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The Influence of Immunopotentiators on Sucking Piglets with Special Reference to the Incidence of Pig Scour
Br. vet. }. ( 1982). 138, 155
THE INFLUENCE OF IMMUNOPOTENTIATORS ON SUCKING PIGLETS WITH SPECIAL REFERENCE TO THE INCIDENCE OF PIG SCOUR
Bv S.
NAMI O KA,
Y.
T. KA\"' ANO, C . T. K. MURAKAMI!
KuMEDA,
WA NG*,
Y.
NAMBAt AND
Department of Veterinary Intema l Medicine, Fa culty of Veterinary Medicine, Hokkaido University, SajJ/Joro 060, Japan
SU MMARY
The prese nt pa per d esci·ibes the effect of the adminis trat ion of peptidoglycan (PG) an d di a li za bl e trans fer factor (TF) upon th e immun ores ponse and the in cidence of diarrhoea amongst sucking piglets . In ex perim en t I, la rger numb ers oflgA-bea ring ce ll s in the la min a propria of the mu cosa of the sma ll intestin es in th e PG and TF-treated groups aged three a nd four wee ks were observed co mpared to those of co ntrol groups, whi le no clear difference of serum IgG leve ls was see n betw een the treated gro ups a nd the controls. Th e co unts of viab le E. coli in various pa rts of the small intestin es of pigl ets trea ted with PG and TF were somewhat lower than those of the co ntrol s. In ex perim ent II , t he in cid ence of di a rrhoea o f PG and TF-treated sucking pi glets was co mp a red with th at of the non-treated controls. Lowe r in cid ences of d ia rrhoea in th e PG (P <0-0025) a nd TF-treated groups (P<0·05) were seen co mp a red to th e res pec tive co ntrol groups . H ae magglutin a tion titres with E. coli 08:K?:H35, whi ch ex isted predo min a ntly in th e small intes tin es of th e pi glets used were hig her in th e PG (P<0·02) a nd TF-treated (P < O·O I ) g roups three a nd four weeks old th a n in the respective control groups . These res ul ts suggest that PG a nd TF play a role in enh a ncing non-specifi c immunopotentiatin g activity in sucking pig lets.
I NT RODUCT ION
It is well known that piglets less than four weeks old a re immunod efi cient (A ll en & Porter, 1973; Bourn e, 1973; Ya biki , K as hiw aza ki & N a miok a, 1974; Kim , 1975; Prese nt a ddresses : • V e te rin a ry Co ll ege, Ta iwan U ni vers it y, Taipei, Taiwa n, Republi c of China. t In s tit ute o f Immuno logy, Esai Co. Lt d., Ko is h ikawa, Bunkyo- ku , To kyo. ! Sa nk yo-Zo hki Co. Ltd ., H onc ho, Niho nbas hi , Chuo- Ku , Tokyo.
BRITISH VETERINARY JOURNAL, 138,2
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Reyero et al., 1978). The poor immunoresponse or the intestinal mucosa or piglets in this period is responsible ror various local inrections (such as colibaci llosis and vira l diarrhoea) despite the presence or a large amount or immunoglobulin (Ig) in colostr um. This immunodeficiency or neonatal piglets is analogous to the hum an baby's variable hypogammag lobu lin ae mi a with B lymp hocytes (Cooper, Lawton & Kinkade, 1972; Lawton, Frank & Cooper, 1975) . R ece ntly , various immunopotent iators have been developed ror human app li cat ion. Peptidoglycan (PG) is one or th em (Chedid et al., 1976), and the dialyzable transrer ractor (TF) is a lso reported to be effective (Spitler, Levin & Fundenburg, 1972). At the same time, levamisole has been appl ied as an immunomodularor to neonatal piglets (Reyero, Stock I & Thalhammer, 1979). I r is also used to control bovine mastitis (Ovadia, Flesh & Nelken, 1978) and calr diarrhoea (F les h, Ovadia & Nelken, 1977) . The purpose or the present study is to observ e the immunological consequences or the adminis t ration or PG and TF to neonatal piglets and th e eOcct iv eness or these substances aga in st pig scour.
MATERIALS AND METHODS
Experiment I Transfer factor (TF}. T he preparation orTF rollowed Lawrence's method (Lawrence & Askali, 197 1) : 500 ml or pooled adult swine venous blood ~ K ept ror I h at 37°C Sedimented
~ Buffy coa t (3 to 4 X 10 9/ ml ) ~ In cubated with DNAse and MgS0 4 ror 30 min at 37°C Lysed by 12 cycles or rreezing and thawi ng ~ Dialyzed aga in st distil led wa ter ror 48 h a t 4°C ~ Lyophilized ~ Reconstituted with distilled water ~ Filtrated with micropore filter One unit consis ts or the substance prepared rrom 10 9 1eukocytes. A hair unit orTF was injected three times subcutaneous ly, a t birth , on the third day and on the firth day respectively . Peptidoglycan (PC) . PG was prepared using Bi.fidobaclerium thennoJ;hilum which exists predominantly in the intestines or adult swine; this subs tance conta in s mura myl dipeptide (Nambaetal., 1981). Bifidobaclerium tlzermophilum strain P2-9 1 was c ulti vated in Brigges liv er broth under anaerob ic co nditi o ns (Mitsuoka, 1969) ror 16 hat 37°C , ha rvested by centriru gation and washed wi th sa lin e . The bacterial ce ll wa ll s were disrupted in a French press.
IMMUNOLOGICAL INFLUENCES ON PIG SCOUR
157
They were centrifuged and washed twice with saline and suspended m phosphate buffer of Sorensen (pH 6·2) in an equal volume of native cells (10 12 cells/ml). Enzymatic digestion of cell walls was performed by adding 0·0 l% egg white lysozymes and 0·05°/., pronase, and then incubating at 37°C for 48 h. The optical density at 64·0 nm of the digested solution was decreased to 5% of the initial value. The supernatant of the f1uid was used as digest.ed cell walls. One ml of the substance was derived from 10 12 of native cells. For more details of the method sec Namba et al. ( 1981). Piglets were given 0·25 ml ofPG orally at birth, two, three, four and five days of age respectively. Animals. Thirty-one Landrace piglets from four sows on farm A were used for this study. Of these, 17 piglets from two sows were used for the TF study: nine piglets were injected subcutaneously with 1·5 units TF as described previously and eight were untreated controls. The remaining 14- piglets were used for the PG study: seven piglets were given five doses of 0·25 ml PG orally, and seven were untreated controls. The piglets in each study were divided randomly. The sanitary condition of farm A was good and no pig scour was seen in the piglets used in the experiment. Serum f[;G level. The test of serum IgG level was carried out by rviancini's method (Mancini, Carbonara & Heremans, 1965) using single radial gel diffusion with antiswine IgG rabbit serum. The rabbit antiserum was prepared by the method of Porter (1969) and Yabiki, Kashiwazaki & Namioka (1973). Count of lgA bearing cells in the lamina j;ropria of the small intestines. The indirect fluorescent isothiocyanate (FITC) method was used. All the samples were collected from the corresponding portions of the small intestines in each piglet. The frozen sections (Sf.Lm) of the cluoclenum, the middle part ofthejejunum and the ileum of piglets aged three and four weeks were attached to glass slides, fixed with chilled acetone for 30 min and then soaked in phosphate buffer (pH 7-4) for several minutes. After drying, the anti-swine a-chain caprine serum (Miles Laboratory Inc.) was mounted and allowed to stand overnight at4 °C. The FITC labelled anti-caprine lgG rabbit serum (Miles Laboratory Inc.) was mounted and then allowed to stand overnight at 4°C. All the samples were examined by a Nikon EF fluorescent microscope equipped with a high mercury larnp (Osram I-lBO 50\'V/AC) under ultraviolet (UV) excitation, with a DM 400 dichroic mirror and a UV 330-380 filter. Cell count was clone using the rnethocl of Allen & Porter ( 1977): counts were made of stained cells present in 20 fields selected at random using an X 4-0 objective with an X 10 eye piece. Photographs were taken with Ektachrome film (ASA 400). Isolation and count of viable E. coli. Immediately after euthanasia, 5-cm long sections of the duodenum, the middle portion of the jejunum and the ileum of the piglets aged three and four weeks were isolated. 'fhcse tissues were opened aseptically and their contents were collected by scraping the mucosa lightly with a surgical knife. The contents were diluted with a 10-tolcl dilution in phosphate butTer (pH 7·4-) and each dilution was streake
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TAl3 L E I SERUM IgG LEVELS (G/ 100 ML) I N PIGLETS TREATED \'V ITI-I PG AND TF AND UNTREATED CO 1TROLS
Week Piglet no.
