- Email: [email protected]
Activity of disaccharidases and dipeptidases of the intestinal mucosa of piglets during mild and severe infections with Strongyloides ransomi
]. c:OMP.
PATH.
XCTIVITY OF THE
1976.
VOL.
OF DISACCHARIDASES AND DIPEPTIDASES INTESTINAL MUCOSA OF PIGLETS DURING MILD AND SEVERE INFECTIONS WITH STRONNGYLOIDES RANSOJIP
K. ENIGK, Institute
243
86.
cf Parasitology.
A. DEY-HAZRA School of bkterinq~
and S. I,. EDUARDO~ Aledirine.
Hannorer,
Federal Republic of Germay
INTKODUCTIOX
Stroncglloidesransomi parasitizes primarily the epithelial layer of the small intestinal mucosa of piglets (Enigk, 1952). The eggsof the parasite are deposited in the epithelial layer and are expelled together with detritus into the lumen of tile gut. The villi of the infected mucosa become flattened by an inflammatory infiltrate in the lamina propria. Previous work has shown marked pathophysiological changes in the small intestine of piglets parasitized Iq this nematode. By labelling plasma albumin with 51Cr iGiese, Dey-Hazra and Enigk, 1972) or g5Nb (Enigk and Dey-Hazra, 1975) a protein-losing cnteropathy associated with hypoalbuminaemia and weight loss has been demonstrated in severely infected piglets. In vitro experiments demonstrated tllat the rate of amino acid absorption was decreased by 55 per cent. due to ,S. ronsomiinfiction (Harmeyer, Birk, blartens, Dey-Hazra and Enigk, 197:j;. To clarify further the pathogenesis of S. ransomi in the pig the levels 01‘ disaccharidase and dipeptidase activity in the intestinal mucosa of piglets c~xposedto milcl or severe infection were measured. MATERIALS
AND
METHODS
Piglets were obtained by caesarean section and raised worm-free. At 14 weeks ot age. the piglets were infected by a subcutaneous injection of infective larvae of .S. rcznsomi, using a strain maintained in our laboratory for several years. Two experiments were carried out. In the 1st 24 piglets were used: 12 animals were infected with 10 000 larvae/kg. body weight and 12 were controls. Four infected and 4 control animals were killed on each of days 7, 14 and 21 after infection (p.i. (groups A, B, C: respectively) and the activities of intestinal disaccharidases wcrc assayed. In the 2nd experiment 12 piglets were divided into 3 groups of 4. Tht animals of group D were infected with 1000 larvae/kg. body weight each, group E with 10 000 larvae/kg. body weight and group F were uninfected controls. All the* animals were killed on the 28th day p,i. and the activities of intestinal disaccharidasca and dipeptidascs were measured. Piglets were killed by i.v. injection of the sodium salt of methyl-thioethyl-2-pentylthiobarbituric acid.: Small and large intestines were opened longitudinally and thr content was removed by washing with ice-cold * Supported by Deutsche Forschungsgemeinschnft. + Present address: College of \.rterinnry l\ledicine. : Thinqcnal. Rlrrck. Darmstadt.
CnivrrAty
l)!liman.
Quczon-City.
PhilippIno.
244
1;. l5KIGK
?I (Ii.
0.15 1~1sodium chlorides and the surfkcc slightly blo~r~tl. Small pi~c~c~~i,4 10 li (‘111. LLC’~‘,‘ cut out from differrnt parts ofsmall and large, intestirlc. and thc.ir II~LICOS~ was scrapc’d scgrri~27rs wcr( sc,lcctcd by lllc,a.surirt,q off with a microscope slide. ‘I%(* intestinal the distance from thr pylorus. ‘I’hc mucnsa was homogcnizcd in ice-cold 0. I5 11 sodium chloridr using an Ultra-‘I’urrax homogrnizcr for 3 rrlin. ‘I’hc homogc-tiaras was left at 4 “C f.or 30 min. for extraction and was centrifuged for 10 min. at
2000g at 4 “C. The supcrnatant was dilutrd with redistilled water IO &rain a suitable enzyme concentration. Substrates i.r. lactose, saccharosc.,trchaloyc, ccllol~iosc and maltose wcrc used in the sarnc c.onccntration a\ raportcd by Dahlqvisc f I Wl-I. L-alanyl-glutamic acid, glycyl-/-lcucinc, glycyl-l-valinc and lht* rcspcctivc amino acids wcrc used in the same concentration Jo&son and Lindberg (1965a, I,, 1966).
arlcl
combination
as de~c~ril,r~tl
1)).
Activities of disaccharidases wcrc dctrrmincd by thck method dcscribrd 1)) Dahlqvist (1964) and cxprcsscd as units of activity per m,~. of protein. Activitic.s of dipeptidases WPI‘Cmrasured by the method of Josefssonand Lindbcrg (I96.5a’~ and expressed
as units of activity
per mg. of total mtrogcn.
Protein was dctermincd by the mrthod of Lowry, Rnsenbrough, Varr and Randall (1951)
with
a commercial
preparation?
as a standard.
Total
nitrogen
was dcterminrd
by the micro-Kjeldahl method. RESULTS
The dose of infection, time of’ slaughter and the number of adult worms recovered are summarized in Table 1. The measurements in the control animals showed that activities of disaccharidases are mainly localized in the mucosa of the small intestine and are only present in very small amounts in the mucosa of the caecum and colon. The distribution of different disaccharidases along the small intestine confirmed that there were two major groups of intestinal disaccharidases, as described previously (Dahlqvist, 196 1j.
