Antibacterial Activity in Colostrum and Milk Associated

Transcription

1 INFECTION AND IMMUNITY, Mar. 1976, p Copyright X) 1976 American Society for Microbiology Vol. 13, No. 3 Printed in U.SA. Antibacterial Activity in Colostrum and Milk Associated with Protection of Piglets Against Enteric Disease Caused by K88-Positive Escherichia li J. M. RUTTER,* G. W. JONES,' G. T. H. BROWN, M. R. BURROWS, AND P. D. LUTHER Agricultural Research Council, Institute for Research on Animal Diseases, Compton, Newbury, Berkshire, England Received for publication 8 December 1975 Piglets suckled by dams that had been vaccinated with K88 antigen were significantly more resistant to deaths caused by neonatal diarrhea after challenge with a large dose of a K88-positive enteropathogenic strain ofescherichia li than piglets suckled by ntrol dams. The factors most likely to be involved in protection of the piglets were investigated by mparing the antibacterial activities of serum and mammary secretions from the two groups of dams. Vaccination stimulated the production of K88 antibodies, which were associated with anti-adhesive activity directed against the adhesive properties of the K88 antigen, and of 08 antibodies; the latter antibodies were attributed to traces of 08 antigen in the vaccine. Neutralizing activity against heat-labile enterotoxin was present in several dams before vaccination but was not stimulated by vaccination and did not appear to ntribute to protection. Bactericidal and bacteriostatic activities were similar in serum and mammary secretions from both groups of dams and appeared to play no significant role in the protective activity of mammary secretions. Increases in the levels of some E. li antibodies after parturition were attributed to exposure of the dams to the challenge strain excreted by the piglets. It was ncluded that neutralization of the adhesive properties of K88 antigen by K88 antibodies in lostrum and in milk ntributed significantly to the protection of piglets from vaccinated dams. However, the ntribution of antibacterial activities associated with the greater levels of 08 antibodies in lostrum from the vaccinated group cannot be entirely excluded. The K88 antigen is an essential virulence determinant of K88-positive strains of Escherichia li that cause neonatal diarrhea in piglets (6, 14). The virulence of these bacteria in nventionally reared pigs depends on the adhesive properties of the K88 antigen, which allow K88-positive strains to attach to the intestinal wall and to proliferate and reach high numbers in the small intestine of piglets. In ntrast, a K88-negative mutant of a virulent K88-positive strain is unable to attach, does not proliferate in the small intestine, and does not cause disease in sucking piglets (6). The attachment of K88-positive bacteria to slices of piglet intestinal tissue is inhibited by K88 antisera (6), and Rutter and Jones (9) reported that litters of piglets from dams vaccinated with K88 survived experimental challenge with a virulent K88-positive strain, whereas the majority of piglets from ntrol dams died with neonatal Present address: Department of Microbiology, Medical School, University of Michigan, Ann Arbor, Mich diarrhea. These results suggest that K88 antibodies produced in the dam by vaccination and transmitted in the lostrum to the small intestine of the piglet reduce the attachment of K88- positive bacteria to the intestinal musa and render the bacteria less virulent. The present paper describes the results of in vitro tests on serum and mammary secretions from vaccinated and nonvaccinated dams to investigate the antibacterial factors most likely to acunt for protection. Reports of some of these results have already been published (7, 9). MATERIALS AND METHODS Bacterial strains. E. li strains used were: Wl [0149:K91(B), K88ac(L):H1Ol and its K88-negative mutant W1(J2) [0149:K91(B):H10] (6); D282 [08:K27(A)-:H-] (15) and two K88-positive strains, D520 [08:K27(A)-, K88ab(L):H-1 (15) and 233/ID [08:K27(A)-, K88ac (L):H-] derived from D282 by njugation with a K88-positive donor strain; and G7 [08:K87(B), K88ab(L)I. Vaccination of dams with K88. Details of the 667