Damno. PG-treated l
2
2
3
3·5
4
*
l 2 4
1·5 1 1·98 1·98
1·03 1·32 1·35
0·75 0·9 1 0·87
0·75
0·68
l 2 3 4 Mean ± SD
1·58 1·45 1·56 1·79 1·69 0·22
0·96 1·04 0·95 1·27 1· 13 0·17
0·63 0·68 0·71 0·92 0·78 0· 12
0·4·2 0·58 0·58 0· 17
0·50 0·66 0·61 0· 10
5 7 8
1·22 0·95 1·74
0·87 0·64 0·99
0·54· 0·57 0·75
0·66
0·68
6 7 8 9 Mean ± SD
1·75 1·49 1·84 1·42 1·49 0·32
0·98 0·95 1· 15 0·93 0·94 0· 15
0·72 0·60 0·80 0·66 0 10
0·62 0·51 0·60 0·08
0·62 0·46 0· 59 0· 11
l 2 3
1·43 2· 19 1·36
1·06 1·42 0·96
0·74 0·84 0·65
0·55
0·68
l 2 3 4 5 Mean ± SD
1·00 1·74 1·64 1·98 l 58 1·64 0·35
0·97 1·32 1·02 0·67 1· 14 l·l l 0·25
0·72 0·64 0·83 0·48 0·77 0·74 0·1 4
0·50 0·76 0·66 0· 16
0·48 0·74 0·62 0 II
5 6 7
1·57 1·95 2·16
1· 15 1·3 1 HI
0·97 0·82 1·03
0·62 0·82
0·62 0·82
6 7 8 9 10 Mean ± SD
0·95 0·33 2·29 1·32 0·68 I ·41 0·7 I
0·59 0·27 1·58 0·81 044 0·94 0·48
0·44· 0·23 0·78 0·60 0·38 0·66 0 ·29
0·58 0· 32 0· 59 0·21
0 56 0·34 0· 59 021
Control l
2
TF-treated 3
4
Control 3
4
* Not tested due
to
sacri fi ce.
OM
TABLE 11 NU I\•IBER• OF lgA-BEARING CE LLS OF T HE LAM INA PROPRIA IN T HE SMALL I NTEST INES OF PIGLETS TREATED W ITH TF AND PC AT FARM A
Portion Three weeks old Duodenum
Damno .
P G-treated
Control
-
3:: Damno.
TF-treated
Control
.,. ';>'
c: z
0 I 2
504(2/462-547t) 409(2/373-445)
4 11 (2/378-444) 530(2/403-657)
3 4
396(2/39 1-400) 460(3/439-482)
218(1 *) 460(3/413-583)
I 2
128(2/108-147) 166(2/114-218)
I 08(2 /60- 155) 45(2/ 40-50)
3 4
116(2/1 06-126) 112(3/91-135)
45(2/40-50) 132(3/ 93-179)
r'
0
0 0
Jejunum
Il eu m
I 2
87 (2/76-98 ) 74(2/59-88)
77 (2/28- 126 ) 18(2/ 16-20)
3 4
48 (2/38-5 7) 65(3/52-85)
24(1) 80(3/79-83)
> r'
z..,
r
c: t"'l
z
0
Four weeks old Duodenum
J ej unum
Ileu m
t"'l
(/)
I 2
545( 1) 557(2/467 -647)
540( 1) 388(2/262 -514)
3 4
46 1(2/4 10-5 11 ) 552(2/543-56 1)
402(2 / 386-4 17) 428(2/398-458)
I 2
130( I) 148(2/128- 167)
125 (1) 141 (2/ 109-172)
3 4
124(2/95- 152) 135(2/1 16-1 53)
83 (2/79-86) 122(2/ 114-129)
3 4
64(2/4 1-87) I 06(2/81-13 1)
I 2
62(1) 80(2/66-93)
32( I ) 63(2 /53 -73)
• Count exp ressed as the mean of the numbers o f lgA-bearing cel ls per 20 field s ( 10 Number of pig le ts exa min ed/ th e ranges of ce ll cou nt.
X
33(2/21-44) 57 (2 /5 4-60)
0
z
-" 0
(/)
0
0
c:
;:o
40) .
t
-
U' <.D
BRITISH VETER!N l\RY.JOURNJ\L, 138,2
160
Experiment II Animals. In al l, 97 pi g le ts ( La ndrace
X La rge White X Duroc) fro m 16 sows o n fa rm B were used. Anim a ls from th e sa m e litter were divided ra ndo ml y in to a treat m e nt g rou p a nd a co ntrol g ro up . Forty- fiv e pigl e ts fro m eight sows were use d for th e PG s tudy, 22 we re gi ven PG a nd 23 we re contro ls. The rema ining 52 p iglets were used fo r the TF st ud y, 25 we re inj ec ted s ubcuta n eously with TF a nd 27 we re co ntrol s . The meth od s of admini s te ring th e s ub sta n ces we re the sa m e as in ex perim ent I . Th e incid e n ce of di a rrh oea on fa rm B h ad been a hout 70% p er yea r . Clinical observation. A st ri ct exa min a tion for di a rrh oea of eac h pi g let was ca rried o ut twi ce a cl ay from birth to th e 28 th day. Th e cha rac te r of di a rrho ea wa s d escr ib ed as so ft ye llowi s h s too l, wa te ry o r white scour , a nd cl ass ifi ed as- A , co ntinu ed so ft ye ll owis h stoo l for more than four cl ays a nd / or co ntinu ed wate ry di a rrh oea for more th a n two clays a nd/o r continued white scour fo r more th a n one clay ; B, co n tinu ed wate ry di a rrhoea for m ore th a n t hree cl ays a nd /o r co nt inu ed white sco ur for m o re th a n one d a y; or C, co ntinued wate ry diarrhoea for m ore th a n four clays a nd /o r co ntinu ed whi te scour fo r more th a n on e cl ay. Indirect lzaemagglutination test. E. coli 08:K ?:H35 , whi ch ex is ted pred o min a ntl y in th e upper portion of th e s m FJ. II intes tin es of most sa mpl e pi g lets o n fa rm B, was used as th e a ntige n . Th e indirect h ae m agglutin a ti o n tes t used was as d escrib ed by Nete r rt al. ( 1952).
RESULTS
It was dete rmin ed that a ll th e pig le ts on fa rm A we re a ppa re ntly hea lth y, w ith no obv ious hi s topatho logica l cha nge.
TABLE Ill
COMPAR I SON Or E. CO U COUN T {I.OGA RITH I'v!I C COUN T /G CONT ENT S) I N THE S :-.·1:\1.1. I NTEST I NES Or PI G L ETS TR EATED WITH PC t\ND T F !\ND UNTRL\TI·: D CON TROLS
Duodenum
J ejunum
i leum
Three weeks old TF-trcatcd Control
7·2(6·8-7-4) 7·0(5 5-7 ·9)
7·6(5·8- 9·4) 8·4(5 ·9--1 0· 7)
10·7(8·8- 11 ·6) 10·8( ! 0·'1- 11·3)
PG -trca ted Control
3· 1(0* -7 ·8) 5·3(3·7-8·0)
6·4(5·9-6·8) 6·3( 4··3-8·4)
8· 7(6·6- 10·6) 9·2(7·0- 11 ·3)
1·6(0--5·9) 2·1 (0-6·6)
2·6(0- 8·5) 3·0(0-6·2)
7·8(6·2-9·5) 9·3(9·0-9·6)
0 0
1·3(0-4·0) 1·7(0-5·1 )
8· 1(7·7-8·6) 8·5(6·0- 10·3 )
Four weeks old TF-trca ted Cont ro l PC- treated Co ntrol
*0
indi cates < 10 3 ' 3 o rga nisms.
TAI3LE I V
INC I DENCE OF DIARRHOEA IN TF-TREATED AND CON TRO L
? I GLI~TS
AT FAR iv! 13
$: $:
Diarrhoea class*
c
B
A
c z 0
r
0
Damno.
