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Transfer of immunoglobulins IgG, IgA and IgM to lacteal secretions in the parturient sow and their absorption by the neonatal piglet
BIOCHIMICA ET BIOPHYSICA ACTA
38I
BBA 35389 T R A N S F E R OF IMMUNOGLOBULINS IgG, IgA AND IgM TO LACTEAL S E C R E T I O N S IN T H E P A R T U R I E N T SOW AND T H E I R A B S O R P T I O N BY THE NEONATAL PIGLET
P. P O R T E R
Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford (Great Britain) (Received March 24th , 1969)
SUMMARY
Porcine immunoglobulins IgG, IgA and IgM have been isolated and used in immunochemical studies of the secretion of these components into colostrum and then intestinal absorption b y the neonatal piglet. Assays on 9 samples of colostrum from different sows indicated that 63.6 ± 8.6 % of the whey protein was accounted for b y the immunoglobulins which comprise IgG (79.7 :E I6.9%), IgA (14.o 5 ~- 7.35%) and IgM (6.27 :E: 2.35%). Immunoglobulins IgG and IgM occur in the colostrum at approx. 2-3 times the level in serum but the level of IgA in colostrum was 3-11 times greater than in serum. Molecular size characteristics of the immunoglobulins in sow serum and colostrum have been obtained in Sephadex G-2oo gel filtration studies. Colostral IgA is eluted in a wide range of molecular sizes in the gel filtration pattern but the high molecular weight forms are predominant. A serum component with a sedimentation constant (s~0' w) of 17.8 which is antigenically distinct from IgM but gives reactions of identity with IgG, also appears in colostrum. Precolostral piglet serum was almost entirely deficient of immunoglobulins. A component having shared antigenic determinants with IgG was present in concentrations less than 50 #g/ml. Studies of 6 litters of piglets indicated that immunoglobulins IgG, IgA and IgM were absorbed from the colostrum. The post colostral serum concentrations of IgG and IgM were mainly similar to adult levels, whilst IgA generally exceeded adult levels, in some cases by as much as 3-4 times.
INTRODUCTION
The serological relationships between proteins of serum and milk have been established for several animal species including the pig 1. The post-partum transport of colostral proteins from mother to offspring is of tremendous immunological importance to the pig, since there is apparently little or no transplacental transfer of immunity. The absorption of ~-globulin into the blood stream of the neonatal piglet Biochim. Biophys. Aeta, 181 (1969) 381-392
382
P. PORTER
via the gut has been demonstrated by the marked change in the serum electrophoretic profile following the ingestion of colostrum, heterologous or homologous 7-globulins2 8. The accent has been on the v-globulins as a whole, without making any distinction between the immunoglobulins IgM, IgA, IgG or placing any emphasis on the nature of passive immunity derived from the colostrum. The 7-S IgG molecule accounts for the greatest proportion of the immunoglobulins in the colostrum of the pig and the studies to date undoubtedly relate almost entirely to this molecule. Some authors 1,7 state that immunoglobulins IgA and IgM are absorbed from colostrum. These findings are based on the appearance of certain precipitin lines in the v-globulin region of immunoelectrophoretograms of postcolostral sera, but without characterizing the components. In view of the finding that IgA in rabbit colostrum migrates on the anodic side of the electrophoretogram s, one cannot safely extrapolate from the well-characterised human electrophoretogram to other species. The intestinal absorption of IgM immunoglobulin is the subject of dispute, since there is evidence of the failure of IgM to be absorbed by the intestine of the newborn pig s. This is also in agreement with results reported for young rats1°, n. In this paper, immunoglobulins IgM, IgA and IgG are isolated, characterized, and assayed in the colostrum and serum of the sow. Quantitative evidence is presented for the intestinal absorption of each of these immunoglobulins b y the neonatal piglet. MATERIALS AND METHODS
Microelectrophoresis Protein samples and fractions were examined by cellulose acetate electrophoresis and immunoelectrophoresis 12. Disc electrophoresis was carried out in polyacrylamide gels 13.
Gelfiltration chromatography Chromatography was carried out in Sephadex G-2oo columns (45 cm × 2.5 cm) and Sephadex G-I5O columns (9° cm × 2.5 cm) using o.I M Tris saline buffer (pH 7.2). Thin-layer gel-filtration studies were made using Sephadex G-2oo superfine 14.
Ion-exchange chromatography Anion-exchange chromatography was carried out on a DEAE-cellulose column (30 cm × 2.0 cm). The stepwise elution entailed the use of four buffers: (I) o.oi M NaH2PO 4 adjusted to pH 7.6 with o.oi M NaOH, (2) 0.02 M NaH~PO 4 adjusted to pH 6.3 with 0.02 M NaOH, (3) 0-05 M NaH2P04, and (4) 0.3 M NaH2PO 4. A fifth buffer, 0. 4 M NaH2PO 4 containing 2 M NaC1, was used to wash firmly bound protein from the column after each run before regenerating with the starting buffer ~5, Cation-exchange chromatography was carried out using CM-cellulose columns (20 cm × 2.0 cm). This technique was used to isolate 7-S immunoglobulin from fractions of pig serum obtained by anion-exchange chromatography. For this purpose a fairly limited stepwise elution procedure using phosphate buffer (pH 5.8) and molarities 0,005 M, o.oi M, 0.02 M, o.04 M in sequence, were found to be adequate. Samples were prepared for ion-exchange chromatography by pressure dialysis against the starting buffer for 24 h or by passing through a Sephadex G-25 column (45 cm × 5 cm) equilibrated with starting buffer. Biochim. Biophys. Acta, 181 (1969) 381-392
IMMUNOGLOBULINS IgG, IgA AND igM IN SOW COLOSTRUM
383
Flow rates up to 2oo ml/h were maintained with the ion-exchange colmnns by use of a suitable hydrostatic head. Eluates were automatically scanned at 28o m# and collected using L K B ultrarac collector and drop counter. Fractions were pooled from the eluates b y reference to the elution pattern.
Isolation of specific immunoglobulins The preparation of purified immunoglobulin components was essential to the investigations. The quantitative studies required the preparation of antisera which reacted specifically with each immunoglobulin. A crude preparation of 7-S IgG was obtained from adult pig serum by precipitation with lO% Na2SO 4. This was further purified b y chromatography on D E A E cellulose. The "fall through" fraction eluted with the first buffer was recovered and subjected to further chromatography on CM-cellulose. The eluates obtained at p H 5.8 with 0.02 M phosphate buffer provided a pure sample of 7-S IgG when examined by immunoelectrophoresis using a rabbit antiserum to whole pig serum. A crude preparation of I9-S IgM is obtained in the eluates with Buffer 4 on chromatography of adult serum on DEAE-cellulose. This fraction contains predominantly albumin and a number of low molecular a-globulins. Chromatography on Sephadex G-2oo separated the macroglobulins in this fraction in eluates obtained at the exclusion limits of the column. Sow colostrum was used as a source of IgA. A Ioo-ml sample of colostral whey was fractionated on Sephadex G-I5O (800 cm × 60 cm) using o.I M Tris-saline buffer (pH 7.2). A crude preparation of colostral IgA is obtained in the first protein peak eluted from the column which also contains IgM. IgA is isolated from this crude fraction by chromatography on DEAE-cellulose. The fraction was applied to the column in o.oi M phosphate buffer pH 7.6 and elution was carried out using a simple increasing salt gradient obtained by the continuous addition of 0.3 M NaC1 to the starting buffer. The greater part of the protein was eluted from the column in a broad tailing peak commencing when the eluting buffer was 0.09 M and terminating when the buffer was 0.22 M. In the first phase of elution of this peak at pH 7.6 a sample of IgA was obtained which was pure as judged b y the criteria of immunoelectrophoresis using rabbit antisera to pig serum and pig colostral whey proteins and also b y ultracentrifugation.
Preparation of rabbit antisera specificfor porcine IgG, IgA and IgM Antisera were prepared b y injection of emulsions of the immunoglobulin isolates with Freunds Bacto-Adjuvant, Incomplete, into New Zealand white rabbits 12. Immunological cross reactivity due to shared antigenic determinants on the immunoglobulins was removed by absorption with the purified nonspecific immunoglobulin. The IgM preparation was contaminated with a2M and this activity was removed from the rabbit antiserum by absorption with precolostral piglet serum.