I
24
A (4+4 U (4.t4 c: (4$4
corm.1 contr.) contr.)
IO 000
21
1000 10 000 Control
II
7 14 ‘8 28 28
37 738 28 267 22 532 2132 17889
Trehalase, lactase and cellobiase were localized mainly in the proximal part, whereas maltase and saccharase were mainly in the distal part of the small intestine. The total activity of maltase was greater in each of the measured areas than the activities of the other enzymes, the highest maltase activit) being in the area between 800 and 1000 cm. from the pylorus. Saccharase activity was very low in the duodenum, but increased in the jejunum. Lactase activity was greater in the proximal than in the distal part of the jejunum and in the duodenum. The distribution of cellobiase activity was nearly the t Lab
Trol,
Dade,
Miami
(U.S.A.).
PI(:I.ET
INFECTIOIiS
WITH
.y. RUL50??2i:
215
BIO(:HEMIS’I’K\’
same as thr lactase activity. The trehalase activity was greatest in thr mucosa of the middle of the ,jejunum. The mucosa of the entire small intestine of the infected animals showed :I reduction in the activity of maltase, saccharase, lactasc, trehalasc and cellobiasc~ (I:ig. 1). Animals killed on days 14 and 21 p.i. showed in general more reduction in intestinal disaccharidases than animals killed on day 7 p.i. The eKect of‘ low-grade and high-grade infections on the disaccharidasc activity is summarized in Table 2. There was a marked reduction in disaccharidasr activit) in thcz mucosa of the heavily infected animals (group Ej. The activities of disaccharidases of the caecum and colon sho\ved that tllcy wt’rc only present in small amounts. The large intestine of the infected animals was f’rrc fiiom .Y. ransomi and the measurement of disaccharidasr acti\.ity showed
Fig.
I.
Average disaccharidases of Strongyloides ransomi/kg. 7, 14 and 21 p.i. (Expt.
* Standard
deviation
is also
activity of the intestinal mucosa of piglets body weight: 4 infected and 4 control 1) q 7 days, C 14 days, q 21 clay? post indicated.
infected piglets infection.
with 10 000 larvnr were killed on da>-s n (Lmtrols.
Maltase
Group D* E F
Saccharase
Cellobiase
Trehalase
50
100
200
985
985
1120
421
475 1074
57.; 1278
925
D E F
28 8.5 112
E” F
35
37
16OO
1868 567 2028
932 512
1637
8.5
127 26
28 12i
17.5
Y
8
1;
13;
i I2
1.7
794 2175
1008
278 180
205
292
308
106 94 148
28
42
en
Ii
13
12
15
21
14 28
13 25
20 12 22
lfi I4 ‘I
2 1 2
-t 3 -I
75 25
35
35
38 52
12 40
12 12 24
2 ” 2
3 2 3
36
40
38
30
23
‘1
56
48
42
2 2 4
t2 I 2
ii:
D
75
220 12.i 212
36
56
105 85
Lactase
* Group
1018 678
129 52 158
D E F
F
1000
42 108
ii
D 1000 larvae
each;
group
9.5
78 i2 88
63
104
E 10 000 larvae;
group
155
F Controls.
no marked difference between control and infected animals except for reduced maltase activity in the infected groups. The dipeptidase activities of the duodenum, jejunum and ileum are shown in Table 3. The activities of the enzymes acting on L-alanyl-Z-glutamic acid, L-glycyl-l-valine, and L-glycyl-l-leucine differed in the different parts of‘ the small intestine. They were low in the duodenum and reached a maximum in
DIPEPTIDASE
L-alanyl-Lglutamic
ACTIVITY
acid
OF INTESTINAL MliCOSA Stronpyloides
OF PIGLETS INFECTED ransomi (Expt. 2)
D ;
L-glycyl-l-leucine
L-glycyl-l-valine
I) E F D E 1,‘
28 DAYS
\v,TII
4.9 3% 3.2
.54
13.4
Iti% 12.1 I r’+l
8.5
13.9
EAKLlER
16.1
.i4 .i.fi
14.9 11.4 15.7
PIGLET
INFECTIOM
WITH
S. ransomi:
BIOCHEMISTRY
247
the jcjunum and the proximal part of the ileum. All severely infected piglets from group E killed on day 28 showed a reduction in the activities of the three dipeptidases, whereas in mild infections dipeptidase activities were scarcel)affected. DISCUSSION
‘The investigation of digestive enzymes in piglets infected with S. ransomi was designed to assess more precisely the effect of the infection on the pathophysiological changes of the intestinal mucosa. The absorptive cells covering the villi of the small intestine are rich in disaccharidases (Dahlqvist, 1961). and dipeptidases (Josefsson and Lindberg, 1965a, b, 1966) which are generally considered to play an essential role in the digestion of carbohydrate and protein. Generalized deficiency of intestinal enzymes appears to be a frequent complication of diseases that cause extensive injury to the intestinal mucosa. Disaccharidase and dipeptidase activities have been found to be depressed in rats infected with Nippostron&us brasiliensis (Symons and Fairbairn, 1963) and in lambs infected with ~ematodirus battus (Coop, hiapes and Angus, 1972: Coop, Angus and Mapes, 1973). The results of the present experiments also show marked deficiencies of the disaccharidases and dipeptidases in the intestinal mucosa of piglets infected with S. ransomi. Digestive enzymes such as pepsin, trypsin and amylase convert protein and polysaccharides to dipeptides and disaccharides. Earlier work has shown tjlat most of the disaccharides and dipeptides are taken up from thr lumen 01’ the small intestine and hydrolyzed in the epithelial cells of the intestinal mucosa (Dahlqvist and Borgstrom, 1961 ; Miller and Crane, 1961 ; Dahlqvist 1963). The brush border and Brun, 1962; Newey, Sandford and Smyth, membrane of the intestinal epithelial cells plays an important role in the final hydrolysis of disaccharidases, as the enzymes are located mainly in this region 01 ttie cpithelial cells (Miller and Crane, 1961; Pcarse and Rieken, 19671. 