Liller size
TF-treated
Control
TF-treated
Control
5
4 4 7 7 8 6
0/ 2t 0/2 1/3 2/3 3/4 0/3 0/4 2/4
2/2 1/2 2/4 4/4 2/4 1/3 1/4 4·/4
0/2 0/2 1/3 2/3 I/4· 0/3 0/4· I/4
2/2 1/2 2/4 3/4 2/4 0/3 1/4 4/4
5/25 20
15/27 55·6
TF-treated
Control
0
2/2 1/2 2/4 3/4 2/4 0/3 0/4 3/4
r
0
6 7 8 9
10 II 15 T o tal 0 /o
x2- tCSl
8
8 52
I 7/27 63
8/25 32 P<0·025
P<0·005
0/2 0/2 1/3 2/3 1/4 0/3 0/4 1/45/25 20 P<0·025
13/27 48·1
• Class ifi cat ion: i\ , continued soft ye ll owish stoo l for >4 days and /o r continu ed wate ry diarrhoea for >2 days and/or continu ed white sco ur for > I day ; 13 , con tinu ed watery diarrhoea for >3 days a nd /or co ntinued whire sco ur fo r >I d ay ; C, co ntinu ed wate ry diarrhoea for >4 cl ays and/or co ntinu ed wh ite sco ur for > I cla y. t Diarrhoea l pigl ets/ piglets examined.
)>
z...,., r
c
L-1
z
0
L-1 [/)
0
z '-::!
0 [/)
0
0
c;;o
BRITISH VETERINARY JO U RNA L, 138, 2
162
Level of senun IgG Th e mea n levels of serum I gG of the a nim a ls treated with TF a nd PG res pectiv ely are shown in Tabl e I. All the IgG leve ls were lowest a t 3·5 wee ks of age because of the turnover o flgG der ived from colostrum. Th e mea n va lues of the IgG co nce ntrati on in both TF a nd PC-treated piglets were so mewh a t hi g her than those of th e co n troi s of simil a r age. H oweve r , thi s difference seems to be not sig nifi ca nt. Count of lgA-bearing cells Th e count of IgA-bea rin g cel ls of th e la min a propri a of the sm a ll intestin es of th e piglets, aged three a nd four weeks, treated with TF a nd PG a rc sho wn in Table If. From the immun o Ou ore~ce nt studi es, it was cl earl y d em onst ra ted that a larger number ofl gA-bea ring ce ll s excited in the la min a propri a of the mu cosa fro m th e TF a nd PC treated gro ups than fro m those of th e co rrespo nding co ntrol g roups. These differences a re sig nifi can t in the four- wee k-old a nim a ls. For both trea ted a nd co ntro l gro up s, th e count oflgA-bcaring ce ll s was hi gher for the du od en um than for either th e jejunum o r the il eum . This result agrees with those of All en & Po rter ( 19 77) . Count of viable E. coli In Tab le III , th e co unt of viable E. coli in various po rti o ns of the sma ll intes tin es of the animals treated with Tf a nd PC respectively arc shown. \t\lith few exce p~i ons, the number of the orga ni sms at a give n loca le of a giv en age gro up was some wh a t less fo r th e Tf an d PG-trcated gro ups tha n for the co n tro ls. This trend see m s to be clea rer at th e age of four weeks. The co unt of E. coli is inv erse ly proportional to th e lgA-bea ring cells in both th e treated gro ups and th e con tro l gro ups.
TA BLE V I NC I DENCE OF C LA SS A D I ARRHOEA I N PC-TREA TE D AND CONT RO L PI GLETS AT FARM 13
Damno.
2 3 4 12 13 16 18
Total %
Liller si;;.e
PG-trealed
Con trol
7 6 6 5 4 4 5 8
1/3* 1/3 1/3 0/2 1/2 0/2 2/3 1/4
3/4 1/3 2/3 2/3 0/2 2/2 2/2 2/4
45
7/22 3 1·8
14/23 60·9
?
X"- lC S l
• Diarrhoea l pigle ts/ piglets exam ined.
P <0·05
l l'vlM UNOLOG ICAL lNFLUE CES ON PIG SCOU R
163
Incidence of dia rrhoea The in cide nce of d iarrh oea among the TF- injected and the control group s is shown in Table IV . A lowe r in cidence of diarrhoea was see n a mongst the treated groups as co mp ared to th at o f th e co ntro ls acco rding to any of the three m et hods of classifi cation: A(P<0·025), B(P< O·OOS) a nd C(P <0·025). According to method A (cf. Table IV ), th e in cid ence of di a rrh oea a m ongst the PC- treated g rou p is lower tha n th at of the co n trol
011110
TF - tree led group
o--c. Control group
40
20
10
0 2 *P
3
4
Age weeks
Fig . J. H acm a gglutinati o n ( H i\) titre of th e sera ofTF-trcat cd pi g le ts with £ . coli 08: K ?: H 35.
g ro up (P
In direct hnemagglutination test The haemagg luti nation (H A) titres of the sera from the treated groups, th ree a nd four weeks of age, aga in st E . coli 08: K ?: H 35 were higher than th ose of th e respective co n tro l g rou ps. Th e results are s hown in Fi gs I an d 2. Since the se ra were not treated with 2- m erca ptoe th a nol, it is not clear whether the an tibody belonged to IgG o·r Ig M . H oweve r, it see m s that th e immun e respo nse of th e treated g ro ups is e nh a n ced , a nd thi s effec t is in ve rse ly propo rti ona l to th e in cide nce of d ia rrh oea, es p ecia ll y a t three a nd four weeks of age.
BRITI SH VETERIN/\RY.JOURNA L, 138,2
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DI SCUSS ION
Tho ugh n eo n a ta l pig lets a r e immu no log ica lly co mpe tent a nd res pond to vario us antigenic stimul a tion s su ch as sh ee p red b lood ce ll a nd ac tinoph age, the un co mmitted multipote nt immuno co mpeten t ce ll s a r e d o min a nt (Kim , 1975) . Since, at thi s s tage, monopotent (monoclon a l) immun oco mpete nt ce ll s a nd m emory ce ll s a r c un a bl e to d eve lop , no r a re the pl as m a ce ll s a bl e to m a ture, th e seco ndary immun e res po nse a nd the produ cin g a bility of Ig are poor (Kim , Bra dl ey & W a tso n , 1966; Binns et al., 1972; o• o PG - treoted group ,_..., Control group
80
0
"'
''
40
'' '' ''
''
''
20
'
r/1*
'' \
10
----o*----- ____
---~()
0 2
3
.fj,
4
Age weeks :~P
** P<0 ·02 Fi g. 2. H aemagg lutill atio ll (Hi\ ) titre o f the se ra of PG- trcatrd pigl e ts w ith 1:. ro/iOil:K ,: J-I:l) .
Bourn e , 1973; Y a biki et al., 1974-; All e n & Po rt er, 1977 ; R cycro et al ., 1979). I n a n ewborn a nim a l, th ere a re very few lymphoid ce ll s in th e la min a pro pri a o f the s m a ll int es tin es, a nd th e ly mph o id fo llicles a re poo rl y d efin ed . These findin gs have a lso been proved w ith hi s tological o bse rva ti o ns (C hu et al., 1979). Th ere a re many agents whi ch en h a nce the hos t d e fence m ech a ni s m s, a nd so m e of th em h ave bee n used for the trea tm e nt of vari o us immunodeficient di seases in m an. H owever, few er s tudi es h ave bee n m ade on th e effec ts of immun o potc nti ato rs o n dom es tic a nim a ls, especia ll y in th e neo n a ta l s tage. It is ve ry inte res ting to no te that PG der ived from Bijidobaclerium thermojJitilum a nd TF from a dult porcine le ukocy tes produ ced a lm os t th e same r esu lts o n th e immun e r espo nse, a nd th ey gave a lower in c id e n ce of pi g scou r co mpared to th ose of the co n tro ls, a lthough th e mod e o f ac tion of th ese two s ubs ta n ces m ay be diffe re nt.
