Immune response to potassium thiocyanate extract and toxoid vaccines of Pasteurella multocida in pigs

Introduction

Pasteurellosis in pig is a highly significant disease of worldwide distribution and accounted alone for heavy economic losses due to its acuteness. The disease is caused by the genus Pasteurella and may occur in acute, septicaemic, subacute and pneumonic form (Krishnamurthy and Kaushik, 1965 [1] ; Chanter and Rutter, 1989) [10] . P. multocida is commonly involved in pig pasteurellosis. The organism rapidly develops resistance to antibiotics and therefore proper and efficient vaccination is the only way of prevention and control of the disease (Cote et al, 1991 [3] ; Sriram et al, 1994). Different extracts of Pasteurella organism like saline and Potassium thiocyanate (KSCN) has also been tried as vaccines against pasteurellosis in animal (Sharma, 1995; Choubey, 2001) [6] . Subcellular vaccines have been introduce (Ayalew et al., 2013) [7] as a promising alternative to the conventional bacterin as well as live Pasteurella vaccines. Currently, the available vaccine against this disease consists of killed P. multocida B: 2 (P52) adjuvanted with alum or oil. One drawback of this vaccine is that its immune response is generally serotype-specific (Carpenter et al., 1991) [8] ; hence it is incapable of inducing adequate protective immunity against other types of P. multocida. Along with the nature of the vaccines, it is important to find out the most immunogenic strain to be used as a vaccine candidate against Pasteurella infection. The pathogenicity of P. multocida is directly related to its antigenicity and the most pathogenic strains of P. multocida are antigenically more complex than the less or nonpathogenic strains.

Pathogencity of P. multocida is also related to the membrane protein and lipopolysaccharide pattern (Lugtenberg et al., 1984) [9] . This indicates that pathogenic strains are more immunogenic. The purified toxin of P. multocida is also immunogenic and harmless if detoxified and antibodies to it are protective (Chanter and Rutter, 1989) [10] . For development of an efficient vaccine against swine pasteurellosis, it is therefore important to identify and select the most pathogenic, immunogenic and toxinproducing strain of P. multocida as a vaccine candidate. However, no attempt has been made to develop an efficient vaccine with these extracts prepared from a suitable vaccine candidate against pneumonic pasteurellosis in pigs. Comparative evaluation of different preparations is also necessary to select an effective antigenic preparation to be used for vaccination against swine pasteurellosis. Keeping the above facts in view, the present study was undertaken with the following objectives: 1. To identify immunogenic strain of Pasteurella multocida of pig origin. 2. To prepare potassium thiocyanate extract and toxoid vaccines of P. multocida. 3. To study the immunogenicity of the vaccines in pigs.

Materials and methods 1. Pasteurella multocida isolates

A total of four predominant pathogenic strains of P. multocida capsular type A recovered from pneumonic lungs of clinically affected pigs (bearing laboratory identification nos. LKD, DP, 17a and 70b) and maintained in the Department of Microbiology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati were taken for the present study. The reference strain (P 52 ) was obtained from the Division of Bacteriology, IVRI, Izatnagar.

Pathogenicity of P. multocida isolates

Pathogenicity of the isolates of P. multocida was studied in mice following the method described by Curtis (1985) [11] with slight modification. The pathogenicity was interpreted on the basis of percentage of mortality instead of pathological lesions. Bacterial concentration was determined by matching with Brown's opacity tube no. 7 to obtain a bacterial suspension containing 10 9 organisms per ml (Wijewardana, 1992) [12] . The group of mice was injected intraperitoneally with 0.1 ml of the inoculum prepared from separate field isolates, containing 10 9 organisms per ml. The inoculated mice were observed at every 8 hours interval for 72 hours post inoculation. Dead mice were subjected to gross pathological examination and samples like heart blood, lungs, liver and spleen were collected for reisolation of P. multocida. Mice of the control group were sacrificed after 72 hours and organ samples were collected for bacteriological examination.

3. Toxigenicity of the P. multocida isolates P. multocida isolates were tested for the toxigenicity trial in mice following the method described by Nakai et al. (1984) [13] . Lethal toxicity was expressed as the number of dead mice divided by the number of mice injected. Toxins were prepared from each selected strain of P. multocida as per the method of Kamp et al. (1987) [14] . The isolates were grown in BHI broth. After 48 hours of incubation in a rotary shaker at 37 C and 200 rpm, it was centrifuged at 8000 rpm for 30 minutes at 4 C. The culture supernatant was filtered through a sterile membrane filter and was used for detection of toxin. Two doses of inoculum (1: 2 and 1: 4 dilution) were prepared from each culture supernatant of P. multocida strain. Dilutions were done with normal saline solution. Mice were inoculated intraperitoneally with 0.2 ml of respective preparation and observed up to 7 days post-inoculation and death of the mice was recorded. Dead mice were subjected to gross pathological examination and samples like heart blood, lungs, liver and spleen were collected for isolation of P. multocida, to ascertain the death of mice was due to the inoculated toxin.

Identification of immunogenic strains

The most immunogenic strain of P. multocida based on pathogenicity and toxigenicity tests was determined in mouse model by protection and cross-protection tests. Bacterin prepared from those strains was used as antigen.

Preparation of bacterin

Formalin inactivated bacterins were prepared from the selected strains of P. multocida as per the method described by Dhanda et al. (1956) [15] with slight modification as proposed by Baxi (1966) [16] . The selected strains of P. multocida were grown in 50 ml of BHI broth for 18 hours at 37 C. Purity of the bacterial strains was tested and broth cultures were spread over a number of BHI agar plates. The plates were incubated at 37 C for 18 hours. Each plate was tested for purity and the growth was harvested by washing with sterile NSS and sterile glass beads. The bacterial suspensions were centrifuged at 3000 x g for 1 hour at 4C. The sediment was washed thrice with sterile NSS. After the last centrifugation, bacterial pellet was resuspended in half of the initial volume with sterile NSS. The viable bacterial count was determined by pour plate method as described by Cruickshank et al. (1975) [17] . The concentration was finally adjusted to 10 9 organisms per ml of suspension (Wijewardana, 1992) [12] . For inactivation of bacteria, formalin [Emark (India) Ltd.] was added at a concentration of 0.25 per cent to a bacterial suspension and incubated overnight at 37C. Viability of the bacterin preparations was tested on blood agar plates.

Formulation of vaccines

The formalin-inactivated bacterins were emulsified with Montanide ISA S7 oil adjuvant (SEPPIC 75, Quai-d'Orsay, Paris). Preparations containing 10 9 cells per ml were emulsified with equal volume of oil adjuvant. Stability of the emulsified preparations was examined at 4 C for 24 hours. Sterility of the antigens was tested by inoculating the preparations on blood agar and incubating at 37 C for 48 hours. Duplicate samples were inoculated on Sabouraud's dextrose agar medium and incubated at 30C for 7-14 days. Safety of the prepared antigens was evaluated in mice. Each preparation was injected at the rate of 0.2 ml subcutaneously to a group of three mice each as per the method of Wijewardana (1992) [12] . The mice were observed up to 3 days for any mortality or development of other abnormalities.

Immunization of mice

Thirty-six healthy Swiss albino mice of identical age group (6-8 weeks) that were free from Pasteurella antibodies were selected and randomly divided into six groups (A 1 , A 2 , B 1 , B 2 , C 1 and C 2 ) with six mice in each group. All the groups of experimental animals were maintained separately without providing any antibiotic either as a treatment or a feed supplement. Groups C 1 and C 2 were kept as control. Mice of groups A 1 and A 2 were immunized subcutaneously with 0.25 ml of bacterin vaccine (strain LKD) per animal containing 10 9 organisms per dose. Similarly, the animals of groups B 1 and B 2 were vaccinated with bacterin prepared from strain DP of P. multocida with the same dose and route as for groups A 1 and A 2 . Animals of all the four groups received booster doses of respective vaccine preparations on 14 th day of post-primary immunization with the same dose and route as for primary vaccination. Mice of control groups (Groups C 1 and C 2 ) were injected with 0.25 ml of sterile NSS subcutaneously.

