Relationships of Intestinal Enzymes and Serum Antitoxin to the Pig Response to Escherichia coli Enterotoxin S. Lariviere and R. Lallier*

ABSTRACT

INTRODUCTION

The intestinal loop technique was used to evaluate the response of three week old piglets to the heat labile (LT) and the heat stable (ST) enterotoxins produced by Escherichia coli F11(P155). The serum anti-LT activity and the lipase, amylase and trypsin activities in the jejunal lumen of these pigs were determined. Piglets responded independently to each toxin and no relationship between these responses and serum anti-LT activity or the enzyme activities of the jejunal content could be demonstrated.

The Escherichia coli strains associated with diarrhea in piglets produce a toxin that causes massive outpouring of fluid into the lumen of the gut (18). This toxin can exist in two forms: one is heat stable, dialysable and apparently nonantigenic. the other is heat labile, nondialysab'e and antigenic (2.2). The enterotoxic activity of E. coli preparations can be demonstrated in pigs by the ligated intestinal loop technique or by oral administration in intact animals (23). Pig to pig variation in sensitivity to E. coli enterotoxin was demonstrated with both techniques (13, 22). The effect of enterotoxin on the intestine will be influenced by three factors: 1) enterotoxin inactivation in the gastrointestinal tract, 2) amount of enterotoxin contact with the reactive site in the intestine and 3) the ease with which a fluid imbalance may be created. Antibodies and enzymes which could be found in the intestine can inactivate the E. coli enterotoxin (7, 23, 24). Antibodies can reach the lumen of the intestine following oral ingestion or passage from the blood through the intestinal mucosa. It has been shown that resistance of the pig to E. coli enterotoxin increases with age (17, 25). Since the enzymatic activity in the gut increases with age (9, 16) and pancreatic enzymatic activity in the gut lumen seems to fall considerably during gastroenteritis (11) a role for the enzymes can be postulated. The objective of this paper is to evaluate the role of intestinal enzymes and serum antitoxic activity in the resistance of the pig to E. coli enterotoxin.

RESUME Les auteurs ont utilise la technique des anses intestinales chez des porcelets de trois semaines, afin d'etudier la reactivite du porc a l'enterotoxine thermolabile (LT) et i l'enterotoxine thermostable (ST) produites par Escherichia coli F11(P155). Ils evaluerent l'activiti anti-LT presente dans le serum de ces porcs, ainsi que l'activite de la lipase, de l'amylase et de la trypsine, dans leur contenu intestinal. Ils demontrerent aussi que ces porcs reagissaient differemment aux deux types d'enterotoxines, mais ils ne purent etablir aucune relation entre la reponse des porcs aux enterotoxines et l'activite anti-enterotoxique du serum ou de l'activite enzymatique du contenu intestinal. *Faculte de

Midecine vit6rinaire, Universite de Montreal, C.P. 5000, Saint-Hyacinthe, Quebec J2S 7C6. Submitted February 6, 1975.

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MATERIALS AND METHODS

they were stored in a deep-freezer at -700 C.

CULTURES

DETERMINATION OF ENZYMATIC ACTIVITIES

E. coli strains Fll and F11(P155) were used in this study and were described earlier (15). The strain F11(P155) was used to produce the enterotoxin(s) and the strain F1l was used as a negative control.

The trypsin and lipase activities of the intestinal washings were determined basically by the method described in Worthington Manual of Enzymes except that the tests were performed at 37°C. Trypsin activity was measured by determining the rate of hydrolysis of p-tosyl-L-arginine methyl ester hydrochloride (TAME)1 based on the method of Hummel (10). One unit is equal to the hydrolysis of one micromole of TAME per minute at 37°C at pH 8.1 in the presence of 0.01M calcium ion. Lipase activity was measured by determining the rate of hydrolysis of an olive oil emulsion using potentiometric titration. One unit of lipase activity is equal to one micromole of acid produced per minute at 370C. The procedure used for the determination. of amylase activity is described by Dah1quist (3). One unit of amylase activity is that liberating one micromole of reducing group as maltose per min at 37°C.

TOXIN PREPARATION

The heat labile (LT) and the heat stable (ST) enterotoxins were obtained from the same broth culture preparation of E. coli F11(P155). The whole cell lysate (WCL) was used as the heat labile enterotoxin. WCL was prepared using the procedure described by Lariviere et al (15). The broth culture supernatant was used as the source of ST. Both preparations were freeze-dried and reconstituted just before use. After reconstitution the broth culture supernatant (ST) was heated at 100°C for ten min to destroy all LT activity. ENTEROTOXIC DETERMINATION The enterotoxic activity of LT and ST was determined by the ligated intestinal segment technique using three week old piglets (14). The 28 suckling piglets used for this study were obtained from eight different litters of the same herd and were milk fed only. The animals were starved for 24 hours before surgery. Toxin preparations were tested in 4 ml volumes. LT was tested at a dose of 40.0 mg per intestinal loop whereas the equivalent of 8 ml of undiluted broth culture supernatant was used as the standard dose of ST. LT was tested in loops #2, #6 and #10 and ST in loops #4 and #8.

