Actinobacillus pleuropneumoniae Vaccines - Europe PMC

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INTRODUCTION. Field trials in a herd ... ANTIGEN PRODUCTION. Anionic subunit .... A. pleuropneumoniae-free herd in. Saskatchewan. ..... fractionating process during the preparation of the .... CORBETT WT, CARTER PB, SCHEY. HM.

Tissue Reaction and Immunity in Swine Immunized with Actinobacillus pleuropneumoniae Vaccines Philip J. Willson, Amalia Rossi-Campos, and Andrew A. Potter

ABSTRACT These studies were done to develop a subunit vaccine for swine that would protect against disease, but not create unacceptable tissue reactions at the immunization site. Swine were used to evaluate the local effects of subunit vaccines prepared from extracts of Actinobacillus pleuropneumoniae serotype 1 containing one of a wide variety of adjuvants. The antigen was an anionic fraction of a saline extract of A. pleuropneumoniae (ANEX). The adjuvants used were vegetable oils (peanut, sesame, canola, or corn oils, vitamin E, or Lipposyn II emulsion); mineral oil (Marcol-52) and other materials (aluminum hydroxide, polyethylene glycol, Quil-A, Amphigen, or EmulsigenPlus). Two types of experiments were done. In the 1st set of experiments, pigs were given multiple simultaneous injections in different sites and euthanized on days 1, 3, 7, 14, 21, or 28. Tissues were examined for gross and histopathological lesions. In the 2nd set of experiments, 48 pigs were allocated to 6 groups and vaccinated twice with a vaccine containing ANEX antigen combined with one of various adjuvants. Antibody responses and protection from challenge were evaluated. Among the adjuvants that were tested, mineral oils induced protective immunity, although the mineral oil Marcol-52 resulted in severe tissue reactions. The vegetable oils induced little protective immunity, and some of them were quite irritating. The response to the other materials ranged from little irritation or protection induced by the vaccine containing aluminum hydroxide to effective protection

without irritation after vaccination with ANEX/Amphigen or ANEX/ Emulsigen-Plus combinations. In conclusion, swine were protected against disease by a subunit vaccine that did not create unacceptable tissue reaction at the immunization site.

immunite' protectrice bien qu'une reaction tissulaire severe aie ete observee avec le Marcol-52. Peu d'immunite protectrice resultait de l'utilisation des huiles vetgetales et quelques unes etaient tres irritantes. Peu d'irritation et de protection resultaient de l'utilisation du vaccin contenant de l'hydroxyde d'aluminium. Par contre, une pro-


tection efficace sans irritation a ete obtenue suite a la vaccination combinant ANEX avec Amphigen ou

Cette etude avait pour but d'evaluer l'effet protecteur d'un vaccin contre la pleuropneumonie porcine prepare a partir d'une fraction anionique d'un extrait salin d'Actinobacillus pleuropneumoniae serotype 1(ANEX) combine a divers adjuvants. Le degre de reaction tissulaire au site d'injection a aussi ete evalue. Les adjuvants utilises ont ete des huiles vegetales (des huiles d'arachide, de sesame, de canola, ou de mais) de la vitamine E, ou une emulsion de Lipposyn II; de l'huile minerale (Marcol-52); ainsi que d'autres produits (hydroxyde d'aluminium, du polyethyl'ene glycol, du Quil-A, de l'Amphigen, ou de l'Emulsigen-Plus). Dans une premiere serie daexperiences, des porcs ont ete vaccines par injections a differents sites et ont ete euthanasies apres 1, 3, 7, 14, 21, ou 28 jours. Les tissus ont ete examines pour la presence de lesions macroscopiques ainsi que microscopiques. Dans la seconde serie d'experiences, 48 porcs ont ete repartis dans six groupes et les animaux dans chaque groupe ont reVu deux injections de l'antigene combine a un des adjuvants choisis. La production d'anticorps et le degre de protection suite a l'infection experimentale ont ete evalues. Parmi les adjuvants utilises, les huiles minerales ont favorise une

Emulsigen-Plus. (Traduit par Docteur Serge Messier)

INTRODUCTION Field trials in a herd endemically infected with Actinobacillus pleuropneumoniae have shown that bacterins containing a mineral oil adjuvant and killed A. pleuropneumoniae induce better protection than do bacterins containing aluminum hydroxide (1). Another study, also in an endemically infected herd, compared vaccination with a killed bacterin combined with either peanut oil or aluminum hydroxide. Although protection from acute pleuropneumonia resulted with either vaccine, pigs vaccinated with these bacterins developed subcutaneous nodules (2). In addition, the level of antibodies induced by a variety of bacterins containing mineral oil, lecithinbased oil, or aluminum hydroxide used in a minimal disease herd (3), showed that lower titers were produced in the pigs that were given bacterins containing aluminum hydroxide. Capsule extracts of A. pleuropneumoniae combined with aluminum hydroxide provided incomplete protection from acute disease (4). Immunogenicity and tissue irritation both result from vaccination with

Veterinary Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, Saskatchewan S7N 5E3. Published with the permission of the director of the Veterinary Infectious Disease Organization as journal series number 192. Submitted January 11, 1995.

Can J Vet Res 1995; 59: 299-305


mineral oil adjuvants, such as Freund's incomplete adjuvant, when used with complex bacterial antigens. This association seems to indicate that vaccine effectiveness is linked to significant tissue irritation. Vaccination with precipitated culture supernatant combined with a commercially prepared adjuvant containing mineral oil may induce immunity to A. pleuropneumoniae without producing any undesirable tissue reaction; however, the tissue reaction was not described (5). Vaccines with mineral oil adjuvants that produce extensive tissue irritation or leave a residue that reduces meat quality are not acceptable to the pork industry. Vitamin E used as an adjuvant in Brucella ovis vaccines given to sheep has resulted in higher antibody titers and no abscess formation at the site of injection when compared with some water-in-oil emulsions (6). The interaction among adjuvants, complex bacterial antigens, host tissue, and immune cells is poorly understood. Some bacterial components may suppress immunity or increase tissue irritation without enhancing immunogenicity. We undertook the studies reported herein to evaluate the immunity and tissue response of pigs immunized with vaccines containing subunit A. pleuropneumoniae antigens and different adjuvants.


Anionic subunit antigen was prepared from A. pleuropneumoniae serotype 1, strain AP37. This strain was isolated from the lung of a pig that died of pleuropneumonia and has previously been described (7, designated 79-9 in 8). Bacteria were grown for 6 h in 3 L of PPLO broth (Difco Laboratories, Detroit, Michigan) supplemented with 1% "IsoVitaleX" (BBL Microbiology Systems, Becton Dickinson, Cockeysville, Maryland), with agitation (180 rpm) at 37°C. The turbid broth was transferred to a centrifuge bottle and spun at 4250 X g for 15 min. The antigens were extracted by a modification of a method previously described (9). The supernatant was decanted, and the cell pellet suspended in 300 mL (10% of the culture volume) of NaCl (2.5%) 300

and transferred to an Erlenmeyer flask containing approximately 100 glass beads (4 mm diameter). Antigens were extracted from the bacteria by vigorous agitation (180 rpm) for 60 min at 60°C. Cellular debris was removed by centrifugation at 20 000 x g for 15 min and filtration through a membrane (0.22 jim pore size). The buffer (2.5% NaCl) was replaced with Bis Tris (20 mM, pH 6.5) containing Lubrol (0.01%) (Pierce Chemical Co., Rockford, Illinois) by passage through a Sephadex G-25 superfine fast desalting column (Pharmacia Canada Ltd., Dorval, Quebec). The anionic fraction was obtained by passing the desalted antigen through an anionic exchange column (Q Sepharose fast flow, Pharmacia Canada Ltd.) and eluting with Bis Tris (20 mM, pH 6.5) containing Lubrol and NaCl (1 M). The volume was adjusted to 300 mL with elution buffer and ANEX stored at -20°C until used to prepare the vaccine. The protein concentration (DC Protein Assay, Bio-Rad Laboratories Ltd., Mississauga, Ontario) was determined to be 508 (±23) mg/L. Each dose of vaccine contained 100 jig of this antigen (diluted to the appropriate volume in sterile distilled water). This is similar to the amount of protein used in other studies of A. pleuropneumoniae subunit vaccines (5). The placebo contained elution buffer without ANEX. The amount of total carbohydrate in ANEX, as determined using a phenol and sulfuric acid assay with glucose as a reference standard was 91 (±5) mg/L. The amount of endotoxin in ANEX, as determined using a Limulus Amebocyte Lysate test (QCL-1000, BioWhittaker, Inc., Walkersville, Maryland) was 4.1 X 106 EU/mL. ADJUVANTS

Vegetable oils (Planter's peanut oil, Hershey Canada Inc., Mississauga, Ontario; Mazola corn oil, Best Foods Canada Inc., Etobicoke, Ontario; West canola oil, Cambra Foods Ltd., Lethbridge, Alberta) were obtained from a local retail outlet. Intravenous fat emulsion (Lipposyn II emulsion, Abbott Laboratories, Montreal, Quebec) was obtained from a pharmacy. Purified saponin (Quil-A 2% dry matter, Superfos Specialty Chemicals a/s, Vedbaek, Denmark) was

obtained from E.M. Sergeant Co., Clifton, New Jersey. Polyethylene glycol, Span 80, Span 85, Tween 80, Tween 85, and vitamin E were obtained from Sigma Chemical Co., St. Louis, Missouri. Sterile sesame oil was kindly provided by Squibb Canada Inc., Montreal, Quebec. The mineral oil (Marcol-52, Texaco) and 2% suspension of aluminum hydroxide (Alhydrogel, Superfos Speciality Chemicals) were kindly supplied by Langford Laboratories, Guelph, Ontario. The 50% oil-in-water emulsion (Emulsigen-Plus, MVP Inc., Ralston, Nebraska) was kindly provided by the manufacturer. The oil Amphigen (SmithKline Beecham Animal Health, Lincoln, Nebraska) was kindly supplied by the manufacturer. VACCINE

Vaccines were prepared to have a hydrophile-lipophile-balance of about 7 (10) by combining the vegetable oil or Amphigen with Span 80 and Tween 80 emulsifiers at ratios of 120:10:3 and 25:7:3 (v/v/v), respectively. These combinations were mixed at high speed (Polytron, Brinkman Instruments, Rexdale, Ontario), and the aqueous antigen was added to the mixture of oil and emulsifiers at ratios of 1:1 and 93:7 respectively. The mixture was homogenized for 1 min. Marcol-52 was combined with Span 85 and Tween 85 at a ratio of 252:15:13 (v/v/v) and mixed. The aqueous antigen was added to the mixture of oil and emulsifiers at a ratio of 11:14 for the 50% mineral oil preparations and 110:14 for the 5% mineral oil preparations while homogenizing the mixture for 1 min, as previously described (11). The vaccines in trial 3 that contained vitamin E (combined with the adjuvant before emulsification) also included Na2SeO3 in the aqueous portion at a final concentration of 3 nM. The volume of diluent added to the antigen was reduced by the volume of the vitamin E used. The other adjuvants were combined with the antigen at the following final concentrations: Emulsigen-Plus, 30%; Lipposyn II, 50%; Quil-A, 0.05%; aluminum hydroxide, 1%; polyethylene glycol, 5%. IMMUNOLOGICAL METHODS

All serum samples were titrated using an ELISA similar to that

described previously (12). The antigen used to coat the plates was ANEX. Briefly, the wells were coated overnight at 4°C with 100 ,uL of a solution containing 10 pug mL-' of ANEX protein in carbonate buffer. The internal standard consisted of a pool of swine sera of pigs that had recovered from A. pleuropneumoniae, serotype 1 infection. Serum dilutions (starting at 1:100) and rabbit antipig peroxidase-conjugated IgG (Organon Teknika Corporation, Durham, North Carolina) were each left to incubate for 1 h at room temperature. Plates were developed for 30 min using 100 ,iL recrystallized 5-aminosalicylic acid (6.5 mM) activated immediately prior to use by the addition of H202 (0.02%, v/v). Both test sera and the standard serum pool were tested in serial dilutions, and regression equations were calculated for all of them. The titers of test sera were determined by the intersection of a least-squares regression of absorbance at 450 nm versus the logarithm of dilution with the standard absorption value. The standard absorption value was calculated by regression analysis for each plate as the value produced by the standard serum pool at a dilution of 1:6400.

TABLE I. Lesion score for pigs at postmortem. A lesion score of 0 indicates normality; 1 indicates discoloration or fascial thickening; 2 indicates discoloration and firm thickening; and 3 indicates a multifocal nodular lesion or abscess

Adjuvant used in injection Pig ID PEG Lipposyn II Emulsion Canola oil Sesame oil Cornoil Peanut oil Marcol-52 Quil-A

Day after vaccination 1 55 1 1

3 56 0 1

7 57 0 1

14 58 1 0

21 59 0 1

21 61 0 0

21 62 0 0

28 60 0 0

28 63 0 0

1 1 1 1 1 3

1 1 1 1 1 3

1 1 2 2 2 3

0 1 1 1 3 3

0 0 2 2 2 2

0 2 0 2 0 3

0 0 2 2 3 0

0 0 0 1 2 0

0 0 1 3 2 0

TABLE II. Lesion score for pigs at postmortem after a second vaccination with various adjuvants. The scoring system is the same as described for Table I Adjuvant used in second injection Pig ID PEG Lipposyn II Amphigen Canola oil Quil-A Marcol-52 (second) Marcol-52 (second) Marcol-52 (first)

Percentage of oil in vaccine

Day after second vaccination

3 Pig 66

none 5% 5% 50% none 5%

1 Pig 67 0 1 0 1 1 1

1 0 0 2 2 2

7 Pig 68 0 0 1 2 2 2

1 3 2

21 Pig 70 0 0 0 1 3 1














Pig 69 0 0 0


Four vaccine trials were done using outbred pigs of both sexes, approximately 6 wk of age, from an A. pleuropneumoniae-free herd in Saskatchewan. All injections were 2 mL in volume and given intramuscularly. Pigs in trial 1 were injected once at 9 wk of age, and pigs in trials 2, 3 and 4 were injected at 6 and 9 wk of age. Pigs were housed indoors, with controlled temperature and ventilation, on vinyl-covered metal flooring with free access to water and commercially prepared feed. They were cared for in accordance with the principles of the Canadian Council on Animal Care (13). Trial 1 was performed to evaluate a wide variety of potential adjuvants expected to produce little tissue reaction. There were 9 pigs (9 wk of age at vaccination) used in this trial. Nine sites were identified in each pig with a tattoo. The injection sites were left or right neck, loin, upper ham, and lower ham, and the control site (not injected) was the left thigh (quadriceps

femoris). Intramuscular deposition of the vaccine was insured by fully inserting an 18 gauge, 1.5 inch needle in the center of each tattoo at a right angle to the body surface. Each vaccine was injected in the same location in each pig (Table I). All pigs were given all injections on d 0, and one or more pigs were euthanized and the injection sites examined on d 1, 3, 7, 14, 21, and 28 after injection. Trial 2 was carried out to evaluate the local tissue response in pigs when given a primary immunization with the vaccine that contained mineral oil (Marcol-52, 50%) on d 21, and a secondary immunization with 7 other preparations (Table II) on d 0. One pig was euthanized and injection sites examined on d 1, 3, 7, 14, and 21 after

