and that weight specific enzyme activity declines with increasing body mass in a similar manner as metabolic rate,
feeding 1979).
rate
and
growth
rate
(Brett
and
Groves,
Comparison between histology and digestive enzyme activities The two methods showed over 95% agreement in diagnosing PD in field-sampled farmed Atlantic salmon smolts. It is not surprising that, with the histological characteristics of the disease showing a complete breakdown of the acinar cells, that it should be accompanied by the total absence of digestive enzyme activity. However, some anomalies are apparent. Firstly, in the transmission trials, detectable levels of chymotrypsin were evident in PD fish. Secondly, of the PD fish found with detectable trypsin activities (< 5%) all came from the “degenerative” classification (Table 1). Two explanations can be put forward for the latter observation; the presence of trypsin could be explained by the possibility that the disease was in its early stages and the exocrine pancreas was still able to produce trypsin, or a second explanation, which seems more likely, is that an “edge effect” (Callanan, personal communication). This is a phenomenon which occurs due to enzymatic degradation of caecal tissue shortly after its excision, usually as a result of poor field sampling or processing. In this event, the three samples could possibly have been from healthy fish as the trypsin assay indicates. The six samples that had no detectable levels of trypsin but were histologically classified as being 100
s 0.24 ,
E
.a
lo
0.21 0.16
f
1
.e %
1
.
:-
*a i. .. l .
0.1
0.03
W
1
o.oo+ . 0
6
.
10
3
15
’ 20
m 25
m 30
m 36
’ 40
45
’ 60
Time, h
Fig. 7. Mean (+ SE) trypsin activities in the caeca of farmed Atlantic salmon smolts (mean weight 265 g) at various times after a meal. Seven fish were sampled prior to feeding at time zero and the following number of animals were sampled after that time: 1 hr (6), 3.5 hr (7), 6 hr (S), 11 hr (6), 23 hr (6), 47 hr (7).
1006
100
10
weight(e)
*
Non”aIRsh
A
q
PD
0 starved
Minimumdti
Fig. 8. Variation in total caecal weights relative to fish weights. The feeding animals with histologically normal parr and smolts are classified as normal. Total caecal weight of starved animals, animals receiving minimal diets or animals diagnosed as having pancreas diseased by histological criteria are included. The regression equations describing the relationships can be found in Table 4.
Salmon pancreas disease
161
Table 4. Relationships between total pyloric caecal weight (g). trypsin activity (pm&s pNA/g,knin) and total trypsin activity (I_tmoles pNA~fish/min) and fish live weight (X, g) using regression equation log Y = log a + b log X Animals
a
Pyloric caecae sea-water All animals PC, starved Trypsin Total trypsin sea-water Total trvusin all animals
b
- I.85 1.17 - 1.77 1.14 -2.72 I .47 -0.54 -0.20 -4.43 1.83 - 2.32 0.94
N
r2
P
89 108 46 108 89 108
0.60 0.90 0.52 0.07 0.44 0.54
Camp.Eiochem.f’iwsiol.Vol. 102A, No. 4. pp. 759-768, 1992 Printed in Great i&in
~~~PA~ATIVE
NUTR~I~N
PAPERS
DIGESTIVE ENZYME LEVELS AND HISTOPATHOLO~Y OF PANCREAS DISEASE IN FARMED ATLANTIC SALMON ~~~~~~ ~~~~~~ G. M.
* K. R. CALLANAN,*A. I. MITCHELL,*R. S. RAYNARD~ NCHJLIHAN, and G.&i. ~~UGHTON~ ~Department of Zoology, University of Aberdeen, AB9 2TN,U.K. Tel.: (0224) 272000; PRINCXE,*
D. F.
Fax: (0224) 272396; @OAFD, Marine Laboratory, PO Box 101, Aberdeen AB9 8DB, U.K.
Abstract--.~I. The pancreatic digestive enzyme act~vjties of tr~psin and chymotry~sin were assessed in vilro and were found to correlate well with the histological changes characteristic of pancreas disease (PD) in farmed Atlantic salmon (S&m s&r). 2. Pancreatic enzyme activity was assessed in Vito using the chymotr~sin specific substrate N-benzoylL-tyrosyl-~-aminobenzo~c acid. In all cases, signi~~ntly less ~-~rn~~o~nzo~c acid was excreted by fish later found to be suffering from PD. 3. It is concluded that the in uitm digestive enzyme assay was effective in djagnosing PD with the irr t&o method, indicating Further promise for assessing exocrine pancreatic function.
