65
Exp. Rep. AHRI., No.38:65~72 (2002)
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 bioassay
＊Corresponding Author
Reprinted from J Chin Soc Vet Sci 27(3):148-155. 2001
66
Ching CHEN et al.
method
INTRODUCTION
CFU/mL and inacti- vated with a final concentration
of 0.2% formalin (Merck, Germany), and preserved
Swine erysipelas (SE) is caused by
with 0.01% Thimerosal (Simga, USA). A 10% (V/V)
Eyrsipelothrix rhusiopathiae (ER.), which is dis-
mixed adjuvant of equal volume of Emulsigen (MVP,
tributed worldwide in pig farms. This organism
USA ) ＋ Al-gel ( prepared in our laboratory ),
causes acute septicemia, urticarial lesions, endo-
which was added to prepare the monovalent bac-
carditis and polyarthritis in pigs [11]. It also
terin, then stored at 4℃ before use.
causes polyarthritis in sheep, lambs and serious
isolated from the body organs of many species of
Five-in-one polyvalent vaccine preparation :
Seed strains used
Bordetella bronchiseptica
wild and domestic mammals and birds [13,14,15].
(Bb, strain 12-1 phase 1), Pasteurella multocida
In humans, Er. causes erysipeloid, a local skin
( Pm-type A and Pm-Type D ), Actinobacillus
lesion that oc- curs mainly as an occupational
pleuropneumoniae ( App-serovar-1 and App-
disease in people
serovar-5 ), Erysipelothrix rhusiopathiae ( Er.
death loss- es in turkey. This organism has been
[6]. The organism can
occasionally be isolated from human cases of
Serovar-1a), pseudorabies virus ( TNL strain ).
endocarditis and rarely cause acute septicemia
Since the attenuated vaccine and antibiotic
Bacterial cells, virus suspension and five-inone inactivated vaccine preparation
The
were developed, the outbreak of swine erysipelas
stains mentioned above were used for bacterial cells
has been remarkably reduced
[12]. Inevitably,
suspension and/or virus suspension preparation.
there are always some cases reported in Taiwan
The manufacturing procedure for each antigen sus-
[2]. On the other hand, according to the report by
pension and the five-in-one inactivated combination
Lu et al . [7], antimicrobial additives containing
vaccine was the same as that described by Chen et
amoxicillin and chloramphenicol used in feed
al . [ 3,4 ].
disease [16].
could be interferred with the immunity effects of
the at- tenuated vaccine in pigs. Therefore, in
Safety and potency tests
order to make the monovalent or polyvalent
tency tests of the monovalent and polyvalent bac-
inactivated bac- terins will be more acceptable to
terins against Erysipelothrix rhusiopathiae. Was
the farmers, the safety and immune efficacy
conducted according to the methods of both the Na-
evaluation and explo- ration of the bacterins are
tional Standard Bioassay Regulations (NSBR) for
essential.
animal drugs [ 1 ] and a modified experimental
The safety and po-
method used by us in this study. In the NSBR
MATERIALS AND HETHODS
method, the mice was vaccine with 0.1 mL ( or
0.2 mL of 1:1 diluted ) vaccine using SC and/or IP
An
injection methods. Our modified procedures were as
erysipelothrix rhusiopathiae strain ( 1a ), which
follows. First, the inactivated vaccine was diluted
was isolated from disease pigs in a pig farm in
10 times, and 0.5 mL of the sample was adminis-
Tai- wan, where an outbreak of erysipelas
tered to each mouse using IP and / or SC. After 14
occurred [2]. The bacterial serotype 1a strain was
days, the experimental and control groups of mice
used for culti- vation in Tyrptose phosphate broth
were challenged with various concentrations of bac-
(Difco) con- taining 0.1 % Tween 80 (Merck,
terial cells suspension and observed for a period of
Germany), with pH adjusted to 7.6 and the culture
two weeks. During that period, the status of the
was shaken at 37℃ for 16 hrs [ 2 ]. The bacterial
immunized and the control groups was recorded.