selected tissues were preserved in Formalin (10%), sectioned, stained with haematoxylin and eosin, and examined for histopathology. The gross lesions were given a lesion score depending on the size and type of lesion in a section transverse to the direction of the injection. A lesion score of 0 indicated normal tissue; 1 indicated a mild lesion (discoloration involving 1 to 4 cm2 of tissue); 2 indicated a moderate lesion (firm thickening and discoloration involving 5 to 10 cm2); and 3 indicated a severe lesion (abscess or multifocal nodular lesion with haemorrhage or fibrosis involving more than 10 cm2). Trial 3 was performed to evaluate the immune response and level of protection in pigs immunized with a vaccine containing ANEX combined with injection. Each injection site was palpated, 1 of 5 adjuvants or no antigen control. and the pigs were observed daily to Forty-eight pigs were randomly allodetect adverse reactions. After cated to 6 groups. The vaccines with euthanasia, each injection site was vitamin E also contained Na2SeO3. examined for gross lesions, and Group 1 received a vaccine containing


ANEX with canola oil and vitamin E as adjuvants. Group 2 received a vaccine containing ANEX and canola oil alone. Groups 3 and 4 received vaccines containing ANEX with Amphigen as the adjuvant with or without vitamin E, respectively. The controls received vaccine containing vitamin E but without ANEX (group 5) or vitamin E with ANEX (group 6). Trial 4 was designed to compare the immunity and protection provided by a variety of other adjuvants with the ANEX/mineral oil combination which provides good immunity, but severe local tissue reactions. Six groups of 8 pigs each were used. Pigs in group 1 received an ANEX/Marcol-52 vaccine, group 2 received an ANEX/Emulsigen-Plus vaccine, group 3 received an ANEX/Quil-A vaccine, group 4 received an ANEX/aluminum hydroxide vaccine, group 5 received an ANEX/Avridine vaccine, and the 6th group received Marcol-52 without antigen as a control. In both trials 3 and 4, pigs were immunized intramuscularly on days 21 and 0, and were challenged on d 7 by aerosol exposure to an A. pleuropneumoniae strain AP37 as described previously (14). Briefly, a nebulizer was used during a 10 min exposure to fill a steel and plexiglass chamber containing 5 or 6 pigs with a mist of AP37. Body temperature and clinical signs of disease were monitored once daily for 3 d following challenge. Each pig received a daily average clinical score based upon the degree of increase in respiratory rate, respiratory effort and lethargy. Clinical scores were described as follows: 0 (normal), 1 (slight increase), 2 (marked increase) or 3 (severe increase). Pigs assessed with a clinical score of 3 were humanely killed. All pigs were examined postmortem for lung lesions and local site reactions. Blood samples were taken at the time of challenge to assess antibody levels. STATISTICAL ANALYSIS

Statistical analysis was done using BMDP Statistical Software (BMDP Statistical Software, Inc., Los Angeles, California). The significance of differences in nominal data (mortality) was determined using contingency table analysis. The significance of differences between groups in ordinal data (tissue score and clinical score)


was determined by Kruskal-Wallis nonparametric analysis of variance. The scores are shown as group medians with quartile deviation to indicate the amount of variation within each group. Antibody titers were logarithmically transformed to normality, and the significance of differences between groups in antibody response and body temperature were determined using one-way analysis of variance and Dunnett's procedure for comparison with a control group. The significance of correlation between antibody titer (independent variable) and clinical signs of disease (dependent variable) was determined using linear regression by groups.