EVTRODUCTION
with measurements at different times after feeding and in animals of different body weight. It was also the aim of this study to investigate the use of an in uivo assessment of pancreatic activity in pancreas-disease fish. This was achieved by adapting a technique normally used to assess human pancreatic function and was based on the orat introduction of ~-benzoyl-L-tyrosyl-~-amino~nzoic acid (Bz-tyPABA) (Dabrowski et al., 1989) which is cleaved by pancreatic ch~otrypsin li~rating ~-amino~nzoic acid (PABA). The amount af PABA absorbed by the intestine and consequently excreted in the urine is used as an index of pancreatic function (Yamato and Kinoshita, 1979; Toskes, 1983). The relationship between the patho~enesis of the disease and enzyme activities were measured in populations of salmon smelts farmed in sea-water and in a transmission experiment carried out in freshwater using salmon parr.
bined
Pancreas disease (PD), a pathogenic condition affecting farmed Atlantic salmon (S&IO s&r), was first recognised in Scotland in 1975 and has since appeared wherever Atlantic salmon have been intensively reared on a significant scale in Europe and North America (~cVjcar and Munro, 1987). The pathological characteristics of the disease include the generalised degeneration of the exocrine pancreas (Munro et al., 1984; McVicar, 1987) and often results in severe ema~iatjon of the affected fish (McVi~ar, 1987). Usually, the condition affects post smelts in their first sea year, aIthougb farmed salmon of alI ages are susceptible to the disease (McVicar and Munro, 1987). Current research suggests an infectious agent is responsible (McVicar and Munro, 1988; MeVicar, I990) and PD has been successfully experimentally transmitted (MeVicar, 1987; McVicar and Munro, 1987; Raynard et ai., 1991). At present, the only reliable pathological diagnosis of PD is by the histopathological examination of the exocrine pancreas, a necessarily slow and laborious tecbn~q~e, but from which it is possible to disti~gujs~ stages in the acinar cell collapse which can be described as normal, pre-acute, acute, chronic and recovery (Munro et al., 1984). The aims of this study were to correlate pancreatic enzyme activities of trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) with the histo~o~~a~ diagnosis of healthy fish and those suffering from PD. Total trypsin and ~hymotrypsin in a fish will depend upon the enzyme activity per gram ofpyloric tissue and the total weight of the pyloric caeca. Pyloric caeca have been found to be variable in mass, and digestive enzyme activity may also vary with time after feeding and composition of the food (Kawai and Ikeda, 1973; Overnell, 1973; Stevens and McLeese, f988). Therefore, studies of digestive enzyme activities were com-
Fish The freshwater transmission experiment was carried out with Atlantic salmon (S~~~~ solar) parr maintained at the SOAFD fish cuitivation unit, Aultbka, Wester Ross, Scotland. Eighty fish were used in the trial (mean weight 24 a). Head k&&y homogenate of known in&ted fish kas us&l to transmit pancreas disease. Protein con~ntrations of infective and control material were measured with the Bradford technique (19%) and injected at constant amounts (3 fig protein/g fish). Forty fish were injected intraperitoneally in the pelvic region with 5.1 ml of infective material. The control group consisted of 45 fish injected with 5.1 ml of non-infective homogenate (Raynard et al., 1991). Before and after the injection the fish were kept in tanks, 50 cm in depth at 14°C. Fish were fed using automatic feeders which supplied food at a rate of 2.5% body weight/day. Ten animals were removed from the control and injected groups at weekly intervals foliowing the injection for 4 weeks. On
759
760
G. M. PRINGLEet al