1)ipcptidase activity is mainly localized in the cytosol fRohdes, Eichholz and (:raIie, 1967; Peters, 1970) and only 5 to 10 per cent. is located in the brush ))orclers. The marked depression of disaccharidase and dipeptidase activities in tile severely infected piglets is, therefore, most probably due to a defect of‘ t hc \,illus epi thelial cells. III other helminth infections of the small intestine the villi become flattenrd. ttlt. brush borders of the epithelial cells on the surface of the villi becomc II:I~I‘OW and the microvilli sparse and distorted (Symons and Fairbairn, 19613. Symons, 1969; Da Costa, Croft and Crcamcr, 1971 ; Coop et al., 1972). ‘1%~. (ausc of such villus changes is direct damage to the intestine by infection and Aso increased turnover of epithelial cells, which are produced in the crypts oL‘ the small intestinal mucosa, migrate up onto the villi and are lost at the tips Leblond and Illessier, 1958; Quastler and Sherman. 1959; Creamer, Shorter :tnd Bamforth, 1961). In the normal intestine there is a constant rate of turnovcl of epithelial cells, but during helminth infection of the small intestine thcrc is a much more rapid turnover of these cells and many epithelial cells from ttlc \.illi of the infected animals appear to be identical with crypt cells (Symons. 196.i ; Symons. Gibbins and Jones, 197 1). Fortin-blagana, Hurw-itz, Herl)st
248
Ii. ENIGIi PI al.
and Kretchmer (~1970)reported that tlie acti\itics of‘ maltase and dipeptitlasc~ in the intestinal crypts of the rat compared wit11 those on the \.illi ~\ere 17 ;III~ 43 per cent. respectively. Consequently, if there are large numl~~~ of (‘I.>pt cells in the infected villi, then the Ic\:el of‘ ciisaccharidascx ;~rrcl clipq~ticlxrs will
be reduced.
In the present experiment, the marked depression of disac.cllaridases antI the reduced levels of dipeptidase activities in the severely infected piglets a~‘(’ due to the presence of larger numbers of adult parasites. Tt is thought tliat their presence causesan increased turnover of epithelial cells with a large populatioii of crypt cells in the intestinal villi. It is worth mentioning that the decrease in mucosal dipeptidase activity in the severely infected piglets is small in comparison with the greater depression of mucosal disaccharidase acti\:ity. This is probably due to the different location of these enzymes in tile epithelial cells. The disaccharidases are predominantly located in tflc lrrush l,order, whereas most of the dipeptidase activity is located in the cytosol. l’he dc,grcc* of the changes in intestinal enzymes is directly correlated with the severity of infection (Tables 2 and 3). We postulate that in mild infection the rate of epithelial cell turnover is not greatly disturbed and ultrastructural changes in the epithelial cells on the villous surface are not extensive. A slight reduction of mucosal disaccharidase activities in mild infection can thus be explained. In contrast, the activities of mucosal dipeptidases were virtually unafl’ectcd by mild infection. It would appear that in a mild infection the changes art: restricted to the brush border of the epithelial cells. SUMMARY
Activities of intestinal disaccharidases and dipeptidases of normal piglets and of piglets parasitized by the nematode Strongyloides ransomi were investigated. A large dose of S. ransomi caused a marked depression of activities of maltasc, saccharase, lactase, trehalase and cellobiase and a slight reduction of dipeptidase activities of intestinal mucosa. The activities of intestinal disaccharidases were reduced more in animals killed on days 14 and 21 after infection, when pathological changes were maximal, than in animals killed on day 7 after infection. In mild infection there was a small fall in the mucosal disaccharidasr activity, whereas dipeptidase activity was unchanged. hlaltase activity in the mucosa of the caecum and colon of infected animals was slightly depressed, whereas saccharase, cellobiase, trehalase and lactase activities were unaffected. REFERENCES
Coop, R. L., Mapes, C. J. and Angus, K. W. (1972). on the distribution 13, 186-188.
of intestinal
enzymes
The effects of’.Nematodirus battus in Iambs. Research in Veterinar_y Science,
Coop, R. L., Angus, K. W., and Mapes, C. J. (1973). ‘The effect of large dosesof Nematodirus battus on the histology and biochemistry of the small lambs. International Journal for Parasitology, 3, 349-361.
intestine
of
Creamer, B., Shorter, R. G., and Bamforth, J. (1961). The turnover and shedding of epithelial
cells. I. The
turnover
in the gastrointestinal
tract.
Gut, 2, 110-I
16.
Da Costa, L. R., Croft, D. N., and Creamer, B. (1971). Protein loss and cell loss from the small intestinal
mucosa.
Gut, 12, 179-183.
PIGLET
INFECTIONS
WITH
s.