Quantitative estimation of immunoglobulins Immunoglobulin levels in sera and colostrum samples were estimated by radial immunodiffusion TM.
Biochim. Biophys. Acta, i81 (1969) 381-392
384.
P. PORTER
Analytical ultracentrifugation Ultracentrifugal analysis was carried out in a Beckman model E centrifuge equipped with phase-plate schlieren diaphram. Sedimentation coefficients were determined at 20 ° employing rotor speeds of 59 780 rev./min.
Preparation of sow's colostral whey Colostrum was collected from pigs at parturition and casein and free fat were removed by centrifugation at 4 ° ooo rev./min at 20 ° (IOO ooo × g) for 30 rain 17. The whey was carefully removed by pipette and recentrifuged under the same conditions to ensure the complete removal of any suspension included in the process of pipetting.
Serum from piglets and sows Piglets were bled from the anterior vena cava and sows from an ear vein. RESULTS
Preliminary studies (using micro techniques) of the nature of sow colostral whey and the intestinal absorption of components by the neonatal piglet In the 2-day-old suckling piglet the considerable absorption of immunolactoglobulin is readily evident from polyacrylamide gel electrophoretie analyses. CornTHEN - LAYER " GEL "tlI[TIIATtQN
COLQSTRUM DIPll IV|T1 [~l~ GAY OLD~
Ct~*t~STRAL WHEY
SUCKLED PIGLET [ 2 IDAYOLD 1
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Fig. I. P o l y a c r y l a m i d e gel electrophoretic studies of c o l o s t r u m a n d sera. (a) Precolostral s e r u m ; (b) postcolostral s e r u m , 2-days-old piglet; (c) colostral w h e y ; (d) sow s e r u m ; (e) s e r u m f r o m suckled piglet, 35 d a y s old; (f) s e r u m f r o m c o l o s t r u m - d e p r i v e d piglet, 35 d a y s old. Fig. 2. T h i n - l a y e r gel-filtration studies of c o l o s t r u m a n d sera i n d i c a t i n g t h e areas of m i g r a t i o n of major components.
Biochim. Biophys. Mcta, 181 (1969) 381-392
IMMUNOGLOBULINSIgG, IgA AND igM IN SOW COLOSTRUM
385
parative studies of sera and colostral whey are shown in Fig. I. The total protein in pooled serum from a litter of newborn piglets was 2.8 g/ioo ml, whilst that of a pooled sample of postcolostral serum at 2 days was 5.4 g/Ioo ml. This represents a nearly 2-fold increase in serum concentration which the polyacrylamide gel electrophoresis indicates is due mainly to the absorption of the 7-S immunoglobulins. Thin-layer gel filtration using Sephadex G-2oo, provides for a separation of proteins which is dependent upon molecular size. Preliminary evidence for the intestinal absorption of high molecular weight immunoglobulins was obtained using this technique. The main groups of proteins involved in the regions of separation are indicated in Fig. 2, which gives a comparative study of colostral whey and sera. For colostral whey a fourth spot is evident in the pattern which is not normally present in chromatograms of serum. This had a more rapid migration than would be expected for peptides and would presumably be prealbumin. In the colostrum the greater proportion of the protein is found in the region involving the immunoglobulins IgA and IgG. This would have been expected from the previous evidence from electrophoresis, and the considerable increase in level of these components in the serum of the suckling pig is readily evident from the comparison of the chromatogram with those of the newborn and colostrum-deprived piglets. Of greater importance from the point of view of the aim of the present studies was the qualitative evidence for an increase in level of high molecular weight components in piglet serum after colostrum ingestion. Components in this region exceeded the exclusion limits for the gel as judged by the migration of Blue Dextran 2000. The IgM immunoglobulins migrate in this region and thus it is evident that components of this size might be involved in the intestinal absorption of immunoglobulins.
Isolation of porcine immunoglobulins and preparation of specific reagentsfor quantitative assays A prerequisite for studies of intestinal absorption of immunoglobulins in the pig is that the individual immunoglobulins should be isolated with sufficient purity to prepare antisera for their detection and also to act as satisfactory protein standards for calibration of the radial immunodiffusion technique. Immunoelectrophoretic analyses of the isolates IgG, IgM and IgA produced by the techniques described in MATERIALSAND METHODSare shown in Fig. 3- The electrophoretic patterns are developed using rabbit antiserum to whole pig serum. These isolates were subsequently used in immunization schedules for the preparation of specific rabbit antisera. Immunoelectrophoresis of the IgG preparation showed multiple precipitation lines which may result from antigenic heterogeneity. Similar observations have been
Fig. 3. Immunoe]ectrophoretic studies of isolated immunoglobulillS IgG (G), IgM (M), IgA (A) compared with whole serum (S). Biochim. Biophys. Acta, 181 (1969) 381-392
386
P. PORTER
reported for human IgO (ref. 18) and bovine IgG (ref. 19). WILLIAMS AND GRABAR18 attributed this to conditions of antibody excess but studies of antigenic fragments prepared from human IgG by papain digestion 2° indicated that the double precipitin line phenomenon was related to the F and S antigenic determinants. Furthermore, mobility differences in [gG preparations are primarily related to differences in the S component. The macroglobulin preparation from sow serum contained at least three components indicated by a strong line for IgM a weak reaction for a2M and a line inside the IgM reaction. The component running closely parallel with IgM was later demonstrated to react with a specific antiserum for IgG and clearly is the high molecular weight IgG studied by KIM et al. 21. Ultracentrifugal analysis of the macroglobulin isolate revealed a single migrating peak with an s~0' w value of 17.8. This material was used to prepare rabbit antiserum which was made specific for IgM by absorption with IgG and IgA preparations. Antibody activity to a2M was removed by absorption with precolostral piglet serum. The isolate provided for a satisfactory preparation of rabbit antiserum. This was a suitable reagent for detection and quantitation of IgM, but the isolate was not entirely satisfactory for calibration of the quantitative radial immunodiffusion technique because of the unknown levels of c~2M and I8-S IgG in the preparation. An improved isolate for calibration was obtained from the euglobulin precipitate of pig serum obtained after dialysis against distilled water for 4 8 h. The precipitate was washed with water, redissolved in Tris saline buffer and the macroglobulin separated by gel filtration on Sephadex G-2oo. The component isolated as IgA had an ultracentrifugal s20,w value of 6.2 and migrated in the fl-globulin region of the imnmnoelectrophoretogram. It reacted with a p o l ~ a l e n t antiserum to IgG and when inoculated into rabbits produced an antiserum which precipitated IgG and IgM. Absorption of this antiserum with isolates of IgG and IgM produced a specific antiserum for a single component in serum. Ultraeentrifugal analysis of the first protein peak of colostrum eluted from Sephadex G-I5O demonstrated a series of peaks with s~0,w values ranging from 6.2 to 17.8. The main amount of tile material appeared in a peak which was partially resolved into two, having a shoulder at lO.8 S and a peak at 8.6 S. By recycling this fraction on Sephadex G-2oo using reverse flow, a double peak was obtained. The IgA component was detected throughout both peaks using the specific antiserum and reactions of identity were obtained with the 6.2-S isolate in immunological double diffusion studies. Thus IgA was present in the initial crude colostrum fraction in a range of molecular sizes. The present communication describes some properties of the component in colostrum and serum; it has subsequently been demonstrated in porcine milk, saliva, intestinal secretions and urine. Antibody reactions against E. coli 0 somatic antigens have been identified with it in colostrum and milk, and it is the major immunoglobulin in sow milk after the first 4 days of lactation. The properties, therefore, seem to be directly analogous to those of human IgA and for the purpose of this communication has been termed IgA. Gel=filtration studies of immunoglobulins of sow serum and colostrum and immunochemical quantitation by radial immunodiffusion Comparative studies of sow serum and colostrum were carried out on Sephadex Biochim. Biophys. dcta, 181 (1969) 381-392
IMMUNOGLOBULINSIgG, IgA AND IgM IN sOW COLOS'rRuM
387
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Fig. 4. Gel filtration of s e r u m and c o l o s t r u m on S e p h a d e x G-coo, giving pooled d a t a and i n d i c a t i n g e l u t i o n of i m r n u n o g l o b u l i n s . I m m u n o e l e c t r o p h o r e t i c analyses for s e r u m fractions using r a b b i t anti-pig s e r u m and for c o l o s t r u m fractions using r a b b i t anti-pig c o l o s t r u m are indicated for each f r a c t i o n to the right of t h e elutioI1 patterns.
