2010,43(10):

Scientia Agricultura Sinica

12

1 2501002 150030

zfb
zfb N--N'--N-MNNG
LD50
Hx

LD50101.83mL-1 104.33mL-1
650 560

Hx
MNNG

Screening an Attenuated Strain and Immunogenicity in Mice of a

Bovine Mastitis Staphylococcus Aureus Mutant
HE Na1,2, WANG Chang-fa1,YANG Hong-jun1, HE Hong-bin1, YANG Shao-hua1, WANG Li-qun2,
GAO Yun-dong1, ZHONG Ji-feng1
(1Research Center of Dairy Cattle, Shandong Academy of Agricultural Science, Jinan 250100; 2College of Life Sciences, Northeast
Agricultural University, Harbin 150030)

Abstract: Objective The objective of the study was to screen an attenuated strain and immunogenicity in mice of a bovine
mastitis Staphylococcus aureus mutant. Method An attenuated mutant, designated Hx, was derived from Staphylococcus aureus
bovine mastitis strain SA zfb after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The characteristics were tested and the
protection experiments were carried out by intraperitoneal administration in mice and challenged with homologous and heterologous
strains. Result Mutant Hx, which was isolated on the basis of hypotoxicity, showed diminished virulence in mice (LD50 of SA Hx:
10-1.83mL-1, LD50 of SA zfb: 10-4.33mL-1). Mutant Hx grew more slowly than its parental strain and showed decreased production of
several exoproteins, such as a- and -hemolysin, DNAse and coagulase. The production of its capsule was induced only under in vivo
growth conditions. In homologous challenge, the LD50 of SA zfb in mice immunized with different doses of the mutant was about 6
to 50 times higher than that of untreated mice. In heterologous challenge, the LD50 of SA 25923 and the LD50 of SA L20-2 in mice
immunized with different doses of the mutant was about 5 and 60 times higher than that of untreated mice. Conclusion The fact
shows that mutant Hx is attenuated and that it confered in mice immunity against homologous and heterologous strains, so it is an
interesting candidate for the preparation of a live-attenuated vaccine against staphylococcal infections.
Key words: Staphylococcus aureus; -hemolysin; MNNG; mutant; LD50; protection test

[1]

150 [2]

2009-05-112010-03-25
Y2007D102006BS060102006YCX028
E-mailhena19842003@yahoo.com.cnTel13153027328E-mailwcf1967@yahoo.com.cn

Staphylococcus aureus

5%50%[3-4][5]

43

Leitner [5] 3

1.1

VLVL8407ZO398 BS449

1.1.1 zfbSA

MASTIVAC I

zfbL20-2SA L20-2

MASTIVAC

Hx SAzfb

9 10

ATCC25923

70%

Str. agalactiae CVCC1886

[6]

10%Bogni

Balb/c 2125

RC122

RC122

1.1.2

RC122

DES

Reinaso

[7]

MNNG

RC122

M-H

RC122

DNA Baird-Parker

RC122

110

mL10-SSA

1.2

1.2.1 LB

zfb 5 mL4 000 r/min 10 min

4 mL 2 mg MNNG 0.05

[3]

mL 2 mL 0.1molL-1 pH 6.0

MNNG

100 gmL-137 30 min

100 L MNNG 700 L

LB 37 4 h 50 L

80%

37 18 h

- -

20

[8]

20%

zfb

-70

CAMP [8,11] 10 mL

[6,9-10]

5% 4 mm

CAMP Str.

agalactiae CVCC1886 zfb

ATCC25923 37

1824 h4 30 min

Hx

1.2.2 20

5 mL4 000 r/min 10

10

min 4 mL

2 min

-1

DES 100 gmL 37

30 min 100 L DES

110

700 L 37 4 h

110

50 L 37

mL10-SSA mL10-SSApH6.0

18 h

[13]

1.2.3 20

mL10-SSA 50 cfu

LB 72 h 4

2%

1 mL

37 24 h

37 72 h

CD

1 mL 4 Balb/c

mL10-SSApH7.0,

8 h 9 mmolL-1 NaCl

1.2.4 LD50

Hx zfb

LB 37 72 h4 000 r/min

K-B

10 min PBS Balb/c

M-H 2%4%NaCl 35

2125 d 6 6

24 h

10

1 mL 10 10 cfu72 h
[12]