UZ?lSWLi:
RIO(:HEMISTKS
24’j
Dahlqvist, A. (1961). The location of carbohydrates in the digrstivc tract of thr pig. Biochemicul Journal, 78, 282-288. disaccharidases. ,.lnalytictrl ljahlqvist, A. ( 1964). Method f or assay of intestinal Biochemists, 7, 18-25. 1 jahlqvist, A., and Borgstrom, B. ( 196 1). Digestion and absorpt ioll of disaccharidayc., in man. Biochemical Journal, 81, 41 l-418. 1 )ahlqvist, A., and Brun, A. (1962). A method for the histochemical dcmonstratiou of disaccharidase activities. Application to invc-rtasc and trchalasr in son11 animal tissue. Journal of Histochemist~y and Qtochemistry, 30, 2+--302. Enigk, K. (1952). Pathogenitat und Therapie des Strongyloidrsl~~1:allcs dcr Haustic*rc,. AIonatshejie fCr praktische Tierheilkunde, 4, 97-- 1 12. Enigk. K., and Dey-Hazra, A. (1975). Intestinal plasma and I)lood loss in pigIt.t\ infected with Strongyloides ransomi. Veterinary Pararitolog, 1, 69 -75. N. (1970). Intestinal l:nrtin-Magana, R.. Hurwitz, R., Hcrbst, J. .J., and Kretchmcr, enzymes: Indicators of proliferation and diffcrcntiation in the +junum. ~S’+IC~~. 167, 16277162%. (;iese, W., Dry-Hazra, A., and Enigk, K. (1972j. Enteral loss of plasma-protein itr Strongyloides infection of pigs. International Journal q/‘ Parasitology, 3, 63 1~ 639. Harmeyrr, J.. Birk, R., Martens, H., Dey-Hazra, A.. and Enigk. K. (1973). Mrssung dcr intcstinalcn Resorptionssttirung clurch Strongyloides-Befall bci Fc’rkrlll ceitsrhriftfir I’arasitenkunde, 41, 47-60. Jost&on, L., and Lindberg, T. (1965a). Intestinal dipeptidases. 1. Spectrophotorrlc.tl-ic, activity in pig intestinal drtermination and characterization of dipeptidasca mucosa. Biochimica et biophysics acta, 105, 149- 16 1. ,Josc,fsson, L., and Lindberg, T. (1965b). Intcsstinal dipeptidasvs. II. IXstributiou of‘ dipeptidascs in the smaIl intestine of thtx pig. Riochimicnet biophwicaat-(c),105, 162. 166. ,Jos~++son, L., and Lindberg, T. (1966). Intestinal dipeptidases. 111. C:haractc.rization and dctcrmination of dipeptidasc activity in adult rat intrstinal mucosa. .lc~cl p/+ologica .scandinavica, 66, 410--4 1%. Lcl)lond, Cl. P., and Messier, B. (1958). Renewal of chief cells and .goblct cells in thtl small intestine as shown by radioautography after injrction of thymidinc-tl:’ into mictx. Anatomical Record, 132, 247- 259. I,owry, 0. H.. Rosenbrough, N. J., Farr, :\. L., and Randall, R. J. (1951 ). I’rotcin measurcm~nt with the Folin ph(znol r.e+gcrlt. ~7r)urnnln/‘ Bioln~qiral (.‘hemi.\/r),.
193, 265. h$ill(*r, I)., and C:rant:, 1~. K. (1961). The di:gcsti\rc func.tion of the cpithcliunl 01‘ thy small intrstine. II. Localization of disaccharide hydrolysis in the isolated brush border portion of intestinal cpithelial cells. Niorhimira PI hioplpica cu./a. .52, 293 298. N(~wry, H., Sanfijrd, P. A., and Smyth, 1). H. (1963). Location of function in 111c absorption. Journal o/ intestinal epithelial cell in relation to carbohydrate I’lpGnlopy, 168, 423-434. PIWSC. A. fi. E.. and Riccken, E. 0. (1967,. Histology and cytochcmistry of the cells of the small intestine, in relation to absorp;ion. I{,-itish .‘lledic-al f?ullr/in. 23, 217-222. P~~tcrs, ‘I’. ,J. (,1970). ‘The subcellular localization of di- and tri-pcptide hydrolasc activity in guinea pig small intestine. Biochemical .Tournal, 120, 195-203. Quastler, H., and Sherman, F. G. (1959). Ccl1 population kinetics in thus int(.htinal cpithelium of the mouse. Experimental Cell Research, 17, 420-438. li<)hdes, J. B., Eichholz, A., and Crane, R. K. (1967). Studies on the organization (11‘ the brush border in intestinal epithclial cells. IV. Aminopeptidase activity in microvillous membranes of hamster intestinal brush horders. Biochimira r/ biop&sica acta, 135, 959-965. Symons, L. E. A. (1965). Kinetics of the epithelial cells and morphology of villi and crypts in the jcjunum of the rat infected by the nematode Nippostron&u,~~ hrasiliensis. Gastroenterology, 49, 158-l 68.
Symons, L. E. A. (1969). Pathology of gastrointestinal helminthiases. /~~/tvm/io~wl Revieut of Tropical illedicine, 3, 49 100. Symons, L. E. .4., and Fairbairn, D. (1963). Biochemical pathology of the racjejunulll parasitized by the ncmatodc .Nip,bostronpylus brasiliensis. &berimental Parasitolq~~.
13,284-304. Symons, L. E. A., Gibbins, J. R., and Jones, W. 0. (1971,). Jejunal malabsorption in the rat infected by the nematode .hfi&ostronglus brasilienk. International Journal for Parasitology, 1, 179-l 87. [Received for publication,
September 1st, 197.51
PATH.
