l'eterinary Microbiology, 29 ( 1991 ) 267-280 Elsevier Science Publist, ers B.V., Amsterdam


Characterization of the immunogenicity of formaldehyde detoxified Pasteurella multocida toxin Anne Bording a'* and Niels T. Foged b ~Ro.ral l'eterinary and Agriculture Universit.;, and Nordisk Droge Ltd.. 30 Tegivaerk~gade. DK-2100 Copenhagen. Denmark bNational l'eterinary Laboratory, Animal Biotechnology Research Center. 27 Biilowsvej. P.O. Box 3 73. DK- 1503 Copenhagen I: Denmark (Accepted 14 May 1991 )

ABSTRACT Bording, A. and Foged, N.T., ! 991. Characterization of the immunogenicity of formaldehyde detoxified Pasteurella multocida toxin. I "et. Microbiol., 29: 267-280. The immunogenicity of the Pasteurella multocida toxin ( PMT ) was studied in murine model systems. Mice were vaccinated with either formaldehyde treated pure PMT (pure toxoid ) or formaldehyde treated crude extract of toxigenic P. multocida (crude toxoid). The corresponding mean antiPMT titres, sero-conversion rates and survival rates after challenge with affinity purified PMT were compared. When assessed both by anti-PMT titres and seroconversion and challenge, pure toxoid was a more potent immunogen than crude toxoid. This greater immunogenic potency ,,'as unaffected by the addition of killed ceil preparations of Bordeteiia bronchiseptica, non-toxigenic P. muitocida and B. pertussis. Increasing anti-PMT titres and seroconversion rates were induced by increasing doses of formaldehyde treated PMT (fPMT) in the pure toxoid vaccines, but not in the vaccines containing crude toxoid. However, improved survival rates were observed for both types of vaccine, when the fPMT content was raised. Immunization of pregnant mice with vaccines containing fPMT induced protection of the offspring against challenge with PM'I': the protection of the offspring corresponded to that of the mother.


The essential role of Pasteurella multocida toxin (PMT) in the pathogenesis of progressive atrophic rhinitis (PAR) in pigs was established during the nineteen eighties (Pedersen et al., 1988 ). The discovery of PMT was followed by the development of a new generation of vaccines against Atrophic Rhinitis (AR). These vaccines contain whole cell preparations of P. multocida, mainly prepared from toxigenic strains, and in some cases the vaccines are fortified *Corresponding author. Present address, Intervet Danmark AS, 5 Roennegade, DK-2100, Copenhagen, Denmark.

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by the addition of either a crude extract of toxigenic P. multocida in a native form (de Jong et al., 1982; Pedersen and Barfod, 1982; Barfod and Pedersen, 1984 ) or a formaldehyde treated crude extract of toxigenic P. multocida (crude toxoid) (Bording and Riising, 1986 ). Field trials in pig herds have been performed to assess the beneficial effects of the vaccines (Bording and Riising, 1986; Voets, 1988; Kobisch and Pennings, 1989), but the complex nature of the vaccines and the lack of data on their methods of preparation and exact contents have limited assessment of the immunogenicity of formaldehyde detoxified PMT (fPMT) itself. However, a recent experimental study in pigs showed that administration to gilts of a single component vaccine, containing formaldehyde treated, affinity purified PMT, protected the offspring from the adverse effects of combined infections with Bordetella bronchiseptica and toxigenic P. multocida (Foged et al., 1989). This observation emphasizes the need for further characterization of the formaldehyde treated PMT as an immunogen. Results obtained during several experiments (Foged et al., 1989; Nielsen et al., accepted; Petersen et al., accepted) with both the pure toxoid vaccine and a recombinant vaccine implied a correlation between the ability of a vaccine to induce protection against intraperitoneal challenge with PMT in mice and its ability to induce protection against the effects of the combined infections with B. bronchiseptica and toxigenic P. multocida in piglets. The systems were essentially designed to assess either passive transfer of immunity from mother to offspring or conventional protection. In the present paper the use of the systems to compare pure and crude toxoid both as single component vaccines and in combination with different killed whole cells is described. MATERIAL AND METHODS

Bacterial strains The following strains were used: 1: B. bronchiseptica strain 1754 B 11-1 (provided by Nordisk Droge Ltd., Copenhagen, Denmark); 2: Non-toxigenic P. multocida type D strain 13684 (provided by National Veterinary Laboratory, Copenhagen, Denmark); 3: Toxigenic P. multocida type D strain 45/78 (NCTC 12178 ); 4: B. pertussis strain 3808 (provided by the Vaccine Dept., State Serum Institute, Copenhagen, Denmark).

Preparation of toxin PMT was purified from a crude extract of the toxigenic P. multocida spp. multocida type D strain 45/78 (NCTC 12178). Purification was done on an



affinity column containing the immobilized anti-PMT monoclonal antibody (mAb), P3FSI, as described previously (Foged, 1988). The purity of the PMT was confirmed by SDS-PAGE and silver staining. The quantification of PMT was done by using a sandwich enzyme-linked immunosorbent assay (ELISA) based on the two anti-PMT mAbs, P3F51 and biotinylated P3F37 (Foged, 1988 ).