Challenge test

Protection conferred by the two bacterin vaccines in mice was studied by challenge test with homologous and heterologous strains of P. multocida (Wijewardana, 1992) [12] , with an infective dose of 100 LD 50 of the organisms (containing 1 x 10 7 organisms in 0.25 ml of broth culture) on 21st day post-primary vaccination. Mice of groups A 1 and B 1 were challenged with homologous strains (strains LKD and DP respectively), and A 2 and B 2 with heterologous strains of P. multocida (strains DP and LKD respectively). The control groups of mice were also challenged with LKD strain of P. multocida (group C 1 ) and DP strain (group C 2 ) of P. multocida.

Immunological study of various vaccine preparations 5.1. Bacterial strain

The most immunogenic strain of P. multocida of swine origin selected on the basis of protection and crossprotection study in mice was used for preparation of KSCN extract, toxoid and combined vaccines.

Preparation of vaccines

Potassium thiocyanate (KSCN) extract, toxoid, combined vaccines were prepared from the selected strain of P. multocida.

Preparation of potassium thiocyanate (KSCN) extract

Potassium thiocyanate extract of the selected strain of P. multocida was prepared as per the method described by Tadayan and Lauerman (1981) [18] and Ringler et al. (1985) [19] with slight modification. Pure culture of P. multocida was grown on BHI agar and incubated for 24 hours at 37 C. The culture was harvested in 6 ml of solution containing equal parts of normal saline and 1 M KSCN (Mol. Wt. 97.18) solution and further incubated at 37 C for 6 hours in a shaking water bath. The whole cells were removed by centrifugation (8000 x g) for 30 minutes at 4 C. The supernatant was collected in a cellophane dialysis tubing (HiMedia) and dialysed against 0.01 M Tris-hydrochloride -0.32 M NaCl -0.01% NaN 3 buffer at 4 C. The preparation was further concentrated by dialysing against 30% (w/v) polyethylene glycol-6000 (PEG-6000) at 4 C. The material thus obtained was designated as the potassium thiocyanate extract and used as vaccine.

Preparation of P. multocida toxoid vaccine

Toxin was extracted from the selected strain of P. multocida as per the method described by Kamp et al. (1987) [14] .

Four sterile conical flasks with 500 ml BHI broth (HiMedia) and 5 ml of an 18-hour culture of the selected strain of P. multocida, were loosely plugged with cotton wool and incubated in a rotary shaker (Water bath shaker, NSW, India) for 65 hours at 37 C at 200 rpm. The cells and celldebris were removed by centrifugation (10,000 x g, 30 minutes, 4 C). Proteins were precipitated at room temperature from the pooled supernatant by slowly adding saturated ammonium sulphate to a final concentration of 35% (w/v). After standing overnight at 4C, the precipitate was collected by centrifugation (30 minutes, 10,000 x g, 4C). The supernatant was dialysed against phosphate buffer saline (PBS) to remove the excess salt. The material thus obtained was designated as the toxin and used for vaccine preparation. The presence of toxin in the supernatant and the LD 50 of the toxin were determined in mice by inoculating 0.2 ml of the two-fold dilution in normal saline solution by intraperitoneal route.

Preparation of toxoid

Toxoid was prepared from the toxin of the selected strain of P. multocida by treating the culture supernatant with 0.5 per cent formalin at 37C for one hour (Jaisunder and Kumar, 2001) [20] . The treated sample was examined for its toxicity in mice.

Estimation of protein concentration

The protein concentration of the KSCN extract and toxin was determined by the method described by Lowry et al. (1951) [21] and were expressed in mg/ml. Bovine serum albumin (100 g/ml) was used as a standard. The optical density (OD) value was determined in UV-visible spectrophotometer at 750 nm.

Formulation of vaccines

Three different types of vaccines were formulated from the KSCN extract and toxin prepared from the most immunogenic porcine strain of P. multocida by emulsifying the respective preparation with equal volumes of Montanide ISA S7 oil adjuvant (SEPPIC 75, Quai-d'Orsay, Paris). The final protein concentration of the formulated KSCN extract and toxoid vaccines was adjusted to 1 mg/ml of inoculum for immunization of pigs. Another vaccine (a combination of KSCN extract and toxoid) was formulated by incorporating 1mg each of KSCN extract and toxoid of P. multocida. Sterility of the formulated vaccines was tested by inoculating them on 10 per cent sheep blood agar and Sabouraud's dextrose agar. Safety of the prepared vaccines was ascertained according to the method described by Wijewardana (1992) [12] . Each preparation was injected at the rate of 0.25 ml subcutaneously to a group of five mice and the mice were observed up to 3 days for any mortality or development of any other abnormality.

Immunization of pigs

A total of 40 weaned piglets of the same age group (2-3 months) and belonging to either sex reared at the 30-Sow unit and AICRP on pigs, College of Veterinary Science, Khanapara, Guwahati were used in the present immunological trial of the prepared vaccines. The piglets were randomly divided (Table 1) into three groups (Groups I, II and III) with 10 animals in each group. The three groups (I, II and III) were utilised for immunization of KSCN extract vaccines, toxoid vaccine and combination of KSCN extract and toxoid vaccine respectively. All the groups were further divided into subgroups (IA, IB, IIA, IIB, IIIA and IIIB), each comprising of five animals. All the groups of experimental animals were maintained separately. Ten animals were kept as unimmunized control.

Prior to immunization, serum samples were collected from each of the animals of all the groups and the samples were tested for the presence of antibodies against the vaccine strain by using indirect haemagglutination (IHA) test and indirect ELISA. The animals that showed no P. multocida antibodies in their serum samples were only used for vaccination trial.

Preparation of antigen

The KSCN extract and the toxin of the selected strain prepared as per the method described by Tadayan and Lauerman (1981) [18] , Ringer et al. (1985) and Kamp et al.

(1987) [14] as described earlier were used as antigen in the IHA test and indirect ELISA. KSCN extract antigen and toxin antigen were used to detect the serum antibody level in the groups vaccinated with KSCN extract vaccine and toxoid vaccine of P. multocida respectively, while both KSCN extract and toxin antigens were used in case of combined vaccine.

Indirect haemagglutination (IHA) test

Humoral antibody in Pasteurella immunized pigs was estimated by IHA test as per the method described by Sawada et al. (1982) [22] . Gluteraldehyde fixed Sheep RBCs (Bing et al., 1967) was mixed with an equal volume of PBS containing 0.005% (w/v) tannic acid and incubated at 37 C for 30 minutes with occasional shaking. The tanned G-SRBCs (T-G-SRBC) were pelleted by centrifugation at 650 x g for 10 minutes at 25 C and washed three times with PBS. After the last wash, the T-G-SRBCs were resuspended in PBS to yield a 10 per cent suspension. The T-G-SRBC was sensitised with KSCN extract and toxin antigens separately (protein concentration 2 mg/ml) and incubated at 37 C for 30 minutes with occasional shaking. The sensitised cells were washed three times with BSA-PBS, containing 0.4 per cent bovine serum albumin in PBS by centrifugation and finally suspended in BSA-PBS to yield a 0.5% (v/v) cell suspension.

Test procedure

The IHA test was performed in 96-well round-bottomed microtitre plates (Laxbro, India). Serial two-fold dilution of antisera was made in BSA-PBS and 25 l of sensitised SRBC was added to 25 l of antiserum dilution in the microtitre plates. The plates were shaken and allowed to stand for 1 to 2 hours at 25 C before SRBC setting patterns were read. The plate was then placed in a refrigerator and a second reading was taken in the following morning. The IHA titre was expressed as the reciprocal of the highest dilution of serum showing a definite positive pattern as compared to the pattern of negative control (button/smooth dot in the centre of the well). The control consisted of unsensitized SRBC in the test serum and sensitised SRBC in the diluent. To inhibit the activity of complement, the serum sample was heated at 56 C for 20 minutes.

Indirect enzyme linked immunosorbent assay (Indirect-ELISA)

The indirect ELISA was performed as per the method described by Engvall and Pearlmann (1971) [23] .

Statistical analysis

The mean antibody titres of all the vaccinated groups as detected by ELISA and IHA tests were subjected to statistical analysis as per the method described by Snedecor and Cochran (1994) [24] to evaluate the comparative efficacy of the three different vaccine preparations of P. multocida and the vaccination schedules. Statistical analysis was also carried out to find out if there was any correlation between the mean ELISA and IHA antibody titres.

Results and Discussion 1. Pathogenicity of Pasteurella Multocida Isolates

The pathogenicity trial showed wide variation among the strains of P. multocida (Tables 2). Variation in pathogenicity of different strains of P. multocida was also reported by other workers (Chandrasekaran and Yeap, 1982 [25] ; JaiSunder and Kumar, 1999, Hazarika et al. 2011) [27] . It was evident from Table 2 that out of the five strains of P. multocida subjected to pathogenicity trial, three (LKD, DP and P52) were found to be highly pathogenic producing 100 per cent mortality in mice. Muller and Krasenmann (1974) [28] , in their study, mentioned that rapidly growing P. multocida were found more pathogenic. Variation in the pathogenicity observed among the strains of P. multocida was in agreement with the findings of other workers (Jaisunder and Kumar, 1999; Dev Sharma, 2001; Hazarika, 2003) [30] .

Toxigenicity of P. multocida isolates

Result of the study indicates that, two isolates (bearing laboratory identification nos. LKD and DP) were found to be highly toxigenic inducing 100 per cent mortality in mice when injected with 1: 2 as well as 1: 4 dilution of the toxins (Table 3).

Identification of immunogenic strains

It was evident from the present finding that the bacterin vaccine prepared from one of the local isolates of P. multocida (LKD) was comparatively more effective than the bacterin prepared from the other (DP) in co nferring 100 percent protection against homologous challenge, while 83.33 per cent of mice inoculated with the same bacterin preparation were protected against heterologous challenge (Table 4). Comparatively less protection (83.33%) and cross-protection (66.67%) were observed in mice inoculated with bacterin prepared from the other strain of P. multocida (DP).

The present observation showed antigenic superiority of LKD and DP strains of P. multocida in terms of protection and cross-protection. Schimmel (1990) [31] could record significant protection and cross-protection in mice immunized with P. multocida inactivated bacterin containing aluminium hydroxide as an adjuvant. Similarly, Sharma (1995) [32] recorded 66.67 per cent protection in mice vaccinated with bacterin of P. multocida type A. Almost a similar finding was also reported by Bora (2002) and Hazarika (2003) [30] .

Immunological study of various vaccine preparations

Details of the immunological studies of the vaccine preparations are shown in Tables 5,6, 7, 8, 9 and 10 and Fig. 1, 2 and 3. The mean serum antibody titres in the vaccinated groups were expressed in log 2 scale.

Response to KSCN extracts vaccine:

The ELISA antibody titre in group IA, reached its peak (10.12  0.20) by the 45th day post-primary vaccination, the titre started to decline gradually from the 60th day (9.92  0.50) onwards to the 120th day (6.92  0.40) post-primary vaccination. In the other group of pigs (group IB) immunized with booster vaccine, there was a gradual rise in antibody titre from 7th day (6.92  0.24) post-primary vaccination and the peak titre (12.12  0.37) was recorded on 60th day post-primary vaccination, thereby gradual decline in the ELISA titre from 75th day (11.52  0.37) to 120th day (7.72  0.24) post-primary vaccination. Similar antibody response was also recorded when the serum samples (Group I) were subjected to IHA test. In the group vaccinated with KSCN extract vaccine of P. multocida (group IA), the mean serum IHA antibody titre reach its peak on 45th day (9.20  0.37) post-primary vaccination and thereafter started to decline gradually from 60th day (8.80  0.48) to 120th day (5.60  0.50) postprimary vaccination. However in group IB that received booster dose on 30th day post-primary vaccination, the mean serum IHA titre started rising as early as first week post-primary vaccination (5.00  0.44). The peak was reached on 60th day (10.40  0.50) and thereafter, the titre showed a steady decline up to 120th day (7.20  0.66) postprimary vaccination (Fig. 1 and2). Increased in antibody titre vaccinated with KSCN extract vaccines were reported in rabbits (Jarvinen et al, 1998) [34] , birds (Gaunt et al, 1977), calves (Mukkur et al, 1978) [36] , mice (Durham et al, 1986) [37] and goat (Choubey and Borah, 2001) [6] . However, a study on immunogenicity of KSCN extract vaccine of P. multocida in pigs is not available.

Response to toxoid vaccine

In the groups of pigs immunized with only primary dose of vaccine (group IIA), there was gradual increase in the ELISA antibody titre from the very first week (6.92  0.24) of immunization and reaching peak titre on the 45th day post-primary vaccination and thereafter started to decline gradually up to the 120 th (7.12  day post-primary vaccination. In group IIB, the mean ELISA antibody titre was reached the peak level at 60 days (11.72  0.24) postprimary vaccination. The peak titre was found to almost persistent up to 75th day (11.32  0.31) and thereafter there was a gradual fall of the titre up to 120th day (8.52  0.37) post-primary immunization (Fig. 1 and2). The same serum samples (Group II), when subjected to IHA test for monitoring the antibody titre, showed almost similar response. In group IIA, the IHA antibody titre was found to increase gradually from the first week (4.80  0.37) of immunization. The antibody titre reached its peak on 45th day (9.40  0.24) post-primary vaccination and thereafter, gradual fall in the antibody titre up to the 120th day (5.80  0.37) of post-primary immunization (Table 8). However, in group IIB, the serum IHA antibody titre reached it peak on the 60th day (10.60  0.24) post-primary vaccination. The titre started to fall gradually from 75th day (9.40  0.24) and the declining trend was observed up to the 120th day postprimary vaccination (Fig. 2). Toxoid vaccines of P. multocida were used by many workers for immunizing animals with promising results (Foged et al, 1989 [43] ; Jaisunder and Kumar, 19999, Pettit et al, 1993 [39] ; Diemen et al, 1994 [40] and Liu et al, 1997).

Response to combined vaccine:

In the present study, a vaccine consisting of a mixture of both toxoid and KSCN extract of P. multocida was also subjected to immunogenicity trial.

ELISA titre against KSCN extract antigen:

The serum samples of the vaccinated group (group IIIA) that received only the primary dose of the vaccine when tested against the KSCN extract antigen revealed its peak on 30th day (10.32  0.31) and remained steady up to 45th day post-primary immunization. A gradual fall of the mean ELISA titre was noticed from the 60th day (9.32  0.31) up to the 120th day (7.32  0.44) post-primary immunization (Table 6). Similar trend of ELISA titre was also observed in the group IIIB that received a booster dose of the same vaccine on 30th day post-primary vaccination. However, in this group, the mean ELISA titre which started to increase gradually from the first week (6.72  0.24) reached its peak on 60th day (13.12  0.20) of primary vaccination and remained steady up to 75th day. A fall of the titre started from the 90th day (10.92  0.24), which continued up to 120th day (9.72  0.24) post-primary vaccination.

ELISA titre against toxin antigen:

The same serum samples of the vaccinated group (Group III) when tested against toxin antigen, in the primaryvaccinated group (group IIIA), mean ELISA titre showed its peak on 30th day (10.32  0.31) and thereafter, gradual fall was observed from 45th day (9.92  0.24) up to the 120th day (6.12  0.37) post-primary immunization. In the other group (Group IIIB) that received a booster dose of the same vaccine, similar trend of ELISA titre was observed and attained its peak on 75th day (12.32  0.31).

IHA titre against KSCN extract antigen

Pigs in the group vaccinated with a single dose of combined vaccine of P. multocida (group IIIA), showed in their sera samples a gradual increase in the mean IHA antibody titre from the first week (6.00  0.44) post-primary immunization. The antibody titre reached its peak on 45th day (9.20  0.66) and thereafter it declined gradually up to 120th day (6.60  0.40) post-primary immunization. In group IIIB, that received a booster dose of the same vaccine on 30th day, the mean serum antibody titre reached its peak on 60th day (10.40  0.40) post-primary immunization. The titre started to decline gradually from 75th day (9.60  0.24) till 120th day post-primary immunization (Fig. 3).

IHA titre against toxin antigen

Similar immune response was observed in the same serum samples when subjected to IHA test using toxin antigen. In the Group IIIA, a gradual increase in mean IHA titre from 1st week (5.60  0.40) post-primary vaccination was recorded. The titre reached its peak on 45th day (9.20  0.20) and thereafter a fall in the titre was recorded up to the 120th day (6.00  0.44) post-primary vaccination. In the other group (group IIIB) that received a booster dose of the same vaccine, the mean serum antibody titre reached its peak on 45th day (10.00  0.54) and thereafter, a gradual decline of the titre was recorded from 60th day (9.60  0.24) up to 120th day (6.60  0.66) post-primary vaccination. In combined vaccinated groups, the peak titre reached on 30 th DPPV (10.32±0.31) in primary vaccinated group and on 60 th day (13.12±0.20) in booster group. The finding is in conjunction with Jarvinen et al (1998) [34] which showed greater protective immunity and higher IgA and IgG antibody titre when immunized with vaccine containing combination of P. multocida toxin and KSCN extract. The present study indicated that the vaccine prepared from local strains of P. multocida was immunogenic and could elicit a good immune response in terms of serum antibody titre. This finding was in agreement with that of Farrington (1986) [42] who observed that pigs vaccinated with homologous strain of P. multocida of the same host origin yielded a strong immunity. Similarly, a booster vaccination was found to enhance the immune response (Foged et al., 1989 [43] ; Choubey, 1997 [44] and Jarvinen et al., 1998) [34] .

Statistical analysis

The statistical analysis of the individual serum antibody titre in the groups vaccinated with different vaccine preparations of P. multocida of swine origin revealed that there were significant (P < 0.01) differences between KSCN extract vaccine, toxoid vaccine and combined vaccine, between the days post immunization as well as between the primary and booster vaccinations (Table 11,12,13 and 14). Information in respect of comparative efficacy of different vaccines prepared from porcine strains of P. multocida could not be traced in the available literature. However, from the above observations on the mean serum antibody titres in pigs and the statistical analysis, it could be suggested that the combined vaccine could induce higher immunity to the animals compared to the KSCN extract vaccine and toxoid vaccine alone. This might be due to the increase in some immunogenic factors when both KSCN extract and toxin were incorporated simultaneously (Jarvinen et al., 1998) [34] . The KSCN extract vaccine was found to elicit significantly higher immunity in animals as compared to toxoid vaccine. The better immune response to the KSCN extract was likely to be due to the multitude of components viz, outer membrane proteins, cell wall fragments, exotoxins and lipopolysaccharide that it contained (Mukkur et al., 1981) [45] . Statistical analysis of the serum antibody titre in pigs immunized with different vaccine preparations of P. multocida revealed that the combined vaccine (KSCN extract + toxoid vaccines) was comparatively more effective than KSCN extract followed by toxoid vaccine. Similar findings were earlier reported by Pedersen and Barfod (1982) [46] , Rutter (1985) [47] and Jarvinen et al. (1998) [34] . However, further study on suitable time for booster vaccination is required before being used as a vaccine candidate in the field condition.

Correlation between ELISA and IHA Titres

Assay of Pasteurella-specific antibody by ELISA and IHA showed a significant (P < 0.01) positive correlation (r = 0.935) between the tests (Fig. 4). Other workers (Solano et al., 1983 [48] ; Chawak et al., 2001) [49] also reported such a positive correlation in antibody titre estimated by ELISA and IHA test. However, considering the higher level of antibody response observed in the present study, ELISA could be advocated as a more suitable test to assay Pasteurella-specific antibodies in pigs.

Conclusion

Based on the results of the present study, it could be concluded that the combined vaccine (KSCN extract + toxoid) prepared from a locally isolated immunogenic strain of P. multocida of swine origin could elicit better and effective serum antibody titre in pigs. However, to enhance the duration of immunity and to ascertain a suitable time of booster vaccination, further studies are required to be conducted with more purified extracts of the organism.

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