XCTIVITY OF THE
1976.
VOL.
OF DISACCHARIDASES AND DIPEPTIDASES INTESTINAL MUCOSA OF PIGLETS DURING MILD AND SEVERE INFECTIONS WITH STRONNGYLOIDES RANSOJIP
K. ENIGK, Institute
243
86.
cf Parasitology.
A. DEY-HAZRA School of bkterinq~
and S. I,. EDUARDO~ Aledirine.
Hannorer,
Federal Republic of Germay
INTKODUCTIOX
Stroncglloidesransomi parasitizes primarily the epithelial layer of the small intestinal mucosa of piglets (Enigk, 1952). The eggsof the parasite are deposited in the epithelial layer and are expelled together with detritus into the lumen of tile gut. The villi of the infected mucosa become flattened by an inflammatory infiltrate in the lamina propria. Previous work has shown marked pathophysiological changes in the small intestine of piglets parasitized Iq this nematode. By labelling plasma albumin with 51Cr iGiese, Dey-Hazra and Enigk, 1972) or g5Nb (Enigk and Dey-Hazra, 1975) a protein-losing cnteropathy associated with hypoalbuminaemia and weight loss has been demonstrated in severely infected piglets. In vitro experiments demonstrated tllat the rate of amino acid absorption was decreased by 55 per cent. due to ,S. ronsomiinfiction (Harmeyer, Birk, blartens, Dey-Hazra and Enigk, 197:j;. To clarify further the pathogenesis of S. ransomi in the pig the levels 01‘ disaccharidase and dipeptidase activity in the intestinal mucosa of piglets c~xposedto milcl or severe infection were measured. MATERIALS
AND
METHODS
Piglets were obtained by caesarean section and raised worm-free. At 14 weeks ot age. the piglets were infected by a subcutaneous injection of infective larvae of .S. rcznsomi, using a strain maintained in our laboratory for several years. Two experiments were carried out. In the 1st 24 piglets were used: 12 animals were infected with 10 000 larvae/kg. body weight and 12 were controls. Four infected and 4 control animals were killed on each of days 7, 14 and 21 after infection (p.i. (groups A, B, C: respectively) and the activities of intestinal disaccharidases wcrc assayed. In the 2nd experiment 12 piglets were divided into 3 groups of 4. Tht animals of group D were infected with 1000 larvae/kg. body weight each, group E with 10 000 larvae/kg. body weight and group F were uninfected controls. All the* animals were killed on the 28th day p,i. and the activities of intestinal disaccharidasca and dipeptidascs were measured. Piglets were killed by i.v. injection of the sodium salt of methyl-thioethyl-2-pentylthiobarbituric acid.: Small and large intestines were opened longitudinally and thr content was removed by washing with ice-cold * Supported by Deutsche Forschungsgemeinschnft. + Present address: College of \.rterinnry l\ledicine. : Thinqcnal. Rlrrck. Darmstadt.
CnivrrAty
l)!liman.
Quczon-City.
PhilippIno.
244
1;. l5KIGK
?I (Ii.
0.15 1~1sodium chlorides and the surfkcc slightly blo~r~tl. Small pi~c~c~~i,4 10 li (‘111. LLC’~‘,‘ cut out from differrnt parts ofsmall and large, intestirlc. and thc.ir II~LICOS~ was scrapc’d scgrri~27rs wcr( sc,lcctcd by lllc,a.surirt,q off with a microscope slide. ‘I%(* intestinal the distance from thr pylorus. ‘I’hc mucnsa was homogcnizcd in ice-cold 0. I5 11 sodium chloridr using an Ultra-‘I’urrax homogrnizcr for 3 rrlin. ‘I’hc homogc-tiaras was left at 4 “C f.or 30 min. for extraction and was centrifuged for 10 min. at
2000g at 4 “C. The supcrnatant was dilutrd with redistilled water IO &rain a suitable enzyme concentration. Substrates i.r. lactose, saccharosc.,trchaloyc, ccllol~iosc and maltose wcrc used in the sarnc c.onccntration a\ raportcd by Dahlqvisc f I Wl-I. L-alanyl-glutamic acid, glycyl-/-lcucinc, glycyl-l-valinc and lht* rcspcctivc amino acids wcrc used in the same concentration Jo&son and Lindberg (1965a, I,, 1966).
arlcl
combination
as de~c~ril,r~tl
1)).
Activities of disaccharidases wcrc dctrrmincd by thck method dcscribrd 1)) Dahlqvist (1964) and cxprcsscd as units of activity per m,~. of protein. Activitic.s of dipeptidases WPI‘Cmrasured by the method of Josefssonand Lindbcrg (I96.5a’~ and expressed
as units of activity
per mg. of total mtrogcn.
Protein was dctermincd by the mrthod of Lowry, Rnsenbrough, Varr and Randall (1951)
with
a commercial
preparation?
as a standard.
Total
nitrogen
was dcterminrd
by the micro-Kjeldahl method. RESULTS
The dose of infection, time of’ slaughter and the number of adult worms recovered are summarized in Table 1. The measurements in the control animals showed that activities of disaccharidases are mainly localized in the mucosa of the small intestine and are only present in very small amounts in the mucosa of the caecum and colon. The distribution of different disaccharidases along the small intestine confirmed that there were two major groups of intestinal disaccharidases, as described previously (Dahlqvist, 196 1j.