G-2oo to obtain information about the molecular size and distribution of the immunoglobulins. The elution patterns, pooling data and immunoelectrophoretic analyses are shown in Fig. 4. In the pattern of elution for colostrum the third peak, which in serum is a major elution band containing mainly albumin, was a very minor elution band and was barely resolved from the large second peak consisting predominantly of 7-S 7-globulins. The fourth peak, peculiar to the colostrum pattern and already mentioned in the thinBiochim. Biophys. Acta, 181 (1969) 381-392
388
P. PORTER
RADIUS(¢m 2 ) 08" 07-
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Fig. 5. Comparative immunodiffusion s t u d y using specific rabbit a n t i s e r u m to IgG d e m o n s t r a t i n g the presence of I8-S IgG. CI and C7 represent colostrum Fractions i and 7 and SI and S 7 represent serum Fractions I and 7 taken from Sephadex G-2oo (Fig. 4). Fig. 6. Q u a n t i t a t i o n of porcine immunoglobulins by radial immunodiffusion. Calibration with purified preparations standardized by biuret. × ×, IgM; 1~--O, IgA; It--It, IgG.
layer analysis, appeared ahead of the normal peptide peak for the colostrum and serum. Analysis by polyacrylamide disc electrophoresis indicated this to be mainly prealbumin with some albumin. The position of elution of IgG, IgA and IgM as identified in immunological double diffusion studies with specific antisera are shown in the elution pattern (Fig. 5). In connection with these observations two points are worthy of note. Firstly, a component reacting with rabbit antiserum specific for porcine 7-S IgG was identified in serum and colostral fractions at the exclusion limits of the Sephadex columns. This component from serum and colostrum showed a line of identity with 7-S IgG in agar gel double-diffusion studies confirming the presence of immunoglobulin IgG in I8-S fractions (Fig. 5). Thus it is clear that the I8-S IgG component is is transferred to the colostrum. Secondly, radial immunodiffusion studies indicated that the distribution of IgA in the elution pattern for colostrum differed from that in the sow serum. Colostral IgA appeared predominantly in the high molecular weight fractions, the peak activity being in Fractions 3 and 4, and falling off through Fractions 4-9. Serum IgA appeared in a two-peak distribution in gel filtration Fractions 3-8 with the peaks of activity in Fractions 4 and 7Typical calibration curves for assay of the three immunoglobulins are shown in Fig. 6. Good linear relationships between area of radial diffusion and amounts of immunoglobulin antigen were obtained. The lower limits for sensitivity of the technique were set on the basis that 2 m m diameter could be measured with 5 % error and on this basis the lower limits for assay of IgG, IgA and IgM corresponded to 0.75, 2 and 3.5 #g, respectively, or concentrations of 5, 13.3 and 23.3 mg/Ioo ml applying 15/zl of solution to the antigen well. Assays of immunoglobulins in colostrum samples taken from sows at parturition are given in Table I. The total immunoglobulin accounts for 49.2-75.9% (mean 63.6%) of the total protein in the colustrum and most of this 65.3-89.6% (mean Biochim. Biophys. Acla, 181 (1969) 381-392
IMMUNOGLOBULINS
IgG, IgA AND IgM IN
SOW COLOSTRUM
389
TABLE I ASSAYS OF IMMUNOGLOBULINS
IgG, IgA
AND
IgM
IN SOW COLOSTRUM
Sample No.
IgG (g/zoo ml)
IgA (g/zoo ml)
IgM (g/Ioo rot)
Total protein (g/zoo ml)
712 873 5 1156 500 794 640 lO6 645
6.42 3.9 ° 5 .8o 5.5 ° 6.95 4 .1° 6.42 7.75 5.95
0.95 o.75 i.ii 0.53 i .98 0.52 i. i i 0.69 1.98
0.25 o.32 o.36 o.21 o. 25 0.29 0.59 o.21 0.36
12.88 8.66 lO.33 8.22 18.66 9.11 13.66 12.66 12.33
Mean -c S.E.
5.87 q- 1.25
1.o 7 ± 0.56
0.32 ± o.12
11.83 4- 3.25
Serum -L S.E. (5 samples)
2.15 ± 0.24
o.18 =E 0.o5
o. i i ± 0.03
6.84 ± 0.33
79.7 %) is in t h e f o r m o f i m m u n o g l o b u l i n IgG. I m m u n o g l o b u l i n a s s a y s in n o r m a l sow s e r u m are g i v e n for c o m p a r i s o n . T h e levels o f t h e i n d i v i d u a l i m m u n o g l o b u l i n s in all t h e c o l o s t r u m s a m p l e s e x c e e d t h o s e in t h e s e r u m . H o w e v e r , t h e a s s a y s w o u l d i n d i c a t e t h a t t h e i m m u n o g l o b u l i n s in t h e c o l o s t r u m do n o t s i m p l y r e p r e s e n t a c o n c e n t r a t e o f t h o s e in t h e s e r u m , since IgA a c c o u n t s for a m u c h h i g h e r p r o p o r t i o n of t h e i m m u n o g l o b u l i n in t h e c o l o s t r u m t h a n in t h e s e r u m . T h e levels o f I g G a n d I g M are a p p r o x . 2 - 3 t i m e s g r e a t e r t h a n t h e m e a n s e r u m c o n c e n t r a t i o n s , w h e r e a s t h e l e v e l o f I g A in t h e c o l o s t r u m in s o m e cases s h o w e d an I I - f o l d i n c r e a s e o v e r t h e m e a n v a l u e d e t e r m i n e d in t h e s e r u m .
Immunoglobulins in the sera of precolostral and suckling pigs Investigations were carried out by immunoelectrophoresis and immunodiffusion r e a c t i o n s u s i n g r a b b i t a n t i s e r a t o a d u l t p i g s e r u m a n d t h e specific a n t i s e r a to t h e t h r e e g r o u p s of i m m u n o g l o b u l i n s . T h e d e f i c i e n c y in i m m u n o g l o b u l i n s in pre-
O@@ @0 Fig. 7. Immunoelectrophoretic studies of piglet sera compared with sow serum and colostrum demonstrating the presence of immunoglobulins IgG, IgA and IgM in postcolostral serum, and a component in precolostral serum reacting with rabbit antiserum to porcine immunoglobulins (indicated by arrow). S, serum; Ab, antibody. Fig. 8. Comparative immunodiffusion study using specific rabbit antiserum (R) to IgG demonstrating the reaction of partial identity between IgG (G) and the component in precolostral piglet .~erum (P),
Biochim, Biophys. Acta, 181 (1969) 381-392
39 °
P, PORTER
colostral sera was readily evident from the immunoelectrophoretogram (Fig. 7)- A reaction in the ),-globulin region was evident when the imnmnoeleetrophoretogram was developed against a composite antiserum to porcine immunoglobulins. Immunological double-diffusion reactions with specific antisera detected a reaction of partial identity for immunoglobulin IgG (Fig. 8), but reactions for IgA and IgM could not be demonstrated. Assays in 8 samples of precolostral sera failed to detect IgG in concentrations greater than 5o #g/ml. This is approx. 5oo times less than is found in the serum of the piglet 24 h after ingestion of colostrum, and is therefore quantitatively unimportant in the consideration of intestinal absorption of immunoglobulins by the neonatal piglet. The presence of the imnmnoglobulins IgG, IgA and IgM is easily recognized in the immunoelectrophoresis pattern of the suckling pig serum (see Fig. 7 in which comparison is also made with adult and precolostral sera, and colostrum). Three lines of reaction against a composite antiserum to the immunoglobulins are identifiable in the immunoelectrophoretogram of postcolostral sera. In studies of 5 litters of piglets at 2 days old, the absorption of all three groups of immunoglobulins has been indicated by immunological double-diffusion studies and immunoelectrophoresis, quantitative assays are given in Table II. The serum concentration of IgG and IgM in the 2-dayold piglets were mainly similar to adult levels. However, the most interesting feature of these studies is the level of IgA in postcolostral sera, this generally exceeded adult levels, in some cases by as much as 3-4 times. TABLE II ASSAYS
OF IMMUNOGLOBULINS
IgG, IgA
AND
IgM
IN SERUM
OF PIGLETS
AT 2 DAYS
Litter No.