the day of sampling the animals were anaesthetized with 10% (w/v) benzocaine and killed bv a sharp blow to the head. ‘Samples of caecal tissue from- each fish were frozen individually in liquid nitrogen and fixed for light microscopy as described below. Salmon smolts in their first year in sea-water, transferred to sea-cages in May, 1990, were used for the field survey. The fish were obtained from a large-scale salmon farming company on the west coast of Scotland (mean weight 261 g). These fish were fed on commercial salmon feeds three times a day at a rate of approximately 2% body weight/day. Variation in trypsin activity with time after feeding was determined with-51 farmed salmon smolts (mean weight 265 n) maintained in 2 m diameter sea-water tanks (125’C) at the Dunstaffnage Marine Laboratory, near Oban, Scot: land. They were all starved, then a group of seven fish was sampled (method described below) and the remaining fish were fed one feed to satiation; these animals were then sampled in groups at various times post feeding. Salmon smolts were obtained from Dunstaffnage Marine Laboratory, which had been maintained in ambient sea-water temperature conditions since May 1990 (mean weight 270 g) in 2 m diameter tanks of 1 m depth. For approximately 6 weeks prior to sampling they had been fed at about “maintenance” ration (approximately 1.0% body weight/day). The fish to be sampled was immediately killed by a sharp blow to the back of the head and the spinal cord was severed just behind the head. Wet weight and length were recorded. The whole pyloric caeca was then removed by cutting immediately at the anterior and posterior ends of the organ (allowing the inclusion of all the caecal fingers) and its wet weight recorded. Two sub-samples of caecal tissue were taken from the same region for histology (light and electron microscopy) before the pyloric caeca was frozen and stored in liquid nitrogen. On return to the laboratory, the frozen caecal samples were stored at -70°C. Histology
Pyloric caeca were fixed in 10% buffered formal saline for light microscopy. Sections (5 pm) of paraffin wax-embedded tissue were stained with haematoxylin and eosin. For electron microscopy, pyloric caeca with pancreas were fixed in 2% paraformaldehyde plus 2.5% gluteraldehyde in 0.1 M Sorenson’s phosphate buffer (pH 7.2), post fixed in 1% osmium tetroxide, dehydrated in graded alcohols and propylene oxide, and embedded in EPON 812 resin, Sections were taken on a Reichert-Jung 0MV3 ultratome, stained with uranyl acetate and lead citrate, and observed with a Philips EM301. Diagonsis of pancreas disease was based on the protocol suggested by Munro er al. (1984) using the wax-embedded sections.
Trypsin activity was measured using the synthetic, a-N-benzoylchromogenic trypsin-specific substrate, DL-arginine-p-nitroanilide (BAPNA), which liberates p-nitroaniline upon hydrolysis by trypsin (Preiser et al., 1975). A IO-fold gain in sensitivity for the trypsin determination was achieved by using BAPNA in combination with the Bratton-Marshall (BM) reaction. The released p-nitroaniline gives a purple colour in the presence of N-lnaththylethylenediamine (Bratton and Marshall, 1939). The reaction was employed by the additions of 0.5 ml each of sodium nitrite, ammonium sulphamate and N-l-naphthylenediamine at 3 min intervals, respectively. The optical density (OD) of the resulting purple coloured samples were read on a Pye UNICAM SP-550 UV/VIS spectrophotometer at 550 nm. Weighed amounts of frozen caecal tissue (approximately 500 mg for smelts and 100 mg for Parr) were homogenized with a Polytron homogeniser in 0.05 M Tris buffer pH 8, containing 0.02 M CaCl, and the homogenate was cen-
trifuged at 0°C for 15 min at 13,000g. The supematant was removed and stored on ice. The remaining pellet was resus~nded in fresh buffer, re-homogeni~ and the supernatant added to the first extract. The incubation conditions were 50 p I of extract plus 50 p 1 of buffer added to 0.7 ml of BAPNA at 15°C for 10 min at pH 8; the reaction was stopped by the addition of 0.8 ml of 0.2 N HCI. The substrate used for the chymotrypsin assays was glutaryl-L-phenylalanine-p-nitroanilide (GPNA) (Sigma, Poole, U.K.) using the methods described by Erlanger et ol. (1966) using incubation conditions of 15°C for 30 min at pH 7.6. Throughout this study, enzyme activities are expressed per gram of caecal tissue. The total protein con~n~ation in the supematant containing the enzyme activities prepared from caecal samples was determined using the- method described by Bradford (1976); no significant differences were found between the samples. In vivo measurement qf’pancreatic function Cylindrical pills 5 mm long by 5 mm diameter, each containing 100mg of the specific synthetic substrate of ch~otrypsin, ~-~nzoyl-L-tyrosyl-p-~ino~nzoi~ acid (By-ty-PABA) (Sigma) were formed using a Gall~kamp pill press. Experiments were carried out on healthy farmed salmon smolts (N = 6, mean weight 302 g) and animals suspected as having PD (N = 11, mean weight 215 g). Each animal received a single pill, placed in the stomach by tube and was kept in 10 I of sea-water at approximately l1.5”C with constant aeration. Water samples (lOm1) were collected at various times after pill administration for up to 48 hr. Each fish was then killed and sampled by the methods described above, in order to determine the condition of the pancreas. For the determination of PABA, 0.5 ml of 1.5 N HCI was added to 1 ml of water sample and heated in boiling water for 15 min. The samples were then allowed to cool before carrying out the BM reaction and subsequent reading of optical density at 550 nm (described above). Statistical methods
The results from enzyme measurements are expressed as pmol of substrate released per g fresh caecal tissue weight per min. Mean _frSE are given throughout. Total pyloric caecal weight, trypsin activities per g of caecal tissue and total trypsin activities per fish were related to total ftsh weight using the allometric equation Y = aXb in the logtransformed form. Regression analysis was carried out by linear least squares analysis and slopes and elevations compared by covariance analysis. Means were compared by one-way analysis of variance and, where appropriate, further analysis was by Student’s f-test for both paired and unpaired data. The 5% level of significance was used throughout. RESULTS Histology
The acinar (i.e. exocrine) pancreas sea-year salmon smolts are organ&d
cells of first
in functional units surrounding a lumen; the organelles are distributed with an orientation around this lumen (Fig. I). The basal region of the cells contains the nuclei and the rough-surfaced endoplasmic reticulum while the apical cytoplasm is normally packed with numerous storage or zymogen granules. The mean acinar cell diameter of normal fish was 14.66pm (kO.715 pm, N = 15) with a mean nuclear diameter of 4.88 ,um (kO.263 pm). The presumed centroacinar cells (Weiss, 1988) found next to the lumen were elongate and characterised by relatively large
Salmon
pancreas
761
disease
Fig. 1. Normal exocrine pancreas of salmon (< I sea-year) showing two cell types: acinar cells predominate and are characterised by the presence of vesicular endoplasmic reticulum (ER), large mitochondria (M), and nucleus (N) and numerous zymogen granules (Zm). The orientation of these granules is towards the lumen. The presumed centro-acinar cell (Be) is elongate and is characterised by its large nucleus.
These cells may function in the production of bicarbonate solution which flushes the trypsinogen out of the ducts (Weiss, 1988). Each acinus was surrounded by a basal lamina and a loose connective tissue matrix. Using the diagnostic protocol for the assessment of histological samples (Table 1) the corresponding electron microscopic appearance of acinar cells in the acute phase of PD was characterised by the endoplasmic reticulum (ER) being condensed into sheets and whorls (Fig. 2). Vacuolation (V) of the cytoplasm was evident. The nuclei (N) were irregular in shape and stained darkly due to condensation of the nuclear components. No zymogen granules were present. In the chronic phase of PD the acinar cells were characterised by the presence of sheets and whorls of collagen (C) (Fig. 3). Large vacuoles were also present. There was an absence of any characteristics typicai of normal acinar cells and it was impossible to differentiate between any cell types. In the acute phase the mean acinar cell diameter was 4.76.um (20.257, N = 15) (significantly smaller than that of normal cells, P < 0.05) although the mean nuclear diameter (3.68 him + 0.245) was similar to those of normal cells. This suggests that the nuclei of these diseased cells remained morphologically intact despite the degenerative condition of the acinar cells. Fish diagnosed as being histologically normal by light microscopy but which did not have any nuclei.
detectable trypsin or chymot~psin activities had two acinar cell types (Fig. 4). The first type, (A), exhibited no signs of necrosis and had the normal acinar cell characteristics. The second cell type, (B), had dark condensation of the endoplasmic reticulum (ER), the mitochondria (M) were enlarged and many were crescent-shaped. The cell membrane, nucleus and zymogen granules appeared to remain intact.