IMM UNO LOG ICAL IN fL UENCES ON PIG SCOUR
165
L a tely , PG (co nta inin g mura m y l dipeptide) has bee n prepared from va ri ous Grampositive bacte ri a, a nd th e res ulting s ubsta nce has been proved to possess th e minimal structure necessa ry for a n adju va nt- ac tive su bsta n ce (Ko ta ni et al., 1975). Th ere a re m a ny reports o n th e re la ti ons hip between th e d eve lo pm ent of immun e responses and the s tab le loca li zat io n of intes tin a l fl o ra (G usta fsso n & La urel! , 1958; Ba uer et al., 1966; Cra bbe et al., 1968; Wost m a nn et al., 1970; Ued a, Y a m aza ki & Someya, 197 3; And erso n , 1977 ; M acDon a ld & C a rter, 1979; Nielson & Friis , 1980). A poor d eve lopment of th e loca l immun e sys te m e nab les E. coli to pro lifera te abno rm a ll y in th e s m a ll intestines of pi g lets at th e s uckin g s tage a nd results in a high in cidence o f pig scour (K e nworth y & Crabbe, 1963). The conditi o n o f the intes tin a l fl o ra ofa very young sucking pi g le t is very un sta ble, a nd th e loca li zat io n o f Bifidobacterium or Lactobacillus a nd the reg res s of E. coli from th e upper po rt ion of th e sm a ll intes tin e to the il e um takes p lace a ft er three wee ks of age (N a mi oka et al., 1965). from these observations, it ca n be inferred th a t PG ac ts as a n immun os timul a nt in neo nates causing a decrease in th e in c id e nce o f di a rrh oea a nd a n in crease in IgA -b ear ing ce ll s in th e la min a propri a co mpared to untrea ted pi g le ts . Howeve r , the in crease of IgAbea ring ce ll s in the PG-treated g ro ups (as we ll as th e Tf-treated g ro ups) compared with those of co ntro l g ro ups w as not a pp a rent befo r e three weeks of age (S .. la m io ka, unpubli shed d a ta). In o th er words, eve n if th ese s ubs ta nces a re ad mi n is te red at the neo na ta l stage, th e cl ea r immunol og ica l effect m ay o n ly ap p ea r a ft e r three weeks o f age. Tf is a sm a ll (6000 m o l. wt.) m o lec ul e extrac ted fro m le uk ocy tes which is reported to have th e rem a rk a b le pro perty of tra nsfe rring to the lymphocytes of the non-se ns iti ve hum a n reci pi ent th e d o nor 's patte rn of d elayed h ype rse nsit ivity a nd , pres um a bl y, immuno logic m e m o ry or m ed iat io n (La wrence, 1969) . The m ec ha ni s m of act ion o fTF is still unknown , a lth o ug h it has e ffi cacy aga in st variou s immun ode fi ciencies, such as th e Vli scott- Aidri ch sy ndrom e o r mu coc ut a neo us cand idiosis in c hi ldren (Sc hu lkind & Ayo ub , 1975). H oweve r , some of th e m echan ism s ofTF have bee n m ade clear: in crease in th e PHA res po nse (G ri sce ll i et al., 1973) , in crease in E-rose tte forming ce ll s (V a ldimarsson et al., 1974), in crease in lymph ocy te co unt (Pabs t & Swanson, 19 72), indu c ti o n of circu la ti o n interfero n (E m odi , Ju st & Grob , 197 3), a nd enh a nce m ent o f ly mph ocyte DNA sy nth es is in res po nse ro a ntige n o r mitoge n (Co hen et al., 1976). The intestin a l tract co m es into direct co nt ac t with a g reate r variety of fl o ra than a ny other o rga n of the body, a nd the loca l immun osys te m m ay receive m ore adj uva ntac tive stimu la ti o ns. In ex perim ent II, a hig her leve l o f a ntibody was o bserved in the HA test in the treated g ro ups th a n in th e co ntro l g ro ups: thi s resu lt differs from th e d a ta on se rum IgG conce ntrat io n in ex per im ent I. On fa rm A no diarrhoe a was see n in a n y o f the piglets used while so m e pig lets o n fa rm B suffe red from severe sco ur . Consequent ly, th ere is a hi g her chance of E. coli bac teraemi a in pi g lets o n far m B than o n farm A. One ca n infer from this tha t the inv ading o rga ni sms stimu la te th e immun e sys tem o f o th er immun oco mpete n t o rga ns o f the host bes id es th e int estines. This may be o ne of th e reaso ns why a diffe rence of HA titre was see n be tw een th e treated a nd co ntro l a nim a ls fro m farm B. It is acce pted that secretory Ig A is o ne of the protec tive s ub sta nces aga in st intest in a l infect io n . H oweve r , it see m s necessa ry to ca rry o ut a s tud y trac in g the se r u m Ig M leve l a nd Ig M-bear in g ce ll s in th e la m ina propria from the o ntoge net ic point o f view in
166
BRITI SH VETERI NA RY JO U R NA L ,
138, 2
connection with immunoresponse. T he resu lts mentioned above, howeve r, sugges t th at PG a nd TF may p lay a role in enh a ncing non-specifi c immunopotenti ating ac tivities at the suckin g stage a nd consequen tly d ecreasing th e in cid ence of pig scour. Th is fac t revea ls a n add itiona l poss ib ili ty for trea ting pig let di a rrh oea ca used by age nts in clud ing E. coli a nd vira l infections . Further studi es a re required to elu cidate th e mechan isms of how PG a nd TF ac tiva te T a nd B lymphocytes in the neo natal stage a nd st im u late immun opote ncy.
ACK NO WLEDGEMENTS
T he authors a re g ra teful to Dr K . Tamu ra , of th e Nat iona l Institute of H ea lth , for se roty pin g of the E. coli. Th e a u thors a re a lso ind ebted to DrY . M aed e, DrS . Im oto a nd Dr T. Ohsugi for th eir tech nica l adv ice. T he st ud y was suppo rted by G ra n t- in-Aid fo r sc ien tific resea rch (G ra nt No . 456208) from the Mini st ry of Educa tion, Sci ence a nd C ulture, J apa n. REFERENCES
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E . ( 1976). Immunology 73, 2472. CHu , R. M., GLOCK, R . D., R oss, R . F. & Cox , D . F. ( 1979). American j ournal of Veterina')' Research 40, I 7 13. Co H EN, L. , H OLZMAN, R. S. , VALE NTI NE, F. T. & L AWRE NC E, H. S. ( 1976). J ou mal of Exf;en'mental Medicine 143, 79 1. COOPER , M . D. , LAWTON , A. R . & KI N KADE, P . \h,l . ( 1972). Clinical and Experimenta l fm munolog;• 11, 143. CRABBE, P. A ., B AZ IN, H ., EYSSEN, H . E . & H EREMAN S, J. F. ( 1968). In temational Archives of Allergy and Applied Immwwlog;• 34, 362 . EMODI, G., j UST, M . & GROll, P . ( 197 3). Lancet 2 , 1382. FI.ESH, j. , 0V AD I A, H . & NELKEN, D. ( 1977). Rejuah velerinarith 34, 97 . GR I SCE LLI , C., REVILLARD, j. P., BETUE L, H ., H ERZOG, C . & TouR,\I NE, .J. L. ( 1973). Biomedicine 18, 220. GU STAI'SSON, B . E. & LAURE LL, C . B . ( 1958). J ournal of Exfm imen tal Medicine 108, 25 1. KE NWO RTHY, R. & CRABBE, W. E. ( 1963) . J oumal of Comparative Patholog;• 73, 2 15. K1 M, Y . B. ( 1975). I n fmmunodeficienCJ' in Man and Animals, p. 549. Sunderla nd , t-.t! ass: C H ED ID ,
AUD I BERT, F ., L EFRANC I ER, P ., C HOAY,
Sin a uer Associates, In c. Ki M, Y. B ., BRADLEY , S. G . & VhTSON, D. V·i . ( 1966) . .Joumal of Im munology 99, 320. KOTAN I , S., WATANABE, Y ., SHIM ONO , T., NARITA, T., K ATO, K ., STEWART-TILL, D . E. S., KI NOS HI TA, S. & T ARU MI , T. ( 1975). Zeitschrifl Jiir fmmun itiitsforschung und exfmimente/le
Therapie 14, 302. ( 1969). Advances in Immunology 11, 195 . L AWR ENCE, H . S. & A SK ALl, A . L. ( 197 I ). l n In- Vitro M ethods in Ce/1-.Medialed Immunity, p. 53 1. L '\ W RENCE, H. S.