Number of pigs
IgG (mg/ioo ml)
IgA (mg/ioo ml)
IgM (mg/ioo ml)
N5
12
65o-187o (I32O ~ 42o)
172-485 (3o4 i 85)
35 - I 4° (87.5 ~ 36.4)
W X 500
6
173o 2500 (218o ± 240 )
250-660 (485 ± 98)
61 162 (96.6 ± 39.5)
W X lO6
8
177o 235 ° (2o90 ± 220)
149-211 (189 i 22)
64-136 (94.6 ± 32.4)
N2
16
186o 236 (2040 ± 16o)
41o 840 (586 ~ 181)
85-16o (118.2 ~ 26.6)
T37
12
52o-244 o (188o ± 690)
85-690 (402 ~ 199)
61-175 (lO9.5 ~ 34.4)
DISCUSSION
The immunoglobulins IgG, IgA and IgM all appear in porcine colostrum at very much higher concentrations than is found for serum. This is contrary to the findings for bovine and human colostrum. IgA and IgM globulins were present in bovine colostrum in concentrations lower than in the serum 2~. Quantitatively the main immunoglobulin in human colostrum is IgA and the IgG molecule, which is the major Biochim. Biophys. Acla, 181 (1969) 381-392
IMMUNOGLOBULINS IgG, IgA AND IgM IN SOW COLOSTRUM
391
immunoglobulin in the colostrum of the pig, accounts for only IO °/o of human colostral immunoglobulin 23. MURPHY et al. 2~ considered their findings in the bovine only from the point of view of selectivity of transport of ?-globulins from the blood serum to the colostrum as determined by the m a m m a r y acinar epithelium. They did not consider the possibility of local synthesis within the gland. This is clearly a possibility, since the assays in Table I indicate that IgA accounts for a much higher proportion of the immunoglobulins of the colostvum than in the serum; also in gel filtration studies (Fig. 5) IgA was found to predominate in higher molecular weight fractions in the colostrum than in the serum. The significance of the wide range of molecular sizes of the IgA molecule is only becoming apparent from recent work. Investigations reviewed by TOMAS124 demonstrate that secretory IgA differs from serum IgA in possessing a complexed nonglobulin component called the transport piece. This component appears to be synthesized in the epithelial cell and has been partially characterized, having a molecular weight of approx. 50 ooo. It is possibly related to the transport of the immunoglobulin to the mucoid surface. In man the secretory IgA has a molecular weight of 39 ° ooo compared with 17o ooo for serum IgA. Clearly the IgA in sow colostrum is not limited to a polymeric form, since IgA appeared in gel filtration fractions over a wide molecular weight range. It is possible that the high molecular weight form is synthesized in the m a m m a r y gland and that lower molecular weight components are transferred directly from the serum. In the newborn pig there is no evidence for the transplacental transfer of IgA and IgM, but a component antigenically related to IgG could be detected. All three specific immunoglobulins, IgG, IgA and IgM, as characterized in the chromatographic and immunological studies with sow serum and colostrum, are absorbed b y the neonatal piglet. IgM plays an important part in the primary immune response and natural antibodies to Gram-negative bacteria are also associated with this immunoglobulin 2~. There is evidence to suggest that in the pig preexperience of antibody is required for its synthesis z6, thus it seems important that the I8-S IgM molecule should be absorbed from colostrum. This would provide for a passive immunity against Gram-negative bacteria in the neonatal pig and satisfy the apparent requirements for subsequent immunocompetence. On this latter point it m a y be significant that colostrum-deprived pigs do not synthesize IgM in their early immune response 27. LOCKE et al. 9 claimed that I8-S antibodies were not absorbed across the intestine of the newborn pig and suggested that the I8-S molecule lacked structures which corresponded to cellular receptors for intestinal transmission of immunoglobulins. Obviously such an hypothesis is unnecessary, since the I8-S IgM molecule is absorbed, and the quantitative assays (Tables I and II) would not suggest any differential absorption of IgM in relation to IgG. It is difficult to understand how LOCKE et al. 9 failed to demonstrate the absorption of the I8-S immunoglobulin, particularly since it appears in postcolostral sera at levels comparable with adult serum. This also has further implications for the work of these authors, since the interpretation of their results was based on the premise that the piglet intestine would not transport I8-S immunoglobulin. It is very interesting that the levels of IgA in postcolostral sera should so far exceed the adult level in so m a n y of the neonatal animals investigated. Possibly this immunoglobulin plays an important role in the immunological defence of the young Biochim. Biophys. Acta,
181 (1969) 381-392
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P. PORTER
piglet. Investigations of this colostral immunoglobulin are continuing in greater detail particularly with reference to antibody to Gram-negative bacteria. REFERENCES i B. W. KARLSSON, Acta Pathol. Microbiol. Scand., 67 (1966) 83. 2 J. F. FOSTER, R. W, FRIEDELL, D. CATRON AND N. R. DICKMAN, Arch. Biochem. Biophys., 31 (1951) lO 4 . 3 J. G. LECCE AND G. MATRONE, J. Nutr., 73 (1961) 167 . 4 J. G. LECCE, D. A. MORGAN AND G. MATRONE, J. Nutr., 77 (1962) 349. 5 L. C. PAYNE AND C. L. MARSH, J. Nutr., 76 (1962) 151. 6 R. H. ]3ARNES, Nutr. Rev., 20 (1962) 316. 7 Y. B. KIM, S. G. ]3RADLEY AND D. N. WATSON, Swine in Biomedical Res. Proc. Symp. BattelleNorthwest, 1965, p. 273. 8 J. J. CEBRA AND J. ]3. ROBBINS, J. Immunol., 97 (1966) 12. 9 R. F. LOCKE, D. SEGRE AND W. L. MYERS, J. Immunol., 93 (1964) 576. io R. HALLIDAY AND R. A. KECKWICK, Proe. Roy. Sue. London, Set. B., 153 (196o) 279. i i I. G. MORRIS, Immunology, 13 (1967) 49. 12 P. PORTER, J. Cutup. Pathol. Therap., 74 (1964) lO8. 13 L. ORSTEIN AND ]3. J. DAVIS, Ann. N . Y . Acad. Sci., 121 (1964) 321. 14 P. PORTER, M. C. PORTER AND J. M. SHANBERGE, Biochemistry, 6 (1967) 1854. 15 R. AUGUSTIN AND M. J. HAYWARD, Immunology, 3 (196o) 45. 16 G. MANCINI, O. CARBONARA AND J. F. HEREMANS, Immunochemistry, 2 (1965) 3517 H. W. ]3OHREN AND V. R. WENNER, J. Dairy Sci., 44 (1961) 1213 . 18 C. A. WILLIAMS AND 1:). J. GRABAR, Immunology, 74 (1955) 158. 19 F. A. MURPHY, O. AALUND, J. W. OSEBOLD A~!D E. J. CARROLL, Arch. Biochem. Biophys., lO8 (1964) 23 ° . 20 G. M. EDELMAN, J. F. HEREMANS, M. T. HEREMANS AND H. G. KUNKEL, J. Exptl. Med., 112 (196o) 203 . 21 Y. ]3. KIM, S. G. ]3RADLEY AND D. N. WATSON, J. Immunol., i o i (1965) 224. 22 F. A. MURPHY, O. AALUND, J. W. OSEBOLD AND E. J. CARROLL, Arch. Biochem. Biophys., lO8 (1964) 230. 23 G. DE MURALT, E. GUGLER AND D. L. A. ROULET, in H. PETERS, Protides of the Biologica~ Fluids, 7th Colloq., Elsevier, A m s t e r d a m , 196o, p. 166. 24 T. ]3. TOMASI, Hospital Practice, 1967, Vol. 2, p. 26. 25 J. G. MICHAEL AND F. S. ROSEN, J. Exptl. Med., 118 (1963) 619. 26 I). SEGRE AND M. L. KAEBER, J. Immunol., 89 (1962) 79 o. 27 Y. 13. KIM, S. G. BRADLEY AND D, N. WATSON, J . Immunol., 97 (1966) 189.