Table 1. Summary of the modified characteristic histopathology (light) used to distinguish the stages of acinar cell collapse during pancreas disease, based on the original protocol suggested by Munro rt crl. (1984) Condition NCXltlal
Degenerative Pre-acute Acute Chronic
Recovery
Histooatholoav
(linht)
None. Dense sheets of acinar cells with zymogen granules, fat and endocrine cells, i.e. islets of Lange&am, ducts, veins, arterioies and bicarbonate cells. Some acinar cells exhibit degenerative changes, but without vacuolatians or signs of necrosis. Some acinar cells develop vacuolatinns along with focal areas of necrosa. Acute and generalised haemorrhagic pancreatic necrosis, deposition of collagen. Total absence of acinar tissue replaced by fibrotic tissue, infiltration with fibrocytes and/or leucocytes. Remaining endocrine pancreatic components remain intact. Small groups of presumed acinar cells can be seen amongst the necrotic tissue, particulariy in assocmtion with islet tissue and/or ducts.
762
6. M.
PRINCLE et it/.
FEg.2. Acute pancreas disease: the endoplasmic reticulum (ER) is condensed into sheets and whorls. vacuolation (V” of the cytoplasm is evident. The nuclei (PI) are irregular in shape and stain darkly due to condensation of the nuclear components. No zymogen granules are present.
The mntral group showed no bistolo~i~~ signs of pancreatic degeneration during the course of the trial (Table 2). The mean try&n and chym~tr~~s~n activities of the control animals declined during the course of the experiment and were signi~cant~y lower by week 4 compared with week 1 in the case of trypsin and by weeks 3 and 4 in the case of chymotryps~n (Fig. 5a and Sb). In animals injected with infective material there were no signs of PD in weeks 1 and 2 but it was diagnosed in some fish in weeks 3 and 4 (Table 2). Mean enzyme activities for trypsin and c~~~~~ry~§i~ were ~igni~~a~~iy lower from the controls in weeks 3 and 4. The majority of the PD diagnosed fish were in the chrome phase with significantly tower trypsin activities compared with the controls in week 3. No trypsin was detected in week 4 (Table 2). Chymotrypsin activities were found to be consistent& lower than trypsin activities but chym~tryps~n activities were measurable in ah the PD fish, although the vatues were s~~n~~~nt~y lower than those of histologically normal animals, There was a significant correlation between the activities of trypsin (X) and chymotrypsin (Y) which can be described by the equation: Y = 0.0085 -I-0.034x (N = 80, Y2= 0.034, P -c 0.05). This means that overall the amounts of the two enzymes are linked afthough chymotrypsin ao tivities were still detestable when trypsin activities were absent in the PD fish.
In f&h sampled from a he&by ~~~~~~~~~~ and having histo~og~ca~~y normal pancreas adnar cells, a greater than IO-fold variation in trypsin activity was found (Fig. Ba): however. most fish had activities close to the mean (0.135 +- 0.0086 ~moles pNA/g/min, N = 68). Thirty-eight fish that were sampted on the same date from a population apparently suffering from PD were found, after histologicat examination using the criteria described in Table I. to have PD. These animals had no detestable Ievefs of trypsin activity. A comparison between the trypsin assay and the conventional histopathoRogica~ method of PD diag nosis from 185samples is summar~sed in Fig.. 6b. The results clearly demonstrate that trypsin actrvtties and histology agree almost completely; pancreas acinar ceils classified as histologically normal almost always exhibited trypsin activities whereas acinar cells &ass&d at various stages of PD bad generally no trypsin activity even when classified to be in the recovery stage {nine out of 80). Of the IO5 samples h~stopaYho~o~ically classified as being normal, 99 (94.3%) had detectable levels of trypsin activity and six (5.7%) had no detectable levels of tryptic activity. Of the 80 samples b~stopathologica~~y classified as having PD, 77 (96.3%) had no detectable levels of tryptic activity and three (3.7%) did have detectable fevels. 1~was found that the three sampIes which had PD with detectable levels of tryptic activity came
Salmon pancreas disease
163
Fig. 3. Chronic pancreas disease: the pancreas tissue is characterised by the presence of sheets and whorls of collagenous fibrotic tissue (F). Large vacuoles are also present. There is an absence of any characteristics typical of normal acinar cells.