I
24
A (4+4 U (4.t4 c: (4$4
corm.1 contr.) contr.)
IO 000
21
1000 10 000 Control
II
7 14 ‘8 28 28
37 738 28 267 22 532 2132 17889
Trehalase, lactase and cellobiase were localized mainly in the proximal part, whereas maltase and saccharase were mainly in the distal part of the small intestine. The total activity of maltase was greater in each of the measured areas than the activities of the other enzymes, the highest maltase activit) being in the area between 800 and 1000 cm. from the pylorus. Saccharase activity was very low in the duodenum, but increased in the jejunum. Lactase activity was greater in the proximal than in the distal part of the jejunum and in the duodenum. The distribution of cellobiase activity was nearly the t Lab
Trol,
Dade,
Miami
(U.S.A.).
PI(:I.ET
INFECTIOIiS
WITH
.y. RUL50??2i:
215
BIO(:HEMIS’I’K\’
same as thr lactase activity. The trehalase activity was greatest in thr mucosa of the middle of the ,jejunum. The mucosa of the entire small intestine of the infected animals showed :I reduction in the activity of maltase, saccharase, lactasc, trehalasc and cellobiasc~ (I:ig. 1). Animals killed on days 14 and 21 p.i. showed in general more reduction in intestinal disaccharidases than animals killed on day 7 p.i. The eKect of‘ low-grade and high-grade infections on the disaccharidasc activity is summarized in Table 2. There was a marked reduction in disaccharidasr activit) in thcz mucosa of the heavily infected animals (group Ej. The activities of disaccharidases of the caecum and colon sho\ved that tllcy wt’rc only present in small amounts. The large intestine of the infected animals was f’rrc fiiom .Y. ransomi and the measurement of disaccharidasr acti\.ity showed
Fig.
I.
Average disaccharidases of Strongyloides ransomi/kg. 7, 14 and 21 p.i. (Expt.
* Standard
deviation
is also
activity of the intestinal mucosa of piglets body weight: 4 infected and 4 control 1) q 7 days, C 14 days, q 21 clay? post indicated.
infected piglets infection.
with 10 000 larvnr were killed on da>-s n (Lmtrols.
Maltase
Group D* E F
Saccharase
Cellobiase
Trehalase
50
100
200
985
985
1120
421
475 1074
57.; 1278
925
D E F
28 8.5 112
E” F
35
37
16OO
1868 567 2028
932 512
1637
8.5
127 26
28 12i
17.5
Y
8
1;
13;
i I2
1.7
794 2175
1008
278 180
205
292
308
106 94 148
28
42
en
Ii
13
12
15
21
14 28
13 25
20 12 22
lfi I4 ‘I
2 1 2
-t 3 -I
75 25
35
35
38 52
12 40
12 12 24
2 ” 2
3 2 3
36
40
38
30
23
‘1
56
48
42
2 2 4
t2 I 2
ii:
D
75
220 12.i 212
36
56
105 85
Lactase
* Group
1018 678
129 52 158
D E F
F
1000
42 108
ii
D 1000 larvae
each;
group
9.5
78 i2 88
63
104
E 10 000 larvae;
group
155
F Controls.
no marked difference between control and infected animals except for reduced maltase activity in the infected groups. The dipeptidase activities of the duodenum, jejunum and ileum are shown in Table 3. The activities of the enzymes acting on L-alanyl-Z-glutamic acid, L-glycyl-l-valine, and L-glycyl-l-leucine differed in the different parts of‘ the small intestine. They were low in the duodenum and reached a maximum in
DIPEPTIDASE
L-alanyl-Lglutamic
ACTIVITY
acid
OF INTESTINAL MliCOSA Stronpyloides
OF PIGLETS INFECTED ransomi (Expt. 2)
D ;
L-glycyl-l-leucine
L-glycyl-l-valine
I) E F D E 1,‘
28 DAYS
\v,TII
4.9 3% 3.2
.54
13.4
Iti% 12.1 I r’+l
8.5
13.9
EAKLlER
16.1
.i4 .i.fi
14.9 11.4 15.7
PIGLET
INFECTIOM
WITH
S. ransomi:
BIOCHEMISTRY
247
the jcjunum and the proximal part of the ileum. All severely infected piglets from group E killed on day 28 showed a reduction in the activities of the three dipeptidases, whereas in mild infections dipeptidase activities were scarcel)affected. DISCUSSION
‘The investigation of digestive enzymes in piglets infected with S. ransomi was designed to assess more precisely the effect of the infection on the pathophysiological changes of the intestinal mucosa. The absorptive cells covering the villi of the small intestine are rich in disaccharidases (Dahlqvist, 1961). and dipeptidases (Josefsson and Lindberg, 1965a, b, 1966) which are generally considered to play an essential role in the digestion of carbohydrate and protein. Generalized deficiency of intestinal enzymes appears to be a frequent complication of diseases that cause extensive injury to the intestinal mucosa. Disaccharidase and dipeptidase activities have been found to be depressed in rats infected with Nippostron&us brasiliensis (Symons and Fairbairn, 1963) and in lambs infected with ~ematodirus battus (Coop, hiapes and Angus, 1972: Coop, Angus and Mapes, 1973). The results of the present experiments also show marked deficiencies of the disaccharidases and dipeptidases in the intestinal mucosa of piglets infected with S. ransomi. Digestive enzymes such as pepsin, trypsin and amylase convert protein and polysaccharides to dipeptides and disaccharides. Earlier work has shown tjlat most of the disaccharides and dipeptides are taken up from thr lumen 01’ the small intestine and hydrolyzed in the epithelial cells of the intestinal mucosa (Dahlqvist and Borgstrom, 1961 ; Miller and Crane, 1961 ; Dahlqvist 1963). The brush border and Brun, 1962; Newey, Sandford and Smyth, membrane of the intestinal epithelial cells plays an important role in the final hydrolysis of disaccharidases, as the enzymes are located mainly in this region 01 ttie cpithelial cells (Miller and Crane, 1961; Pcarse and Rieken, 19671. 