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LAWTON, A ., FR,\NK, L. Y . & CoOPER, M.D . (1975). In Birth Defects, ed. D . Bergs m a & R. A . Good, p. 28. Original Article Series 11 ( I ), The Nat iona l Found a tion . M ACDONALD, T. T. & CARTER, P . B. ( 1979). Journal of Immunology 122, 2624. M ANC INI, G ., CARBONARA , A. 0 . & H EREMANS, j. E. ( 1965). l mmunochemistT)' 12, 235 . M ITSUO KA , T. ( 1969). Zentralb!att fiir Bakteriologie, Parasitenkunde, h ifektionskrankheiten und H]•giene A b teilung I Origi n a le 210 , 52. NAMBA, Y ., HIDAKA , Y ., TAK I, K . & MOR IMOTO, T. (198 1). Infection and Immunity 31, 580. NAM IOKA , S., M URATA, M ., OSADA, H ., I SHIZA~\' A, T. & K uROOKA, R . (1965). J apanese
J ournal of Veterinary Science 27, 221. NETER, E., B ERTRAM, L. F ., ZAK, D. A ., MARDOCK, M . R. & ARBf-:SMAN , C . E. ( 1952).
J ournal of Experimental Medicine 96, I. NIELSEN , E. & F RII S, C. W. ( 1980). Acta pathologica et microbiologica scandinavica 88, I 2 I. 0V ADIA, H. , FLES H, ]. & NELKEN, D . ( 1978). Israel J ournal of M edical Science 14, 394. PABST, H . F . & SWANSON, R. ( 1972). British MedicalJournal 2, 442 . PORTER, P . ( 1969). Acta Biochemica et BiojJII)•Sica , Amsterdam 181 ,381. R EYERO, C., THALHAMM ER, J. G., R ESZLER, G. & STOCK!., W. ( 1978). Zeitschrifl fiir
l mmuniliitsforsclumg tmd experimentclle TherajJie 154, 409 . R EYERO, C., ST6CKL, \A/. & THALI-JAMMER, G. ( 1979). British VeterinaryJoumal 135, 17. SCHULK IND, M . L. & AYOUB, E. M . ( 1975) . In Immunodeficiency in Man and Animals, p. 436 . Sunderland, M ass: Sinau e r Associates , In c. SPIT LER, L. E., LE VIN, A. S . & FuDENBURG, H . H . ( I972).Joumal of Clinical Investigation 51, 32 16. UEDA, K., YAMA ZAK I, S. & SOMEYA , S. ( l 973) . J oumal of the Reticuloendothelial Society 12, 545. VALD IMARSSON, H ., HAMB LETON, G ., H ENRY, K. & McCONNELL, I. ( 1974). Clinical and
Experimental Immunology 16, 14 I. \'\'OSTMANN, B.S ., PL EASANTS, j . R ., BEALME R, P . & KI NECADE, D . W . ( 1970). Immunology
19, 443. YAIJIKI, T ., KA SHI WAZAK I, M . & NAM IOKA, S. ( 1973) . J ajmnese J oumal of Veterinary Science
35, 199. YAIJ IKI , T. , K AS HI WAZAK I, M. & NAM IOKA, S. ( 1974) . American Joumal of VeterinaT)' Research
35, 1483. {AccejJ!edfor publication 11 Jul]• 1981)
THE INFLUENCE OF IMMUNOPOTENTIATORS ON SUCKING PIGLETS WITH SPECIAL REFERENCE TO THE INCIDENCE OF PIG SCOUR
Bv S.
NAMI O KA,
Y.
T. KA\"' ANO, C . T. K. MURAKAMI!
KuMEDA,
WA NG*,
Y.
NAMBAt AND
Department of Veterinary Intema l Medicine, Fa culty of Veterinary Medicine, Hokkaido University, SajJ/Joro 060, Japan
SU MMARY
The prese nt pa per d esci·ibes the effect of the adminis trat ion of peptidoglycan (PG) an d di a li za bl e trans fer factor (TF) upon th e immun ores ponse and the in cidence of diarrhoea amongst sucking piglets . In ex perim en t I, la rger numb ers oflgA-bea ring ce ll s in the la min a propria of the mu cosa of the sma ll intestin es in th e PG and TF-treated groups aged three a nd four wee ks were observed co mpared to those of co ntrol groups, whi le no clear difference of serum IgG leve ls was see n betw een the treated gro ups a nd the controls. Th e co unts of viab le E. coli in various pa rts of the small intestin es of pigl ets trea ted with PG and TF were somewhat lower than those of the co ntrol s. In ex perim ent II , t he in cid ence of di a rrhoea o f PG and TF-treated sucking pi glets was co mp a red with th at of the non-treated controls. Lowe r in cid ences of d ia rrhoea in th e PG (P <0-0025) a nd TF-treated groups (P<0·05) were seen co mp a red to th e res pec tive co ntrol groups . H ae magglutin a tion titres with E. coli 08:K?:H35, whi ch ex isted predo min a ntly in th e small intes tin es of th e pi glets used were hig her in th e PG (P<0·02) a nd TF-treated (P < O·O I ) g roups three a nd four weeks old th a n in the respective control groups . These res ul ts suggest that PG a nd TF play a role in enh a ncing non-specifi c immunopotentiatin g activity in sucking pig lets.
I NT RODUCT ION
It is well known that piglets less than four weeks old a re immunod efi cient (A ll en & Porter, 1973; Bourn e, 1973; Ya biki , K as hiw aza ki & N a miok a, 1974; Kim , 1975; Prese nt a ddresses : • V e te rin a ry Co ll ege, Ta iwan U ni vers it y, Taipei, Taiwa n, Republi c of China. t In s tit ute o f Immuno logy, Esai Co. Lt d., Ko is h ikawa, Bunkyo- ku , To kyo. ! Sa nk yo-Zo hki Co. Ltd ., H onc ho, Niho nbas hi , Chuo- Ku , Tokyo.
BRITISH VETERINARY JOURNAL, 138,2
156
Reyero et al., 1978). The poor immunoresponse or the intestinal mucosa or piglets in this period is responsible ror various local inrections (such as colibaci llosis and vira l diarrhoea) despite the presence or a large amount or immunoglobulin (Ig) in colostr um. This immunodeficiency or neonatal piglets is analogous to the hum an baby's variable hypogammag lobu lin ae mi a with B lymp hocytes (Cooper, Lawton & Kinkade, 1972; Lawton, Frank & Cooper, 1975) . R ece ntly , various immunopotent iators have been developed ror human app li cat ion. Peptidoglycan (PG) is one or th em (Chedid et al., 1976), and the dialyzable transrer ractor (TF) is a lso reported to be effective (Spitler, Levin & Fundenburg, 1972). At the same time, levamisole has been appl ied as an immunomodularor to neonatal piglets (Reyero, Stock I & Thalhammer, 1979). I r is also used to control bovine mastitis (Ovadia, Flesh & Nelken, 1978) and calr diarrhoea (F les h, Ovadia & Nelken, 1977) . The purpose or the present study is to observ e the immunological consequences or the adminis t ration or PG and TF to neonatal piglets and th e eOcct iv eness or these substances aga in st pig scour.
MATERIALS AND METHODS
Experiment I Transfer factor (TF}. T he preparation orTF rollowed Lawrence's method (Lawrence & Askali, 197 1) : 500 ml or pooled adult swine venous blood ~ K ept ror I h at 37°C Sedimented
~ Buffy coa t (3 to 4 X 10 9/ ml ) ~ In cubated with DNAse and MgS0 4 ror 30 min at 37°C Lysed by 12 cycles or rreezing and thawi ng ~ Dialyzed aga in st distil led wa ter ror 48 h a t 4°C ~ Lyophilized ~ Reconstituted with distilled water ~ Filtrated with micropore filter One unit consis ts or the substance prepared rrom 10 9 1eukocytes. A hair unit orTF was injected three times subcutaneous ly, a t birth , on the third day and on the firth day respectively . Peptidoglycan (PC) . PG was prepared using Bi.fidobaclerium thennoJ;hilum which exists predominantly in the intestines or adult swine; this subs tance conta in s mura myl dipeptide (Nambaetal., 1981). Bifidobaclerium tlzermophilum strain P2-9 1 was c ulti vated in Brigges liv er broth under anaerob ic co nditi o ns (Mitsuoka, 1969) ror 16 hat 37°C , ha rvested by centriru gation and washed wi th sa lin e . The bacterial ce ll wa ll s were disrupted in a French press.
