Nghiên cứu một số đặc điểm sinh học của vi khuẩn streptococcus suis gây bệnh ở lợn tại tỉnh thái nguyên và chế tạo autocvaccine phòng bệnh tt

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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

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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: ...............................................

Reviewer 3: ...............................................

The thesis will be defended at the university examination committee
At: Thai Nguyen University of Agriculture and Forestry
Thai Nguyen University
At ..... hour, on date ..... month ......year.....

The thesis can be found at Libraries:

-

2

National library of Viet Nam


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-

3

Thai Nguyen University of Learning Resource Center
Library of Thai Nguyen University of Agriculture and
Forestry


LIST OF PUBLICATION RELATED TO THE THESIS

1. Nguyen Manh Cuong, To Long Thanh, Nguyen Van Quang, Do
Hong Anh (2018), "Determination of some biological characteristics

of Streptococcus suis caused disease in pigs in Thai Nguyen
province", Veterinary Sciences and Techniques Magazine, 25(6), pp.
36 - 42.
2. Nguyen Manh Cuong, To Long Thanh, Nguyen Van Quang, Nguyen
Quang Tuyen, Do Hong Anh (2019), "Isolation, serotype
determination and toxicity test of Streptococcus suis strains caused
disease in pigs in Thai Nguyen province", Veterinary Sciences and
Techniques Journal, 26(2), pp. 67 - 73.
3. Nguyen Manh Cuong, To Long Thanh, Nguyen Van Quang, Nguyen
Quang Tuyen, Do Hong Anh (2018), "Results of experimental
inactivated vaccine preventing pneumonia, athritis diseases for pigs
in Thai Nguyen province", Science and Technology Journal of
Agriculture and Rural Development, November's volume, pp. 130 135.

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INTRODUCTION
1. Urgency of the thesis
Pig production in Thai Nguyen province in recent years has
developed rapidly quantity and improved quality, meeting the demand
of pig product consumption in the province and export-orientation,
contributing to job creation and increasing income for animal producers
in the province, therefore it plays an important role in the economic
development of the province and has become a leading livestock
industry to be interested in developing. Pig production in Thai Nguyen

is mostly small scale farms, so it faces many difficulties, especially in
the field of disease control, which greatly affects the efficiency of
livestock production. In addition to infectious diseases that occur in pigs
such as foot and mouth disease, swine fever, PRRS, pneumonia,
arthritis in pigs caused by Streptococcus suis (S. suis) also occurs quite
commonly leading to economic loss for farmers. The disease is caused
by S. suis in pigs with pathological manifestations such as hemorrhage,
pneumonia, arthritis, encephalitis...resulting in death, especially in the
period of post-weaning piglets, affecting weight gain, animal breed
quality and increasing the rate of breeding program.
S. suis plays an important role in respiratory disease and arthritis
in pigs. Investigation of Nguyen Thi Noi and Nguyen Ngoc Nhien
(1993) on the respiratory bacterial flora of 162 pigs with respiratory
infection accompanied with coughing showed that Streptococcus
accounted for 74.0%. Investigation results of Do Ngoc Thuy et al.
(2009b) from healthy pigs raised at pig farms, which were taken to
slaughter houses which expressed symptoms of pneumonia, PRRRS or
arthritis, encephalitis, S. suis was isolated from all of them with
different rates. Among the S. suis strains isolated, important and
common pathogenic serotypes 2 ,7 and 9 were found.
S. suis not only causes infection in pigs, it also causes dangerous
diseases in humans. In Vietnam, many cases and deaths have been
recorded due to streptococcal infection. According to statistics of the
general department of Preventive Medicine (Ministry of Health) in
2017, in the country 171 cases of S. suis in pigs were recorded in the
whole country, 14 of which died. In particular, in june -2018 in Thai
Nguyen province, there were 3 cases suffering from S. suis infection