ANTITOXIC EVALUATION OF THE PIGLET'S SERUM

The sera were obtained from the piglets which were used for the enzymatic determination. The antitoxic activity was determined by neutralization of LT in rabbit jejunal loops. The rabbit jejunal loop techniaue has been described earlier (14). One ml containing 1.0 mg of WCL was mixed with 1.0 ml of physiological saline or 1.0 ml of serum diluted 1:100 in physiological saline. The mixture was incubated at 37°C for 30 min before injection into a loop. The tests were conducted in pairs with control and serum containing inocula in adjacent jejunal loops.

INTESTINAL WASHING

STATISTICAL METHODS

Ten ml of sterile 0.85% NaCl solution were injected into loops #1, #3, #5, #7 and #9 immediately after the ligatures were secured. One minute later the washing was collected with a syringe and distributed in three tubes. The tubes were maintained in ice cold water for not more than two hours before

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Statistical tests described by Siegel (21) were used throughout this paper. The value expressed in Tables I, II and III were compared by the t-test. Using the 'Calbiochem,

San Diego, California.

Can. J. comp, Med.

TABLE I. Enzymatic Activities of Jejunal Washings of Piglets Tested with Escherichia coli Fll (P155) Heat Labile Enterotoxin Gut Loop Responsea 0.0 - 0.7

No. of Segments 20

Enzymatic Determination (units/ml)b

Amylase

Trypsin

84.4 i 54.20

26.8 i 22.5

72.2 ± 34.3

37.9 + 26.4

1.9 - 3.2 19 120.8 i 76.5 aExpressed as the ratio of fluid in ml to length of loop in cm bEnzyme units are described in Materials and Methods cMean ± S.E.

58.3 i 33.0

0.8- 1.8

32

Fischer exact test the following were analyzed: the relation between the pig response to LT and the pig response to ST and the relation between the pig response to each of the enterotoxin and the serum antitoxic activity.

RESULTS Using 28 piglets, a mean loop response of 1.3 ± 0.5 ml/cm was obtained in the 71 loops injected with LT and 0.7 ± 0.4 ml/cm in the 56 loops injected with ST. In Table I and II, the loops were divided into three groups according to their loop response to LT and ST respectively. The first group included all the segments which gave a loop response lower than the mean minus one standard error (S.E.), the second group included those which gave a response between the mean minus one S.E. and the mean plus one S.E. and the third group included those which gave a response greater than the mean plus one S.E. The enzymatic activities of each toxin injected loop were estimated by calculating the mean value of each enzyme activity of the two adjacent loops. In Table I and Table II

Lipase 1.5 ± 0.6

1.0 ± 0.3 0.9 i 0.4

the mean ± S.E. of the estimated values of each enzyme activity are recorded for each group of segments. When each enzymatic activity is considered individually no significant difference in activity could be observed between the groups of loops injected with LT (Table I) or ST (Table II) (P > 0.05). In Table III the loop response to E. coli enterotoxins and the enzyme activities of three different portions of the jejunum are expressed. No significant difference in enterotoxic and enzyme activities was observed between the three portions of the

jejunum. As more than one loop per pig was injected with each toxin the mean loop response of each pig to each toxin was used to determine the reactivity of the piglets to the enterotoxin. The mean response of the piglets to LT was 1.4 ± 0.6 ml per cm whereas a mean response of 0.8 ± 0.5 ml per cm was obtained for ST. Figure 1 shows the distribution of the piglets in relation to their reactivity to each toxin. No correlation between the response to LT and to ST could be found when the correlation test was applied (P > 0.05). The figure shows also the distribution of the pigs whose sera diluted 1:100 neutralized completely a LT preparation. Using the Fischer exact test we were not

TABLE II. Enzymatic Activities of Jejunal Washings of Piglets Tested with Escherichia coli Fll (P155) Heat Stable Enterotoxin Gut Loop

Responsea 0.0 - 0.2

No. of

Segments 19

Enzymatic Determination (units/ml)b

Amylase 101 i 86.4c 20 1.1 0.3 102 i 84.1 17 1.2 - 2.3 19.3 + 15.2 -Expressed as the ratio of fluid in ml to length of loop in cm bEnzyme units are described in Materials and Methods eMean S.E.

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Trypsin 58.2 ± 34.6 33.8 i 22.5 65.2 i 37.0

Lipase 0.7 ± 0.4 1.7 ± 1.0

0.7 ± 0.3

373

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Loop response to ST (ml/cm) Fig. 1. Distribution of the piglets in relation to their loop respoases to the heat labile (LT) and the heat stable (ST) enterotoxins. Eaeh piglet is represented by a letter and a different letter was usd for eaeh litter. The piglets designated by a capital lettr had stronger anti-LT activity in their serum than these with a man letter. The mean loop responses to LT and ST are represented by the dotted lines.