IMMUNOLOGICAL INFLUENCES ON PIG SCOUR
157
They were centrifuged and washed twice with saline and suspended m phosphate buffer of Sorensen (pH 6·2) in an equal volume of native cells (10 12 cells/ml). Enzymatic digestion of cell walls was performed by adding 0·0 l% egg white lysozymes and 0·05°/., pronase, and then incubating at 37°C for 48 h. The optical density at 64·0 nm of the digested solution was decreased to 5% of the initial value. The supernatant of the f1uid was used as digest.ed cell walls. One ml of the substance was derived from 10 12 of native cells. For more details of the method sec Namba et al. ( 1981). Piglets were given 0·25 ml ofPG orally at birth, two, three, four and five days of age respectively. Animals. Thirty-one Landrace piglets from four sows on farm A were used for this study. Of these, 17 piglets from two sows were used for the TF study: nine piglets were injected subcutaneously with 1·5 units TF as described previously and eight were untreated controls. The remaining 14- piglets were used for the PG study: seven piglets were given five doses of 0·25 ml PG orally, and seven were untreated controls. The piglets in each study were divided randomly. The sanitary condition of farm A was good and no pig scour was seen in the piglets used in the experiment. Serum f[;G level. The test of serum IgG level was carried out by rviancini's method (Mancini, Carbonara & Heremans, 1965) using single radial gel diffusion with antiswine IgG rabbit serum. The rabbit antiserum was prepared by the method of Porter (1969) and Yabiki, Kashiwazaki & Namioka (1973). Count of lgA bearing cells in the lamina j;ropria of the small intestines. The indirect fluorescent isothiocyanate (FITC) method was used. All the samples were collected from the corresponding portions of the small intestines in each piglet. The frozen sections (Sf.Lm) of the cluoclenum, the middle part ofthejejunum and the ileum of piglets aged three and four weeks were attached to glass slides, fixed with chilled acetone for 30 min and then soaked in phosphate buffer (pH 7-4) for several minutes. After drying, the anti-swine a-chain caprine serum (Miles Laboratory Inc.) was mounted and allowed to stand overnight at4 °C. The FITC labelled anti-caprine lgG rabbit serum (Miles Laboratory Inc.) was mounted and then allowed to stand overnight at 4°C. All the samples were examined by a Nikon EF fluorescent microscope equipped with a high mercury larnp (Osram I-lBO 50\'V/AC) under ultraviolet (UV) excitation, with a DM 400 dichroic mirror and a UV 330-380 filter. Cell count was clone using the rnethocl of Allen & Porter ( 1977): counts were made of stained cells present in 20 fields selected at random using an X 4-0 objective with an X 10 eye piece. Photographs were taken with Ektachrome film (ASA 400). Isolation and count of viable E. coli. Immediately after euthanasia, 5-cm long sections of the duodenum, the middle portion of the jejunum and the ileum of the piglets aged three and four weeks were isolated. 'fhcse tissues were opened aseptically and their contents were collected by scraping the mucosa lightly with a surgical knife. The contents were diluted with a 10-tolcl dilution in phosphate butTer (pH 7·4-) and each dilution was streake
BR IT ISH VETERINARY JOURNAL, 138, 2
158
TAl3 L E I SERUM IgG LEVELS (G/ 100 ML) I N PIGLETS TREATED \'V ITI-I PG AND TF AND UNTREATED CO 1TROLS
Week Piglet no.
Damno. PG-treated l
2
2
3
3·5
4
*
l 2 4
1·5 1 1·98 1·98
1·03 1·32 1·35
0·75 0·9 1 0·87
0·75
0·68
l 2 3 4 Mean ± SD
1·58 1·45 1·56 1·79 1·69 0·22
0·96 1·04 0·95 1·27 1· 13 0·17
0·63 0·68 0·71 0·92 0·78 0· 12
0·4·2 0·58 0·58 0· 17
0·50 0·66 0·61 0· 10
5 7 8
1·22 0·95 1·74
0·87 0·64 0·99
0·54· 0·57 0·75
0·66
0·68
6 7 8 9 Mean ± SD
1·75 1·49 1·84 1·42 1·49 0·32
0·98 0·95 1· 15 0·93 0·94 0· 15
0·72 0·60 0·80 0·66 0 10
0·62 0·51 0·60 0·08
0·62 0·46 0· 59 0· 11
l 2 3
1·43 2· 19 1·36
1·06 1·42 0·96
0·74 0·84 0·65
0·55
0·68
l 2 3 4 5 Mean ± SD
1·00 1·74 1·64 1·98 l 58 1·64 0·35
0·97 1·32 1·02 0·67 1· 14 l·l l 0·25
0·72 0·64 0·83 0·48 0·77 0·74 0·1 4
0·50 0·76 0·66 0· 16
0·48 0·74 0·62 0 II
5 6 7
1·57 1·95 2·16
1· 15 1·3 1 HI
0·97 0·82 1·03
0·62 0·82
0·62 0·82
6 7 8 9 10 Mean ± SD
0·95 0·33 2·29 1·32 0·68 I ·41 0·7 I
0·59 0·27 1·58 0·81 044 0·94 0·48
0·44· 0·23 0·78 0·60 0·38 0·66 0 ·29
0·58 0· 32 0· 59 0·21
0 56 0·34 0· 59 021
Control l
2
TF-treated 3
4
Control 3
4
* Not tested due
to
sacri fi ce.
OM
TABLE 11 NU I\•IBER• OF lgA-BEARING CE LLS OF T HE LAM INA PROPRIA IN T HE SMALL I NTEST INES OF PIGLETS TREATED W ITH TF AND PC AT FARM A
Portion Three weeks old Duodenum
Damno .
P G-treated
Control
-
3:: Damno.
TF-treated
Control
.,. ';>'
c: z
0 I 2
504(2/462-547t) 409(2/373-445)
4 11 (2/378-444) 530(2/403-657)
3 4
396(2/39 1-400) 460(3/439-482)
218(1 *) 460(3/413-583)
I 2
128(2/108-147) 166(2/114-218)
I 08(2 /60- 155) 45(2/ 40-50)
3 4
116(2/1 06-126) 112(3/91-135)
45(2/40-50) 132(3/ 93-179)
r'
0
0 0
Jejunum
Il eu m
I 2
87 (2/76-98 ) 74(2/59-88)
77 (2/28- 126 ) 18(2/ 16-20)
3 4
48 (2/38-5 7) 65(3/52-85)
24(1) 80(3/79-83)
> r'
z..,
r
c: t"'l
z
0
Four weeks old Duodenum
J ej unum
Ileu m
t"'l
(/)
I 2
545( 1) 557(2/467 -647)
540( 1) 388(2/262 -514)
3 4
46 1(2/4 10-5 11 ) 552(2/543-56 1)
402(2 / 386-4 17) 428(2/398-458)
I 2
130( I) 148(2/128- 167)
125 (1) 141 (2/ 109-172)
3 4
124(2/95- 152) 135(2/1 16-1 53)
83 (2/79-86) 122(2/ 114-129)
3 4
64(2/4 1-87) I 06(2/81-13 1)
I 2
62(1) 80(2/66-93)
32( I ) 63(2 /53 -73)
• Count exp ressed as the mean of the numbers o f lgA-bearing cel ls per 20 field s ( 10 Number of pig le ts exa min ed/ th e ranges of ce ll cou nt.
X
33(2/21-44) 57 (2 /5 4-60)
0
z
-" 0
(/)
0
0
c:
;:o
40) .
t
-
U' <.D
BRITISH VETER!N l\RY.JOURNJ\L, 138,2
160
Experiment II Animals. In al l, 97 pi g le ts ( La ndrace
X La rge White X Duroc) fro m 16 sows o n fa rm B were used. Anim a ls from th e sa m e litter were divided ra ndo ml y in to a treat m e nt g rou p a nd a co ntrol g ro up . Forty- fiv e pigl e ts fro m eight sows were use d for th e PG s tudy, 22 we re gi ven PG a nd 23 we re contro ls. The rema ining 52 p iglets were used fo r the TF st ud y, 25 we re inj ec ted s ubcuta n eously with TF a nd 27 we re co ntrol s . The meth od s of admini s te ring th e s ub sta n ces we re the sa m e as in ex perim ent I . Th e incid e n ce of di a rrh oea on fa rm B h ad been a hout 70% p er yea r . Clinical observation. A st ri ct exa min a tion for di a rrh oea of eac h pi g let was ca rried o ut twi ce a cl ay from birth to th e 28 th day. Th e cha rac te r of di a rrho ea wa s d escr ib ed as so ft ye llowi s h s too l, wa te ry o r white scour , a nd cl ass ifi ed as- A , co ntinu ed so ft ye ll owis h stoo l for more than four cl ays a nd / or co ntinu ed wate ry di a rrh oea for more th a n two clays a nd/o r continued white scour fo r more th a n one clay ; B, co n tinu ed wate ry di a rrhoea for m ore th a n t hree cl ays a nd /o r co nt inu ed white sco ur for m o re th a n one d a y; or C, co ntinued wate ry diarrhoea for m ore th a n four clays a nd /o r co ntinu ed whi te scour fo r more th a n on e cl ay. Indirect lzaemagglutination test. E. coli 08:K ?:H35 , whi ch ex is ted pred o min a ntl y in th e upper portion of th e s m FJ. II intes tin es of most sa mpl e pi g lets o n fa rm B, was used as th e a ntige n . Th e indirect h ae m agglutin a ti o n tes t used was as d escrib ed by Nete r rt al. ( 1952).
RESULTS
It was dete rmin ed that a ll th e pig le ts on fa rm A we re a ppa re ntly hea lth y, w ith no obv ious hi s topatho logica l cha nge.
TABLE Ill
COMPAR I SON Or E. CO U COUN T {I.OGA RITH I'v!I C COUN T /G CONT ENT S) I N THE S :-.·1:\1.1. I NTEST I NES Or PI G L ETS TR EATED WITH PC t\ND T F !\ND UNTRL\TI·: D CON TROLS
Duodenum
J ejunum
i leum
Three weeks old TF-trcatcd Control
7·2(6·8-7-4) 7·0(5 5-7 ·9)
7·6(5·8- 9·4) 8·4(5 ·9--1 0· 7)
10·7(8·8- 11 ·6) 10·8( ! 0·'1- 11·3)
PG -trca ted Control
3· 1(0* -7 ·8) 5·3(3·7-8·0)
6·4(5·9-6·8) 6·3( 4··3-8·4)
8· 7(6·6- 10·6) 9·2(7·0- 11 ·3)
1·6(0--5·9) 2·1 (0-6·6)
2·6(0- 8·5) 3·0(0-6·2)
7·8(6·2-9·5) 9·3(9·0-9·6)
0 0
1·3(0-4·0) 1·7(0-5·1 )
8· 1(7·7-8·6) 8·5(6·0- 10·3 )
Four weeks old TF-trca ted Cont ro l PC- treated Co ntrol
*0
indi cates < 10 3 ' 3 o rga nisms.
TAI3LE I V
INC I DENCE OF DIARRHOEA IN TF-TREATED AND CON TRO L
? I GLI~TS
AT FAR iv! 13
$: $:
Diarrhoea class*
c
B
A
c z 0
r
0
Damno.
Liller size
TF-treated
Control
TF-treated
Control
5
4 4 7 7 8 6
0/ 2t 0/2 1/3 2/3 3/4 0/3 0/4 2/4
2/2 1/2 2/4 4/4 2/4 1/3 1/4 4·/4
0/2 0/2 1/3 2/3 I/4· 0/3 0/4· I/4
2/2 1/2 2/4 3/4 2/4 0/3 1/4 4/4
5/25 20
15/27 55·6
TF-treated
Control
0
2/2 1/2 2/4 3/4 2/4 0/3 0/4 3/4
r
0
6 7 8 9
10 II 15 T o tal 0 /o
x2- tCSl
8
8 52
I 7/27 63
8/25 32 P<0·025
P<0·005
0/2 0/2 1/3 2/3 1/4 0/3 0/4 1/45/25 20 P<0·025
13/27 48·1
• Class ifi cat ion: i\ , continued soft ye ll owish stoo l for >4 days and /o r continu ed wate ry diarrhoea for >2 days and/or continu ed white sco ur for > I day ; 13 , con tinu ed watery diarrhoea for >3 days a nd /or co ntinued whire sco ur fo r >I d ay ; C, co ntinu ed wate ry diarrhoea for >4 cl ays and/or co ntinu ed wh ite sco ur for > I cla y. t Diarrhoea l pigl ets/ piglets examined.
)>
z...,., r
c
L-1
z
0
L-1 [/)
0
z '-::!
0 [/)
0
0
c;;o
BRITISH VETERINARY JO U RNA L, 138, 2
162
Level of senun IgG Th e mea n levels of serum I gG of the a nim a ls treated with TF a nd PG res pectiv ely are shown in Tabl e I. All the IgG leve ls were lowest a t 3·5 wee ks of age because of the turnover o flgG der ived from colostrum. Th e mea n va lues of the IgG co nce ntrati on in both TF a nd PC-treated piglets were so mewh a t hi g her than those of th e co n troi s of simil a r age. H oweve r , thi s difference seems to be not sig nifi ca nt. Count of lgA-bearing cells Th e count of IgA-bea rin g cel ls of th e la min a propri a of the sm a ll intestin es of th e piglets, aged three a nd four weeks, treated with TF a nd PG a rc sho wn in Table If. From the immun o Ou ore~ce nt studi es, it was cl earl y d em onst ra ted that a larger number ofl gA-bea ring ce ll s excited in the la min a propri a of the mu cosa fro m th e TF a nd PC treated gro ups than fro m those of th e co rrespo nding co ntrol g roups. These differences a re sig nifi can t in the four- wee k-old a nim a ls. For both trea ted a nd co ntro l gro up s, th e count oflgA-bcaring ce ll s was hi gher for the du od en um than for either th e jejunum o r the il eum . This result agrees with those of All en & Po rter ( 19 77) . Count of viable E. coli In Tab le III , th e co unt of viable E. coli in various po rti o ns of the sma ll intes tin es of the animals treated with Tf a nd PC respectively arc shown. \t\lith few exce p~i ons, the number of the orga ni sms at a give n loca le of a giv en age gro up was some wh a t less fo r th e Tf an d PG-trcated gro ups tha n for the co n tro ls. This trend see m s to be clea rer at th e age of four weeks. The co unt of E. coli is inv erse ly proportional to th e lgA-bea ring cells in both th e treated gro ups and th e con tro l gro ups.
TA BLE V I NC I DENCE OF C LA SS A D I ARRHOEA I N PC-TREA TE D AND CONT RO L PI GLETS AT FARM 13
Damno.
2 3 4 12 13 16 18
Total %
Liller si;;.e
PG-trealed
Con trol
7 6 6 5 4 4 5 8
1/3* 1/3 1/3 0/2 1/2 0/2 2/3 1/4
3/4 1/3 2/3 2/3 0/2 2/2 2/2 2/4
45
7/22 3 1·8
14/23 60·9
?
X"- lC S l
• Diarrhoea l pigle ts/ piglets exam ined.
P <0·05
l l'vlM UNOLOG ICAL lNFLUE CES ON PIG SCOU R
163
Incidence of dia rrhoea The in cide nce of d iarrh oea among the TF- injected and the control group s is shown in Table IV . A lowe r in cidence of diarrhoea was see n a mongst the treated groups as co mp ared to th at o f th e co ntro ls acco rding to any of the three m et hods of classifi cation: A(P<0·025), B(P< O·OOS) a nd C(P <0·025). According to method A (cf. Table IV ), th e in cid ence of di a rrh oea a m ongst the PC- treated g rou p is lower tha n th at of the co n trol
011110
TF - tree led group
o--c. Control group
40
20
10
0 2 *P
3
4
Age weeks
Fig . J. H acm a gglutinati o n ( H i\) titre of th e sera ofTF-trcat cd pi g le ts with £ . coli 08: K ?: H 35.
g ro up (P
In direct hnemagglutination test The haemagg luti nation (H A) titres of the sera from the treated groups, th ree a nd four weeks of age, aga in st E . coli 08: K ?: H 35 were higher than th ose of th e respective co n tro l g rou ps. Th e results are s hown in Fi gs I an d 2. Since the se ra were not treated with 2- m erca ptoe th a nol, it is not clear whether the an tibody belonged to IgG o·r Ig M . H oweve r, it see m s that th e immun e respo nse of th e treated g ro ups is e nh a n ced , a nd thi s effec t is in ve rse ly propo rti ona l to th e in cide nce of d ia rrh oea, es p ecia ll y a t three a nd four weeks of age.