2 668 RUTTER ET AL. preparation of the vaccine and the vaccination procedure have been described (9). K88 was prepared from strains D520 and 233/ID; these strains are laboratory derived and do not produce enterotoxins. The K88 preparation ntained 98% (wt/wt) protein (predominantly K88 antigen), 0.5% (wt/wt) carbohydrate (estimated as neutral hexoses), and 1.5% (wt/ wt) nucleic acids. Dams were vaccinated twice during pregnancy; the first injection was given by the intramammary route with a water-in-oil emulsion as adjuvant; the send injection was given subcutaneously without adjuvant. Control dams received injections of adjuvant followed by saline. Infection procedure. The method described by Rutter and Anderson (8) was used, except that a fresh suspension of strain Wl was harvested from buffered gluse nutrient agar (6) for each experiment. Each piglet was given 1 ml of a suspension ntaining 10"' viable organisms by stomach tube at birth. Agglutinin tests. A microtiter method with live bacterial cultures (8) was used for agglutination tests. Precipitin tests. Precipitins were detected against a pool of extracted K88 antigen (6) or against whole bacterial cultures frozen and thawed 10 times or heated at 60 C for 30 min. The identity of the K88 precipitin line was nfirmed by standard absorption procedures. Tanned cell hemagglutination tests. The method described by Cruickshank (3) was used. The K88 antigen did not agglutinate goat erythrocytes and these cells were used in the test. Each sample was titrated in microtiter trays and tested in duplicate against erythrocytes ated with K88ab or K88ac antigen extracted from K-12 strains into which the K88 plasmids had been introduced (kindly provided by H. W. Smith). Erythrocytes ated with K88 antigens and titrated against known K88ab or K88ac rabbit antiserum served as positive ntrols. Negative ntrols included unated cells with and without antiserum and ated cells in saline. Anti-adhesion tests. A suspension of Wl bacteria (0.9 ml) was incubated for 30 min at 37 C with 0.1 ml of sample, and the adhesion test was performed (6). Percentage of specific adhesion was calculated (7). Anti-enterotoxin test. Enterotoxin neutralization was detected in pigs by the ligated intestinal loop test (13); the results in this test system were mpared with a cell culture test (10). In the ligated loop test, 2 ml of a bacteria-free lysate from strain Wl was incubated for 30 min at room temperature (18 C) with an equal volume of sample diluted 1 in 5 with saline. The mixture was injected into alternate ligated intestinal loops prepared in specific pathogen-free pigs; a positive ntrol with saline in place of the sample was injected into the remaining loops. The fluid accumulating in the loops was measured the following day and mpared with the positive ntrol loop that was immediately anterior. In the cell culture test, bacteria-free lysates of strain Wl were incubated with dilutions of each sample for 1 h at room temperature (10) and added to monolayers of a pig thyroid cell line for 2 h at 37 C. After absorption each monolayer was washed with maintenance INFECT. IMMUN. medium to remove the residual mixture, and 0.15 ml of medium, buffered at ph 7.2 with 20 mm N-2- hydroxyethylpiperazine-n'-2-ethanesulfonic acid, was added. PHA tests. The microtiter method described by Rutter and Anderson (8) was used for passive hemagglutination (PHA) tests. AGHA tests. The antiglobulin hemagglutination test (AGHA) test described by Buxton and Thomlinson (2) was used, with rabbit anti-pig serum (Hoechst Laboratories, Mill Hill, U.K.) diluted 1 in 10, and antigens were prepared from strains Wl and D282. Bacteriostatic and bactericidal tests. Bacteriostatic and bactericidal tests described by Brandenburg and Wilson (1) and Jones and Rutter (7) were used. RESULTS Mortality. Deaths attributed to neonatal diarrhea in piglets from vaccinated and nonvaccinated dams are shown in Table 1. Within 18 h of infection, 27 of 29 piglets from nonvaccinated dams and 27 of 31 piglets from vaccinated dams had diarrhea. In the four litters from nonvaccinated dams, the piglets lost weight and excreted large numbers of strain Wl, and 20 of the 29 piglets died within 72 h. Large numbers of strain Wl (range, 109 to 10"' per g of ntents) were revered from the anterior small intestine of the dead piglets. In the surviving piglets killed 7 days after infection, strain Wl was absent, or present in low numbers (range, 10: to 104 per g of ntents) in the anterior small intestine. In ntrast, the majority of piglets from the vaccinated dams gained weight throughout the experiments; only 10 of the piglets had diarrhea 48 h after infection and diarrhea ceased within 72 h, as did excretion of strain Wl. However, four of the 31 piglets died within 72 h and strain Wl was revered from the anterior small intestine of these piglets (range, 10 to 109 per g of ntents). In the TABLE 1. Mortality attributed to neonatal diarrhea after oral administration of E. li strain Wl to piglets from vaccinated and nonvaccinated dams Piglets that Dam no. Sire no. died/no. challenged Vaccinated / / / /8 Nonvaccinated / / / /8