Biochim. Biophys. Acta, 171-(1969 ) 381-392
38I
BBA 35389 T R A N S F E R OF IMMUNOGLOBULINS IgG, IgA AND IgM TO LACTEAL S E C R E T I O N S IN T H E P A R T U R I E N T SOW AND T H E I R A B S O R P T I O N BY THE NEONATAL PIGLET
P. P O R T E R
Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford (Great Britain) (Received March 24th , 1969)
SUMMARY
Porcine immunoglobulins IgG, IgA and IgM have been isolated and used in immunochemical studies of the secretion of these components into colostrum and then intestinal absorption b y the neonatal piglet. Assays on 9 samples of colostrum from different sows indicated that 63.6 ± 8.6 % of the whey protein was accounted for b y the immunoglobulins which comprise IgG (79.7 :E I6.9%), IgA (14.o 5 ~- 7.35%) and IgM (6.27 :E: 2.35%). Immunoglobulins IgG and IgM occur in the colostrum at approx. 2-3 times the level in serum but the level of IgA in colostrum was 3-11 times greater than in serum. Molecular size characteristics of the immunoglobulins in sow serum and colostrum have been obtained in Sephadex G-2oo gel filtration studies. Colostral IgA is eluted in a wide range of molecular sizes in the gel filtration pattern but the high molecular weight forms are predominant. A serum component with a sedimentation constant (s~0' w) of 17.8 which is antigenically distinct from IgM but gives reactions of identity with IgG, also appears in colostrum. Precolostral piglet serum was almost entirely deficient of immunoglobulins. A component having shared antigenic determinants with IgG was present in concentrations less than 50 #g/ml. Studies of 6 litters of piglets indicated that immunoglobulins IgG, IgA and IgM were absorbed from the colostrum. The post colostral serum concentrations of IgG and IgM were mainly similar to adult levels, whilst IgA generally exceeded adult levels, in some cases by as much as 3-4 times.
INTRODUCTION
The serological relationships between proteins of serum and milk have been established for several animal species including the pig 1. The post-partum transport of colostral proteins from mother to offspring is of tremendous immunological importance to the pig, since there is apparently little or no transplacental transfer of immunity. The absorption of ~-globulin into the blood stream of the neonatal piglet Biochim. Biophys. Aeta, 181 (1969) 381-392
382
P. PORTER
via the gut has been demonstrated by the marked change in the serum electrophoretic profile following the ingestion of colostrum, heterologous or homologous 7-globulins2 8. The accent has been on the v-globulins as a whole, without making any distinction between the immunoglobulins IgM, IgA, IgG or placing any emphasis on the nature of passive immunity derived from the colostrum. The 7-S IgG molecule accounts for the greatest proportion of the immunoglobulins in the colostrum of the pig and the studies to date undoubtedly relate almost entirely to this molecule. Some authors 1,7 state that immunoglobulins IgA and IgM are absorbed from colostrum. These findings are based on the appearance of certain precipitin lines in the v-globulin region of immunoelectrophoretograms of postcolostral sera, but without characterizing the components. In view of the finding that IgA in rabbit colostrum migrates on the anodic side of the electrophoretogram s, one cannot safely extrapolate from the well-characterised human electrophoretogram to other species. The intestinal absorption of IgM immunoglobulin is the subject of dispute, since there is evidence of the failure of IgM to be absorbed by the intestine of the newborn pig s. This is also in agreement with results reported for young rats1°, n. In this paper, immunoglobulins IgM, IgA and IgG are isolated, characterized, and assayed in the colostrum and serum of the sow. Quantitative evidence is presented for the intestinal absorption of each of these immunoglobulins b y the neonatal piglet. MATERIALS AND METHODS
Microelectrophoresis Protein samples and fractions were examined by cellulose acetate electrophoresis and immunoelectrophoresis 12. Disc electrophoresis was carried out in polyacrylamide gels 13.
Gelfiltration chromatography Chromatography was carried out in Sephadex G-2oo columns (45 cm × 2.5 cm) and Sephadex G-I5O columns (9° cm × 2.5 cm) using o.I M Tris saline buffer (pH 7.2). Thin-layer gel-filtration studies were made using Sephadex G-2oo superfine 14.
Ion-exchange chromatography Anion-exchange chromatography was carried out on a DEAE-cellulose column (30 cm × 2.0 cm). The stepwise elution entailed the use of four buffers: (I) o.oi M NaH2PO 4 adjusted to pH 7.6 with o.oi M NaOH, (2) 0.02 M NaH~PO 4 adjusted to pH 6.3 with 0.02 M NaOH, (3) 0-05 M NaH2P04, and (4) 0.3 M NaH2PO 4. A fifth buffer, 0. 4 M NaH2PO 4 containing 2 M NaC1, was used to wash firmly bound protein from the column after each run before regenerating with the starting buffer ~5, Cation-exchange chromatography was carried out using CM-cellulose columns (20 cm × 2.0 cm). This technique was used to isolate 7-S immunoglobulin from fractions of pig serum obtained by anion-exchange chromatography. For this purpose a fairly limited stepwise elution procedure using phosphate buffer (pH 5.8) and molarities 0,005 M, o.oi M, 0.02 M, o.04 M in sequence, were found to be adequate. Samples were prepared for ion-exchange chromatography by pressure dialysis against the starting buffer for 24 h or by passing through a Sephadex G-25 column (45 cm × 5 cm) equilibrated with starting buffer. Biochim. Biophys. Acta, 181 (1969) 381-392
IMMUNOGLOBULINS IgG, IgA AND igM IN SOW COLOSTRUM
383
Flow rates up to 2oo ml/h were maintained with the ion-exchange colmnns by use of a suitable hydrostatic head. Eluates were automatically scanned at 28o m# and collected using L K B ultrarac collector and drop counter. Fractions were pooled from the eluates b y reference to the elution pattern.
Isolation of specific immunoglobulins The preparation of purified immunoglobulin components was essential to the investigations. The quantitative studies required the preparation of antisera which reacted specifically with each immunoglobulin. A crude preparation of 7-S IgG was obtained from adult pig serum by precipitation with lO% Na2SO 4. This was further purified b y chromatography on D E A E cellulose. The "fall through" fraction eluted with the first buffer was recovered and subjected to further chromatography on CM-cellulose. The eluates obtained at p H 5.8 with 0.02 M phosphate buffer provided a pure sample of 7-S IgG when examined by immunoelectrophoresis using a rabbit antiserum to whole pig serum. A crude preparation of I9-S IgM is obtained in the eluates with Buffer 4 on chromatography of adult serum on DEAE-cellulose. This fraction contains predominantly albumin and a number of low molecular a-globulins. Chromatography on Sephadex G-2oo separated the macroglobulins in this fraction in eluates obtained at the exclusion limits of the column. Sow colostrum was used as a source of IgA. A Ioo-ml sample of colostral whey was fractionated on Sephadex G-I5O (800 cm × 60 cm) using o.I M Tris-saline buffer (pH 7.2). A crude preparation of colostral IgA is obtained in the first protein peak eluted from the column which also contains IgM. IgA is isolated from this crude fraction by chromatography on DEAE-cellulose. The fraction was applied to the column in o.oi M phosphate buffer pH 7.6 and elution was carried out using a simple increasing salt gradient obtained by the continuous addition of 0.3 M NaC1 to the starting buffer. The greater part of the protein was eluted from the column in a broad tailing peak commencing when the eluting buffer was 0.09 M and terminating when the buffer was 0.22 M. In the first phase of elution of this peak at pH 7.6 a sample of IgA was obtained which was pure as judged b y the criteria of immunoelectrophoresis using rabbit antisera to pig serum and pig colostral whey proteins and also b y ultracentrifugation.
Preparation of rabbit antisera specificfor porcine IgG, IgA and IgM Antisera were prepared b y injection of emulsions of the immunoglobulin isolates with Freunds Bacto-Adjuvant, Incomplete, into New Zealand white rabbits 12. Immunological cross reactivity due to shared antigenic determinants on the immunoglobulins was removed by absorption with the purified nonspecific immunoglobulin. The IgM preparation was contaminated with a2M and this activity was removed from the rabbit antiserum by absorption with precolostral piglet serum.