1)ipcptidase activity is mainly localized in the cytosol fRohdes, Eichholz and (:raIie, 1967; Peters, 1970) and only 5 to 10 per cent. is located in the brush ))orclers. The marked depression of disaccharidase and dipeptidase activities in tile severely infected piglets is, therefore, most probably due to a defect of‘ t hc \,illus epi thelial cells. III other helminth infections of the small intestine the villi become flattenrd. ttlt. brush borders of the epithelial cells on the surface of the villi becomc II:I~I‘OW and the microvilli sparse and distorted (Symons and Fairbairn, 19613. Symons, 1969; Da Costa, Croft and Crcamcr, 1971 ; Coop et al., 1972). ‘1%~. (ausc of such villus changes is direct damage to the intestine by infection and Aso increased turnover of epithelial cells, which are produced in the crypts oL‘ the small intestinal mucosa, migrate up onto the villi and are lost at the tips Leblond and Illessier, 1958; Quastler and Sherman. 1959; Creamer, Shorter :tnd Bamforth, 1961). In the normal intestine there is a constant rate of turnovcl of epithelial cells, but during helminth infection of the small intestine thcrc is a much more rapid turnover of these cells and many epithelial cells from ttlc \.illi of the infected animals appear to be identical with crypt cells (Symons. 196.i ; Symons. Gibbins and Jones, 197 1). Fortin-blagana, Hurw-itz, Herl)st
248
Ii. ENIGIi PI al.
and Kretchmer (~1970)reported that tlie acti\itics of‘ maltase and dipeptitlasc~ in the intestinal crypts of the rat compared wit11 those on the \.illi ~\ere 17 ;III~ 43 per cent. respectively. Consequently, if there are large numl~~~ of (‘I.>pt cells in the infected villi, then the Ic\:el of‘ ciisaccharidascx ;~rrcl clipq~ticlxrs will
be reduced.
In the present experiment, the marked depression of disac.cllaridases antI the reduced levels of dipeptidase activities in the severely infected piglets a~‘(’ due to the presence of larger numbers of adult parasites. Tt is thought tliat their presence causesan increased turnover of epithelial cells with a large populatioii of crypt cells in the intestinal villi. It is worth mentioning that the decrease in mucosal dipeptidase activity in the severely infected piglets is small in comparison with the greater depression of mucosal disaccharidase acti\:ity. This is probably due to the different location of these enzymes in tile epithelial cells. The disaccharidases are predominantly located in tflc lrrush l,order, whereas most of the dipeptidase activity is located in the cytosol. l’he dc,grcc* of the changes in intestinal enzymes is directly correlated with the severity of infection (Tables 2 and 3). We postulate that in mild infection the rate of epithelial cell turnover is not greatly disturbed and ultrastructural changes in the epithelial cells on the villous surface are not extensive. A slight reduction of mucosal disaccharidase activities in mild infection can thus be explained. In contrast, the activities of mucosal dipeptidases were virtually unafl’ectcd by mild infection. It would appear that in a mild infection the changes art: restricted to the brush border of the epithelial cells. SUMMARY
Activities of intestinal disaccharidases and dipeptidases of normal piglets and of piglets parasitized by the nematode Strongyloides ransomi were investigated. A large dose of S. ransomi caused a marked depression of activities of maltasc, saccharase, lactase, trehalase and cellobiase and a slight reduction of dipeptidase activities of intestinal mucosa. The activities of intestinal disaccharidases were reduced more in animals killed on days 14 and 21 after infection, when pathological changes were maximal, than in animals killed on day 7 after infection. In mild infection there was a small fall in the mucosal disaccharidasr activity, whereas dipeptidase activity was unchanged. hlaltase activity in the mucosa of the caecum and colon of infected animals was slightly depressed, whereas saccharase, cellobiase, trehalase and lactase activities were unaffected. REFERENCES
Coop, R. L., Mapes, C. J. and Angus, K. W. (1972). on the distribution 13, 186-188.
of intestinal
enzymes
The effects of’.Nematodirus battus in Iambs. Research in Veterinar_y Science,
Coop, R. L., Angus, K. W., and Mapes, C. J. (1973). ‘The effect of large dosesof Nematodirus battus on the histology and biochemistry of the small lambs. International Journal for Parasitology, 3, 349-361.
intestine
of
Creamer, B., Shorter, R. G., and Bamforth, J. (1961). The turnover and shedding of epithelial
cells. I. The
turnover
in the gastrointestinal
tract.
Gut, 2, 110-I
16.
Da Costa, L. R., Croft, D. N., and Creamer, B. (1971). Protein loss and cell loss from the small intestinal
mucosa.
Gut, 12, 179-183.
PIGLET
INFECTIONS
WITH
s.