3 VOL. 13, 1976 surviving piglets killed 7 days after infection, strain Wl was absent, or present in small numbers (range, 102 to 101 per g) in the anterior small intestine. The difference in mortality between the two groups is statistically significant (P < 0.01 in a one-tailed t test). Agglutinins. Agglutinins against strain Wl (K88ac) increased in serum from three of four dams after vaccination (Table 2). These agglutinins did not increase in dam 40 after vaccination but increased after parturition. Agglutinins against strain D520 (K88ab) were greater mpared with agglutinins against strain Wl (K88ac) in serum from both groups before vaccination and did not increase after vaccination. High titers of agglutinins against strain D282 (08) were present in serum from the vaccinated group, which may have influenced the agglutination results with strain D520. In the nonvaccinated group, there was an increase in agglutinins against strain Wl in serum after parturition, which was presumably attributable to infection of the dams with the challenge strain Wl excreted by their piglets. Agglutinins against the K88-positive strains Wl and D520 were greater in lostrum mpared with serum llected at parturition in both groups of dams; in ntrast, agglutinins against the K88-negative strains Wl (J2) and D282 were absent or decreased in lostrum. These results suggest that the increased agglutinating activity in lostrum against the K88- positive strains is directed against the K88 antigen. However, the high levels of K88 agglutinins in mammary secretions from nonvaccinated dams suggests that part of the agglutinating activity in lostrum and milk may not be attributable entirely to antibodies. Agglutination may also be caused by substances ANTIBACTERIAL ACTIVITY AND ENTERIC DISEASE 669 such as glyproteins or oligosaccharides in mammary secretions (5), which may be capable of reacting with the K88 antigen on the bacterial cell. Because the levels of agglutinins against strain Wl in lostrum from both vaccinated and nonvaccinated dams were similar, it appears that agglutinating activity was not involved in protection of the piglets from vaccinated dams. Antibodies against the K88 antigen. K88 precipitins were absent from serum llected before vaccination (Table 3). In tests with heated and freeze-thawed bacterial extracts as antigen, K88 precipitins were present in serum and lostrum from all vaccinated dams. In tests with the K88 pool as antigen, K88 precipitins were detected in serum from vaccinated dams 106 and 114, and in lostrum from all of the vaccinated dams. These antibodies disappeared during the transition from lostrum to milk and were present 7 days postpartum only in samples from dam 114. Precipitin tests with heated or with freeze-thawed bacterial extracts and lostrum revealed additional weak lines of precipitation; these precipitins were presumably directed against intracellular bacterial antigens liberated by the heating or freezing procedures but were not characteristic of either the vaccinated or the nonvaccinated group. Production of K88 antibodies was also detected with the tanned erythrocyte hemagglutination test (Table 4). K88 antibodies were present in serum at parturition and in lostrum from all the vaccinated dams, and high titers were present in serum from dams 106 and 114. These antibodies decreased during the transition from lostrum to milk and were present 7 days postpartum only in samples from dam 114. K88 antibodies were present in milk 2 and 7 TABLE 2. Agglutinins in serum, lostrum, and milk from vaccinated and nonvaccinated dams Means and ranges of titers" in: Groups Test Serum Mammary secretions of dams strainb After vaccina- Before vacci- tion (parturi- 7 days postpar- Parturition - Milk 2 days Milk 7 days nation tion) tum lostrum postpartum postpartum Vacci- WI 16 (<2-32) 184 (32-512) 160 ( ) 352 ( ) 192 ( ) 144 (64-256) nated Wl (J2) <2 <2 <2 <2 <2 <2 D ( ) 288 ( ) 256 (256) 768 (256-2,048) 176 (64-512) 368 (64-1,024) D (64-2,048) 848 (64-2,048) 353 (128-1,024) 272 (64-512) 72 (16-256) 30 (8-64) Nonvac- Wi 12 (<2-32) 6 (4-8) 60 (16-128) 384 ( ) 132 (16-256) 192 ( ) cinated Wl (J2) <2 <2 <2 <2 <2 <2 D ( ) 112 (64-128) 192 ( ) 512 (256-1,024) 384 (128-1,024) 560 (64-1,024) D (32-256) 96 (32-256) 320 (64-1,024) 22 (4-64) 21 (4-32) 8 (4-16) a The reciprocal of the last dilution causing mplete bacterial agglutination was used; means were calculated from the results of tests with samples examined twice from four different dams. b Wl = 0149:K91(B), K88ac(L):H1O. Wl (J2) = 0149:K91(B):H1O. D520 = 08:K27(A)-, K88ab (L); H-. D282 = 08:K27 (A)-:H-.

