Satety and Potency of Five-In-One Inactivated Vaccine Against Erysipelothrix Rhusiopathiae

Safety and potency of erysipelas vaccine. Corresponding author 67

Satety and Potency of Five-in-one Inactivated Vaccine Against Erysipelothrix rhusiopathiae Challenge in Mice

*Ching CHEN, Hao-Jan KO, Tgy-Fong CHIOU, Jiun-shong LAI,

Bao-Ren JIANG and Shiu-Yuh LIN

National Institute for Animal Health, Council of Agriculture, Executive Yuan Tansui, Taipei, Taiwan 251, ROC (Received: October 5, 2000. Accepted: April 17, 2001. )

ABSTRACT A five-in-one inactivated vaccine consisting of Bordetella bronchiseptica (Bb),Pasteurella multocida (Pm), Actinobocillus pleuropneumoniae (App), Erysipelothrix rhusiopathi- ae (Er) and pseudorabies virus was used to immunize mice. In this research, both the five-in-one inactivated vaccine and the monovalent Erysipelothrix rhusiopathiae bacterin were tested with two methods: the National Standard Bioassay Regulations (NSBR) and our modified method. Po- tency tests in duplicate against Erysipelothrix rhusiopathiae with 0.5 mL of the 10 times diluted five-in-one vaccine in mice, administered by intraperitoneal inject (IP) method had protection indexes of 102.42 (263) and 102.7(501.2), respectively. However, the same dosage injected subcu- taneously had very low or undetectable protection. When using the NSBR method of administer- ing with 0.1 mL by subcutaneous injection (SC) resulted in a protection index of 100.82 (6.6) and 100.9 (7.9). The protection indexes were increased to 101.31 (20.4) and 104.16 (14454.4) respective- ly. When the same dosage was administered by IP method. Obviously, these results indicated that vaccination using the IP method was more effective than the SC method. There were some losses of mice (21%) and unstable of safety, that might be caused by the higher concentration of the bacterin. On the other hand, the potency test in mice for the trial of Eysipelothrix rhu- siopathiae monovalent bacterin with 10-1 0.5mL administered by IP method had a protection index of 102.99(977.2).The same dosage administered by SC method had a protection index of only 101.15(1.41). When using the NSBR procedure with a dosage of 0.2 mL (1:1 diluted), the potency test resulted from the IP method was also better than that from the Sc method. Based on these find- ings, we suggest that the potency test procedure for the Erysipelothrix rhusiopathiae bacterin needs to be modified according to the injection method and the dosage used. The suggested pro- cedures are as follows. First, the inactivated bacterin should be diluted 10 times. Second, ad- minister 0.5 mL of the diluted bacterin by using the IP method. After 14 days, challenge both the experimental and control groups of mice and observe for two weeks. During this period, record the results of both the immunized and the control groups. Then, calculate the LD50 using the Reed & Muench method. The result of protection index for the immunize group should be equal to or more than 101.0 (10). This modified method is not only easier to follow but more accurate to ob- tain the results. [*Chen C, Ko HJ, Chiou TF, Lai JS, Jiang BL, and Lin SY. Safety and potency of five- in-one inactivated vaccine against erysipelothrix rhusiopathiae challenge in mice. J Chin Soc Vet Sci 27(3): 148-155,2001. * Corresponding author TEL: 02-2621 2111 ext. 230, FAX: 02-2622 5345, E-mail :Bioprod@ mail.tahri.gov.tw]

Key words: Erysipelothrix rhusiopathiae, Monovalent bacterin, Five-in-one vaccine, Modified bioassaymethod

Safety and potency of erysipelas vaccine. Corresponding author 67

INTRODUCTION

Swine erysipelas (SE) is caused by Eyrsipelothrix rhusiopathiae (ER.), which is dis- tributed worldwide in pig farms. This organism causes acute septicemia, urticarial lesions, endo- carditis and polyarthritis in pigs [11]. It also causes polyarthritis in sheep, lambs and serious death loss- es in turkey. This organism has been isolated from the body organs of many species of wild and domestic mammals and birds[13,14,15]. In humans, Er. causes erysipeloid, a local skin lesion that oc- curs mainly as an occupational disease in people [6]. The organism can occasionally be isolated from human cases of endocarditis and rarely cause acute septicemia disease [16].