None of the vaccinated pigs showed antemortem evidence of reaction to immunization. However, the pig sacrificed 1 d after vaccination had gross and histologic evidence of irritation at all injection sites (Table I). Gross reactions ranged from 1 cm3 of pale discoloration where Lipposyn II was injected, to 6 cm3 of white streaking where mineral oil was injected. The histologic reaction was characterized by acute myositis with multifocal, linear suppurative accumulations of neutrophils. Most of the sites showed focal haemorrhage and necrosis. Injection sites examined 3 d after vaccination showed generally less extensive gross reactions. The largest reaction (3 cm3) occurred at the site of the Quil-A (Saponin) injection. The histologic appearance of the reaction at d 3 was characterized by subacute myositis with multifocal, linear accumulations of necrotic neutrophils and macrophages. Hemorrhage was not a feature of the local reaction at d 3, but necrosis was greater than that observed on d 1. Macrophages and subacute myositis were apparent at all injection sites at this time point. On d 7 postinjection, the site where polyethylene glycol was injected was the only site with no visible gross lesions. The sites where vegetable oils were injected showed ill-defined pallor or mottled red and white areas in the fascia overlying the muscle. The site of mineral oil injection showed 1 cm3 of distinct pallor in the muscle

with white linear streaking, and the Quil-A injection site showed a 6 cm3 region of pallor and linear streaking. There was no histopathology where polyethylene glycol was injected. The other histologic lesions indicated subacute to chronic myositis or fascitis of regenerative or fibrosing nature or an early granulomatous reaction. On d 14 after injection, there were either no grossly visible lesions or illdefined pallor in the muscle except the peanut oil (1 cm3 of pale yellow muscle) and Quil-A sites. One abscess from which Actinomyces pyogenes was isolated had formed at the site of Quil-A injection. This was apparently introduced from the skin surface since the vaccine was bacteriologically sterile. Histologically, myositis and/or fascitis persisted until d 14. Reactions were of a suppurative and granulomatous type at that time. Reactions observed 21 d following vaccination were less severe. There was no gross lesion at the site where PEG had been injected. Injections with vegetable oil showed either no reaction or only nodular thickening in the fascia. The reaction to the mineral oil injection was characterized grossly by streaks of pallor and histologically by multiple small foci of epithelioid cells and occasional lymphocytes or plasma cells. Appreciable gross reactions 28 d after injection only occurred at the site where mineral oil was injected and resulted in focal linear streaking. Histologic reactions caused by mineral oil showed multiple vacuoles surrounded by collagen and fibrocytes with a few epitheloid cells and lymphocytes. No histologic lesions were observed at the sites where polyethylene glycol, Lipposyn II, or sesame oil were included in the vaccine. Reactions at the peanut oil, corn oil, and canola oil sites were characterized by clear spaces or vacuoles surrounded by collagen and fibrocytes as well as epithelioid cells and occasional giant cells. Occasional foci of eosinophilic remnants of myofibrils, lymphocytes, and macrophages were also observed in sites where vegetable oils were injected. The Quil-A site was characterized by occasional necrotic fibres and extensive deposition of collagen and fibrocytes, as well as a few swollen and occasionally vacuolate macrophages.


Local tissue reactions caused by vaccines containing ANEX with Amphigen, PEG, or Lipposyn II administered 21 d following a primary immunization with ANEX in Marcol-52 resulted in a mild tissue reaction within 7 d of the booster. Tissue responses to vaccines that contained the other adjuvants using the same injection protocol were similar to those observed in trial 1 in which no primary vaccine was given. The vaccine containing Quil-A produced a mild reaction on d 1, a moderate reaction on d 3 and 7, and a severe reaction on d 14 and 21. The canola oil adjuvant resulted in moderate reactions on d 3 and 7 and only mild tissue response on d 1, 14, and 21 postsecondary vaccination. Mineral oil used at 5% produced moderate tissue reactions on d 1 through 14 and a mild reaction on d 21. The mineral oil used at 50% in the secondary immunizations produced severe tissue reactions on d 3 through 14 and moderate reaction on d 21. The site of the priming immunizations with 50% mineral oil was also examined, and severe reactions were observed at each time point (22 to 42 d after the priming injection). This confirmed previous observations that local reaction to ANEX in mineral oil tended to become more severe for at least 6 wk following vaccination. TRIAL 3

Groups of pigs vaccinated with ANEX combined with either canola oil and vitamin E, Amphigen and vitamin E, or Amphigen alone, developed high antibody titers and showed significantly (P < 0.05) less mortality than the control group (group 5). Antibody titers, tissue reaction scores, and clinical results following challenge are shown in Table III. A. pleuropneumoniae was cultured from lung tissue of each challenged pig. Pigs given a vaccine containing vitamin E adjuvant developed undesirable tissue reactions at the site of injection. Pigs vaccinated with ANEX and Amphigen alone had little reaction at the injection site and significantly less clinical disease than the controls.

