1

THAI NGUYEN UNIVERSITY
THAI NGUYEN UNIVERSITY OF AGRICULTURE AND FORESTRY

NGUYEN MANH CUONG

INVESTIGATING SOME BIOLOGICAL
CHARACTERISTICS OF STREPTOCOCCUS SUIS CAUSING
PIG DISEASES IN THAI NGUYEN PROVINCE AND
MANUFACTURING AUTOVACCINE FOR PREVENTION
Major: Parasitology & Veterinary Microbiology
Code : 9.64.01.04

SUMMARY OF DOCTORAL THESIS

1


2
THAI NGUYEN - 2019
This thesis was completed at:
THAI NGUYEN UNIVERSITY OF AGRICULTURE AND FORESTRY

Scientific advisors:

1. Assoc. Prof. Dr. To Long Thanh
2. Dr. Nguyen Van Quang

Reviewer 1: ...............................................
Reviewer 2: ...............................................

Ampicillin

168

122

5

Ofloxaxin

168

90

6

Tetracyclin

168

71

7

Gentamicin

168

43


1
53.5
7
42.2
6
25.5
9
12.5
0
10.1

(+)

(%)

(+)

(%)

15

8.92

11

6.54

17

10.1

76

45.23

88

52.38

98

58.33

18
25
49
59
52

10.7
1
14.8
8
29.1
6
35.1
1
30.9


17


3

1.78

26

5
22.6
1
19.0
4
15.4
7

121

72.02

132

78.57

139

82.73

Table 3.5 showed that S. suis strains were highly susceptible to
ceftiofur (84.52%), florfenicol (81.54%), amoxicillin (80.35%),
ampicillin (72.61%) and were resistant to several antibiotics such as

Gilts, sows
( ≥ 6 months
old)
total

17

Total
strain
s

Serotype 2
Numbe
r of
Percen
positive
t
strains
(%)
(+)

Serotype 7
Numbe
r of
Percen
positive
t
strains
(%)
(+)


Unidentified
Numbe
r of
Percen
positive
t
strains
(%)
(+)

39

23

58.97

2

5.13

6

15.39

0

0

1


12.00

41

21

51.21

3

7.32

9

21.96

1

2.44

1

2.44

6

14.64

23


11

7.19

25

16.34

3

1.96

4

2.61

21

13.72


18
The results of our thesis were in agreement with those of some
authors such as Cu Huu Phu (2011), identified S. suis serotype 2 strains in
pigs with PRRS at the rate of 58.33. % and Le Van Duong et al. (2013)
when determinating serotype of S. suis isolated from samples of pigs with
PRRS in Bac Giang, serotype 2 accounted for the highest rate (56.29%),
followed by serotype 9 (17.03%) and the lowest was serotype 7 (5.18%).
3.2.6. The results of determination of gene encoding virulent factors of

Well 3: sly Gene Well 4: epf Gene
Figure 1: Results of PCR used for determining genes encoding
virulence of S. suis strains isolated
3.2.7. Result of investigating some biochemical characteristics of S.
suis strains selected after 5 passage cultures
From different kinds of pigs such as sucking piglets, postweaning piglets, porkers, gilts and sows, we selected 9 S. suis strains
representing the pathogenic serotypes in pigs, including: 3 serotype 2
strains (S.TN2-1, S.TN2-2, S.TN2-3); 3 strains of serotype 7 (S.TN7-4,
S.TN7-5, S.TN7-6) and 3 strains of serotype 9 (S.TN9-7, S.TN9-8,
S.TN9-9) distributed in 8 districts/cities of Thai Nguyen province.
The selected S. suis strains selected after 5 passage cultures, were
found to be stable in biochemical characteristics and in accordance with
the evaluation criteria according to the S. suis disease diagnosis
procedure in pigs (TCVN 8400-2: 2010). These are important
characteristics of a master seed in propagation for working seed because
they can ensure and retain their biological characteristics through
different working seed batches.
3.2.7. Results of virulent examination of some selected S. suis strains
after 5 passage cultures
Table 3.7 showed that after 5 passage cultures of S. suis strains
belong to serotype 2, 100% of the experimental mice died after 24
hours, of which 66.67% of mice died from 12 -24 hours and 33.33%
strains from 18-24 hours. The strains of S. suis serotype 9 also killed
100% of experimental mice in the period of 12-24 hours, of which
33.33% of strains caused 12-24 hours of rat death and 66.67% of strains 1824 hours. Meanwhile, only 33.33% of tested serotype 7 strains killed 100%
of experimental mice after 24-36 hours of injection and 66.67% of strains
killed 50% of mice within the previous 36-48 hours after injection.
Table 3.7: Results of virulent examination of some selected S. suis
strains after 5 passage cultures