4 670 RUTTER ET AL. days postpartum from nonvaccinated dam 51; this was presumably caused by infection of the dam with the K88-positive challenge strain excreted by the piglets. The pattern of results in tanned erythrocyte hemagglutination tests is similar to that obtained in precipitin tests with the K88 pool as antigen, nfirming that both tests detect the same mponent. However, it appears that the precipitin test is slightly less sensitive than the hemagglutination test for detecting K88 antibodies. It is clear that K88 antibodies were characteristic only of lostrum from the vaccinated group, suggesting that these antibodies play a significant role in protection. Antibodies against 0 antigens. Antibodies directed against the 08 antigen of vaccine strains D520 and 233/ID and the 0149 antigen of the challenge strain Wl were detected in PHA tests and AGHA tests (Table 5). In general, the PHA titers were lower than the AGHA titers, but the trends in both tests were similar. Antibodies in serum against the 08 antigen increased as a result of vaccination, particularly in dams 106 and 114; this pattern of results is similar to that obtained in tests for K88 antibodies. Antibodies in lostrum directed against the 08 antigen, particularly AGHA antibodies, were greater in the vaccinated group mpared with the nonvaccinated group and decreased in both groups during the transition from lostrum to milk. The greater level of 08 PHA antibodies in lostrum from the vaccinated mpared with the nonvaccinated group was attributable to individual variations; in ntrast, the greater level of 08 AGHA antibodies was characteristic-of the vaccinated group. Antibodies in serum directed against the 0149 antigen increased after vaccination; this was attributable to increased titers in dams 40 and 114, which is difficult to explain. These antibodies increased in serum from both groups after parturition, which is presumably attributable to infection of the dams with the 0149 challenge strain excreted by the piglets. Although the 0149 antibody levels were greater in lostrum from the vaccinated mpared with the nonvaccinated group, this merely reflected the greater prevaccination titers of indi- TABLE 3. K88 precipitins in serum, lostrum, and milk from vaccinated and nonvaccinated dams Serum K88 precipitins in: Mammary secretions Groujs of dams Before vaccina- Afte(parturia- 7 days postpar- Parturition - Milk 2 days Milk 7 days ttion ttion) tum lostrum postpartum postpartum a" b" a b a b a b a b a b Vaccinated 0/4 0/4 2/4 4/4 3/4 4/4 4/4 4/4 1/4 3/4 1/4 1/4 Nonvaccinated 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 a a, K88 pool as antigen; b, heated and freeze-thawed extracts of Wl, D520, and 233/1D as antigen. TABLE 4. Group of dams K88 antibodies detected by tanned erythrocyte hemagglutination in serum, lostrum, and milk from vaccinated and nonvaccinated dams vidual animals and was not characteristic of the vaccinated group. In general there was a decrease in antibody levels in milk from both groups between parturition and 2 days postpar- Anti- Anti- Serum Means and ranges of titersa in: Mammary secretions INFECT. IMMUN. Before After vaccina- 7 days post- Parturition Milk 2 days Milk 7 days nation tion) partum lostrum postpartum postpartum Vaccinated K88ab < (<20-640) 200 ( ) 450 (40-1,280) 365 (<20-1,280) <20 (<2040) K88ac < (20-1,280) 220 (80-320) 360 (80-640) 185 (<20-640) 20 (<20-80) Nonvaccinated K88ab <20 <20 <20 <20 <20 (<20-40) <20 (<2040) K88ac <20 <20 <20 <20 <20 (<2040) <20 (<20-40) a The reciprocal of the last dilution causing mplete hemagglutination was used; means were calculated from the results of tests with samples for four different dams.