UZ?lSWLi:
RIO(:HEMISTKS
24’j
Dahlqvist, A. (1961). The location of carbohydrates in the digrstivc tract of thr pig. Biochemicul Journal, 78, 282-288. disaccharidases. ,.lnalytictrl ljahlqvist, A. ( 1964). Method f or assay of intestinal Biochemists, 7, 18-25. 1 jahlqvist, A., and Borgstrom, B. ( 196 1). Digestion and absorpt ioll of disaccharidayc., in man. Biochemical Journal, 81, 41 l-418. 1 )ahlqvist, A., and Brun, A. (1962). A method for the histochemical dcmonstratiou of disaccharidase activities. Application to invc-rtasc and trchalasr in son11 animal tissue. Journal of Histochemist~y and Qtochemistry, 30, 2+--302. Enigk, K. (1952). Pathogenitat und Therapie des Strongyloidrsl~~1:allcs dcr Haustic*rc,. AIonatshejie fCr praktische Tierheilkunde, 4, 97-- 1 12. Enigk. K., and Dey-Hazra, A. (1975). Intestinal plasma and I)lood loss in pigIt.t\ infected with Strongyloides ransomi. Veterinary Pararitolog, 1, 69 -75. N. (1970). Intestinal l:nrtin-Magana, R.. Hurwitz, R., Hcrbst, J. .J., and Kretchmcr, enzymes: Indicators of proliferation and diffcrcntiation in the +junum. ~S’+IC~~. 167, 16277162%. (;iese, W., Dry-Hazra, A., and Enigk, K. (1972j. Enteral loss of plasma-protein itr Strongyloides infection of pigs. International Journal q/‘ Parasitology, 3, 63 1~ 639. Harmeyrr, J.. Birk, R., Martens, H., Dey-Hazra, A.. and Enigk. K. (1973). Mrssung dcr intcstinalcn Resorptionssttirung clurch Strongyloides-Befall bci Fc’rkrlll ceitsrhriftfir I’arasitenkunde, 41, 47-60. Jost&on, L., and Lindberg, T. (1965a). Intestinal dipeptidases. 1. Spectrophotorrlc.tl-ic, activity in pig intestinal drtermination and characterization of dipeptidasca mucosa. Biochimica et biophysics acta, 105, 149- 16 1. ,Josc,fsson, L., and Lindberg, T. (1965b). Intcsstinal dipeptidasvs. II. IXstributiou of‘ dipeptidascs in the smaIl intestine of thtx pig. Riochimicnet biophwicaat-(c),105, 162. 166. ,Jos~++son, L., and Lindberg, T. (1966). Intestinal dipeptidases. 111. C:haractc.rization and dctcrmination of dipeptidasc activity in adult rat intrstinal mucosa. .lc~cl p/+ologica .scandinavica, 66, 410--4 1%. Lcl)lond, Cl. P., and Messier, B. (1958). Renewal of chief cells and .goblct cells in thtl small intestine as shown by radioautography after injrction of thymidinc-tl:’ into mictx. Anatomical Record, 132, 247- 259. I,owry, 0. H.. Rosenbrough, N. J., Farr, :\. L., and Randall, R. J. (1951 ). I’rotcin measurcm~nt with the Folin ph(znol r.e+gcrlt. ~7r)urnnln/‘ Bioln~qiral (.‘hemi.\/r),.
193, 265. h$ill(*r, I)., and C:rant:, 1~. K. (1961). The di:gcsti\rc func.tion of the cpithcliunl 01‘ thy small intrstine. II. Localization of disaccharide hydrolysis in the isolated brush border portion of intestinal cpithelial cells. Niorhimira PI hioplpica cu./a. .52, 293 298. N(~wry, H., Sanfijrd, P. A., and Smyth, 1). H. (1963). Location of function in 111c absorption. Journal o/ intestinal epithelial cell in relation to carbohydrate I’lpGnlopy, 168, 423-434. PIWSC. A. fi. E.. and Riccken, E. 0. (1967,. Histology and cytochcmistry of the cells of the small intestine, in relation to absorp;ion. I{,-itish .‘lledic-al f?ullr/in. 23, 217-222. P~~tcrs, ‘I’. ,J. (,1970). ‘The subcellular localization of di- and tri-pcptide hydrolasc activity in guinea pig small intestine. Biochemical .Tournal, 120, 195-203. Quastler, H., and Sherman, F. G. (1959). Ccl1 population kinetics in thus int(.htinal cpithelium of the mouse. Experimental Cell Research, 17, 420-438. li<)hdes, J. B., Eichholz, A., and Crane, R. K. (1967). Studies on the organization (11‘ the brush border in intestinal epithclial cells. IV. Aminopeptidase activity in microvillous membranes of hamster intestinal brush horders. Biochimira r/ biop&sica acta, 135, 959-965. Symons, L. E. A. (1965). Kinetics of the epithelial cells and morphology of villi and crypts in the jcjunum of the rat infected by the nematode Nippostron&u,~~ hrasiliensis. Gastroenterology, 49, 158-l 68.
Symons, L. E. A. (1969). Pathology of gastrointestinal helminthiases. /~~/tvm/io~wl Revieut of Tropical illedicine, 3, 49 100. Symons, L. E. .4., and Fairbairn, D. (1963). Biochemical pathology of the racjejunulll parasitized by the ncmatodc .Nip,bostronpylus brasiliensis. &berimental Parasitolq~~.
13,284-304. Symons, L. E. A., Gibbins, J. R., and Jones, W. 0. (1971,). Jejunal malabsorption in the rat infected by the nematode .hfi&ostronglus brasilienk. International Journal for Parasitology, 1, 179-l 87. [Received for publication,
September 1st, 197.51