Antibody production by the pig colon during infection with Treponema hyodysenteriae

Research in Veterinary Science 1989, 47, 263-269

Antibody production by the pig colon during infection with Treponema hyodysenteriae A. S. REES, Department of Veterinary Medicine, University of Bristol, Langford, Bristol, Avon BS18 7DU, R. J. LYSONS, AFRC Institute for Animal Health, Compton, Newbury, Berkshire RG16 ONN, C. R. STOKES, F. J. BOURNE, Department of Veterinary Medicine,

University of Bristol, Langford, Bristol, A von BS18 7DU

When 47 pigs were dosed orally with cultures of Treponema hyodysenteriae, 44 (94 per cent) developed swine dysentery. Of those which recovered and were rechallenged, nine of 21 (43 per cent) showed clinical signs, as did one of 10 (10 per cent) challenged on a third occasion. Clinical disease was associated with development of specific IgG, IgA and IgM antibodies in serum and the local production of IgA in gut mucosal tissues. The appearance of antibody was not directly related to protection but rather indicated either prolonged exposure (in the case of serum IgG) or recent exposure to T hyodysenteriae (for secretory IgA). Infection also resulted in the appearance of IgG and IgA memory cells in gutassociated lymphoid tissue. However, these studies indicated th.\lt humoral immunity alone is not responsible for the onset of a protective response to T hyodysenteriae in the colon. SWINE dysentery is associated with infection by the spirochaete Treponema hyodysenteriae which colonises only the large intestine. Pigs recovering from the disease show varying degrees of resistance to subsequent rechallenge with this organism (Olson 1974, Joens et al 1979) and the presence of specific anti- T hyodysenteriae antibody has been demonstrated in the serum and colon of convalescent pigs (Joens et al 1984). The origin and protective significance of antibody to T hyodysenteriae appearing in the large gut has not been investigated. While a number of studies have identified immune effector mechanisms that operate against small intestine enteropathogens (review Newby 1984), little information is available on large intestinal immunity. The purpose of the present investigation was to utilise the restricted site of colonisation of T hyodysenteriae to investigate immune effector mechanisms stimulated by a pathogen of the large intestine.

Materials and methods

Bacteria T hyodysenteriae strain P 18A was grown in a liquid medium (Lemcke et al 1979). For infection of pigs, broth cultures of bacteria grown to log phase were administered by stomach tube. Animals A total of 47 pigs from a swine dysentery-free herd were used in this study. They were fed an antibioticfree ration. Three experiments were performed in which pigs were orally dosed with log phase T hyodysenteriae broth culture containing on average lOS cfu mt ", allowed to recover, and challenged a second and third time. The pigs were treated with tiamulin in between challenges to eliminate any residual T hyodysenteriae. To ensure that the rechallenge was adequate, control pigs from the swine dysentery-free herd were dosed at the same time and included as 'one-challenge animals'. The challenge dose was dependent on bodyweight. Antibody levels were determined in pigs slaughtered after one, two or three challenges. Slaughter times are shown in Table I.

Experiment 1 Ten pigs were challenged at four, 18 and 31 weeks old when they weighed approximately 5, 50 and 120 kg, respectively. They were dosed with 20 ml broth culture on four successive days for the first challenge, 100 ml on two successive days for the second challenge and 250 ml on two successive days for the third challenge.

263

Experiment 2 The pigs used in this experiment were the offspring

A. S. Rees, R. J. Lysons, C. R. Stokes, F. J. Bourne

264

TABLE 1: Experimental protocol and clinical signs of pigs challenged with T hyodysenter/ae. Letters in italics identify the pig groups used in Fig 1