5 VOL. 13, 1976 ANTIBACTERIAL ACTIVITY AND ENTERIC DISEASE 671 TABLE 5. PHA and AGHA antibodies in lostrum and milk from vaccinated and nonvaccinated dams Means and ranges of titers" in: Antigen ex- Serum Mammary secretions Group of dams tract Test from 0 After vacci- 7 d t Patuiti Milk 2 Milk 7 days group Befon nation (par- pays lostrum days post- Mikt ays cination turition) partum os rum partum postpartum Vaccinated 149b PHA (32-512) ( ) (128-1,204) (8-512) (4-512) (8-32) AGHA 285 1,140 2,986 1, (80-640) (80-2,560) (1,280-2,560) (320-2,560) (40-120) (40-1,280) 8' PHA (64-256) ( ) ( ) (32-2,048) (16-2,048) (16-128) AGHA 825 2,400 6,000 13,700 3, (100-1,600) (1,600-3,200) (1,600-12,800) (3,200-25,600) (800-6,400) ( ) Nonvaccinated 149 PHA (16-64) (32-256) (64-1,024) (16-32) (8-32) (8-256) AGHA , (80-640) ( ) (320-5,120) ( ) (40-160) (40-1,280) 8 PHA (32-64) (64-128) (64-128) (32-128) (8-64) (8-16) AGHA <100 (100400) ( ) ( ) (100400) (< ) (< ) athe reciprocal of the last dilution causing mplete hemagglutination was used; means were calculated from the results of tests with samples from four different dams antigen prepared from strain Wl [0149:K91(B), K88ac (L):H1lO. '08 antigen prepared from strain G7 108:K87(B), K88ab (L)]. days post-partum; this occurred particularly with dams 22, 90, and 55. It can be ncluded that the 08 AGHA antibodies in lostrum from vaccinated dams were nsistently and nsiderably greater mpared with nonvaccinated dams, and that these antibodies may ntribute to survival of the challenged piglets. However, differences in the 08 PHA, 0149 PHA, and 0149 AGHA antibodies in lostrum from the two groups were attributable to individual variations and were not characteristic of only the vaccinated group. Anti-adhesive antibodies. Anti-adhesive antibodies were stimulated by vaccination and were detected in serum and lostrum llected at parturition from vaccinated dams (Table 6). These antibodies decreased in serum after parturition and during the transition from lostrum to milk and were present in milk 7 days postpartum from only dam 114. Significant anti-adhesive activity was detected 2 and 7 days postpartum in milk from nonvaccinated dam 51. These results agree closely with the tanned erythrocyte hemagglutination test results with the K88ac antigen, indicating that the antiadhesive antibodies are directed against the K88 antigen. It is clear that vaccination with K88 stimulated nsiderable amounts of antiadhesive activity in the lostrum of all these animals, whereas the lostrum from nonvaccinated dams ntained no anti-adhesive activity. These results support the view that K88 anti-adhesive activity plays a significant role in the survival of challenged piglets from vaccinated dams. Anti-enterotoxin activity. Neutralization of heat-labile enterotoxin was detected in ligated intestinal loops of pigs (Table 7). The enterotoxin activity was mpletely neutralized by prevaccination serum of dams 55, 106, and 114 in the vaccinated group, and dam 90 of the nonvaccinated group. Neutralizing activity was present in lostrum from the same animals and decreased during the transition from lostrum to milk. Neutralizing activity was not detected in lostrum from dam 40 of the vaccinated group, indicating that antibodies were not stimulated by vaccination. Furthermore, neutralizing activity was not detected in milk after natural exposure of the dams that responded by producing other E. li antibodies (Tables 4-6). Neutralizing activity was also detected in a pig thyroid cell culture system (Table 7). As in the ligated loop test, neutralizing titers were present in the prevaccination serum from dams 55, 106, 114, and 90. The titers in serum did not increase after vaccination, but there was a 10- fold increase in lostrum mpared with serum at parturition. The neutralizing titers decreased during the transition from lostrum to milk but remained high in milk 7 days postpartum from dam 106. Neutralizing titers increased in serum after paturition from dams 90,

6 672 RUTTER ET AL. INFECT. IMMUN. TABLE 6. Anti-adhesive activities in serum, lostrum, and milk from vaccinated and nonvaccinated dams Groups of dams Means and ranges of percentage-specific adhesion in: Serum Mammary secretions Before vacci- After vaccina- 7 days postpar- Parturition - Milk 2 days Milk 7 days nation tion (parturi- tum lostrum postpartum postpartum Vaccinated (88-113) (32-58) (0-11) (7-137) (49-135) Nonvaccinated (88-140) (59-160) (96-116) (88-109) (56-120) (8-97) Mean result of samples from four dams, with range of values in parentheses. tum, followed by an increased between 2 and 7 55, and 114. Thus, there was reasonable agreement between the results in the two test systems, and the discrepancies may have been associated with the greater sensitivity of the cell culture test. Although enterotoxin-neutralizing activity was present in lostrum from four of the dams, this activity did not influence the onset of diarrhea, which occurred for up to 72 h after infection in piglets from both groups and was presumably attributable