Since the attenuated vaccine and antibiotic were developed, the outbreak of swine erysipelas has been remarkably reduced [12]. Inevitably, there are always some cases reported in Taiwan [2]. On the other hand, according to the report by Lu et al. [7], antimicrobial additives containing amoxicillin and chloramphenicol used in feed could be interferred with the immunity effects of the at- tenuated vaccine in pigs. Therefore, in order to make the monovalent or polyvalent inactivated bac- terins will be more acceptable to the farmers, the safety and immune efficacy evaluation and explo- ration of the bacterins are essential.

MATERIALS AND HETHODS

Monovalentbacterinpreparation Anerysipelothrix rhusiopathiaestrain (1a), which was isolated from disease pigs in a pig farm in Tai- wan, where an outbreak of erysipelas occurred [2]. The bacterial serotype 1a strain was used for culti- vation in Tyrptose phosphate broth (Difco) con- taining 0.1 % Tween 80 (Merck, Germany), with pH adjusted to 7.6 and the culture was shaken at 37℃ for 16 hrs [ 2 ]. The bacterial cell concentra- tion was adjusted to 3 × 1011 CFU/mL and inacti- vated with a final concentration of 0.2% formalin (Merck, Germany), and preserved with 0.01% Thimerosal (Simga, USA). A 10% (V/V) mixed adjuvant of equal volume of Emulsigen (MVP, USA ) ＋ Al-gel ( prepared in our laboratory ), which was added to prepare the monovalent bac- terin, then stored at 4℃ before use.

Five-in-one polyvalent vaccine preparation :Seed strains used Bordetella bronchiseptica (Bb, strain 12-1 phase 1), Pasteurella multocida ( Pm-type A and Pm-Type D ), Actinobacillus pleuropneumoniae ( App-serovar-1 and App- serovar-5 ), Erysipelothrix rhusiopathiae ( Er. Serovar-1a), pseudorabies virus ( TNL strain ).

Bacterial cells, virus suspension and five-in- one inactivated vaccine preparation The stains mentioned above were used for bacterial cells suspension and/or virus suspension preparation. The manufacturing procedure for each antigen sus- pension and the five-in-one inactivated combination vaccine was the same as that described by Chen et al . [ 3,4 ].

Safety and potency tests The safety and po- tency tests of the monovalent and polyvalent bac- terins against Erysipelothrix rhusiopathiae. Was conducted according to the methods of both the Na- tional Standard Bioassay Regulations (NSBR) for animal drugs [ 1 ] and a modified experimental method used by us in this study. In the NSBR method, the mice was vaccine with 0.1 mL ( or 0.2 mL of 1:1 diluted ) vaccine using SC and/or IP injection methods. Our modified procedures were as follows. First, the inactivated vaccine was diluted 10 times, and 0.5 mL of the sample was adminis- tered to each mouse using IP and / or SC. After 14 days, the experimental and control groups of mice were challenged with various concentrations of bac- terial cells suspension and observed for a period of two weeks. During that period, the status of the immunized and the control groups was recorded. Then, the LD50 was calculated, using the Reed & Muench method. The protection index for the im- munized group should be equal to or greater than 101.0.

Titration of growth agglutination titers The growth agglutination (GA) test for the antisera collected from immunized mice, at various immu- nization periods (week) was carried out according to the method described by Sawada et al . [10] and Chen et al . [2]. A locally isolated Er. Strain (1a) was used as the antigen for these tests.