Quantitative estimation of immunoglobulins Immunoglobulin levels in sera and colostrum samples were estimated by radial immunodiffusion TM.
Biochim. Biophys. Acta, i81 (1969) 381-392
384.
P. PORTER
Analytical ultracentrifugation Ultracentrifugal analysis was carried out in a Beckman model E centrifuge equipped with phase-plate schlieren diaphram. Sedimentation coefficients were determined at 20 ° employing rotor speeds of 59 780 rev./min.
Preparation of sow's colostral whey Colostrum was collected from pigs at parturition and casein and free fat were removed by centrifugation at 4 ° ooo rev./min at 20 ° (IOO ooo × g) for 30 rain 17. The whey was carefully removed by pipette and recentrifuged under the same conditions to ensure the complete removal of any suspension included in the process of pipetting.
Serum from piglets and sows Piglets were bled from the anterior vena cava and sows from an ear vein. RESULTS
Preliminary studies (using micro techniques) of the nature of sow colostral whey and the intestinal absorption of components by the neonatal piglet In the 2-day-old suckling piglet the considerable absorption of immunolactoglobulin is readily evident from polyacrylamide gel electrophoretie analyses. CornTHEN - LAYER " GEL "tlI[TIIATtQN
COLQSTRUM DIPll IV|T1 [~l~ GAY OLD~
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SUCKLED PIGLET [ 2 IDAYOLD 1
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Fig. I. P o l y a c r y l a m i d e gel electrophoretic studies of c o l o s t r u m a n d sera. (a) Precolostral s e r u m ; (b) postcolostral s e r u m , 2-days-old piglet; (c) colostral w h e y ; (d) sow s e r u m ; (e) s e r u m f r o m suckled piglet, 35 d a y s old; (f) s e r u m f r o m c o l o s t r u m - d e p r i v e d piglet, 35 d a y s old. Fig. 2. T h i n - l a y e r gel-filtration studies of c o l o s t r u m a n d sera i n d i c a t i n g t h e areas of m i g r a t i o n of major components.
Biochim. Biophys. Mcta, 181 (1969) 381-392
IMMUNOGLOBULINSIgG, IgA AND igM IN SOW COLOSTRUM
385
parative studies of sera and colostral whey are shown in Fig. I. The total protein in pooled serum from a litter of newborn piglets was 2.8 g/ioo ml, whilst that of a pooled sample of postcolostral serum at 2 days was 5.4 g/Ioo ml. This represents a nearly 2-fold increase in serum concentration which the polyacrylamide gel electrophoresis indicates is due mainly to the absorption of the 7-S immunoglobulins. Thin-layer gel filtration using Sephadex G-2oo, provides for a separation of proteins which is dependent upon molecular size. Preliminary evidence for the intestinal absorption of high molecular weight immunoglobulins was obtained using this technique. The main groups of proteins involved in the regions of separation are indicated in Fig. 2, which gives a comparative study of colostral whey and sera. For colostral whey a fourth spot is evident in the pattern which is not normally present in chromatograms of serum. This had a more rapid migration than would be expected for peptides and would presumably be prealbumin. In the colostrum the greater proportion of the protein is found in the region involving the immunoglobulins IgA and IgG. This would have been expected from the previous evidence from electrophoresis, and the considerable increase in level of these components in the serum of the suckling pig is readily evident from the comparison of the chromatogram with those of the newborn and colostrum-deprived piglets. Of greater importance from the point of view of the aim of the present studies was the qualitative evidence for an increase in level of high molecular weight components in piglet serum after colostrum ingestion. Components in this region exceeded the exclusion limits for the gel as judged by the migration of Blue Dextran 2000. The IgM immunoglobulins migrate in this region and thus it is evident that components of this size might be involved in the intestinal absorption of immunoglobulins.
Isolation of porcine immunoglobulins and preparation of specific reagentsfor quantitative assays A prerequisite for studies of intestinal absorption of immunoglobulins in the pig is that the individual immunoglobulins should be isolated with sufficient purity to prepare antisera for their detection and also to act as satisfactory protein standards for calibration of the radial immunodiffusion technique. Immunoelectrophoretic analyses of the isolates IgG, IgM and IgA produced by the techniques described in MATERIALSAND METHODSare shown in Fig. 3- The electrophoretic patterns are developed using rabbit antiserum to whole pig serum. These isolates were subsequently used in immunization schedules for the preparation of specific rabbit antisera. Immunoelectrophoresis of the IgG preparation showed multiple precipitation lines which may result from antigenic heterogeneity. Similar observations have been
Fig. 3. Immunoe]ectrophoretic studies of isolated immunoglobulillS IgG (G), IgM (M), IgA (A) compared with whole serum (S). Biochim. Biophys. Acta, 181 (1969) 381-392
386
P. PORTER
reported for human IgO (ref. 18) and bovine IgG (ref. 19). WILLIAMS AND GRABAR18 attributed this to conditions of antibody excess but studies of antigenic fragments prepared from human IgG by papain digestion 2° indicated that the double precipitin line phenomenon was related to the F and S antigenic determinants. Furthermore, mobility differences in [gG preparations are primarily related to differences in the S component. The macroglobulin preparation from sow serum contained at least three components indicated by a strong line for IgM a weak reaction for a2M and a line inside the IgM reaction. The component running closely parallel with IgM was later demonstrated to react with a specific antiserum for IgG and clearly is the high molecular weight IgG studied by KIM et al. 21. Ultracentrifugal analysis of the macroglobulin isolate revealed a single migrating peak with an s~0' w value of 17.8. This material was used to prepare rabbit antiserum which was made specific for IgM by absorption with IgG and IgA preparations. Antibody activity to a2M was removed by absorption with precolostral piglet serum. The isolate provided for a satisfactory preparation of rabbit antiserum. This was a suitable reagent for detection and quantitation of IgM, but the isolate was not entirely satisfactory for calibration of the quantitative radial immunodiffusion technique because of the unknown levels of c~2M and I8-S IgG in the preparation. An improved isolate for calibration was obtained from the euglobulin precipitate of pig serum obtained after dialysis against distilled water for 4 8 h. The precipitate was washed with water, redissolved in Tris saline buffer and the macroglobulin separated by gel filtration on Sephadex G-2oo. The component isolated as IgA had an ultracentrifugal s20,w value of 6.2 and migrated in the fl-globulin region of the imnmnoelectrophoretogram. It reacted with a p o l ~ a l e n t antiserum to IgG and when inoculated into rabbits produced an antiserum which precipitated IgG and IgM. Absorption of this antiserum with isolates of IgG and IgM produced a specific antiserum for a single component in serum. Ultraeentrifugal analysis of the first protein peak of colostrum eluted from Sephadex G-I5O demonstrated a series of peaks with s~0,w values ranging from 6.2 to 17.8. The main amount of tile material appeared in a peak which was partially resolved into two, having a shoulder at lO.8 S and a peak at 8.6 S. By recycling this fraction on Sephadex G-2oo using reverse flow, a double peak was obtained. The IgA component was detected throughout both peaks using the specific antiserum and reactions of identity were obtained with the 6.2-S isolate in immunological double diffusion studies. Thus IgA was present in the initial crude colostrum fraction in a range of molecular sizes. The present communication describes some properties of the component in colostrum and serum; it has subsequently been demonstrated in porcine milk, saliva, intestinal secretions and urine. Antibody reactions against E. coli 0 somatic antigens have been identified with it in colostrum and milk, and it is the major immunoglobulin in sow milk after the first 4 days of lactation. The properties, therefore, seem to be directly analogous to those of human IgA and for the purpose of this communication has been termed IgA. Gel=filtration studies of immunoglobulins of sow serum and colostrum and immunochemical quantitation by radial immunodiffusion Comparative studies of sow serum and colostrum were carried out on Sephadex Biochim. Biophys. dcta, 181 (1969) 381-392
IMMUNOGLOBULINSIgG, IgA AND IgM IN sOW COLOS'rRuM
387
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Fig. 4. Gel filtration of s e r u m and c o l o s t r u m on S e p h a d e x G-coo, giving pooled d a t a and i n d i c a t i n g e l u t i o n of i m r n u n o g l o b u l i n s . I m m u n o e l e c t r o p h o r e t i c analyses for s e r u m fractions using r a b b i t anti-pig s e r u m and for c o l o s t r u m fractions using r a b b i t anti-pig c o l o s t r u m are indicated for each f r a c t i o n to the right of t h e elutioI1 patterns.