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(Phuong Nam, 2018). The affected people confirmed that they had
contact with sick pigs in feeding, slaughtering, eating pork or blood
pudding infected with Streptococcus suis.
Derived from the practical situation of production and food safety
at present, aiming at better understanding of pneumonia and arthritis in
pigs associated with S. suis, which is a scientific basis for thesising
measures of effective prevention and treatment, an urgent requirement,
making contribution to improvement of production efficiency,
increasing income for pig producers and protecting public health, we
carried out the project: "Investigating some biological characteristics of
Streptococcus suis causing pig diseases in Thai Nguyen province and
manufacturing Autovaccine for prevention"
2. Objective of thesis
- Identifying some biological characteristics of S. suis causing
pneumonia and arthritis in pigs raised in Thai Nguyen province.
- Study on production of Autovaccine using S. suis strains isolated for
prevention of pneumonia and arthritis in pigs and trials of effective
treatment regimens for pneumonia and arthritis caused by S. suis, making
contribution to promoting pig production in Thai Nguyen province
3. The scientific and practical significance of the thesis
3.1. Scientific significance
- It is a systematic investigation of biological characteristics of S. suis
strains in pigs raised in Thai Nguyen.
- Investigation of manufacturing Autovaccine using S. suis strains
isolated for effective prevention of pneumonia, arthritis of pigs raised
in Thai Nguyen and recommendation of effective treatment regimens of
pneumonia, arthritis associated with S. suis
- Supplement and enrichment of scientific data, using as reference in

teaching and thesising of pathogenic S. suis in pigs.
3.2. Practical significance
- Trials of autovaccination of pigs raised in Thai Nguyen province has
contributed to decreasing the rate of pigs suffering from pneumonia,
arthritis caused by S. suis.
- Applying the treatment regimens for pneumonia, arthritis in pigs
reaches high efficiency, contributing to increased income for animal
producers and sustainable development of pig husbandry in Thai
Nguyen province.

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- The results of the thesis are a scientific basis to formulate effective
measures for prevention and treatment of diseases caused by S. suis in pigs.
4. New contributions of the thesis:
- A systematic and complete investigation of S. suis causing pig diseases
in Thai Nguyen province.
- Autovaccine that has been successfully manufactured by isolated S.
suis strains can be effectively used for prevention pigs from pneumonia
arthritis; the treatment regimens of pigs can be applied in practice of pig
production in Thai Nguyen.
Chapter 1
REVIEW OF LITERATURE
Chapter 2
CONTENTS, MATERIALS AND METHODS
2.1. Objects and location
2.1.1. Objects of the thesis
- S. suis isolated from pigs that infected by pneumonia and arthritis.

- S. suis Autovaccine to prevent pigs from pneumonia and arthritis.
2.1.2. Study location
- Pig farms and farmer household in Thai Nguyen province which were
suspicious of being infected by S. suis.
- Division of Microbiology, National Veterinary Diagnostic Center,
Department of Animal Health.
- Institute of Life Science, Thai Nguyen University.
2.1.3. Study period -From 2015 to 2018.
2.2. Contents of the thesis.
2.2.1. Investigating the situation of pneumonia and arthritis in pigs
raised in Thai Nguyen
- Identifying prevalence of affected pigs and motarlity of pigs with
pneumonia and arthritis over the years in some districts and cities of
Thai Nguyen province from 2015-2017.
- Investigating the rate of prevalence and mortality of pigs caused by
pneumonia, arthritis in pigs raised in Thai Nguyen from 2015-2017 at
different ages and seasons.

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2.2.2. Investigating some biological characteristics of isolated S. suis
strains
- Determining some biological and chemical characteristics of S. suis
strains isolated.
- Testing susceptibility of S. suis strains isolated to some antibiotics.
- Serotyping isolated S. suis strains
- Determining virulent factors of isolated S. suis strains.
2.3.3. Investigating exprimental inactivated Autovaccine manufacture