Number of pigs

Clinical signs

4a

SO SO

1 challenge Experiment Experiment Experiment Experiment

1 2 3 3

2 challenges Experiment Experiment Experiment Experiment

2 2 2 3

21 1g 4h

None Diarrhoea SO None

3 challenges Experiment Experiment Experiment Experiment Experiment

1 1 1 2 2

2/ 2j 1k 2/ 3m

None Soft faeces Diarrhoea None None

3b 1c 2d

3e

so

SO

Slaughter time (weeks after Days duration challenge) 14-28 3-14 14-28

4

7 9 6 6

1 10

1 4

2

5 5 4 4 4 1 2

In vitro antibody production Tissues. A piece of proximal colon approximately 5 ern x 2 ern was taken from each pig, avoiding the areas of lymphoid aggregates, rinsed gently under a running tap and placed in RPM) 1640 - Dutch modification (Flow) supplemented with 2 per cent fetal calf serum (FCS; Gibco). The pieces of tissue were rinsed in two further changes of this medium. Using a scalpel, the lamina propria was sliced off the underlying muscle and connective tissue, and chopped into pieces approximately 2 mm x 2 mm. 300 mg of these pieces were placed in each of the two 10 ml volumes of tissue culture medium (RPMI 1640Dutch modification, supplemented with 10 per cent FCS, 2 mM glutamine, 50 lAg ml- I gentamicin, I lAg ml- I fungizone [Flow] and 2x 10- 5 M 2-mercaptoethanol [DDH]). One of these 10 ml volumes was stored at - 20°C immediately, as a control for preformed antibody; the other was incubated in a tissue

SO Swine dysentery

of sows from a previous experiment. These sows were challenged on three occasions with 500 ml of culture but did not develop swine dysentery. They were given antibiotics before farrowing. Specific IgG and IgA levels in their milk and colostrum were not raised above control values, and serum IgG titres in the offspring were one in 16 or less at two weeks old. The II offspring were challenged at three, 12 and 21 weeks old (weights approximately 5,30 and 80 kg) with 50 ml on two successive days, 75 ml on two successive days and 150 ml on two successive days, respectively.

Experiment 3 Eight pigs were challenged with 125 ml on two days at eight weeks old. Four were challenged a second time, along with two susceptible controls, at 20 weeks old with 450 ml on two successive days, 250 ml on one day and 300 ml on two successive days.

4

Appearance of clinical signs of infection • none A once • •

twice three times

3

j.Am

"E

'" s

i •• i

2

"0

Aa

C


~ Cl

o

--J

Serum and secretions The pigs were slaughtered by electrical stunning and exsanguination. Blood was collected, allowed to clot at room temperature, and the serum removed and stored at - 20°C until assayed. Bile was collected from the gall bladder. Gut washings were taken from pieces of gut, rinsed in tap water to remove large debris, by scraping gently with a glass microscope slide. Washings and faeces were placed in a solution of enzyme inhibitors (Evans et al 1980) then sonicated for 20 seconds at I· 5 amps and spun at 30,000 g for 15 minutes. Antibody levels in the supernatants, and in the bile and sera, were determined by ELISA.

.h

'0

o

-1

a .. a

••• o

.f e

1

2

Number of times challenged

1I . . . m

3

FIG 1: Serum IgG antibody to T hyodysenteriae in pigs challenged on one, two or three occasions. The symbols indicate how many challenges were required before the pigs became resistant. The letters beside symbols refer to the groups in Table 1

Colon antibody to T hyodysenteriae TABLE 2: Serum IgG. IgA and IgM antibody to T hyodysanta,ma (mean ± SO) in unchallenged control pigs and pigs challenged on one or three occasions Anti- T hvodvsenterise antibody in experiment 1 pig sera (% of standard serum) IgG IgA IgM Controls (n ; 3) 1 x challenged (n ;4) 3 x challenged (n; 5)

0·07 ± 0·05

0·05 ± 0·00

75·0 ± 35·4

13·5 ± 24·3

0·20 ± 0·29

1650 ± 2263

1273 ± 3288

40·8 ± 89·0

1900 ± 894

culture flask for 18 hours at 37°C with 5 per cent carbon dioxide then stored at - 20°C. Both 10 ml cultures were thawed to disrupt the tissue and release antibody. The suspensions were centrifuged (1500 g for 10 minutes) and antibody levels in the supernatants determined by ELISA. Small intestine antibody production was determined by the same method (modified from Svennerholm and Holmgren 1977).

Cells. Small pieces of lymph node from the colonic mesenteries and pieces of spleen were disrupted gently in glass homogenisers (Jencons), The resulting cell suspensions were washed three times in RPM) and resuspended in the complete culture medium at 107 ml I. 2 ml volumes were incubated in six-well tissue culture plates overnight at 37°C in 5 per cent carbon dioxide, frozen and thawed (control plates were frozen without the incubation period), centrifuged and the supernatants assayed for antibody by ELISA. In vitro antigen challenge Spleen and lymph node cell suspensions were adjusted to 3 x I()6 cells ml I. 10 ml cell suspension were placed in each of three tissue culture flasks with the sonicated antigen preparation at a final concentration of 0, O' I and I /Ag ml : I. The flasks were incubated for four days at 37°C in a carbon dioxide incubator. The cells were then spun down gently, washed three times and resuspended at 5 x 1()6 cells ml I in culture medium. 2 ml of each cell suspension were placed in six-well tissue culture dishes and reincubated without antigen at 37°C overnight in a carbon dioxide incubator. The suspensions were

265

frozen and thawed to disrupt the cells and release endogenous antibody, debris was spun down, and the supernatants assayed for antibody by ELISA (method modified from Hammerberg and Schurig 1984). ELISA

Coating antigen was prepared from T hyodysenteriae cells by sonication in carbonate coating buffer. Debris was removed by centrifugation and the protein concentration of the supernatant was determined. For use, the antigen preparation was diluted to 5 /Ag protein ml : 1 of carbonate coating buffer, and 150 /AI well- 1 were used to coat ELISA plates (Dynatech) overnight at 4°C. The plates were then washed with phosphate buffered saline + O' 5 per cent Tween 20 (PBS - T20). A high titre serum from a pig with swine dysentery was used as a standard in all assays. Culture supernatants and secretions were diluted I in 5; sera were diluted by three la-fold dilutions. All dilutions were in PBS - T20, and 100/AI were applied to each well. After two hours at 37°C, plates were washed in PBS - T20, and 150/A1 of mouse monoclonal antibody to pig IgG, IgA or IgM (Bristol Veterinary School) at 10 /Ag ml" PBS - T20 were applied to appropriate wells and incubated for two hours at 37°C. After washing, 150/A1 sheep anti-mouse IgG (Bristol Veterinary School) conjugated with alkaline phosphatase (Sigma) and diluted I in 500 in PBS - T20 were applied to each well. The plates were incubated and washed as before, then 150/A1 of phosphatase substrate (Sigma) at I mg ml 1 carbonate buffer were added toeach well. The plates were read on a Titertek ELISA reader at optical density450nm when a suitable amount of yellow colour had developed in the positive control (usually two to three hours at 37°C). For total as opposed to specific antibody, ELISA plates were coated with sheep anti-pig IgG or IgA at 10 /Ag ml 1 instead of with the bacterial sonicate. All other stages of the ELISA were identical, except for standards which consisted of purified pig immunoglobulins (Bristol Veterinary School) at fivefold dilutions starting with 20 /Ag ml I. Serum samples were read off the standard serum dilution curve and expressed as a percentage of standard serum. In order to allow for sampling differences, results

TABLE 3: Antibody in intestinal washings of pigs from experimant 1 Gut washings: specific activities Colon IgA :gG. Controls (n; 3) 1 x challenged (n; 4) 3 x challenged (n; 5)

0·3 2·2 13·0

0·4 2·1 14·0

9·3

2488 139

15·4 3159 163

1·9

84 6·0

IgM 1·4 113 7·4

Small intestine IgA 31·0 ± 1·0 137 ± 74 57 ± 39

266

A. S. Rees, R. J. Lysons, C. R. Stokes. F. J. Bourne variation between individual pigs (Fig I). Serum antibody levels correlated with the amount of exposure/duration of clinical signs rather than with protection; for example, the highest serum IgG antibody level was found in the pig which developed diarrhoea on third challenge, and pigs which showed no clinical signs on second and third challenge had the lowest levels. The rise in amount of specific antibody with each exposure would therefore appear to be a response to challenge and is not necessarily indicative of protection. Changes in serum IgG, IgA and IgM antibody levels in the pigs from experiment I are shown in Table 2. Mean levels of serum IgG and IgA antibody were higher in pigs challenged three times compared to once, although there was considerable variation between individuals. In contrast there was little difference in the mean IgM levels between those challenged once and three times, although mean IgM levels increased on first exposure from prechallenge levels.

TABLE 4: Specific anti-T hyodysenteriae activity in faeces and bile Faeces Controls 1 x exposed 3x exposed

IgG

IgA

Bile IgA

0 0 0

0·75 ± 1·5 6·75 ± 9·0 8'40 ± 9·7

0·39 ± 0·16 1·13 ± 1·53 2·43 ± 2·03

for secretions and faeces were standardised by expressing them as specific activities: Specific activity

lI70 of standard serum x 100 = ----:--,-.;....:.;.--.,-------

,..g ml : I total IgG or IgA

Results

Development of clinical swine dysentery Of the 47 pigs dosed with cultures of T hyodysenteriae, 44 (94 per cent) developed clinical swine dysentery. When 21 of these were rechallenged after having recovered from the disease, nine (43 per cent) developed clinical disease, as did one of 10 (10 per cent) challenged on a third occasion. The second and third challenges were severe, producing disease in all 18 control pigs dosed for the first time. The occurrence and duration of clinical disease following repeated challenge is shown in Table I.

Gut antibody Antibody levels in colon washings (Table 3) showed a different pattern: there was a less dramatic increase in IgG from first to third challenge, and IgA levels were on average higher after the first challenge, suggesting that gut IgA may be a transient response to infection and that it is not continually produced by resistant pigs. IgM was highest on first exposure. The small intestine is not colonised by T hyodysenteriae but washings contained specific IgA at

Serum antibody Levels of serum IgG antibody to T hyodysenteriae rose with each infection, though there was great

0·8

(b)

-.

0·7 1·2

(a)

1·0

E 0·8 c

~ 0 0

0·6 0'4 0·2 0

===

0·6

i~1-

~~ -

2:=

::::---

0123 Number of times challenged

E

c 0·5 In

~ 0 0

0·4 0·3 0·2 0·1 0

-

-......

'>

~~ =-~

---

-: ~ ~

0123 Number of times challenged

FIG 2: Immunoglobulin production in cultures. Pieces of colon from control pigs 10) and from pigs challenged on one, two or three occasions were either frozen immediately or cultured at 37°C for 16 hours. Each line represents the results from an individual pig Oeft-hand end of line, frozen; right-hand end, 37°C). Levels of antibody in culture supernatants were determined by ELISA and results expressed as optical density for a 1 in 5 dilution. (a) IgA production in culture; tb) IgG production in culture

267

Colon antibody to T hyodysenteriae Colon lymph node cells

1·0

IgG

IgA

0·8

E

c

0·6

~ 0

0

0·4

0·2

I a

10

0·1

1'0110

Recovered

0·4

"0,

Controls

,a

0·1 Recovered

1·0 II

a

0,'

1,0,

Controls

"g rnl-' Spleen cells

1·0

IgA

IgG

0·8

E

c

0·6

c

o

0·4

0·2

a

,a

0,' Recovered

1,0,,0

0·4

"0 110

Controls

0·1 Recovered

"0,,0

0·1

"0,

Controls

IJg rnl:'

FIG 3: IgA and IgG antibody production in culture by spleen and lymph node cells from recovered and control pigs, after stimulation in vitro with 0·1 or '·0 "g ml 1 of sonicated antigen. Each of the three pigs in the 'recovered' group is identified by its own symbol

lower levels than in the colon (Table 3). IgA levels in the colon and small intestine of individual pigs showed significant positive correlation (P
In vitro antibody production Antibody levels in gut washings reflect not only

locally produced antibody, but also antibody transudated or actively transported from serum. By incubating pieces of colon tissue and observing the rise in antibody levels in the supernatant, it is possible to detect local antibody production separately. Fig 2a shows that the colon of some animals was capable of synthesising considerable amounts of specific IgA in response to infection, but again the levels did not correlate with the number of challenges or apparent

268

A. S. Rees, R. J. Lysons, C. R. Stokes, F. J. Bourne

protection. The increase in specific IgA was not reflected by a significant change in the total IgA level in the culture fluid (controls 52 ± 44, 1 X challenged 70 ± 87, 3 x challenged 129 ± 111 ug IgA 100 rng" J colon). IgO (Fig 2b) was present in larger amounts in exposed pigs than in controls, but there was little increase in in vitro culture, suggesting that much of the IgO detected in washings is serurn-transudated. Cell suspensions from spleen and lymph node did not produce enough antibody to be detectable by ELISA. In vitro antigen challenge Cells from pigs that had recovered from one exposure to swine dysentery produced specific antibody in response to in vitro rechallenge with T hyodysenteriae (Fig 3). In the absence of antigen, no IgA antibody was detectable and IgO was only produced by one of the recovered pigs, in both lymph node and spleen cells. When antigen-stimulated, however, cells from recovered pigs produced specific IgO and IgA while those from control pigs did not, showing that the cells from recovered pigs had been primed in vivo and had the capacity to respond to a second challenge in vitro. It also appears that for each of the three recovered pigs studied, the optimal challenge dose was the same for IgO and IgA production, by both lymph node cells and spleen cells. Discussion The present authors have shown that infection with the spirochaete T hyodysenteriae resulted in the appearance of significant levels of antibody in serum and gastrointestinal tract secretions. Earlier studies have shown that repeated infection resulted in varying degrees of protection as judged by reappearances of clinical disease (Olson 1974). The present study has confirmed that resistance to swine dysentery requires stimulation resulting from an extended period of clinical disease (several weeks) and often requires a number of exposures to the organism. Repeated infection usually resulted in high levels of antibody, but serum IgO tended to be correlated with the duration of clinical signs rather than with the degree of protection and secretory IgA was indicative of recent exposure to the organism. Disruption of the mucosa during the disease may allow bacterial antigen to penetrate deeper tissues and blood vessels, so the serum antibody response is not necessarily entirely due to mucosal stimulation. Pigs infected with T hyodysenteriae produced significant levels of IgA antibody in both large and small intestinal secretions, faeces and bile. While in intestinal secretions all three isotypes were present, in the faeces of infected pigs only IgA antibody could be

detected, possibly because it is more resistant to proteolysis. The IgA antibody response in gut secretions was not persistent, levels after the primary infection being greater than after three challenges. It would thus appear that IgA antibody is not continually produced by resistant pigs. Such findings are consistent with the view that while there is clear evidence for memory in the secretory IgA system (Andrew and Hall 1982), the response in the gut is of short duration (Evans et al 1980, Burr et al 1987). Thus the presence of IgA antibody in gut secretions is indicative of recent exposure rather than solid immunity. Cells isolated from the colonic lymph nodes or spleen of pigs which had recovered from one infection with T hyodysenteriae responded to in vitro challenge with antigen. This again demonstrates that mucosal infection stimulates the production of memory cells, in this case for both IgA and IgO production. Antibody appearing in mucosal secretions may do so as a result of local production or by transudation from serum. The demonstration that isolated colonic tissue from infected pigs could synthesise considerable amounts of IgA antibody in vitro, but not IgO, confirms the potential of the colon for local production of IgA rather than IgO. The presence of high levels of IgO antibody in colonic tissue before culture and its failure to increase during culture would suggest that it is largely derived from serum. This finding reflects the numbers of immunoglobulin producing cells found in the porcine colon (Brown and Bourne 1976) where both IgA and IgM cells are more numerous than IgO. T hyodysenteriae lipopolysaccharide has been used by others as the coating antigen in ELlSAs for detecting antibody to T hyodysenteriae (Joens et aI1982). In the present study a sonicate of T hyodysenteriae was used in the ELlSAs and antibodies to a range of specificities are likely to have been detected, including antibodies to T hyodysenteriae lipopolysaccharide, heat-stable proteins, cell wall antigens, intracellular proteins and extracellular proteins as shown by an inhibition ELISA (A. S. Rees, unpublished observations). The relative importance of these antigens is not known since the pathogenesis of disease associated with T hyodysenteriae infection is unclear (Wilcock and Olander 1979), and thus the antigens involved in the disease process or in the protective response remain to be characterised. However, the present data would suggest that antibody alone is not solely responsible for the protection afforded following repeated infection. In recent years a number of cellular mechanisms have been implicated in small intestinal defence (Huntley et al 1979, Cepica and Derbyshire 1984, Tagliabue et al 1984, Sellwood et aI1986). Their involvement in immunity to a large gut pathogen such as T hyodysenteriae remains to be determined.

Colon antibody to T hyodysenteriae Acknowledgements

This work was funded by a grant from the Agricultural and Food Research Council to the late Dr T. J. Newby. The authors would like to thank Mr L. G. Murray for his technical assistance and Mrs J. Maudlin for preparing the manuscript. References ANDREW, E. & HALL, J. G. (1982) Immunology 45,177-182 BROWN, P. J. & BOURNE, F. J. (1976) American Journal of Veterinary Research 37, 9-13 BURR, D. H., KERNER, D. T., BLANCO, C. S., BOURGEOIS, A. L. & WISTAR, Jr, R. (1987) Journal of Immunological Methods 99, 277-281 CEPICA, A. & DERBYSHIRE, J. B. (1984) Canadian Journal of Comparative Medicine 48, 258-261 EVANS, P. A., NEWBY, T. J., STOKES, C. R., PATEL, D. & BOURNE, F. J. (1980) Scandinavian Journal of Immunology 11, 419-429 HAMMERBERG, C. & SCHURIG, G. C. (1984) Veterinary Immunology and Immunopathology 7,139-152 HUNTLEY, J., NEWBY, T. J. & BOURNE, F. J. (1979) Immunology 37, 225-230

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JOENS, L. A., DeYOUNG, D. W., CRAMER, J. C. & GLOCK, R. D. (1984) Proceedings of the 8th Congress of the International Pig Veterinary Society. p 1.87 JOENS, L. A., HARRIS, D. L. & BAUM, D. H. (1979) American Journal of Veterinary Research 40, 1352-1354 JOENS, L. A., NORD, N. A., KINYON, J. M. & EGAN, I. T. (1982) Journal of Clinical Microbiology IS, 249-252 LEMCKE, R. M., BEW, J., BURROWS, M. R. & LYSONS, R. J. (1979) Research in Veterinary Science 26, 315-319 NEWBY, T. J. (1984) Local Immune Responses of the Gut. Eds T. J. Newby and C. R. Stokes. Florida, CRC Press. pp 143-198 OLSON, L. D. (1974) Canadian Journal of Comparative Medicine 38,7-13 SELLWOOD, R., HALL, G. & ANGER, H. (1986) Research in Veterinary Science 40, 128-135 SVENNERHOLM, A.-M. & HOLMGREN, J. (1977) Infection and Immunity IS, 360-369 TAGLIABUE, A. D., BORASCHI, D., VILLA, L., KEREN, D. F., LOWELL, G. H., RAPPUOLI, R. & NENCIONI, L. (1984) Journal of Immunology 133, 988-992 WILCOCK, B. P. & OLANDER, H. J. (1979) Veterinary Pathology 16,567-573

Received February 19, 1988 Accepted January 9, 1989