G-2oo to obtain information about the molecular size and distribution of the immunoglobulins. The elution patterns, pooling data and immunoelectrophoretic analyses are shown in Fig. 4. In the pattern of elution for colostrum the third peak, which in serum is a major elution band containing mainly albumin, was a very minor elution band and was barely resolved from the large second peak consisting predominantly of 7-S 7-globulins. The fourth peak, peculiar to the colostrum pattern and already mentioned in the thinBiochim. Biophys. Acta, 181 (1969) 381-392
388
P. PORTER
RADIUS(¢m 2 ) 08" 07-
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Fig. 5. Comparative immunodiffusion s t u d y using specific rabbit a n t i s e r u m to IgG d e m o n s t r a t i n g the presence of I8-S IgG. CI and C7 represent colostrum Fractions i and 7 and SI and S 7 represent serum Fractions I and 7 taken from Sephadex G-2oo (Fig. 4). Fig. 6. Q u a n t i t a t i o n of porcine immunoglobulins by radial immunodiffusion. Calibration with purified preparations standardized by biuret. × ×, IgM; 1~--O, IgA; It--It, IgG.
layer analysis, appeared ahead of the normal peptide peak for the colostrum and serum. Analysis by polyacrylamide disc electrophoresis indicated this to be mainly prealbumin with some albumin. The position of elution of IgG, IgA and IgM as identified in immunological double diffusion studies with specific antisera are shown in the elution pattern (Fig. 5). In connection with these observations two points are worthy of note. Firstly, a component reacting with rabbit antiserum specific for porcine 7-S IgG was identified in serum and colostral fractions at the exclusion limits of the Sephadex columns. This component from serum and colostrum showed a line of identity with 7-S IgG in agar gel double-diffusion studies confirming the presence of immunoglobulin IgG in I8-S fractions (Fig. 5). Thus it is clear that the I8-S IgG component is is transferred to the colostrum. Secondly, radial immunodiffusion studies indicated that the distribution of IgA in the elution pattern for colostrum differed from that in the sow serum. Colostral IgA appeared predominantly in the high molecular weight fractions, the peak activity being in Fractions 3 and 4, and falling off through Fractions 4-9. Serum IgA appeared in a two-peak distribution in gel filtration Fractions 3-8 with the peaks of activity in Fractions 4 and 7Typical calibration curves for assay of the three immunoglobulins are shown in Fig. 6. Good linear relationships between area of radial diffusion and amounts of immunoglobulin antigen were obtained. The lower limits for sensitivity of the technique were set on the basis that 2 m m diameter could be measured with 5 % error and on this basis the lower limits for assay of IgG, IgA and IgM corresponded to 0.75, 2 and 3.5 #g, respectively, or concentrations of 5, 13.3 and 23.3 mg/Ioo ml applying 15/zl of solution to the antigen well. Assays of immunoglobulins in colostrum samples taken from sows at parturition are given in Table I. The total immunoglobulin accounts for 49.2-75.9% (mean 63.6%) of the total protein in the colustrum and most of this 65.3-89.6% (mean Biochim. Biophys. Acla, 181 (1969) 381-392
IMMUNOGLOBULINS
IgG, IgA AND IgM IN
SOW COLOSTRUM
389
TABLE I ASSAYS OF IMMUNOGLOBULINS
IgG, IgA
AND
IgM
IN SOW COLOSTRUM
Sample No.
IgG (g/zoo ml)
IgA (g/zoo ml)
IgM (g/Ioo rot)
Total protein (g/zoo ml)
712 873 5 1156 500 794 640 lO6 645
6.42 3.9 ° 5 .8o 5.5 ° 6.95 4 .1° 6.42 7.75 5.95
0.95 o.75 i.ii 0.53 i .98 0.52 i. i i 0.69 1.98
0.25 o.32 o.36 o.21 o. 25 0.29 0.59 o.21 0.36
12.88 8.66 lO.33 8.22 18.66 9.11 13.66 12.66 12.33
Mean -c S.E.
5.87 q- 1.25
1.o 7 ± 0.56
0.32 ± o.12
11.83 4- 3.25
Serum -L S.E. (5 samples)
2.15 ± 0.24
o.18 =E 0.o5
o. i i ± 0.03
6.84 ± 0.33
79.7 %) is in t h e f o r m o f i m m u n o g l o b u l i n IgG. I m m u n o g l o b u l i n a s s a y s in n o r m a l sow s e r u m are g i v e n for c o m p a r i s o n . T h e levels o f t h e i n d i v i d u a l i m m u n o g l o b u l i n s in all t h e c o l o s t r u m s a m p l e s e x c e e d t h o s e in t h e s e r u m . H o w e v e r , t h e a s s a y s w o u l d i n d i c a t e t h a t t h e i m m u n o g l o b u l i n s in t h e c o l o s t r u m do n o t s i m p l y r e p r e s e n t a c o n c e n t r a t e o f t h o s e in t h e s e r u m , since IgA a c c o u n t s for a m u c h h i g h e r p r o p o r t i o n of t h e i m m u n o g l o b u l i n in t h e c o l o s t r u m t h a n in t h e s e r u m . T h e levels o f I g G a n d I g M are a p p r o x . 2 - 3 t i m e s g r e a t e r t h a n t h e m e a n s e r u m c o n c e n t r a t i o n s , w h e r e a s t h e l e v e l o f I g A in t h e c o l o s t r u m in s o m e cases s h o w e d an I I - f o l d i n c r e a s e o v e r t h e m e a n v a l u e d e t e r m i n e d in t h e s e r u m .
Immunoglobulins in the sera of precolostral and suckling pigs Investigations were carried out by immunoelectrophoresis and immunodiffusion r e a c t i o n s u s i n g r a b b i t a n t i s e r a t o a d u l t p i g s e r u m a n d t h e specific a n t i s e r a to t h e t h r e e g r o u p s of i m m u n o g l o b u l i n s . T h e d e f i c i e n c y in i m m u n o g l o b u l i n s in pre-
O@@ @0 Fig. 7. Immunoelectrophoretic studies of piglet sera compared with sow serum and colostrum demonstrating the presence of immunoglobulins IgG, IgA and IgM in postcolostral serum, and a component in precolostral serum reacting with rabbit antiserum to porcine immunoglobulins (indicated by arrow). S, serum; Ab, antibody. Fig. 8. Comparative immunodiffusion study using specific rabbit antiserum (R) to IgG demonstrating the reaction of partial identity between IgG (G) and the component in precolostral piglet .~erum (P),
Biochim, Biophys. Acta, 181 (1969) 381-392
39 °
P, PORTER
colostral sera was readily evident from the immunoelectrophoretogram (Fig. 7)- A reaction in the ),-globulin region was evident when the imnmnoeleetrophoretogram was developed against a composite antiserum to porcine immunoglobulins. Immunological double-diffusion reactions with specific antisera detected a reaction of partial identity for immunoglobulin IgG (Fig. 8), but reactions for IgA and IgM could not be demonstrated. Assays in 8 samples of precolostral sera failed to detect IgG in concentrations greater than 5o #g/ml. This is approx. 5oo times less than is found in the serum of the piglet 24 h after ingestion of colostrum, and is therefore quantitatively unimportant in the consideration of intestinal absorption of immunoglobulins by the neonatal piglet. The presence of the imnmnoglobulins IgG, IgA and IgM is easily recognized in the immunoelectrophoresis pattern of the suckling pig serum (see Fig. 7 in which comparison is also made with adult and precolostral sera, and colostrum). Three lines of reaction against a composite antiserum to the immunoglobulins are identifiable in the immunoelectrophoretogram of postcolostral sera. In studies of 5 litters of piglets at 2 days old, the absorption of all three groups of immunoglobulins has been indicated by immunological double-diffusion studies and immunoelectrophoresis, quantitative assays are given in Table II. The serum concentration of IgG and IgM in the 2-dayold piglets were mainly similar to adult levels. However, the most interesting feature of these studies is the level of IgA in postcolostral sera, this generally exceeded adult levels, in some cases by as much as 3-4 times. TABLE II ASSAYS
OF IMMUNOGLOBULINS
IgG, IgA
AND
IgM
IN SERUM
OF PIGLETS
AT 2 DAYS
Litter No.