mice
(hour)
(mouse)
2/2
18 - 24
100

Reisolation

+


20
S-TN2-2
S-TN2-3
S-TN7-4
S-TN7-5
S-TN7-6
S-TN9-7
S-TN9-8
S-TN9-9

2
2
7
7
7
9
9
9

< 48
24 - 36
< 36
12 - 24
18 - 24
18 - 24

100
100
50
100
50
100
100
100

+
+
+
+
+
+
+
+

All of dead mice slaughtered showed that the lesions were
similar, for example presence of abscess at the injection sites,
pneumonia and lung congestion, swollen soft, heart and pericardial
effusion. When the bacteria were isolated again, it was possible to
obtain pure S. suis from heart blood.

3.3.2.1. Results of cultivating S. suis in broth to produce experimental
Autovaccine
The serotypes 2, 7 and 9 were cultured and shaken (300-500
times/minute) separately for 24 hours, the minimum density was 1.64 x
108 bacteria in 1 ml of broth. After that, it was inactivated with formalin
at the percentage of 0.3% and aluminum adjuvant was added at the rate
of 1/5 (20%) according to the procedure of manufacturing inactivated
vaccine with aluminum adjuvant of the Veterinary Institute. In order to
test experimental Autovaccine procedure of testing inactivated vaccine
with aluminum adjuvant was used according to TCVN 8684: 2011 and
QCVN 01: 187-2018.
Table 3.8: Results of bacteria counting in S. suis broth used
for experimental Autovaccine production
Autovaccine
batch
Batch I (1.5 liters)
Batch II (1.5 liters)
Batch III (1.5 liters)

Number of bacteria /1 ml of broth
Total
Average
Broth 2
Broth 7
Broth 9
Average number Average number Average number number of
of bacteria /1ml of of bacteria /1ml of of bacteria /1ml of bacteria/1m
l of broth
broth
broth



22
Autovaccine batches met the criteria of purity. In media such as meat
broth, liver broth, blood agar, MacConkey agar on which were
cultured 3 different S. suis strains (S-TN2-2, S-TN7-5 and S-TN9-7)
in all 3 experimental Autovaccine batches: I, II and III were found
only S. suis growth.
3.3.2.3. Results of sterility testing of the experimental Autovaccine
The results showed that the experimental Autovaccine batches
met the criteria for sterility at all stages after inactivation with 0.3%
formalin, after adding 20% aluminum adjuvant and after bottling.
3.3.2.4. Results of safe testing for experimentally manufactured
Autovaccine:
The results showed that Autovaccine was twice as high as the
recommended injection dose after 21 days, all experimental pigs
survived and did not result in any local or systemic reactions to the pigs,
none of pigs showed any local signs such as inflammation or red ness
and swelling at the injection site or systemic symptoms such as fever
and anorexia. The experimental pigs were healthy, eating normally,
none of pigs expressed local reactions such as inflammation, swelling,
and redness. Thus, S.suis inactivated Autovaccine with aluminium
adjuvant reached safety standards as stipulated.
3.3.2.5. Results of effective experimental Autovaccine testing for
laboratory animals
- Challenged with S. suis serotype 2:
21 days after the first Autovaccine injection, all experimental and
control mice were challenged with S. suis serotype 2 at dose of 10LD50
(LD50 = 4.5 x 107 CFU). The results showed that there were10/10
mice surviving in all 3 groups vaccinated with Autovaccine after 10

Table 3.9: Results of antibody titre determination of the
experimental pigs 30 days after autovaccination
Antigen
used for
testing