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Can. J. comp. Med.

TABLE III. Enzymatic Activities and Gut Loop Response to Eacherichia coli Fll (P155) Enterotoxin(s) in Three Different Parts of the Piglet Jejunum

Jejunal Loops Anterior Median Posterior

Gut Loop Response

Enzymatic Determination (units/ml)b

LT ST Amylase Trypsin Lipase 1.6 4 0.5& (27)d 0.9 ± 0.4^ (28)d 50.3 i 31.5c 32.3 ± 21.3 0.8 1 0.3 1.2 + 0.5 (17) 79.8 ± 45.1 52.7 : 26.4 1.1 4 0.6 1.2 + 0.4 (27) 0.7 4 0.4 (28) 116.4 i 57.4 44.4 i 24.7 1.3 ± 0.4 *Expressed as the mean ±fi S.E. of the ratio of fluid in ml to length of loop in cm bEnzyme units are described in Materials and Methods eMean ± S.E. dNo. of segments (one segment in the anterior jejunum and one in the posterior jejunum are not included because they ruptured)

able to show a relation between the presence of anti-LT activity in the sera of the piglets and their loop response to either LT or ST (P = 0.6). Most piglets from litters B, E, F and G had a relatively high anti-LT activity in their sera compared with those from litters a, c and d.

DISCUSSION This study shows that piglets react independently to LT and ST (Fig. 1). This observation would substantiate the hypothesis that LT possesses a different mechanism of action than ST. Other arguments in favor of this hypothesis are: reactivity of cells in vitro to LT but not to ST (5, 6, 12), dilatation of the intestine of newborn mice by ST only (20) and induction of a response by ST in a shorter time than by LT (2, 25). It has been shown that, like choleragen, LT increases adenyl cyclase activity (5, 6). It seems that ST does not stimulate appreciably the adenyl cyclase (6). We have found that all the piglets used possessed some anti-LT activity in their serum and that the level of antitoxin was closely associated with the litter. The relationship between the level of antitoxin and the litters probably reflects that of the sows' colostrum since the antitoxic activity of the serum resides probably in the immunoglobulin fraction which is acquired passively from the dam's colostrum. Since we observed variation in toxin sensitivity within certain litters it appears that the antitoxic activity in the sera does not reduce appreciably the intestinal loop response and therefore does not account for

Vol. 39 - October, 1975

the pig to pig variation in response to E. coli enterotoxin. Most of the other studies seem to indicate that serum anti-enterotoxin does not influence appreciably the loop response (1, 4, 8, 19, 24). As in other studies we have found large variations in enzyme activities in the intestine of the piglets (3, 9, 11). This variation was not only observed between the piglets but also between different segments within the same piglet. Although LT can be inactivated in vitro by lipase (Lariviere, Ph.D. Thesis, University of Guelph, Ontario) this study shows that the pig response to LT is independent of the amylase, lipase or trypsin which is present in the intestinal lumen. In addition to lipase, amylase can inactivate ST in vitro (Lariviere, Ph.D. Thesis). However the only enzyme which could have had an effect on ST activity in vivo is amylase but the relation between amylase and the sensitivity of piglets to ST is weak (P < 0.20). The apparent discrepancy between the sensitivity of LT and ST to the different enzymes in vivo and in vitro could be explained by several factors. First, the inactivation of the E. coti enterotoxins in vitro is incomplete after 24 hours of incubation (in this study the time of exposure of the enterotoxins to the intestinal content was probably insufficient to reduce appreciably the effect of the toxins). Second, it may be that the enterotoxins, particularly LT, became insensitive to the effect of the intestinal enzymes because of their rapid association with the intestinal mucosa (6). Third, the conditions of pH and enzyme concentrations set up in vitro are probably different to those obtained in vivo. Finally, although the mean enzymatic values of the adjacent loops were used to calculate the enzymatic activities of the toxin injected loops these values could be very different from the

375

true values. It seems that there is less enzyme activity in the anterior loops than in the posterior loops and that it coincides with a slight decrease in loop response to both toxins but the differences are not statistically significant (Table III).

CONCLUSIONS This study supports the hypothesis that LT and ST act by a different mechanism since we have shown that piglets react independently to both "forms" of the enterotoxin. We have also been unable to show that pig to pig variation in reactivity to E. coli enterotoxin is dependent on the serum anti-LT activity and the lipase, amylase and trypsin activities present in the intestinal lumen of the pig.

ACKNOWLEDGMENTS This work was supported by the Conseil des Recherches agricoles du Quebec, Grant MMV-72-502. It is a pleasure to acknowledge Miss Annette Jodoin, Miss Clarisse Desautels and Mr. Daniel Perron for their valuable technical assistance.

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