Toxin for challenge Affinity purified PMT was diluted to the desired concentrations in sterile filtered phosphate buffered saline (PBS) containing 0.02% normal mo~jse serum (Dakopatts, Copenhagen, Denmark). The normal mouse serum was added to prevent the binding of PMT to plastic tubes and syringes, since some of the concentrations of PMT used for challenge were as low as 40 ng/ml.

Preparation of vaccines Pure toxoid. Purified PMT was dialyzed against 0.37% formaldehyde in 0.01 M phosphate buffered saline (PBS), pH 7.2 at 20°C for 48 h before the addition to the dialysis buffer of lysine-hydrochloride to 0. 1% (w/v). After the final dialysis against water: PBS (3: l ), the concentration of the fPMT was determined by a sandwich ELISA similar to that used for quantifying the PMT. Only two different mAbs were used, D l F47 and biotinylated D l F37. Unlike P3F51 and P3F37 these two mAbs react with epitopes on the PMT which are only .~li~htly a f f a . e t a d hy t h a t r ~ , a t m ~ n t Lvith t',~,-~!,~,,h,,de (in "~"'~"*"~*;'~"~ up to 1% at 20°C for two days).The pure toxoid was adsorbed to a 20% suspension of aluminium hydroxide gel (Alhydrogel Grade A, Superfos, Denmark) according to the guidelines given by the manufacturer. Finally, thiomersal (0.02% w/v) was added as a preservati'-e and the vaccine was stored at 4°C.

Crude toxoid. A vaccine based on a crude toxoid preparation of the P. multocida (NCTC 12178 ) was used similarly in mos*.experiments. Treatment with formaldehyde was done as described for the purified PMT. The quantification of PMT in the crude extract, and of the fPMT in the crude toxoid, was performed as described for the purified PMT and pure toxoid. The crude toxoid was adsorbed to a 20% Alhydrogel suspension and added thiomersal (0.02% w/v) and stored at 4°C. Before the pure and the crude toxoid preparations were used for immunization the required concentrations were prepared by dilution with a 20% Alhydrogel suspension.



Bacterial preparations Jbr immunization The bacterial preparations were made from broth cultures. They were treated with 0.37% formaldehyde for 48 hours at 20°C. When mixed with the the pure toxoid or the crude toxoid the cell concentrations of B. bronchiseptica ( 1754) and non-toxigenic P. multocida (13684) were adjusted at l × 109 cells per mi and that of B. pertussis (3808) at l × l0 s cells per ml. The mixtures ofpure toxoid or crude toxoid and bacterial preparations were adsorbed to a 20% Alhydrogel suspension. The following bacterial antigens were prepared and pure or crude toxoid added in the following combinations: 1" B. bronchiseptica ( 1754); 2: B. bronchiseptica (1754) and non-toxigenic P. multocida ( 13684); 3: B. bronchiseptica ( 1754), non-toxigenic P. multocida (13684) and B. per-

tussis (3808). Sq/bty testing. In order to ascertain non-toxicity, samples of the pure and crude toxoid were obtained before and after adsorption to Alhydrogel and tested for cytopathic effect on embryonic bovine lung cells (Rutter and Luther, 1984) or for dermonecrotic effect in guinea pigs (de Jong et al., 1980), or for lethal effect in mice (Foged, 1988 ).

Placebo vaccine. A placebo vaccine consisted ofbovine serum aibumine (BSA, Sigma, St. Louis, Minnesota) formaldehyde treated and adsorbed to a 20% Alhydrogel suspension as described for the pure and crude toxoid vaccines.

Experimental design Two model systems, A and B, based on BALB/c mice inbred over eight generations, were used for the evaluation of the immunogenicity of fPMT. Model system A had vaccination schemes designed to be as similar as possible to the vaccination of sows used for the prevention of atrophic rhinitis. Protection of both the progeny as well as that of the breeding animals was assessed. At day 0 mature ~ were mixed with d' mice for mating. Simultaneously, the ~ mice were immunized s.c. for the first time with 0.3 ml of vaccine. At day 21, approx, l week before expected birth, the d' mice were removed and the ~ mice were given the second immunization. Ten days after birth the baby mice were challenged. For challenge, affinity purified PMT was used in doses from 4-750 ng per mouse. Three quarters of the baby mice were challenged i.p. with 20/zl of the varying challenge suspensions and one quarter of the baby mice served as non-challenged controls. The number of surviving baby mice was registered over a period of 5 d. On day 15 after the birth of the baby mice, the adult ~ mice were challenged and bled. This challenge was done by i.p. injection of 0.5 ml of affinity purified PMT in doses from 20 to 16 000 ng per mouse. The number of surviving mice



TABLE I Experimental design for groups of mice tested by the model system A Vaccine t

No. of mother mice

Challenge (ng P M T )

No. of baby mice

Challenge (ng PMT )

Pure toxoid Pure toxoid Pure toxoid Crude toxoid Crude toxoid Placebo Placebo Placebo