from a “degenerative” normal and pre-acute. Variation
category,
in trypsin activity
diagnosed
between
in normalJish
In view of the variation in trypsin activity found in histologically normal animals, experiments were carried out to determine whether this variation was due to the site of caecal sampling or time since feeding. The caecal mass consisted of a clearly distinguishable anterior section which has caeca both dorsally and ventrally and a posterior section with only dorsal caeca. The caeca of seven animals from the same population were divided up into six sections and trypsin activity determined. The mean levels of activity in each section were found to be similar (Table 3) and no significant difference was found between any section (paired r-test). In feeding animals, the mean trypsin activities declined significantly by 5 hr post feeding. Mean activities returned to close to pre-feeding levels by 47 hr after the last meal (Fig. 7). Eflects of body size The weight of the pyloric caeca of histologically normal, fed fish were positively correlated with total fish weight (Fig. 8) (Table 4); combining the results from parr and sea-water smolts did not significantly alter the slope or intercept of the regression equations. The results from sea-water animals that were histologically normal but which had been main-
tained on minimal diets or without food gave a regression equation which was significantly lower than that of the fed fish (Fig. 8). The pyloric caecal weights of the PD fish were almost all within the range found for the minimal ration or starved fish. Regression analysis of all the low ration, starved and PD fish had a similar slope to that from the fed fish but had a significantly lower elevation (Table 4). Trypsin activity per g was also correlated with fish weight in normal, fed fish (Table 4). Animals which had been starved for 12 days had trypsin activities which were within the range found from normal feeding animals (Fig. 9). Total trypsin activity (from the product of total caecal weight and weightspecific trypsin activity) was correlated with total fish weight (Fig. 10); in fed sea-water smelts the slope was greater than one but when the parr were included, the slope was not significantly different from one (Table 4). Bz-ty-PABA function
and in vivo measurement
of‘pancreatic
In salmon smolts which were later found to have histologically normal pancreas cells and normal in vitro chymotrypsin activities (0.0052 pmoles pNA/g/min) (Fig. ll), the appearance of the breakdown product of the chymotrypsin-sensitive substrate followed a sigmoidal curve with time. After 44 hr 67 + 2.6% of the calculated dose had been recovered. Control experiments where the pill was left in tank water gave 6.22% recovery after 48 hr. Animals
763
G.
M.
F'RINGLE~I al
Fig. 4. Fish diagnosed as being hist~logicaily normal by light microscopy but having trypsin activities of zero, show two acinar cell types. The first type, (A), exhibits no signs of necrosis and has the normal acinar cell characteristics. The second cell type, (B), shows dark condensation of the endoplasmic reticulum (ER), the mitochondria (M) are enlarged and many are crescent shaped. The cell membrane, nucleus and zymogen granules appear to remain intact.
which were later determined to have PD from histological ex~~mination of their pancreas cells and had zero in vitrochymotrypsin activities had significantly lower levels of breakdown products at all time points and the excretion rate appeared to be linear; percentage recovery was only 36 + 2.8% after 44 hr. Taking the 24-hr time point, the normal fish had excreted 47.1 rt 6.6% of the calculated dose whereas the PD fish had excreted 17.4 i: 3.4% (P < 0.0001~.
Previous, unpublished studies into the pathology of PD have also investigated the levels of trypsin activity, but a non-specific protease assay method was used. It is possible, therefore, that some of the results obtained may have been erroneous due to contamination by other proteolytic enzymes. The trypsin assay used in this study was found to be highly specific and repeatable; it does not
Table 2. Percentage of fish in different histological categories of the 40 infected fish from the ~~ansrnissiantrials with salmon parr in fresh water Enzyme Week (E) ____ -._..---
i
% _--.. ~.
Normal .._..E:_
Acute
% ~~_____
Chronic E
oio
E .____..._..I^
Recovery 91. ..___.. _. .,_..-. E
100 :
2
0.232 5 0.024 D.Ol52 ri:o.w9 100
7 e 3
o.zau & 0.040 0.0113 + 0.0014 60
4
10 0.140 & 0,025 0.008 i: 0.0015
T c so T c
30
0.058
0.007 +- 0.0004* 0.0001 1.0.0001*
0.0046 40
0.062 4 0.01 L 0.0067 f 0.0009
IO 0.000 0.00009 4 o.O0005*
Ten animals were sampled at each week. The mean + SE trypsin CT) and c~ymot~~jn (C) levels fpmoles ~~gh~~rni~~ in the different categories of pancreas-diseased animals are indicated. *P < O.Oal compared with mean confrol values.