BRITI SH VETERIN/\RY.JOURNA L, 138,2
164
DI SCUSS ION
Tho ugh n eo n a ta l pig lets a r e immu no log ica lly co mpe tent a nd res pond to vario us antigenic stimul a tion s su ch as sh ee p red b lood ce ll a nd ac tinoph age, the un co mmitted multipote nt immuno co mpeten t ce ll s a r e d o min a nt (Kim , 1975) . Since, at thi s s tage, monopotent (monoclon a l) immun oco mpete nt ce ll s a nd m emory ce ll s a r c un a bl e to d eve lop , no r a re the pl as m a ce ll s a bl e to m a ture, th e seco ndary immun e res po nse a nd the produ cin g a bility of Ig are poor (Kim , Bra dl ey & W a tso n , 1966; Binns et al., 1972; o• o PG - treoted group ,_..., Control group
80
0
"'
''
40
'' '' ''
''
''
20
'
r/1*
'' \
10
----o*----- ____
---~()
0 2
3
.fj,
4
Age weeks :~P
** P<0 ·02 Fi g. 2. H aemagg lutill atio ll (Hi\ ) titre o f the se ra of PG- trcatrd pigl e ts w ith 1:. ro/iOil:K ,: J-I:l) .
Bourn e , 1973; Y a biki et al., 1974-; All e n & Po rt er, 1977 ; R cycro et al ., 1979). I n a n ewborn a nim a l, th ere a re very few lymphoid ce ll s in th e la min a pro pri a o f the s m a ll int es tin es, a nd th e ly mph o id fo llicles a re poo rl y d efin ed . These findin gs have a lso been proved w ith hi s tological o bse rva ti o ns (C hu et al., 1979). Th ere a re many agents whi ch en h a nce the hos t d e fence m ech a ni s m s, a nd so m e of th em h ave bee n used for the trea tm e nt of vari o us immunodeficient di seases in m an. H owever, few er s tudi es h ave bee n m ade on th e effec ts of immun o potc nti ato rs o n dom es tic a nim a ls, especia ll y in th e neo n a ta l s tage. It is ve ry inte res ting to no te that PG der ived from Bijidobaclerium thermojJitilum a nd TF from a dult porcine le ukocy tes produ ced a lm os t th e same r esu lts o n th e immun e r espo nse, a nd th ey gave a lower in c id e n ce of pi g scou r co mpared to th ose of the co n tro ls, a lthough th e mod e o f ac tion of th ese two s ubs ta n ces m ay be diffe re nt.
IMM UNO LOG ICAL IN fL UENCES ON PIG SCOUR
165
L a tely , PG (co nta inin g mura m y l dipeptide) has bee n prepared from va ri ous Grampositive bacte ri a, a nd th e res ulting s ubsta nce has been proved to possess th e minimal structure necessa ry for a n adju va nt- ac tive su bsta n ce (Ko ta ni et al., 1975). Th ere a re m a ny reports o n th e re la ti ons hip between th e d eve lo pm ent of immun e responses and the s tab le loca li zat io n of intes tin a l fl o ra (G usta fsso n & La urel! , 1958; Ba uer et al., 1966; Cra bbe et al., 1968; Wost m a nn et al., 1970; Ued a, Y a m aza ki & Someya, 197 3; And erso n , 1977 ; M acDon a ld & C a rter, 1979; Nielson & Friis , 1980). A poor d eve lopment of th e loca l immun e sys te m e nab les E. coli to pro lifera te abno rm a ll y in th e s m a ll intestines of pi g lets at th e s uckin g s tage a nd results in a high in cidence o f pig scour (K e nworth y & Crabbe, 1963). The conditi o n o f the intes tin a l fl o ra ofa very young sucking pi g le t is very un sta ble, a nd th e loca li zat io n o f Bifidobacterium or Lactobacillus a nd the reg res s of E. coli from th e upper po rt ion of th e sm a ll intes tin e to the il e um takes p lace a ft er three wee ks of age (N a mi oka et al., 1965). from these observations, it ca n be inferred th a t PG ac ts as a n immun os timul a nt in neo nates causing a decrease in th e in c id e nce o f di a rrh oea a nd a n in crease in IgA -b ear ing ce ll s in th e la min a propri a co mpared to untrea ted pi g le ts . Howeve r , the in crease of IgAbea ring ce ll s in the PG-treated g ro ups (as we ll as th e Tf-treated g ro ups) compared with those of co ntro l g ro ups w as not a pp a rent befo r e three weeks of age (S .. la m io ka, unpubli shed d a ta). In o th er words, eve n if th ese s ubs ta nces a re ad mi n is te red at the neo na ta l stage, th e cl ea r immunol og ica l effect m ay o n ly ap p ea r a ft e r three weeks o f age. Tf is a sm a ll (6000 m o l. wt.) m o lec ul e extrac ted fro m le uk ocy tes which is reported to have th e rem a rk a b le pro perty of tra nsfe rring to the lymphocytes of the non-se ns iti ve hum a n reci pi ent th e d o nor 's patte rn of d elayed h ype rse nsit ivity a nd , pres um a bl y, immuno logic m e m o ry or m ed iat io n (La wrence, 1969) . The m ec ha ni s m of act ion o fTF is still unknown , a lth o ug h it has e ffi cacy aga in st variou s immun ode fi ciencies, such as th e Vli scott- Aidri ch sy ndrom e o r mu coc ut a neo us cand idiosis in c hi ldren (Sc hu lkind & Ayo ub , 1975). H oweve r , some of th e m echan ism s ofTF have bee n m ade clear: in crease in th e PHA res po nse (G ri sce ll i et al., 1973) , in crease in E-rose tte forming ce ll s (V a ldimarsson et al., 1974), in crease in lymph ocy te co unt (Pabs t & Swanson, 19 72), indu c ti o n of circu la ti o n interfero n (E m odi , Ju st & Grob , 197 3), a nd enh a nce m ent o f ly mph ocyte DNA sy nth es is in res po nse ro a ntige n o r mitoge n (Co hen et al., 1976). The intestin a l tract co m es into direct co nt ac t with a g reate r variety of fl o ra than a ny other o rga n of the body, a nd the loca l immun osys te m m ay receive m ore adj uva ntac tive stimu la ti o ns. In ex perim ent II, a hig her leve l o f a ntibody was o bserved in the HA test in the treated g ro ups th a n in th e co ntro l g ro ups: thi s resu lt differs from th e d a ta on se rum IgG conce ntrat io n in ex per im ent I. On fa rm A no diarrhoe a was see n in a n y o f the piglets used while so m e pig lets o n fa rm B suffe red from severe sco ur . Consequent ly, th ere is a hi g her chance of E. coli bac teraemi a in pi g lets o n far m B than o n farm A. One ca n infer from this tha t the inv ading o rga ni sms stimu la te th e immun e sys tem o f o th er immun oco mpete n t o rga ns o f the host bes id es th e int estines. This may be o ne of th e reaso ns why a diffe rence of HA titre was see n be tw een th e treated a nd co ntro l a nim a ls fro m farm B. It is acce pted that secretory Ig A is o ne of the protec tive s ub sta nces aga in st intest in a l infect io n . H oweve r , it see m s necessa ry to ca rry o ut a s tud y trac in g the se r u m Ig M leve l a nd Ig M-bear in g ce ll s in th e la m ina propria from the o ntoge net ic point o f view in
166
BRITI SH VETERI NA RY JO U R NA L ,
138, 2
connection with immunoresponse. T he resu lts mentioned above, howeve r, sugges t th at PG a nd TF may p lay a role in enh a ncing non-specifi c immunopotenti ating ac tivities at the suckin g stage a nd consequen tly d ecreasing th e in cid ence of pig scour. Th is fac t revea ls a n add itiona l poss ib ili ty for trea ting pig let di a rrh oea ca used by age nts in clud ing E. coli a nd vira l infections . Further studi es a re required to elu cidate th e mechan isms of how PG a nd TF ac tiva te T a nd B lymphocytes in the neo natal stage a nd st im u late immun opote ncy.
ACK NO WLEDGEMENTS
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