!0 I0 !0 12 I0 I0 !0 I0

20-330 I I 0 - ! 2,800 4000- i 6 000 20-300 100-800 20-300 100-400 50- i 000

20 15 !5 0 10 !2 !0 16

4-160 20-500 30-750 N.D.-" 20-500 4-24 20- 100 30-750

~Toxoid vaccines contained 1.5 gg fPMT and placebo vaccines contained 1.5 gg BSA treated with 0.37% formaldehyde for 48 hours at 20-'C. -'Not done.

was registered over a period of 5 d and the serum samples were analysed for anti-PMT antibodies in the blocking sandwich ELISA (see below). Model system A was used for comparing the immunogenicity of fPMT in pure toxoid and crude toxoid. The anti-PMT titres and seroconversion rates and protection against challenge with PMT obtained by vaccinating with the two preparations were compared to the effects of the placebo vaccine. The concentration of fPMT or BSA in all three vaccines was adjusted to 1.5/tg per dose. in each of three repeated similar trials l 0-12 adult female mice were used for each vaccination group, and a total of 98 baby mice were included in the challenge of the offspring (Table l ). Model system B was used to assess the immunogenicity of fPMT by s.c. immunizations of ~ mice weighing 18-20 g at days 0 and 14 with 0.5 ml of vaccine. A group of non-immunized mice served as negative controls. At day 24 the mice were bled then challenged i.p. with PMT in concentrations varying from 100 to 40 000 ng. The number of surviving mice was recorded over a period of 6 d and the serum samples were analysed for anti-PMT antibodies in the blocking sandwich ELISA (see below). Model B was used for determining the anti-PMT titres and the seroconversion rates and the protection against challenge with PMT by vaccinating mice with: 1" pure toxoid vaccines containing 0.02 to 2.5 gg per dose of fPMT; 2: crude toxoid vaccines containing 0.055 to 2.4 gg per dose of fPMT; 3: pure toxoid (6/tg fPMT per dose) in the described combinations with


A. B O R D I N G A N D N.T. F O G E D

preparations of B. bronchiseptica, non-toxigenic P. multocida and B. pertuss•; 4: crude toxoid (6/zg fPMT per dose) in identical combinations of bacterial antigens. For studies 1 and 2 above 108 mice were used and for each of studies 3 and 4 four vaccination groups each consisting of 16-23 mice were used.

Serological tests All adult mice were bled immediately before challenge, and the sera were tested for the presence of antibodies to PMT by the anti-PMT ELISA previously described (Foged et al., 1988; Foged et al., 1990). The anti-PMT ELISA used was similar to the sandwich ELISA designed for quantification of PMT, only instead of incubating with a PMT sample, a preincubated mixture of 100 ng/ml PMT and serum was added to the mAb-coated well. A mouse was rated as seroconverted when serum blocked the binding of PMT to the coating mAb, so that the absorbance was reduced to less than 50% of the absorbance obtained when buffer was used instead of serum. For each seroconverted mouse, the 50% blocking titre to PMT of the serum sample was estimated by using three-fold dilutions and interpolation on a semi-logarithmic plot of absorbance (linear scale) versus dilution factor (logarithmic scale ). Statistical analysis All titre values were transformed into natural logarithms in order to ensure normal distribution and variance homogenicity. For each group of titres the logarithmic mean and standard deviation were calculated (proc. MEANS, SAS Institute Inc., 1987). For statistical analyses .........

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variance was used

(proc. ANOVA, SAS Institute Inc., 1987) and in unbalanced experiments proc. GLM [ SAS Institute Inc., 1987 ) was used. Analysis of survival data was done by using a modified X-" test, the MantelHaenzel test. RESULTS

Model system A Anti-PMT titres after vaccination of pregnant mice with pure or crude toxoid Using model system A sera from all adult mice vaccinated with the placebo BSA-vaccine were seronegative in the anti-PMT ELISA, whereas the seroconversion rates were 100% and 59% for the adult mice :.'nmunized with pure toxoid and crude toxoid, respectively.



- -II-



2 73








& 0


!00 1000 10000 Cnal:er, ge dose (ng of PMT)


Fig. 1. Survival (%) of vaccinated mature mice after challenge with PMT. The mice were vaccinated s.c. twice at 43 and 22 days before i.p. challenge with varying doses of PMT. The vaccines consisted of either formaldehyde treated BSA ( O , ,z= 30 mice), crude toxoid ( A , n = 2 2 mice ) or pure toxoid ( I , n = 30 mice). The vaccines contained 1.5 gg of either formaldehyde treated BSA or f P M T per dose. Mice from all 3 groups survived challenge with 20 ng of PMT.

For the three groups of adult mice vaccinated with pure toxoid the mean logarithmic anti-PMT titres_+SD were 6.91-+0.49, 6.79-+0.66, and 5.58 _+0.93. Significantly lower anti-PMT titres (P

Characterization of the immunogenicity of formaldehyde detoxified Pasteurella multocida toxin.

The immunogenicity of the Pasteurella multocida toxin (PMT) was studied in murine model systems. Mice were vaccinated with either formaldehyde treated...
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