with alumminium adjuvant from isolated S. suis strains.
- Selecting S. suis strains used for experimental Autovaccine
manufacture.
- Investigating Autovaccine produced from selected S. suis strains.
- Determining immune response of pigs to experimental Autovaccine.
2.2.4. Investigation of prevention and treatment of arthritis,
pneumonia in pigs caused by S. suis
- Experimenting Autovaccine produced from isolated S. suis for
prevention of pneumonia and arthritis in pigs.
- Using treatment regimens of pneumonia and arthritis in pig caused by
S. suis.
2.3. Materials
- Samples were collected from lung, retropharyngeal fluid, synovial
fluid, cerebrospinal fluid. of sick or dead pigs with symptoms and
lesions suspicious of pneumonia or S. suis infection such as fever,
anorexia , dyspnea, cough, swollen joints, nerve signs...
- Healthy white mice (18-20g/mouse);
- Healthy piglets at the age of 5-6 weeks were not vaccinated with
Autovaccine against pneumonia, arthritis caused by S. suis.
- Media and chemicals used according to the thesis procedure of the
division of Microbiology of the Veterinary Institute and the National
Veterinary Diagnostic Center and the Animal Health Department.
- API 20 Strep kit system for determining biochemical characteristics
and nomenclature of S. suis (BioMérieux, Marcy l’Etoile, France).
- Antibotic assay paper for testing antibiotic sustibility produced by
Oxoid argency (UK).
- Chemicals and bio-products used for agglutination reactions, serology
and PCR technique, IHA reaction ...

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- Antibiotics: Cefanew-LA, Marflo-45%, Marphamox-LA and Gluco-K-CNamin produced by Marphavet Veterinary phar maceutical Company.
- Standard strains of S. suis and corresponding standard sera were
provided by the National Veterinary Diagnostic Center, department of
Animal Health, used for reference of strains isolated using PCR and
serology.
- Common laboratory instruments, inoculation chamber, autoclaves,
centrifuges, shaking machines, PCR technique used for investigation of
bacteria belonging to the Central Veterinary Diagnostic Center, Animal
Health Department and Institute of Life Sciences of Thai Nguyen University.
2.4. Methods of the thesis
2.4.1. Method of epidemiological thesis
Using methods of descriptive epidemiology and practical
epidemiology of Nguyen Nhu Thanh (2001).
2.4.2. Sampling method
- Samples were collected from retropharyngeal fluid, syncvial fluid:
Using a sterile cotton swab to place deeply in the throat, and the
inflamed joint of sick pigs then place in cotton swab tube, stored at 4 0C
and transferred to the laboratory for cultture and isolation of bacteria.
- Lung samples from sick or dead pigs that expressed signs and lesions
suspicious of S. suis infection were placed in sterile nylon bags, stored
at 40C and brought to the laboratory immediately for culture and
isolation of bacteria.
2.4.3. Methods of the thesis
- Methods of culture, isolation and testing of bacteria were carried out
according to the routine methods of Department of Microbiology,
National Veterinary Institute, National Veterinary Diagnostic Center and
Department of Animal Health .

- PCR technique was used to identify genes encoding some virulence
factors and S. suis pathogenic serotypes according to Wisselink H.J. et
al. (2002b), Silva L.M. et al.. (2006), Do Ngoc Thuy and Le Thi Minh
Hang (2009a).
- LD50 calculation method according to Reed L.J. & Muench H. (1938).
- Determination of virulence of S. suis isolated in experimental animals
of Sawade (1985).
- Determination of susceptibility to antibiotics of S. suis isolated from
laborratory animals according to Bauer A.W. (1966).

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- Experimenting treatment regimens for pigs infected by pneumonia,
arthritis based on the determination of susceptibility to antibiotics of S.
suis bacteria strains to select highly sensitive antibiotic drugs, being
allowed in Vietnam.
- The method of manufacturing Autovaccine is tested from isolated S.
suis bacteria strains according to the process of Department of
Microbiology, Institute of Veterinary Medicine.
- Testing sterile, safe and effective of Autovaccine tested according to
TCVN 8684: 2011 and QCVN 01: 187-2018.
- Method of antibody titre determination in pigs injected with
Autovaccine by Indirect Haemaglunation (IHA) according to the OIE
vaccine evaluation procedure (2018).
Chapter 3
RESULTS AND DISCUSSION
3.1. The situation of pigs affected and died of pneumonia, arthritis
from 2015 to 2017 in Thai Nguyen province

3.1.1. Prevalence of pigs affected and motarlity of pigs with
pneumonia, arthritis from 2015-2017
Table 3.1: Prevalence of affected pigs and mortality rates of pigs
associated with pneumonia and arthritis from 2015-2017
Year