S. suis
serotype 2
S. suis
serotype 7
S. suis
serotype 9

Number of pigs
tested by
serums
(pig)
Exprimental
Control
Exprimental
Control
Exprimental
Control

35
5
35
5
35
5

0
100
0
100
0

Antibody titre
1/32
Number
of
Percen
positive
t
samples
(%)
(+)
27
77.1
0
0
26
74.2
0
0
27
77.1
0
0

Number

0
6
0
7
0

Percen
t
(%)
22.8
0
17.1
0
20.0
0

Table 3.9 showed that there was a good immune response in all
pigs immunized with Autovaccine to the antigens of three bacterial
strains used for vaccinne production including S. suis serotype 2, 7 and
9 antigens. Agglutination occured in 100% of serum samples at dilution
of 1/6 and aglutination was determinated in serum samples of many
pigs at dilution of 1/32 and 1/64. Specifically: With serotype 2 antigen:
At dilution of 1/16, agglunating antibody titre was determinated in100%
of pigs , at dilution of 1/32, in 77.1% and at dilution of 1/64 in 22.8%.
With serotype 7 antigen: At dilution of 1/16, agglutinating antibody titre
was determinated in 100% of pigs tested; at dilution of 1/32, in 74.2%
and at dilution of 1/64, in 17.1%. With serotype 9 antigen: At the
dilution of 1/16, agglutinating antibody titre was determinated in 100%

23

Exprimental

Control

Control

Control

3
5
5
3
5
5
3
5
5

1/16
Number Number
of
of
serum
positive
samples samples
tested
(+)

Percen
t

35

35

100

35

32

91.4

35

12

5

0

0

5

0

0

5



0

0

5

0

0

35

35

100

35

31

88.5

35

10

28.5

5

8.3%. With the serotype 9 antigen: At dilution of 1/16 agglutinating
antibody titre was determinated in 100% of pigs; at dilution of 1/32, in
88.5% increasing by 11.4%; at dilution of 1/64, in 28.57%, increasing
by 8.0%.
3.3.3.3. Results of antibody titre determination of the experimental pigs
90 days after autovaccination
Table 3.11: Results of antibody titre determination of the
experimental pigs 90 days after autovaccination

24


25
1/8
Antigen used
for testing

S. suis
serotype 2
S. suis
serotype 7
S. suis
serotype 9

Number of pigs
tested by serums
(pig)

Exprimental
Control

100
0

Antibody titre
1/16
1/32
Number
Number
of
Percen
of
Percen
positive
t
positive
t
samples
(%)
samples
(%)
(+)
(+)
33
94.2
22
62.8
0
0
0
0

11.4
0
0
5
14.2
0
0

Table 3.11 showed that at the time of 90 days after
autovaccination of pigs, aglutination reaction occured in 100% of pigs
tested at serum dilution 1/ 8 and from dilution of 1/16 to 1/64 the
agglutination rate of serum antibody in experimental pigs decreased
gradually compared to the second test after 60 days. Specifically: With
serotype 2 antigen: At serum dilution of 1/8, agglutinating antibody titre
was determiated in 100% of pigs; at serum dilution of 1/16, in4.2%,
decreasing by 5.8%; at the dilution of 1/32, in 62.8% decreasing by
28.6%; at the the dilution of 1/64, in 17.1% decreasing by 8.6%. With
serotype 7 antigen:
At serum dilution of 1/8, agglutinating antibody titre was
determiated in 100% of pigs; at the dilution 1/16, in 88.5% decreasing
by 11.5%; at the the dilution of 1/32, in 54.2% decreasing by 31.5%; at
the dilution of 1/64, in 11.4%, decreasing by 14.3%. With serotype 9
antigens: At the dilution of 1/8 agglutinating antibody titre was
determiated in 100%; at the dilution of 1/16, in 91.4% decreasing by
8.6%; at the dilution of 1/32, in 57.1% decreasing by 31.4%; at the
dilution of 1/64 in 14.2% decreasing by 14.3%.
3.3.3.4. Results of antibody titre determination of the experimental pigs
120 days after autovaccination
Table 3.12 showed that 120 days after immunization with
Autovaccine, the antibody titre in the experimental pigs from serum