Karber

LD50

19 mm MRSA European
Antimicrobial Resistance Surveillance System, 2002

1.2.5 zfb

1.2.6

ATCC25923 Hx

1315 g 6 2

15 d104106cfu

SAzfb SA25923

Hx 0.5 mL 7

37 12 h

0.5 mL
3 zfb
104106cfu

Hx 24 h

104106cfu

Hx LB 37 24 h

Hx 0.5 mL 7

ATCC25923104106cfu

http://bio.biox.cn/Biology/200701/

L20-2

20070106230847_25557.shtml

37 18 h
-DNA
100 15 min

2.1

50 mL -DNA

MNNG zfb

374 h 5

20

mm

Hx 37

0.5 mL
EDTA
37

18 h 4 4 h
1
CAMP Hx

CAMP zfb

ATCC25923 2 MNNG

2.2

72 h Hx

zfb 3

2.3

Table 1

SA zfb Hx72h
3 d 4
1 3

43

zfb Hx

Dilution

LD50 of mice infected with SAzfb and SAHx

respectively

Hx

zfb

Animal mount

Mortuus/survival

Mortuus/survival

10-1

4/2

6/0

10

-2

3/3

5/1

10

-3

1/5

6/0

10

-4

0/6

2/4

10

-5

0/6

2/4

37 18 h

10

-6

0/6

2/4

Total

36

9/27

23/13

37 72 h

LD50

1.83

4.33

DES Hx
168 SAzfb

3 d

2.4 LD50
Karber :LogLD50 = L + dS-0.5

Hx 24 h
SA zfb
Hx 2

L
LD50

SAHx

d S

SAzfb LD50 4.33 Hx LD50 1.83

SAzfb SA25923

2.5

SAzfb

SAzfb SA25923

37 12 h

Hx mL10-SSA

zfb Hx 25923

Table 2

Phenotypic characteristics of mutant Hx and parental strain zfb

Characteristic

zfb

Hx

25923

- -hemolysin

- -hemolysin

Coagulase

Clumping factor

CAMP CAMP factor

Lipase

Thermonuclease

Mannitol utilization

Pigment production

at pH 7.0

at pH 6.0

pH 7.0 at pH 7.0 after in vivo growth

pH 6.0 at pH 6.0 after in vivo growth

Resistance to methicillin

Colonial morphology in m l 10-SSA

D = Diffuse; C = Compact (colonial morphology in mL 10-SSA medium); R = resistant to antibiotics; W= without resistant
to antibiotics

10

A: SA 25923B: Hx Mutant strain HxC: SA zfb

Fig. 1 Negative hemolysis status of mutant strain Hx in blood

37 18 h CAMP

Fig. 2

agar

Phenomenon of CAMP after incubated of 18 h at 37

A: Hx Mutant strain Hx; B: SA zfb

3
Fig. 3

Comparison between negative hemolytic status of mutant strain Hx and parent strain zfb on microscope

A: Hx Mutant strain HxB: SAzfbC: Type strain 25923

4
Fig. 4

Identification of lipase and thermonuclease

Hx

2.6

mL10-SSA pH 7.0

SA25923 mL10-SSApH 6.0

7 SAzfb

Hx
7

43

mL10-SSA pH 7.0

10 cfu SAzfb

3 Hx

104 cfu 3

CAMP

SAzfb

CAMP

Hx 4

5 50 3

SA 25923 SA L20-2

5 60

[14-15]

Hx

Hx

Table 3 Protection experiments with mice immunized with

mutant Hx and challenged with the homologous
strain SA zfb

LD50(cfu) of mice

1)

[16] Hx

challenged with SA zfb

Ratio

Immunization dose of Hx

105

4.23105

SAzfb

106

2.50106

107

2.16107

50

10

1)

4.0410

[8]

LD50 LD50

LD50 of immunized mice versus LD50 of unimmunized mice. The same as

below

Table 4 Protection experiments with mice immunized with

mutant Hx and challenged with the heterologous
strain SA 25923 and SA L20-2
LD50

HL
Larsen [18]

Hx

Matsunaga [17]

462 97% -HL

LD50

SAzfb

Ratio

Hx

LD50(cfu) of

LD50(cfu) of

unimmunized mice

immunized mice

SA 25923

7.7106

3.9107

Huseby [19]

SA L20-2

4.2105

2.5107

60

Strain

Hx

CAMP Christie 1944

CAMP

1 24 h

2 Hx

[11]

BHI 37

2 Hx

MNNG

72 h zfb

MNNG

Hx mL10-SSA

[6] MNNG

Hx

Hx DES

10

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