to enterotoxins Ḃactericidal and bacteriostatic activities. Bactericidal and bacteriostatic activities were detected in serum, lostrum, and milk from both vaccinated and nonvaccinated dams (Table 8). The bactericidal activity in serum did not increase significantly after vaccination or after parturition. Bactericidal activity in lostrum and 2-day milk from vaccinated and nonvaccinated dams was nsiderably greater mpared with serum, but there was no difference in activity between the two groups. However, milk 7 days postpartum from the vaccinated group had nsiderably more bactericidal activity than rresponding samples from the nonvaccinated group. Bactericidal tests with other O groups ofe. li demonstrated that the bactericidal activity in mammary secretions was not specific for the 0149 group; however, bactericidal tests with rabbit antisera raised against the different antigens of strain Wl indicated that bactericidal activity was directed against only 0149 antigen. Bacteriostatic activity was not detected in serum and mammary secretions, but the bacteriostatic test did reveal bactericidal activity. The trends of these results were mparable to those obtained in the bactericidal test. In serum, the activities were similar in both groups of dams; in mammary secretions, the activities were similar in both groups of dams, but the samples were less bactericidal mpared with the bactericidal test. The similar levels of bactericidal activity in lostrum and milk from vaccinated and nonvaccinated dams indicates that this mechanism played no significant specific role in the protection of piglets from vaccinated dams against disease caused by K88-positive E. li; however, bactericidal activity may nstitute a "background" antibacterial activity of porcine mammary secretions. DISCUSSION Two essential virulence determinants have been regnized in K88-positive enteropathogenic strains of E. li; these are enterotoxins that cause fluid loss into the alimentary tract resulting in diarrhea, dehydration, and death (13, 14) and the K88 antigen, which is associated with bacterial adhesion and lonization of the small intestine (6, 14). The adhesive properties of K88 are neutralized by K88 antiserum in vitro (6, 16), and the results of infection experiments with piglets suckled by dams vaccinated with K88 antigen suggest that protection may result from K88 antibodies in mammary secretions inhibiting the adhesive activity of K88 in the gut of piglets. Vaccination of dams with K88 antigen stimulated the production of K88 antibodies (detected in the tanned erythrocyte hemagglutination test and in the precipitin test) and anti-adhesive antibodies. The rrelation between the results in the anti-adhesive test and in the tanned erythrocyte test, together with our previous observations (6), suggests that anti-adhesive antibodies are directed against the K88 antigen. The presence of anti-adhesive antibodies in lostrum from all the vaccinated dams, and the absence of similar activity in the nonvaccinated dams, support the view that anti-adhesive K88 antibodies produced by vaccination are involved in protection. In three of the vaccinated dams, the levels of the K88 and anti-adhesive antibodies decreased during the transition from lostrum to milk 7 days postpartum. The short duration

7 VOL. 13, 1976 ANTIBACTERIAL ACTIVITY AND ENTERIC DISEASE 673 r- 0. C) C', C; V v v :3-0 t: c-4 Q oo C4 cq to -1 (D Cd OD Cd cq C) S. cq C) OD rn S. OD cq eq 4) Cd C; OD v cq cc ad OD ea um c-i C4 00 all m C4 C'i 03 ad Ci > C14 cq C13 Cj CJ C) OD la :t. Cd t-i t- CID C14 C'i v U 4J, Cd C) 75 ci 1-1 t- r,7 c" cq Q v C14 S. Cd v cd Ca C14 Cd Lim -4 M Co 00 C; t- OD z C) Cq 00 =I 00 Ca CM v ci 16. 4) e. > z :3 44 Cq x E bid 40 S. cys cla 00,. cis V 8 -A Mt m r. C; C) tj ci 40 > C.) lx > cd E- Co > N o6 (L) I.. ci Ci Co f*.. tgd 4J, 10 CD 4.) C; C; rn Cd Co > C-0 Cd bid 4) bid 0 10 Cs A. z OD CO 4) r..2..: > -ij 4i cis Q ci t... as r. ci r. cis CJ r. C's bo ci > 4) > ci r. z r. z 0 as 0 ;. > z > z

8 674 RUTTER ET AL. of the secretion of K88 antibodies may be sufficient to protect piglets during the critical first few days of life, after which other factors such as bactericidal activity and the development of the gut flora may play a protective role. Alternatively, antibodies accumulated by absorption to the intestinal muus membrane may be active in vivo and reduce intestinal lonization. Significant amounts of K88 and anti-adhesive antibodies were detected in milk 7 days postpartum from vaccinated dam 114 and nonvaccinated dam 51. The presence of these antibodies appears to be attributable to infection of the dams with the Wl challenge strain excreted by the piglets. Support for this suggestion is provided by (i) the increases in 0149 antibody titers in serum (dams 22, 54, 55, and 114) and in milk (dams 22, 55, and 90) after parturition, in ntrast to the general trend of decreasing antibody levels, and (ii) revery of strain Wl from rectal swabs from several of the dams after parturition. Infection of the dams uld be either by intestinal