11
Then, the LD50 was calculated, using the Reed &
Monovalent
bacterin
preparation
cell concentra- tion was adjusted to 3 × 10
Safety and potency of erysipelas vaccine. Corresponding author
Muench method. The protection index for the im-
67
groups. The detailed results are shown in Table 2.
munized group should be equal to or greater than
1.0
10 .
Titration of growth agglutination titers
Potency of inactivated Erysipelothrix rhusiopathiae, five-in-one combination and
monovalent vaccines in mice According to
The growth agglutination (GA) test for the
the experimental results, the IP immunized group
antisera collected from immunized mice, at
had higher potency indexes than those by SC im-
various immu- nization periods (week) was
munized groups. In addition, the dosage had some
carried out according to the method described by
influence. The detailed results are listed in Table 3.
Sawada et al . [10] and Chen et al . [2]. A locally
On the other hand, the monovalent vaccine of
isolated Er. Strain (1a) was used as the antigen for
Eysipelothrix rhusiopathiae was as potent as the
these tests.
polyvalent vaccine when challenged with living cells
of E. rhusiopathiae ( Table 4).
RESULTS
Experimental result from the safety test of
both inactivated five-in-one Polyvalent
vac- cine and adjuvants alone Following the
Growth agglutination titers of sera of mice
inoculated
with
Erysipelothrix
rhusiopathiae,
five-in-one
and
monovalent vaccines The pool sera of a
inocu- lation of various concentrations and volumes
random sampling of mice at vari- ous immunized
of vac- cine and immunization routes of the
stages (week) were used for growth agglutination
inactivated Bordetella, Pasteurlla, Actinobacillus,
tests. The results indicated that the
Erysipe- lothrix and pseudorabies virus five-in-one
the mice vaccinated by intraperitoneal injection were
combina- tion vaccine, the mice were observed for
higher than those subcutaneous injec- tion with the
two
same dosage. The details are listed in Table 5.
weeks. The
results indicated that the mouse
GA titers of
casual- ties were different among the four groups of
mice inoculated. The casualty in mice which were
inocu- lated with 0.5 mL of
10-1
DISCUSSION
diluted vaccine by
in- traperitoneal injection ( group A ) was the lowest.
According to the federal regulations No.
On the other hand, mice of groups C and D were
113.67 governing animal and animal products of the
inoculated using IP and/or SC according to the NS-
United States, The Erysipelothrix rhusiopathiae
BR, their casualties were higher than those of
(Er .) vaccine shall be prepared as a desiccated live
groups A and B. The results are detailed in Table
culture of an avirulent or modified strain of Er.
1. In the absorption tests, mice which were inject-
The Master Seed which has been established as
ed SC with five-in-one combination vaccine and ad-
pure, safe, and immunogenic shall be used for vac-
juvants
hardening,
cine production . On the other hand, regulations
swelling or ulcer at the inoculation sites. Side reac-
No. 113.119 indicates that the bacterin shall be
tion was not observed in the mice injected IP either
produced from a culture of Er. ,that has been inac-
with 0.2 mL of 1:1 diluted by physiological saline
tivated and is nontoxic (9 CFR) [5]. In USA,
alone,
showed
or with 0.5 mL of
10-1
apparent
diluted vaccines. But, the
the Er. Bacterin potency test uses mouse protection
mice vaccinated with high concentration of vaccine
test method. Dosage required for mouse is 1/10 of
had higher casualty rate. On the other hand, the
the least dosage recommended on the label for
mice inoculated with Al-gel or Emulsigen adjuvants
swine. If the relative potency ( RP ) of the un-
alone did not show any observable side reaction.
known group was more than 0.6 ( the reciprocal of
Swelling or incomplete absorption were seen at the
50 percent endpoint dilution of unknown / recipro-
site of mice inoculated with the Al-gel ( orginal )
cal of 50 percent endpoint of standard ), the serial
and the mixed ( Al-gel ＋ Emulsigen ) adjuvants
being tested would be considered as satisfactory
68
Ching CHEN et al.
[5]. Therefore, it is different from the regulations
ture, Forestry and Fisheries [8] stipulates that (1)
used in Taiwan.
the potency test of inactivated Er. Bacterin
On the other hand, in Japan, the No. 1424(9/
should use a mouse model of 10 mice ; each mouse
12 1997) announcement of the Ministry of Agri-culTable 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
Route of
Test
No. of mice
No. of mice
Mortality
(mL)
vaccination
No.
used
casualty
(%)
a
50
0
0
50
b
55
1
1.8
54
a
50
2
4.0
48
b
55
3
5.5
52
a
50
13
26.0
37
b
55
9
16.4
46
a
50
5
10.0
45
b
55
7
12.7
48
1:10
A
0.5 mL
IP
1:10
B
0.5 mL
SC
1:1
C
0.2 mL
IP
1:1
D
0.2 mL
SC
a : Date of the first experiment
IP : Intraperitoneal injection
b : Date of the second experiment
SC : Subcutaneous injection
Table 2.
No. of mice
Dosage
used for next
challenge
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
Period of
No. of mice
mice
observation
Challenged for
tested
(days)
potency test
Results
hard at inoculated site
Five-in-one combined
1:1
0.2
105
14
93
vaccine by SC
Five-in-one combined
vaccine by IP
Al-gel
(33 mg/mL)
Contorl
SC
Emulsigen
swelling or ulcer,
casualty rate of mice up to
11.4
hard at inoculated site
1:10
0.5
105
14
100
1:1
0.2
105
14
83
1:10
0.5
105
14
104
1:10
0.5
10
14
－
Original
0.5
10
14
－
1:10
0.5
10
14
－
good absorption
Original
0.5
10
14
－
good absorption
1:10
0.5
10
14
－
good absorption
Original
0.5
10
14
－
swelling, incomplete
swelling, casualty rate 4.8%
no side reaction was observed,
casualty rate up to 21%
no side reaction was observed,
casualty rate 1%
good absorption
no hardening or swelling
swelling, incomplete
absorption
(Oil-in water
Adjuvant)
Mixed
Afjuvant
(Al-gel +
Safety and potency of erysipelas vaccine. Corresponding author
Emulsigen)
69
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
A
Dosage
Route of
Time of
(mL)
vaccination
tested
1:10
IP
0.5 mL
B
1:10
SC
0.5 mL
C
1:1
IP
0.2 mL
D
1:1
SC
0.2 mL
－
Control
※
Protection
LD50
index
a
10-6.75
102.99
b
10-6.75
101.15
a
＜10-9
－
b
10-9.35
100.11
a
10-7.86
101.31
b
10-5.29
104.16
a
10-8.35
100.82
b
10-8.55
100.9
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
Route of
(mL)
vaccination
Concentration of bacterial suspension used
For challengea and survival of mice
LD50
10-5
10-6
10-7
10-8
10-9
10-10
Protection
index
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
－
－
0/10
0/10
0/10
2/10
5/10
10-9.82
－
Control
a
Dosage
－
: 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.
Should be subcutaneously immunized with 0.5 mL
group should be challenged with 0.1 mL of 10 3
of material (bacterin) twice at two week interval ;
CFU/mL of Fujizawa strain (1a) broth culture sus-
another 10 mice should be used as a control group ;
pension subcutaneously; (3) the two groups of mice
(2) two weeks later, the experimental and control
should be recorded for 7 days postchallenge; and
70
Ching CHEN et al.
(4) the resulting survivors of the experimental
1999) of Minisry of Agriculture, Forestry and
group must be more than 70%, and mortality of
Fisheries [9] contains a new bioassay regulation for
the control group must be more than 90%. Two
inactivated Erysipelas bacterin (Tocopherol Acetate
years later, another announcement No. 1247 (9/30
adjuvant). In this method, the experimental group
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)
Five-in-one
vaccine
Monovalent
bacterin
Route of
vaccination
Growth agglutination titer(1:X)
at various
immunized stages(week) of mice
2
3
4
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
1:10
0.5
SC
＜2
＜2
2
1:1
0.2
IP
2
4
8
1:1
0.2
SC
＜2
＜2
4
＜2
＜2
＜2
Control
A local strain (1a) was used to prepare the bacterial suspension as antigen for GA tests.