0.000 0.0005 @A!$
fresh
765
Salmon pancreas disease
a very beneficial effect in greatly reducing the impact
of PD. Lowered ration levels or starvation also resulted in the same wide variations in trypsin levels but there was a marked reduction in caecal mass compared with feeding animals. Nevertheless, the total trypsin activity per fish of animals starved for 12 days appears to be within the normal range of total trypsin activities for feeding animals (Fig. IO). Reduction in
week 1
Week 2
weak 9
week4
Week number
.,. .c E
0.018 ,
b)
pyloric caecal size has been reported previously and found to be associated with reduced digestibility (Windell, 1967). The small differences in the levels of tryptic activity from the different areas of the pyloric caeca were not found to he significant (Table 3), suggesting that dispersed pancreatic tissue is homogeneously
0.016
‘;gJ) 0.014 2
0.012
1
0.010
8 1
0.006
‘;
0.006
6
0.004
E 3 0
0.(x12 0.060 week 1
w&k 2
week 3
Week number
Fig. 5 (a) Mean (&SE) trypsin activities in the caeca of the control and infected groups of salmon parr in the transmission trial with time. (b) Mean (&SE) chymotrypsin activities of the same animals. Ten animals from each group were analysed for each time point.
0.e.n
@xl. O.oal. O.?Ol- 0.151-OPl- 0.251. om- om- 0.4w o.oso o.tw O.,JO 0.200 0.250 0.300 0.350 0.400 0.450
n
PDFish
n
Healthy Fish
differentiate between trypsin and its inactive precursor trypsinngen. The assay conditions ensure that all the stored trypsinogen was converted to trypsin, thus achieving a measure of the total potential tryptic activity. Trypsin actim2ie.s
There is clearly a great deal of variation in trypsin activities in normaily feeding salmon smolts. Part of this variation has been found to be due to a decrease in trypsin activities after feeding (Fig. 7). As the trypsin method detected total trypsin it can be inferred that the pancreas had been stimulated by the arrival of the meal to secrete trypsinogen/trypsin from the pancreas into the gut and therefore the enzyme activity was reduced in the region of caecal tissue that was sampled (Love& 1989). What effect repeated meals have on trypsin activities is not known, although if combined with the indicated resynthesis time for the enzyme of around 48 hr (Fig. 7), it can be speculated that the pancreas is being placed under increased physiological pressure {stress) to produce sufficient enzyme to be able to fully digest each meal. This stress may increase the pancreas’s susceptibility to illness. Such speculation is further reinforced by findings from within the salmon farming industry itself, where lowered feeding rations and even starvation at the first signs of PD appear to have
Pancrease disease
HietopethoiogicdClassification TtypeinAssay
Fig. 6 (a) The distribution in trypsin activities from 68 histologically normal farmed Atlantic salmon smelts (mean weight 283 g) randomly sampled in early October 1990. Also included is the data from 38smolts his~oiogic~ly confirmed to be suffering from PD sampled from a different nooulation on the same date. (b) Comp&son of the pancreatic trypsin activities with the histological assessment for PD. The data for histologically normal farmed salmon smelts were taken from 105 animals sampled between late August and November 1990. The 80 farmed salmon smolts diagnosed as having PD were sampled over the same period.
166
G.
Table 3. Distribution
of trypsin activities Anterior
M.
et al.