lonization or possibly subclinical infection of the mammary glands. Of particular interest is the observation that 0149 antibodies were detected in the absence of K88 antibodies, suggesting that production or detection of 0149 antibodies may be more efficient mpared with K88 antibodies. Presumably the passive protection that we observed in the infection experiment results from K88 antibody occluding the active sites of the K88 adhesin and preventing bacterial attachment to the musa. Other mechanisms such as persistalsis and microbial antagonism may then assist to remove and prevent the pathogen from establishing in the intestine. Because the majority of piglets died within 48 h of infection, and because a K88 antibody response was detected in milk 2 days after parturition from nonvaccinated dam 51, when four of her five piglets had died, it seems reasonable to nclude that the lostrum plays a vital role in protection in our test system. The preliminary results of fractionation experiments suggest that the anti-adhesive activity in lostrum from dams vaccinated by the intrammary route is predominantly in the immunoglobulin G fraction (Rutter, Bourne, Jones, and Burrows, unpublished data). In the very young piglet and in the presence of a developing gut flora (4) immunoglobulin G may be sufficiently stable to exert a significant protective effect. Our demonstration of K88 precipitins in gut ntents from piglets suckled by dams vaccinated with K88 antigen supports this suggestion (unpublished data). The presence of similar amounts of bacterial INFECT. IMMUN. agglutinins in lostrum from both vaccinated and nonvaccinated dams is also of interest. If milk glyproteins or oligosaccharides ntribute to the agglutination reaction as we suggest, then glyproteins in lostrum and milk may bind to the K88 antigen on the surface of bacterial cells and prevent attachment to musal receptors and thus provide additional protection to the musal surface. Neutralizing activity against heat-labile enterotoxin was detected in prevaccination serum from four of the eight dams and the activity was not stimulated by vaccination, nfirming that heat-labile enterotoxin was not a mponent of the vaccine. In the vaccinated group, lostrum from dam 40 had no anti-enterotoxin activity, and because this litter was protected it seems that neutralizing activity against heat-labile enterotoxin is not essential for protection. Indeed, the initial diarrhea induced in all piglets by the massive challenge dose indicates that enterotoxin-neutralizing activity had little effect on the urse of the disease. High titers of 08 antibody were detected in lostrum from the vaccinated group by the AGHA test. Presumably traces of 08 antigen in the vaccine stimulated the 08 antibody response, and this may have ntributed to protection. If so, then protection was not mediated by a bactericidal response to a mmon antigenic mponent, since the bactericidal activities in lostrum from both groups were similar. However, the ability of endotoxin to induce increased resistance to infection should be nsidered in this respect. The appearance of K88 and 0149 antibodies in serum and milk, presumably as a result of natural exposure of the dam to the challenge strain, has interesting implications. Dams vaccinated orally with K88-positive, non-enterotoxin-producing strains nfer passive protection on their piglets; however, in vitro antiadhesive activity is not nsistently associated with oral vaccination (Rutter, Jones, Burrows, and Anderson, unpublished data), and further studies are necessary to investigate the protective mechanism following this route of vaccination. Since the mpletion of the present investigation, the important observation that K88-positive E. li do not attach in vitro to intestinal tissue from all piglets has been made. These results have now been extended to demonstrate that there are at least two phenotypes of pig designated "adhesive" and "nonadhesive" for the K88 antigen (11, 12). These characters are inherited in a simple Mendelian manner, adhesive being dominant over nonadhesive. Fur-

9 VOL. 13, 1976 thermore, the virulent K88-positive strain Wl does not establish in the intestines of nonadhesive piglets, with the result that such piglets are genetically resistant to infection. In ntrast, the K88-positive pathogen attaches and establishes in the small intestine of adhesive piglets, and these piglets beme severely diseased and die (8a). Clearly, these observations have an important bearing on the interpretation of infection experiments. Although the present experiment was designed so that the boars were similar in both groups, our recent results indicate that boar 3 (Table 1) is homozygous nonadhesive: thus, litters sired by this boar should be phenotypically 100% nonadhesive if the dam is homozygous nonadhesive, or 50% adhesive if the dam is heterozygous, or 100% adhesive if the dam is homozygous adhesive. Because some piglets from dams 106 and 114 died of neonatal diarrhea and were presumably adhesive, these dams must be