Number
of
investigate
d pigs

2015
2016
2017
Total

3.850
4.120
3.170
11.140

Pneumonia
Infected pigs
Mortality
Numbe
Numbe
Percen
Percen
r of
r of

t (%)
t (%)
pigs
pigs
496
12.88
56
11.29
565
13.71
68
12.03
518
16.34
80
15.44
1.579
14.17
204
12.91

Arthritis
Infected Pigs
Mortality
Numbe
Numbe
Percen
Percentag
r of
r of

t (%)
e (%)
pigs
pigs
366
9.50
21
5.73
434
10.53
28
6.45
440
13.88
36
8.18
1.240
11.13
85
6.85

Pneumonia and Arthritis
Infected Pigs
Mortality
Numbe
Numbe
Percen
Percen
r of
r of

t (%)
t (%))
pigs
pigs
153
3.97
4
2.61
207
5.02
6
2.89
218
6.87
8
3.66
578
5.18
18
3.11

The data in table 3.1 showed that the prevalence of pigs infected
by pneumonia and arthritis in Thai Nguyen province is quite high. In
particular, pigs with pneumonia made up an average rate of 14.17% and
motarlity was 12.91%; pigs with arthritis accounted for average
percentage 11.13% and mortality was 6.85%. Pigs infected and died by
both diseases had different rates among the years of investigation. With
pneumonia, in 2017 the herds of pigs accounted for the highest
morbidity and mortality rates (16.34% and 15.44% respectively),
followed by 2016 (the rates was 13.71% and 12.03% respectively) and

lowest was in 2015 (the rates were 12.88% and 11.29% respectively).

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For arthritis, the highest prevalence and mortality rates were in 2017
(13.88% and 8.18% respectively), followed by 2016 (10.53% and
6.45% respectively) and the lowest were in 2015 (9.50% and 5.73%).
Similarly, the highest number of pigs infected and died of pneumonia
and arthritis was in 2017 (6.87% and 3.66%, respectively), followed by
2016 (5.02% and 2.89% respectively) and the lowest were in 2015
(3.97% and 2.61% respectively).
In 2017, pneumonia and arthritis in pigs in Thai Nguyen were
higher morbidity and mortality prevalences than in the previous years
because at that time the price of porkers was the lowest since the past
10 years so it had a great influence on the pig industry.. commercial
pigs that were not able to be sold led to farms and family farms suffer
from heavy losses. Animal producers almost neglected management and
care of animals, sanitation, prevention and treatment of animal diseases
so a lot of diseases occured.
3.1.2. Morbidity and mortality rates of pigs associated with
pneumonia and arthritis in different seasons
Table 3.2: Morbidity and mortality rates of pigs associated with
pneumonia and arthritis different seasons
Seasons
Summer
-Autumn
Winter Spring
Total

Number of
investigate
d pigs

Pneumonia
Morbidity
Mortality
Number
of pigs

Percen
t (%)

Number
of pigs

Percen
t (%)

4.357

395

9.06

32

8.10

6.783

1.184

17.45

172

11.140

1.579

14.17

204

Arthritis
Morbidity
Mortality
Numbe Percen
Percen
Number of
r of
t
t
pigs
pigs
(%)
(%)
415

9.52

24

5.78

14.52

825

12.16

61

7.39

12.91

1.240

11.13

85

6.85

The data of Table 3.2 showed that prevalences of morbidity and
mortality of pneumonia and arthritis in pigs raised in Thai Nguyen in
Winter - Spring seasons were higher than in Summer - Autumn seasons.