RESULTS

Experimental result from the safety test of both inactivated five-in-one Polyvalent vac-cine and adjuvants aloneFollowing the inocu- lation of various concentrations and volumes of vac- cine and immunization routes of the inactivated Bordetella, Pasteurlla, Actinobacillus, Erysipe- lothrix and pseudorabies virus five-in-one combina- tion vaccine, the mice were observed for two weeks. The results indicated that the mouse casual-ties were different among the four groups of mice inoculated. The casualty in mice which were inocu- lated with 0.5 mL of 10-1diluted vaccine by in- traperitoneal injection ( group A ) was the lowest. On the other hand, mice of groups C and D were inoculated using IP and/or SC according to the NS- BR, their casualties were higher than those of groups A and B. The results are detailed in Table 1. In the absorption tests, mice which were inject- ed SC with five-in-one combination vaccine and ad- juvants alone, showed apparent hardening, swelling or ulcer at the inoculation sites. Side reac- tion was not observed in the mice injected IP either with 0.2 mL of 1:1 diluted by physiological saline or with 0.5 mL of 10-1 diluted vaccines. But, the mice vaccinated with high concentration of vaccine had higher casualty rate. On the other hand, the mice inoculated with Al-gel or Emulsigen adjuvants alone did not show any observable side reaction. Swelling or incomplete absorption were seen at the site of mice inoculated with the Al-gel ( orginal ) and the mixed ( Al-gel＋ Emulsigen ) adjuvants groups. The detailed results are shown in Table 2.

Potency of inactivated Erysipelothrix rhu- siopathiae, five-in-one combination and monovalent vaccines in mice According to the experimental results, the IP immunized group had higher potency indexes than those by SC im- munized groups. In addition, the dosage had some influence. The detailed results are listed in Table 3. On the other hand, the monovalent vaccine of Eysipelothrix rhusiopathiae was as potent as the polyvalent vaccine when challenged with living cells of E. rhusiopathiae ( Table 4).

Growth agglutination titers of sera of mice inoculated with Erysipelothrix rhusiopathi- ae, five-in-one and monovalent vaccines The pool sera of a random sampling of mice at vari- ous immunized stages (week) were used for growth agglutination tests. The results indicated that the GA titers of the mice vaccinated by intraperitoneal injection were higher than those subcutaneous injec- tion with the same dosage. The details are listed in Table 5.

DISCUSSION

According to the federal regulations No. 113.67 governing animal and animal products of the United States, The Erysipelothrix rhusiopathiae (Er .) vaccine shall be prepared as a desiccated live culture of an avirulent or modified strain of Er. The Master Seed which has been established as pure, safe, and immunogenic shall be used for vac- cine production . On the other hand, regulations No. 113.119 indicates that the bacterin shall be produced from a culture of Er. ,that has been inac- tivated and is nontoxic (9 CFR) [5]. In USA, the Er. Bacterin potency test uses mouse protection test method. Dosage required for mouse is 1/10 of the least dosage recommended on the label for swine. If the relative potency ( RP ) of the un- known group was more than 0.6 ( the reciprocal of 50 percent endpoint dilution of unknown / recipro- cal of 50 percent endpoint of standard ), the serial being tested would be considered as satisfactory [5]. Therefore, it is different from the regulations used in Taiwan.

On the other hand, in Japan, the No. 1424(9/ 12 1997) announcement of the Ministry of Agri-cul- ture, Forestry and Fisheries [8] stipulates that (1) the potency test of inactivated Er. Bacterin should use a mouse model of 10 mice ; each mouse

Safety and potency of erysipelas vaccine. Corresponding author 69

Table 1. safely tests in mice after inoculated with different concentrations of vaccine and immunization routes of Bordetella bronchiseptica, Pasteurella multocida, Actinobacillus pleuropneumoniae, Erysipelothrix rhusiopathiae and pseudorabies virus five-in-one inactivated vaccine.

Group / Dosage
(mL) / Route of
vaccination / Test
No. / No. of mice
used / No. of mice
casualty / Mortality
(%) / No. of mice
used for next
challenge
1:10 / a / 50 / 0 / 0 / 50
A / 0.5 mL / IP / b / 55 / 1 / 1.8 / 54
1:10 / a / 50 / 2 / 4.0 / 48
B / 0.5 mL / SC / b / 55 / 3 / 5.5 / 52
1:1 / a / 50 / 13 / 26.0 / 37
C / 0.2 mL / IP / b / 55 / 9 / 16.4 / 46
1:1 / a / 50 / 5 / 10.0 / 45
D / 0.2 mL / SC / b / 55 / 7 / 12.7 / 48

a : Date of the first experiment IP : Intraperitoneal injection

b : Date of the second experiment SC : Subcutaneous injection

Table 2. Results of the absorption test in mice for the Bordetella, Pasteurella, Actinobacillus, Erysipelothrix and pseudorabies virus five-in-one inactivated vaccine and adjuvant alone.

Kind of vaccine
or adjuvant used / Dosage (mL) / No. of
mice
tested / Period of
observation
(days) / No. of mice
Challenged for
potency test / Results
Five-in-one combined
vaccine by SC / 1:1 / 0.2 / 105 / 14 / 93 / hard at inoculated site
swelling or ulcer,
casualty rate of mice up to
11.4
1:10 / 0.5 / 105 / 14 / 100 / hard at inoculated site
swelling, casualty rate 4.8%
Five-in-one combined
vaccine by IP / 1:1 / 0.2 / 105 / 14 / 83 / no side reaction was observed,
casualty rate up to 21%
1:10 / 0.5 / 105 / 14 / 104 / no side reaction was observed,
casualty rate 1%
Al-gel
(33 mg/mL) / 1:10 / 0.5 / 10 / 14 / － / good absorption
no hardening or swelling
Original / 0.5 / 10 / 14 / － / swelling, incomplete
absorption
Contorl
SC / Emulsigen
(Oil-in water
Adjuvant) / 1:10 / 0.5 / 10 / 14 / － / good absorption
Original / 0.5 / 10 / 14 / － / good absorption
Mixed
Afjuvant
(Al-gel +
Emulsigen) / 1:10 / 0.5 / 10 / 14 / － / good absorption
Original / 0.5 / 10 / 14 / － / swelling, incomplete
absorption

－ : Not done, SC : Subcutaneous injection, IP : Intraperitioneal injection.

Table 3. Results of the potency tests for Bordetella, Pasteurella, Actinobacillus, Erysipelothrix and psedorabies virus five-in-one inactivated vaccine against Erysipelothrix rhusiopathiae challenge in mice.

Group / Dosage
(mL) / Route of
vaccination / Time of
tested / LD50 / Protection※
index
A / 1:10 / IP / a / 10-6.75 / 102.99
0.5 mL / b / 10-6.75 / 101.15
B / 1:10 / SC / a / ＜10-9 / －
0.5 mL / b / 10-9.35 / 100.11
C / 1:1 / IP / a / 10-7.86 / 101.31
0.2 mL / b / 10-5.29 / 104.16
D / 1:1 / SC / a / 10-8.35 / 100.82
0.2 mL / b / 10-8.55 / 100.9
Control / － / a / 10-9.17 / －
b / 10-9.45 / －

※The Erysipelothrix rhusiopathiae strai (1a) isolated from Chia-yi county was cultured in Tryptose phos- phate boroth at 37℃ for 16 hrs used for challenge, each mouse inoculated with 0.2 mL SC.

Protection index ＝ LD50% of immunized group/ LD50% of normal control group

Table 4. Results of the potency test for Erysipelothrix rhusiopathiae monovalent inactivated bacterin with various dosages and routes in mice.