TABLE III. Vaccine and challenged trial 3 Group 1 2 3 4

5 6

Adjuvant Canola and vitamin E Canola

Amphigen and vitamin E Amphigen VitaminE (no antigen) Vitamin E

Antibody titer' l9ooab

400bc 3200ab

(0.0) 0.0ab (1.8) 2.5ab (0.9)

Clinical Body score4 temp.3 40.6 ± 0.5ab 0Sab (0.6) I Ob 41.1 ±0.28c (0.4) 0.4ab 40.0 ± 0.3ab (0.4) 0. a 40.2 ±0.4ab (0.2) 1.5b 41.4 ±0.2c (0.5) 0.6ab 40.1 ± 0.2b (0.8)

Tissue score2 ND 0.0

Body temp.3 40.0 ± 0.5 40.2 ± 0.7

Tissue score2 3 0b (1.0) 0.Oa (0.0) 4.5b (1.2)


10800a lOOC 2700ab

Mortality' 0/8a 4/8bc


0/8a 7/8c 3/8abc

TABLE IV. Vaccine and challenged trial 4 Group 1 2

Adjuvant Marcol-52 EmulsigenPlus Quil-A

Antibody titer' 63800a 8600b

Clinical score4 0.25a 0.375ab

Mortality5 0/8a 2/88b

(0.0) 1.1 25ab 3/8abC 40.4 ± 0.6 0.0 (0.0) 4/8bc 0.875ab 0.0 40.6 ± 0.7 4 I000C Alhydrogel (0.0) 6/8k 1.25ab 40.4 ± 0.9 5 0.0 4200b Avridine (0.5) (0.0) I 7/8c 39.7 I.5b 0.0 OOd 6 Marcol-52 (0.3) (no antigen) 'Geometric mean antibody titer (to the nearest 100) at the time of challenge was determined by ELISA for ANEX antigens. Pigs that received no antigen had only background levels of antibodies. Within each column, numbers with different letter superscripts are significantly different (P < 0.05) 2 Sum of the lesion score for the 2 injection sites per pig (median of group and quartile deviation) at postmortem. Scoring system is described in Table I. Vaccination sites of pigs in Table IV, group I were not examined Arithmetic mean and standard deviation of body temperature (C) for survivors on the 2nd day after challenge. In Table IV there was only I survivor in group 6 4 Median clinical score (and quartile deviation) for all survivors during the 3 days after challenge. A clinical score of 0 incicates normality; I indicates increased respiratory rate, alert, active; 2 indicates increased respiratory rate labored breathing, depressed, inactive; and 3 indicates moribund severely dyspneic, cyanotic. Table IV, group 2 median clinical score is less than group 6 (P < 0.1) Number of pigs that died or were euthanized in extremis over the total in the group 3


titers, the lowest clinical scores, and no mortality (Table IV). All vaccinated groups showed antibody titers significantly (P < 0.05) greater than those of the control group. All pigs immunized with any of the ANEX and adjuvant combinations developed an antibody response, but the magnitude of the response depended on the type of adjuvant included in the vaccine. Antibody titers, mean body temperature on the 2nd d after challenge, median clinical scores, and mortality rates are shown in Table IV. Antibody titers were TRIAL 4 inversely related to the mean daily Pigs receiving ANEX with mineral clinical scores (multiple r2 = 0.5361; oil adjuvant had the highest antibody P

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