Number of pigs
IgG (mg/ioo ml)
IgA (mg/ioo ml)
IgM (mg/ioo ml)
N5
12
65o-187o (I32O ~ 42o)
172-485 (3o4 i 85)
35 - I 4° (87.5 ~ 36.4)
W X 500
6
173o 2500 (218o ± 240 )
250-660 (485 ± 98)
61 162 (96.6 ± 39.5)
W X lO6
8
177o 235 ° (2o90 ± 220)
149-211 (189 i 22)
64-136 (94.6 ± 32.4)
N2
16
186o 236 (2040 ± 16o)
41o 840 (586 ~ 181)
85-16o (118.2 ~ 26.6)
T37
12
52o-244 o (188o ± 690)
85-690 (402 ~ 199)
61-175 (lO9.5 ~ 34.4)
DISCUSSION
The immunoglobulins IgG, IgA and IgM all appear in porcine colostrum at very much higher concentrations than is found for serum. This is contrary to the findings for bovine and human colostrum. IgA and IgM globulins were present in bovine colostrum in concentrations lower than in the serum 2~. Quantitatively the main immunoglobulin in human colostrum is IgA and the IgG molecule, which is the major Biochim. Biophys. Acla, 181 (1969) 381-392
IMMUNOGLOBULINS IgG, IgA AND IgM IN SOW COLOSTRUM
391
immunoglobulin in the colostrum of the pig, accounts for only IO °/o of human colostral immunoglobulin 23. MURPHY et al. 2~ considered their findings in the bovine only from the point of view of selectivity of transport of ?-globulins from the blood serum to the colostrum as determined by the m a m m a r y acinar epithelium. They did not consider the possibility of local synthesis within the gland. This is clearly a possibility, since the assays in Table I indicate that IgA accounts for a much higher proportion of the immunoglobulins of the colostvum than in the serum; also in gel filtration studies (Fig. 5) IgA was found to predominate in higher molecular weight fractions in the colostrum than in the serum. The significance of the wide range of molecular sizes of the IgA molecule is only becoming apparent from recent work. Investigations reviewed by TOMAS124 demonstrate that secretory IgA differs from serum IgA in possessing a complexed nonglobulin component called the transport piece. This component appears to be synthesized in the epithelial cell and has been partially characterized, having a molecular weight of approx. 50 ooo. It is possibly related to the transport of the immunoglobulin to the mucoid surface. In man the secretory IgA has a molecular weight of 39 ° ooo compared with 17o ooo for serum IgA. Clearly the IgA in sow colostrum is not limited to a polymeric form, since IgA appeared in gel filtration fractions over a wide molecular weight range. It is possible that the high molecular weight form is synthesized in the m a m m a r y gland and that lower molecular weight components are transferred directly from the serum. In the newborn pig there is no evidence for the transplacental transfer of IgA and IgM, but a component antigenically related to IgG could be detected. All three specific immunoglobulins, IgG, IgA and IgM, as characterized in the chromatographic and immunological studies with sow serum and colostrum, are absorbed b y the neonatal piglet. IgM plays an important part in the primary immune response and natural antibodies to Gram-negative bacteria are also associated with this immunoglobulin 2~. There is evidence to suggest that in the pig preexperience of antibody is required for its synthesis z6, thus it seems important that the I8-S IgM molecule should be absorbed from colostrum. This would provide for a passive immunity against Gram-negative bacteria in the neonatal pig and satisfy the apparent requirements for subsequent immunocompetence. On this latter point it m a y be significant that colostrum-deprived pigs do not synthesize IgM in their early immune response 27. LOCKE et al. 9 claimed that I8-S antibodies were not absorbed across the intestine of the newborn pig and suggested that the I8-S molecule lacked structures which corresponded to cellular receptors for intestinal transmission of immunoglobulins. Obviously such an hypothesis is unnecessary, since the I8-S IgM molecule is absorbed, and the quantitative assays (Tables I and II) would not suggest any differential absorption of IgM in relation to IgG. It is difficult to understand how LOCKE et al. 9 failed to demonstrate the absorption of the I8-S immunoglobulin, particularly since it appears in postcolostral sera at levels comparable with adult serum. This also has further implications for the work of these authors, since the interpretation of their results was based on the premise that the piglet intestine would not transport I8-S immunoglobulin. It is very interesting that the levels of IgA in postcolostral sera should so far exceed the adult level in so m a n y of the neonatal animals investigated. Possibly this immunoglobulin plays an important role in the immunological defence of the young Biochim. Biophys. Acta,
181 (1969) 381-392
392
P. PORTER
piglet. Investigations of this colostral immunoglobulin are continuing in greater detail particularly with reference to antibody to Gram-negative bacteria. REFERENCES i B. W. KARLSSON, Acta Pathol. Microbiol. Scand., 67 (1966) 83. 2 J. F. FOSTER, R. W, FRIEDELL, D. CATRON AND N. R. DICKMAN, Arch. Biochem. Biophys., 31 (1951) lO 4 . 3 J. G. LECCE AND G. MATRONE, J. Nutr., 73 (1961) 167 . 4 J. G. LECCE, D. A. MORGAN AND G. MATRONE, J. Nutr., 77 (1962) 349. 5 L. C. PAYNE AND C. L. MARSH, J. Nutr., 76 (1962) 151. 6 R. H. ]3ARNES, Nutr. Rev., 20 (1962) 316. 7 Y. B. KIM, S. G. ]3RADLEY AND D. N. WATSON, Swine in Biomedical Res. Proc. Symp. BattelleNorthwest, 1965, p. 273. 8 J. J. CEBRA AND J. ]3. ROBBINS, J. Immunol., 97 (1966) 12. 9 R. F. LOCKE, D. SEGRE AND W. L. MYERS, J. Immunol., 93 (1964) 576. io R. HALLIDAY AND R. A. KECKWICK, Proe. Roy. Sue. London, Set. B., 153 (196o) 279. i i I. G. MORRIS, Immunology, 13 (1967) 49. 12 P. PORTER, J. Cutup. Pathol. Therap., 74 (1964) lO8. 13 L. ORSTEIN AND ]3. J. DAVIS, Ann. N . Y . Acad. Sci., 121 (1964) 321. 14 P. PORTER, M. C. PORTER AND J. M. SHANBERGE, Biochemistry, 6 (1967) 1854. 15 R. AUGUSTIN AND M. J. HAYWARD, Immunology, 3 (196o) 45. 16 G. MANCINI, O. CARBONARA AND J. F. HEREMANS, Immunochemistry, 2 (1965) 3517 H. W. ]3OHREN AND V. R. WENNER, J. Dairy Sci., 44 (1961) 1213 . 18 C. A. WILLIAMS AND 1:). J. GRABAR, Immunology, 74 (1955) 158. 19 F. A. MURPHY, O. AALUND, J. W. OSEBOLD A~!D E. J. CARROLL, Arch. Biochem. Biophys., lO8 (1964) 23 ° . 20 G. M. EDELMAN, J. F. HEREMANS, M. T. HEREMANS AND H. G. KUNKEL, J. Exptl. Med., 112 (196o) 203 . 21 Y. ]3. KIM, S. G. ]3RADLEY AND D. N. WATSON, J. Immunol., i o i (1965) 224. 22 F. A. MURPHY, O. AALUND, J. W. OSEBOLD AND E. J. CARROLL, Arch. Biochem. Biophys., lO8 (1964) 230. 23 G. DE MURALT, E. GUGLER AND D. L. A. ROULET, in H. PETERS, Protides of the Biologica~ Fluids, 7th Colloq., Elsevier, A m s t e r d a m , 196o, p. 166. 24 T. ]3. TOMASI, Hospital Practice, 1967, Vol. 2, p. 26. 25 J. G. MICHAEL AND F. S. ROSEN, J. Exptl. Med., 118 (1963) 619. 26 I). SEGRE AND M. L. KAEBER, J. Immunol., 89 (1962) 79 o. 27 Y. 13. KIM, S. G. BRADLEY AND D, N. WATSON, J . Immunol., 97 (1966) 189.
Biochim. Biophys. Acta, 171-(1969 ) 381-392