PRINGLE
@moles pNA/g fresh weight caecal tissue/min) caeca of salmon smelts
pyloric region
in various regions of the pyloric Posterior
Dorsal
Ventral
Anterior
Posterior
Anterior
0.0883 k 0.0119
0.0883 f 0.0135
0.0746 i 0.0082
region
Anterior
Posterior
0.0831 k 0.0114
0.0770 * 0.010
Posterior 0.0794 f 0.01 I4
pyloric Dorsal
The caeca were divided into an anterior region which has both dorsal and ventral caeca and a posterior region which only has dorsal caeca. Each region was in turn divided into anterior and posterior subsections. Each mean is from seven animals sampled from the same population
throughout the pyloric caeca. This agrees with the findings of Stevens and McLeese (1988) for rainbow trout (Onchovhynchus mykiss). Pancreatic tissue is also found around the stomach, bile duct, anterior spleen and upper intestine (Munro er al., 1984), although it is not yet clear whether these cells add significantly to the total trypsin activity determined from the caecal tissue. The amount and activity of the digestive enzymes has previously been related to the time of feeding (Steffens, 1989). In carp, the level of protease activity in the intestine reaches a maximum 5 hr after feeding (Onishi et al., 1976) with the largest rate of enzyme secretion per unit of time being observed during the first 5 hr post feeding (Steffens, 1989). In the European eel (Anguilla anguillu), the highest enzyme secretion and activity levels were recorded 12 hr after feeding; at 46 hours after feeding the rate of rise of total activity was found to be at its highest (Steffens, 1989). There are clearly two separate aspects to digestive enzyme activities in salmon; trypsin and chymotrypsin activities per g and the total weight of the caecal tissue. Although it is recognised that the pyloric caeca increase the intestinal surface area (Buddington and Diamond, !987) most studies have concentrated on the number of caeca. The number of caeca has been found to be correlated with size of fish in rainbow trout (Babushkin, 1974; Bergot et al., 1981). Results from the present measurements clearly demonstrate that caecal mass is related to body weight in an isometric fashion and that weight specific enzyme activity declines with increasing body mass in a similar manner as metabolic rate,
feeding 1979).
rate
and
growth
rate
(Brett
and
Groves,
Comparison between histology and digestive enzyme activities The two methods showed over 95% agreement in diagnosing PD in field-sampled farmed Atlantic salmon smolts. It is not surprising that, with the histological characteristics of the disease showing a complete breakdown of the acinar cells, that it should be accompanied by the total absence of digestive enzyme activity. However, some anomalies are apparent. Firstly, in the transmission trials, detectable levels of chymotrypsin were evident in PD fish. Secondly, of the PD fish found with detectable trypsin activities (< 5%) all came from the “degenerative” classification (Table 1). Two explanations can be put forward for the latter observation; the presence of trypsin could be explained by the possibility that the disease was in its early stages and the exocrine pancreas was still able to produce trypsin, or a second explanation, which seems more likely, is that an “edge effect” (Callanan, personal communication). This is a phenomenon which occurs due to enzymatic degradation of caecal tissue shortly after its excision, usually as a result of poor field sampling or processing. In this event, the three samples could possibly have been from healthy fish as the trypsin assay indicates. The six samples that had no detectable levels of trypsin but were histologically classified as being 100
s 0.24 ,
E
.a
lo
0.21 0.16
f
1
.e %
1
.
:-
*a i. .. l .
0.1
0.03
W
1
o.oo+ . 0
6
.
10
3
15
’ 20
m 25
m 30
m 36
’ 40
45
’ 60
Time, h
Fig. 7. Mean (+ SE) trypsin activities in the caeca of farmed Atlantic salmon smolts (mean weight 265 g) at various times after a meal. Seven fish were sampled prior to feeding at time zero and the following number of animals were sampled after that time: 1 hr (6), 3.5 hr (7), 6 hr (S), 11 hr (6), 23 hr (6), 47 hr (7).
1006
100
10
weight(e)
*
Non”aIRsh
A
q
PD
0 starved
Minimumdti
Fig. 8. Variation in total caecal weights relative to fish weights. The feeding animals with histologically normal parr and smolts are classified as normal. Total caecal weight of starved animals, animals receiving minimal diets or animals diagnosed as having pancreas diseased by histological criteria are included. The regression equations describing the relationships can be found in Table 4.
Salmon pancreas disease
161
Table 4. Relationships between total pyloric caecal weight (g). trypsin activity (pm&s pNA/g,knin) and total trypsin activity (I_tmoles pNA~fish/min) and fish live weight (X, g) using regression equation log Y = log a + b log X Animals
a
Pyloric caecae sea-water All animals PC, starved Trypsin Total trypsin sea-water Total trvusin all animals
b
- I.85 1.17 - 1.77 1.14 -2.72 I .47 -0.54 -0.20 -4.43 1.83 - 2.32 0.94
N
r2
P
89 108 46 108 89 108
0.60 0.90 0.52 0.07 0.44 0.54