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
ACKNOWLEDGEMENT
strain culture suspension subcutaneously. Two
groups of mice should be recorded for 7 days
This study was supported partly by the re-
postchallenged. The resulting survivors for the ex-
search grant from 88-BT-2.3-BAPHIQ-01 (1).
perimental group must be more than 80%, and
The authors would like to thank Dr. Chung Po for
mortality for the control group must be more than
his review and correction of the manuscript.
90%.
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1. Council fo Agriculture, Executive Yuan. In: The National
istrated IP in mice developed high antibody titers
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(Table 5), the potency tests showed that the pro-
hsiang Print Co. Ltd. Taipei Taiwan Taipei. 31-33, 99-100,
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might be due to the different immune responses of
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72
Ching CHEN et al.
豬博德氏菌、巴氏桿菌、胸膜肺炎放線桿菌、丹毒絲狀菌
及假性狂犬病五合一不活化混合疫苗免疫小鼠對
豬丹毒絲狀菌攻擊之安全與免疫效力
*陳
清
柯浩然
邱資峰
賴俊雄
姜寶仁
林士鈺
行政院農業委員會家畜衛生試驗所
(收稿日期: 89 年 10 月 5 日。接受日期: 90 年 4 月 17 日)
摘要 豬博德氏菌、巴氏桿菌、放線桿菌、丹毒絲狀菌及假性狂犬病五合一不活化混合疫苗，接種試驗
小鼠，其結果免疫注射後小鼠會損失。在本試驗中試製五合一不活化菌苗及單價豬丹毒不活化菌苗，依
目前國家檢定標準及筆者等之改進方式作一比較試驗。五合一不活化菌苗，使用小鼠為材料，重複測試
2.24
其抗豬丹毒效力之結果，得知如以 10 倍稀釋菌苗 0.5 mL 腹腔免疫注射方法，其防禦指數分別為 10
( 263 )
2.7
及 10 ( 501.2 )，而相同劑量如以皮下注射免疫方法其防禦指數卻很低，或無法測知。以目前國家檢定劑
0.82
量 0.1 mL 皮下注射方法免疫者，其防禦指數分別為 10
1.31
射者，其防禦指數分別提升為 10
4.16
( 20.4 )及 10
0.9
( 6.6 )及 10 ( 7.9 )，但同劑量如改以腹腔免疫注
( 14454.4 )，雖較皮下注射表現佳，惟因濃度高供試小鼠
有損失( 21% ) 與不穩定。至於試製單價不活化丹毒菌苗之力價試驗，10 倍稀釋菌苗 0.5 mL 腹腔注射免
2.89
疫者其防禦指數可達 10
1.15
( 977.2 )，相同劑量皮下注射免疫者則僅有 10
( 14.1 )。以國家檢定劑量 0.1 mL
腹腔免疫者之效力亦較皮下免疫注射者為佳。至於免疫各組小鼠血清之發育凝集抗體價如表所示成績。
根據本試驗成績，建議修正豬丹毒不活化菌苗檢定標準時，將效力試驗皮下免疫注射方法及免疫劑量修
正為將不活化菌苗 10 倍稀釋液 0.5 mL 腹腔免疫注射 14 日後攻毒，觀察二週，試驗組及對照組分別加以
1
記錄，依 Reed & Muench 方法計算其 LD50，結果試驗組之防禦指數須≧10 ( 10 )。本改進之檢定方法易於
實施且結果將更為穩定與確實。[*陳清、柯浩然、邱資峰、賴俊雄、姜寶仁、林士鈺。豬博德氏支氣管敗
血症桿菌、巴氏德敗血症桿菌、胸膜肺炎放線桿菌、丹毒絲狀桿菌及假性狂犬病不活化混合疫苗免疫小
鼠對豬丹毒之安全性與免疫效力。中華獸醫誌 27 (3): 148-155，2001。 *聯絡人 TEL: 02-2621 2111 ext. 230，
FAX: 02-2622 5345，E-mail: Bioprod@mail.tahri.gov.tw]
鍵詞: 丹毒絲狀菌，單價不活化菌苗，五合一不活化疫苗，改進檢定方法