Group / Dosage
(mL) / Route of
vaccination / Concentration of bacterial suspension used
For challengea and survival of mice / LD50 / Protection
index
10-5 / 10-6 / 10-7 / 10-8 / 10-9 / 10-10
1 / 1:10 / 0.5 mL / IP / 7/15b / 4/10 / 6/10 / 3/10 / 6/10 / － / 10-6.83 / 102.99
2 / 1:10 / 0.2 mL / SC / － / 2/10 / 5/10 / 2/10 / 5/10 / 5/10 / 10-8.67 / 101.15
3 / 1:1 / 0.2 mL / IP / 9/10 / 10/10 / 7/10 / 9/10 / 8/10 / － / ＜10-5 / ＞104.82
4 / 1:1 / 0.2 mL / SC / － / 6/10 / 6/10 / 6/10 / 7/10 / 8/10 / 10-7.11 / 102.71
Control / － / － / － / 0/10 / 0/10 / 0/10 / 2/10 / 5/10 / 10-9.82 / －

a : The Erysipelothrix rhusiopathiae (serovar 1a) isolated from Chia-yi county was cultured in Tryptose phos- phate broth at 37℃ for 16 hrs used for challenge, each mouse was inoculated with 0.2 mL SC.

b : No. of survival / No. of tested.

Safety and potency of erysipelas vaccine. Corresponding author 69

Should be subcutaneously immunized with 0.5 mL of material (bacterin) twice at two week interval ; another 10 mice should be used as a control group ; (2) two weeks later, the experimental and control group should be challenged with 0.1 mL of 103 CFU/mL of Fujizawa strain (1a) broth culture sus- pension subcutaneously; (3) the two groups of mice should be recorded for 7 days postchallenge; and (4) the resulting survivors of the experimental group must be more than 70%, and mortality of the control group must be more than 90%. Two years later, another announcement No. 1247 (9/30 1999) of Minisry of Agriculture, Forestry and Fisheries [9] contains a new bioassay regulation for inactivated Erysipelas bacterin (Tocopherol Acetate adjuvant). In this method, the experimental group

Safety and potency of erysipelas vaccine. Corresponding author 69

Table 5. Growth agglutination titers for Bordetella, Pasteurella, Actinobacillus, Erysipelothrix and pseudorabies virus five-in-one inactivated vaccine and Erysipelothrix monvalent bacterin against Erysipelothrix rhusiopathiae in mice.

Group / Dosage (mL) / Route of
vaccination / Growth agglutination titer(1:X)
at various immunized stages(week) of mice
2 / 3 / 4
Five-in-one
vaccine / 1:10 / 0.5 / IP / ＜2 / ≦2 / 2
1:10 / 0.5 / SC / ＜2 / ＜2 / ≦2
1:1 / 0.2 / IP / ＜2 / ≦2 / 4
1:1 / 0.2 / SC / ＜2 / ＜2 / ≦2
1:10 / 0.5 / IP / ＜2 / 2 / 4
Monovalent
bacterin / 1:10 / 0.5 / SC / ＜2 / ＜2 / 2
1:1 / 0.2 / IP / 2 / 4 / 8
1:1 / 0.2 / SC / ＜2 / ＜2 / 4
Control / ＜2 / ＜2 / ＜2

A local strain (1a) was used to prepare the bacterial suspension as antigen for GA tests.

Safety and potency of erysipelas vaccine. Corresponding author 71

should consist of 10 mice and each mouse has to be vaccinated with 0.5 mL of bacterin. Three weeks later, the experimental and control groups will be challenged with 0.1 mL of approximately 100 LD50/ 0.1 mL of Fujizawa strain or the same virulent strain culture suspension subcutaneously. Two groups of mice should be recorded for 7 days postchallenged. The resulting survivors for the ex- perimental group must be more than 80%, and mortality for the control group must be more than 90%.

Based on ou experimental results, although high concentration of vaccine (1:1 diluted admin- istrated IP in mice developed high antibody titers (Table 5), the potency tests showed that the pro- tection indexes were unstable (Table 3), which might be due to the different immune responses of the individual reaction. Therefore, our experiment- tal results indicated that the growth agglutination titers and potency tests in mice by intraperitoneal injection (10-1 diluted bacterin) method were not only better than those administered by subcuta- neous injection, but also more stable and accurate to obtain the results.

ACKNOWLEDGEMENT

This study was supported partly by the re- search grant from 88-BT-2.3-BAPHIQ-01 (1). The authors would like to thank Dr. Chung Po for his review and correction of the manuscript.

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