either heterozygous or homozygous adhesive. Thus, it can be predicted that at least 50% of their litters should have been adhesive and susceptible to infection; in fact, only 3 of 16 piglets from these two litters died. Furthermore, we believe that boar 2 is heterozygous, so that even if mated to a homozygous nonadhesive female at least 50% of his progeny should be adhesive and susceptible to infection. The litter from dam 90 is probably in this category, but the results with the litter from dam 55 can only be explained by postulating that passive protection of at least 50% of the litter occurred. On the basis of these results, it seems reasonable to nclude that the difference in mortality between the two groups was not attributable to an unfortunate choice of matings, but that K88 vaccination did ntribute significantly to protection. Studies of the role of K88 antigen in porcine neonatal diarrhea have clarified a particular pathogenic mechanism in K88-positive strains of E. li, but it is clear that subsequent work has revealed many interesting but mplicating factors, which increase the mplexity of the in vivo situation. For example, are K88 antibodies in mammary secretions for 2 days postpartum adequate to explain protection and what other factors render older animals resistant to disease? Why are anti-adhesive K88 antibodies detected only in some orally stimulated animals? In this respect, it is interesting to speculate whether the K88-positive strains are able to establish sufficiently in nonadhesive animals to provide the antigenic stimulation necessary for the production of K88 antibodies. Clearly, assumptions cannot be made, and it is essential ANTIBACTERIAL ACTIVITY AND ENTERIC DISEASE 675 to nsider the effects of, firstly, the virulence mechanisms of the pathogen, sendly, the type of host-produced antibacterial activity that may modify these mechanisms, and, thirdly, the existence of host phenotypes that may modify disease susceptibility before the influence of the immune response on the host-pathogen relationship can be evaluated and understood. ACKNOWLEDGMENTS We are indebted to C. Jay and J. Manning for expert technical assistance. We thank F. Orskov, W. J. Sojka, and H. W. Smith for providing us with various strains ofe. li. LITERATURE CITED 1. Brandenburg, A. C., and M. R. Wilson Immunity to Escherichia li in pigs: IgG immunoglobulin in passive immunity to Escherichia li enteritis. Immunology 24: Buxton, A., and J. R. Thomlinson The detection of tissue sensitizing antibodies to Escherichia li in oedema disease and haemorrhagic gastroenteritis in normal pigs. Res. Vet. Sci. 2: Cruickshank, R Medical microbiology, 11th ed., p E. S. Livingstone Ltd., London. 4. Fubara, E. S., and R. Freter Availability of locally synthesized and systemic antibodies in the intestine. Infect. Immun. 6: Gibbons, R. A., G. W. Jones, and R. Sellwood An attempt to identify the intestinal receptor for the K88 adhesion by means of a haemagglutination inhibition test using glyproteins and fractions from sow lostrum. J. Gen. Microbiol. 86: Jones, G. W., and J. M. Rutter Role of the K88 antigen in the pathogenesis of neonatal diarrhoea caused by Escherichia li in piglets. Infect. Immun. 6: Jones, G. W., and J. M. Rutter Contribution of the K88 antigen of Escherichia li to enteropathogenicity; protection against disease by neutralizing the adhesive properties of the K88 antigen. Am. J. Clin. Nutr. 27: Rutter, J. M., and J. C. Anderson Experimental neonatal diarrhoea caused by an enteropathogenic strain of Escherichia li in piglets; a study of the disease and the effect of vaccinating the dam. J. Med. Microbiol. 5: a. Rutter, J. M., M. R. Burrows, R. Sellwood, and R. A. Gibbons A genetic basis for resistance caused by E. li. Nature (London) 257: Rutter, J. M., and G. W. Jones Protection against enteric disease caused by Escherichia li - a model for vaccination with a virulence determinant? Nature (London) 242: Rutter, J. M., and P. D. Luther Cytopathic factors in bacteria-free lysates of Escherichia li. J. Med. Microbiol. 6: Sellwood, R., R. A. Gibbons, G. W. Jones, and J. M. Rutter A possible basis for the breeding of pigs relatively resistant to neonatal diarrhoea. Vet. Rec. 95: Sellwood, R., R. A. Gibbons, G. W. Jones, and J. M. Rutter Adhesion of enteropathogenic Escherichia li to pig intestinal brush borders: the existence of two pig phenotypes. J. Med. Microbiol. 8: Smith, H. W., and C. L. 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10 676 RUTTER ET AL. 14. Smith, H. W., and M. A. Linggood Observations on the pathogenic properties of the K88, Hly and Ent plasmids ofescherichia li with particular reference to porcine diarrhoea. J. Med. Microbiol. 4: Stirm, S., F. Orskov, I. 0rskov, and B. Mansa Episome-carried surface antigen K88 of Escherichia INFECT. IMMUN. li. II. Isolation and chemical analysis. J. Bacteriol. 93: Wilson, M. R., and A. W. Hohmann Immunity to Escherichia li in pigs: adhesion of enteropathogenic Escherichia li to isolated intestinal epithelial cells. Infect. Immun. 10: