Neutralization of Pasteurella haemolytica leucotoxin by bovine immunoglobulins

Br. vet. J . (1987) . 143, 439

NEUTRALIZATION OF PASTEURELLA HAEMOLYTICA LEUCOTOXIN BY BOVINE IMMUNOGLOBULINS

J . K . O'BRIEN* and W. P . H . DUFFUS Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES

SUMMARY Purified bovine immunoglobulin (Ig) G2 neutralized Pasteurella haemolytica Al leucotoxin in vitro more efficiently than IgG1 ; IgM did not neutralize . A significant correlation was found between the neutralization titre of unfractionated immune serum and the specific binding of IgG2 only, when measured in an antiglobulin ELISA using P. haemolytica culture filtrate as the coating antigen .

INTRODUCTION The production by Pasteurella haemolytica of an exotoxin which is cytolytic for bovine (Shewen & Wilkie, 1982), ovine (Sutherland, 1985) and caprine (Kaehler et al., 1980) leucocytes may be an important virulence factor in the pathogenesis of pneumonic pasteurellosis . Field (Shewen & Wilkie, 1983) and experimental (Cho et al., 1984 ; Gentry et al., 1985) studies in North America demonstrated that leucotoxin serum neutralization titres of cattle may provide a better indication of resistance to the disease than titres to P . haemolytica cell wall antigens . Crude bovine immunoglobulin (Ig) G (Gentry et al., 1985) and purified IgG2 (Cho et at, 1984) from serum, and purified IgA and `IgG' from pulmonary lavage fluid (Moore et al., 1985) will neutralize the toxin in vitro. The neutralizing ability of other Ig isotypes and subisotypes of known purity has not been reported . Contamination of any supposedly `pure' Ig iso- or subisotype preparation by an heterologous Ig may produce misleading results in biological assays (Perlmann et at, 1981) . Thus it is essential that some quantitative measure of the degree of contamination is available . Immunization with crude leucotoxin provided some resistance to experimental pneumonic pasteurellosis (Shewen, 1983) . Development of a leucotoxin vaccine would be aided by more detailed information on the relative neutralizing abilities of bovine Ig isoand subisotypes in order that the most appropriate route of immunization and immunization schedule may be devised . IgA is the predominant immunoglobulin in secretions of the upper bovine respiratory tract ; in the lower tract roughly equivalent amounts of IgA and IgG are present, and *Present address : Department of Veterinary Medicine, University of Bristol, Langford House, Langford, Bristol BS18 7DU, UK .



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there are low levels of IgM (Wilkie & Markham, 1981) . Most of the IgG in lower respiratory tract secretions is IgGI (Walker et al., 1980). The present study examined the ability of bovine complement and purified IgM, IgG1 and IgG2 of known purity to neturalize the leucotoxin and determined which of these isotypes/subisotypes in immune serum mediated neutralization .

MATERIALS AND METHODS The source and purification of the macrophages used in the cytotoxicity assay have been described previously (O'Brien & Duffus, 1987) . Briefly, bronchoalveolar macrophages were obtained from healthy unanaesthetized 4-8 week old Ayrshire calves by bronchoalveolar lavage with phosphate buffered saline (PBS) which was infused and retrieved via a 4 mm polyethylene tube passed through one nostril until it lodged in a major bronchus . Mammary macrophages were obtained from the mammary quarters of a maiden Ayrshire heifer four or five days after instillation of 25 ug Escherichia coli lipopolysaccharide (Difco), as described by Wardley et al. (1976) . Following centrifugation of the recovered cells on ficoll-hypaque (Flow) gradients, the interface layer was harvested, washed three times and resuspended in RPMI 1640 medium (Flow) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 0 . 1 M sodium bircarbonate, 25 mM HEPES, 100,ug streptomycin/ml and 100 units penicillin/ml (MED). The final cell preparations were >95% viable by trypan blue exclusion, and contained >95% macrophages as determined by morphology in Giemsastained smears, staining for non-specific esterase, and ingestion of latex beads . Leucotoxic culture filtrate was prepared by a modification of the method of Shewen & Wilkie (1982) as previously described (O'Brien & Duffus, 1987) . Briefly, pelleted bacteria from a 5 . 5 hour tryptose phosphate broth culture of P. haemolytica biotype A serotype 1 (isolated from a fatal case of pneumonic pasteurellosis) were used to inoculate MED (antibiotics omitted) and incubated for one hour . These log-phase bacteria were then pelleted and the supernatant filtered through a 0 .22 µm filter (Millipore), dialysed extensively against distilled water, divided into 5 ml aliquots and lyophilized . For the cytotoxicity assay each aliquot was resuspended in 5 ml MED . A six-month old Ayrshire calf was immunized on three occasions at two-week intervals by intramuscular injection of 2 X 10 10 washed formalin-inactivated P. haemolytica (from a stationary phase culture) in PBS . Serum (designated hyperimmune) was collected two weeks after the last inoculation and stored at -20 °C. Sera were also obtained from healthy cattle of various ages and from calves suffering from chronic undifferentiated clinical pneumonia, and stored at -20 °C . As a source of bovine complement, blood was obtained from two adult cows, allowed to clot and centrifuged at 4°C . The serum was removed, pooled and immediately stored at -70 °C in 0 . 5 ml amounts . Fractionation of the hyperimmune serum into isotypes and subisotypes IgG1, IgG2 and IgM was achieved by gel filtration through Sephacryl S300 (Pharmacia) followed by ion-exchange chromatography as described by Mach & Pahud (1971) for IgM and by Townsend et al. (1982) for the IgG subisotypes . All fractions were absorbed against heterologous monospecific antisera (described below), adjusted to 2 mg/ml and stored at -20°C.



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An enzyme-linked immunosorbent assay (ELISA), a modification of that described by Townsend et al. (1982) was used to confirm the purity of the Ig fractions . Monospecific antisera diluted in carbonate-bicarbonate buffer were coated overnight at 4 °C on PVC microtitre ELISA plates (Falcon) at previously determined optimal dilutions . These antisera were as follows (coating dilution in parenthesis) : monoclonal mouse antisera specific for bovine IgG1 (1 :100 000) and IgM (1 :10 000), and polyclonal rabbit antibovine IgG2 (1 :10000). Serial dilutions of the Ig fractions were made in PBS plus 0 . 05% Tween 20 and added to the washed plates in duplicate 100,ul aliquots, along with representative reference fractions of known purity . After incubation for two hours at 37°C and washing, affinity purified rabbit anti-bovine Ig light chain alkaline phosphate conjugate at a previously determined optimal dilution of 1 :800 was added to each well and incubated as before . After washing, the substrate was added, incubated for 30 min, and the optical density (OD) of each well was measured to 405 nm in an automatic ELISA reader (Dynatech) . The percentage contamination of each fraction was determined by comparison of their respective end-point titres (OD>0 . 01) against each monospecific antiserum . For example, in the ELISA plate coated with monospecific anti-IgG2, both the purified IgG2 fraction and reference pure IgG2 had reciprocal log o titres of 5 . 5, whereas the IgG1 and IgM fractions had titres of 3 and 3 . 5 respectively, representing 0 . 32% contamination of IgG1 and 1% contamination of IgM by IgG2 . In ELISA tests used to measure the end-point titres of Ig fractions and various sera to both formalin-inactivated bacterial cells and the culture filtrate, the latter were used as coating antigens . The optimal coating concentrations of bacterial cells (10 6 ' 5 /well) and culture filtrate (1 Rg dry weight/well) were first determined by chequerboard titrations (Fig . 1) . The OD 40' readings of reference negative sera were always less than 0 . 02 at these optimal coating concentrations. A batch of culture filtrate was prepared for use in the ELISA after incubation in tryptose phosphate broth for 6 . 5 h only, ie, omitting the final incubation step in MED, in order to avoid binding of the enzyme conjugate to traces of Ig present in the FCS supplement . This filtrate was found to be slightly less leucotoxic than that produced by the complete method . For use in the ELISA the filtrate was resuspended in coating buffer . An antiglobulin ELISA (Townsend et al, 1982) using the same monospecific antisera and appropriate anti-species immunoglobulin enzyme conjugates was used to measure the specific anti-culture filtrate component(s) activity to each isotype/subisotype in unfractionated sera (C-ELISA) . Titres obtained with pure fractions using a direct and antiglobulin C-ELISA titres were similar . The possibility that competitive binding between iso- and subisotypes in serum would produce misleading results in the antiglobulin C-ELISA was investigated by comparing the titres obtained with single Ig fractions with those obtained when similar fractions were mixed with an heterologous fraction, as described by Townsend et al. (1982) for Trypanosoma theikri . No competitive binding was found to occur in this assay, and it was therefore unnecessary to use an antibody `capture' assay (Tedder, 1984) . The cytotoxicity assay has been previously described (O'Brien & Duffus, 1987) . Briefly, triplicate 100,ul samples of serum or Ig fractions (diluted in MED) and 100,ul culure filtrate (1 :2 dilution) were incubated for 45 min at room temperature in LP2 tubes (Luckham). Then 100 ,d amounts of 51 Cr-labelled macrophages (10 4 /ml) were added to each tube, the tubes vortexed and incubated at 37 ° C for two hours . Control tubes contained cells and medium or cells and toxin only .



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BRITISH VETERINARY JOURNAL, 143, 5 • Bacteria

0 Culture filtrate

1 .5 -

0

0

a

1

0

Culture filtrate concn .(Ng/ml) 7.5 9.5 r 6.5 8. 5 Bacterial concn .(Iog1o

Fig . 1 . Dose response curve of formalin-inactivated P. haemolytica (hyperimmune serum diluted 1 :320) and P. haemolytica culture filtrate (serum diluted 1 :100) binding to the solid phase . The arrows indicate the concentrations used in subsequent assays .

Following vortexing and centrifugation, 150

µl

supernatant was removed to a second

tube and both pellet and supernatant were counted in a gamma counter (Wallac) . The percentage isotope released from the cells was calculated as follows : % release = counts per second (cps) of the supernatant X 2 X 100 cps of pellet + cps of supernatant The natural logarithms of the per cent isotope release were analysed by analysis of variance and tests were then made to determine the least significant difference at a 5% confidence level by multiple range testing (Franks & Bradley, 1977) . The reciprocal log10 of the highest dilution of serum or Ig fraction which prevented a significant (P < 0 . 05) release of the isotope was defined as the neutralization titre . The neutralizing capacity of each dilution of serum or Ig fraction was defined as follows : specific neutralization = %

release without antibody-% test release

release without antibody-% release with medium only



NEUTRALIZATION OF

P. HAEMOLYTICA

LEUCOTOXIN

443

RESULTS AND DISCUSSION While the culture filtrate used to coat the ELISA plates in the C-ELISA doubtless contained many bacterial products other than the leucotoxin, a significant correlation was nevertheless found between the C-ELISA and neutralization titres (Fig . 2) . Fetal and gnotobiotic calf sera did not bind in the C-ELISA and did not neutralize . In contrast, sera from 30 cattle of ages ranging from two weeks to adult cows gave reciprocal log o C-ELISA titres from 1 . 5 to 4 . 5 .

5• Mammary macrophages

O Bronchoalveolar macrophages

3-

1-

1

C-ELISA titrellogl0 )

5

Fig . 2 . Relationship between reciprocal log o neutralization titres and titres obtained in the ELISA using culture filtrate as the coating antigen . Sera from eight animals (including three pneumonic calves) with high C-ELISA titres (3 . 5-4 . 5) were selected for further study . No significant correlation was found between neutralization titres and titres of these sera to whole cells (Table I) . Similarly, in the immunized calf, although the anti-whole bacterial cell titre increased by 2 . 5 log o dilutions after immunization, both the neutralization and C-ELISA titres remained unchanged (Fig . 3) . Antibodies to formalin-killed P. haemolytica are directed mainly against bacterial lipopolysaccharide (LPS) (Confer et aL, 1986) ; supernatants from log phase cultures, used here as the crude C-ELISA antigen, were found to be free of LPS (Baluyut et al., 1981). These results are in agreement with other studies (Gentry et al., 1985) and indicate that inactivated stationary phase bacterial preparations given by parenteral routes do not immunize against the leucotoxin . Similar results were found in a single calf immunized subcutaneously with a commercial formalin-inactivated whole cell vaccine, absorbed onto aluminium hydroxide (results not shown) . Comparison of the (antiglobulin) specific iso- and subisotype C-ELISA titres of the eight high-titred sera with their neutralization titres demonstrated a significant cor-



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BRITISH VETERINARY JOURNAL, 143, 5

relation between the IgG2 antiglobulin titres only (Table I) . Confirmation of the efficiency of IgG2 was obtained using the purified Ig fractions (Fig . 4). The end point neutralizing titre of IgGI was 1 . 5 log 10 dilutions less than that of IgG2 . Thus it appears that IgGI may have some weak neutralizing activity, which may be accounted for in part in the present study by the 0 . 32% contamination with IgG2 demonstrated by the ELISA used to examine the purity of the Ig fractions. Moore et al. (1985) found that IgG1 was roughly one third as efficient as IgG2, but the purity of either preparation was not reported . Table I Comparison of leucotoxin neutralization and ELISA titres (reciprocal log o ) of whole sera from eight cattle

Animal

1 2 3 4 5 6 7 8

Clinical status

Normal Normal Normal Normal Normal Pneumonic Pneumonic Pneumonic

Neutralization

ELISA litre (whole

titre

cells)

IgG1

a 2.5 2.5 2 2.5 3.5

b 2.5 2.5 2. 5 2 2.5

c 1.5 2

4 3 2.5

3 2

1 .5 2.5 3.5 2

2.5

3

Correlation coefficient

ab 0-44

Antiglobulin C-ELISA litre

4

a°0 . 11

IgG2

IgM

d 2 2 0.5 2

e 3

2. 5 2.5

3. 5 0 3 3 2 2 2

ad0 . 89 *

-0 . 21

3 3 .5

*Student's t test, 0-01>P>0-001 . Although IgM was found to be 1% contaminated with IgG2, this fraction did not neutralize . Contamination may have arisen during the gel filtration stage and may therefore have involved aggregated, ie, non-functional, IgG2 . However, binding of this contaminating IgG2 in the C-ELISA was not examined . All fractions were free of contamination by IgA, as measured in an ELISA assay using a mouse monoclonal antibovine IgA (Dr K. Stephens, personal communication) . Surprisingly, all Ig fractions bound strongly in the C-ELISA ; the log o titres of IgG1, IgG2, and IgM (assuming the highest concentration of each was 1 mg/ml) were 2, 2 . 5 and 3, respectively. The titres against whole bacterial cells were 3, 4, and 3, respectively . The complement source was found to have a 50% haemolytic activity (CH 50 ) of 32 . This was reduced to zero by heating to 56 °C for 30 min . Heat treatment however had no effect on the neutralizing capacity of the complement source, indicating no demonstrable role for complement in toxin neutralization (Fig . 5) . It is clear from the present study that IgG2 is highly efficient in neutralizing the P . haemolytica leucotoxin . As IgA has previously been shown to neutralize (Moore et al.,



NEUTRALIZATION OF

P. HAEMOLYTICA

LEUCOTOXIN

445

•Bacteria

ELISA IC-ELISA ∎Neuvalisation assay

5, f

4

0 0

O-O-~

•

•

∎

0

0-0

∎-∎

2

1-f t f 4 weeks

6

8

Fig. 3 . Effect of immunization with formalin-inactivated P. haemolytica on neutralization titres and ELISA titres to the culture filtrate and whole bacteria . The arrows indicate when the calf was immunized . 0-0 Ig G1

100-

8-0-0-0

"IgG2 HIgN

c 0

0

S

S

4) 50a O O

m a to 02 Fig . 4 .

t 0 .2 '0 .62 '0 .002 0 .0002 Ig concn .(mg/mI)

Titration of leucotoxin neutralizing activity of purified IgG1, IgG2 and IgM .



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BRITISH VETERINARY JOURNAL, 143, 5

100

U 0

m n 41 aR

0

1 :3

Fig. 5.

1 :300 serum dilution

1 :30,000

Effect of heat inactivation of immune serum on neutralizing activity .

1985), effective immunization against pneumonic pasteurellosis may require, at least in part, stimulation of high levels of leucotoxin neutralizing IgA and IgG2 in respiratory secretions . Stimulation of an efficient mucosal immune response in the respiratory tract to non-replicating agents is difficult (Clancy & Bienenstock, 1984) . Development of a leucotoxin vaccine may therefore require careful consideration of the route of immunization and the use of effective adjuvants or carriers .

ACKNOWLEDGEMENTS The authors are grateful to Dr J . Morris, CVL, Weybridge, and the late Dr T . Newby, Langford, for gifts of the monoclonal antisera ; to Mr B . Hunt, Cambridge Veterinary Investigation Centre, for supplying the bacterial isolate and bacteriological advice, and to the staff of Merton Hall Farm, Cambridge . J . K . O'Brien was supported by an AFRC Veterinary Schools Fellowship .

REFERENCES BALUYCT, C . S ., SIMONSON, R . R., BEMRICK, W . J . & MAHESWARAN, S. K . (1981) . American Journal of Veterinary Research 42, 1920 . CHO, H . J ., BOHAC, J . G ., YATES, W . D. G . & BIELEFELDT OHMANN, H . (1984) . Canadian Journal of Comparative Medicine 48, 151 . CLANCY, R . & BIENENSTOCK, J . (1984) . In Immunology of the Lung and Upper Respiratory Tract, ed . J . Bienenstock, pp . 216-231 . New York : McGraw-Hill . CONFER, A . W., PANCIERA, R. J . & MOSIER, D . A . (1986) . American Journal of Veterinary Research 47, 1134. FRANKS, D . & BRADLEY, B . A . (1977) . Tissue Antigens 10, 1 . GENTRY, M . J ., CONFER, A . W . & PANCIERA, R . J . (1985) . Veterinary Immunology and Immunopathology 9,239 .



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KAEHLER, K . L ., MARKHAM, R. J . F ., MUSCOPLAT, C . C . & JOHNSON, D . W . (1980) . Infection and

Immunity 30, 615 . MACH, J . P . & PAHUU, J . J . (1971) . Journal of Immunology 106, 552 . MOORE, R . N ., WALKER, R . D ., SHAW, G . A ., HOPKINS, F . M . & SHULL, E . P . (1985) . American

Journal of Veterinary Research 46, 1949 . O'BRIEN, J . K . & DUFFUS, W . P . H . (1987) . Veterinary Microbiology 13, 321 . PERLMANN, H ., PERLMANN, P ., MOREITA, L . & RONNHOLM, M . (1981) . Scandinavian Journal of

Immunology 14, 47 . SHEWEN, P. E . (1983) . Thesis, University of Guelph . SHEWEN, P . E . & WILKIE, B . N . (1982) . Infection and Immunity 35, 91 . SHEWEN, P. E . & WILKIE, B . N . (1983) . Canadian Journal of Comparative Medicine 47, 497 . SLIHERLANU, A . D . (1985). Veterinary Microbiology 10, 431 . TEDDER, R . S . (1984) . In Recent Advances in Virus Diagnosis, ed . M . S . McNulty & J . B . McFerran, p . 61 . Boston/The Hague/ Dordrecht/Lancaster : Martinus Nijhoff. TOWNSEND, A . J ., DuFFus, W . P . H . & L,\MMAs, D . A . (1982) . Research in Veterinary Science 33, 319 . WALKER, R . D ., CORSI VET, R . E ., LESSLEY, B . A . & PANCIERA, R . J . (1980) . American Journal of

Veterinary Research 41, 1015 . WARDLEY, R . C ., RousE, B . T . & BABIUK, L . A . (1976) . Journal of the Reticuloendothelial Society 19, 29 . WILKIE, B . N . & MARKHAM, R . J . F. (1981) . American Journal of Veterinary Research 42, 241 . (Accepted for publication 25 February 1987)