For pneumonia, the prevalences were 17.45% and 14.52% of pigs
infected and motarlity in Winter-Spring seasons was higher than those
in Summer-Autumn seasons, whose prevalences were 9.06% and 8.10%
respectively; for arthritis 12.16% and 7.39% compared with 9.52% and
5.78% respectively;.
The results of the thesis showed that pneumonia and arthritis in
pigs in Thai Nguyen province often occured throughout the year, but in

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the months of the Winter-Spring seasons, it is cold, and drizzly and humid,
adversely affecting the health of animals, creating favourite condition for
diseases occur, so both the diseases occured at a higher rate.
3.1.3. Morbidity and mortality rates of pigs infected by pneumonia
and arthritis at different ages
Table 3.3 showed that prevalence of pigs raised in Thai Nguyen
province suffered and died of pneumonia and arthritis was various
among different ages. With pneumonia, the highest prevalence was in
pigs of post-weaned age (> 1-3 months of age) (16.79%); followed by
piglets less than 1 month of age (15.47%) and the lowest was in gilts
and reproductive sows (8.18%). The highest mortality of pigs with
pneumonia was in post-weaning pigs, followed by piglets and the
lowest was in gilts and sows (15.35, 14.11) and 5.31% respectively).
For arthritis, the highest prevalence was in post-weaning pigs (16.02%),
followed by piglets under 1 month of age (12.02%) and the lowest was
in gilts and sows (3.04%).
Table 3.3: Morbidity and mortality rates of pigs infected by
pneumonia and arthritis at different ages

Ag
e
(m
ont
hs
old
)

N
u
m
b
er
of
in
ve
st
ig
at
e
d
pi
gs
(p
ig
)

Pneumon
ia

Mo Mo
rbi rtal
dity ity
N
u
m
b
e
r
o
f
p
i
g
s

N
P u
e m
r b
c e
e r
n o
t f
( p
%i
) g
s

Arthritis

Mo Mo
rbi rtal
dity ity

N
P u
e m
r b
c e
e r
n o
t f
( p
%i
) g
s

N
P u
e m
r b
c e
e r
n o
t f
( p
%i
) g
s

P
e
r
c
e
n
t
(
%
)

Pig
lets
(≤
1
mo
nth
old
)
Pos
t
we

13

1
1
1
3.
6

5 5
4 4 2
4
7
2 .
0 .
. 1 .
2
1
8 7
3 4
1 2 0
5
9
7
1
2
4. 6 1 1 1 6 1 5 7
1 9 6 0 5 6 6 2 .
5 7 . 7 . 5 .
8


14
ane
d
pig
lets
(>1
7

3
0
-3 0
9
5
2
mo
nth
s
old
)
Por
ker
(>3
1
2.
7
5
–
2 1
1
4
2 .
.
mo
8 .
2
1
1 3
2

nth
5 7
8
6
6
9
s
9
old
)
Gil
ts,
so
ws
1.
8
5
3
(>
1 9 .
. 3 .
6
5
4 4 1
3 5 0
mo
9
8
1
4

nth
s
old
)
1 1
1 1 1
11
. 4 2 2 . 1
Tot .1
5 . 0 . 2 .
al 4
7 1 4 9 4 1
0
9 7
1 0 3

1

4

3
.
1
2

1

2
.
8

5

6
8 .
5 8
5

The highest mortality of pigs with arthritis was in post-weaning
pigs, followed by piglets and the lowest was in gilts and sows (7.81;
6.79 and 2.85%, respectively). Our results were in agreement with those
of Dutkiewicz J and et al. (2017) showed that in pigs with streptococcal
disease, most pigs were infected during the period from 5 to 10 weeks
of age, few pigs were infected over 32 weeks of age or several hours
after birth.
Investigation results of survival of S.suis in some areas of Thua
Thien Hue province in spring-summer season of 2015 by Bui Thi Hien
et al. (2016) also showed that the prevalence of S. suis infection in
healthy pigs was 11.4%.
3.2. Results of some biological characteristics of isolated S. suis strains

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3.2.1. Isolation of S. suis bacteria from pigs with pneumonia and
arthritis in Thai Nguyen
Table 3.4 showed that in 403 samples from pigs with pneumonia
and arthritis at different ages, there were 212 samples that S. suis
isolated accounting for 52.60%; The highest prevalence was that of
post-weaning pigs> 1 - 3 months of age (58.91%) and the lowest was

that of piglets less than 1 month of age (45.79%). Our results were in
agreement with those of S. suis isolated from specimens of pigs infected
with PRRS in Bac Giang by Truong Quang Hai and et al. (2012), which
was 51.67% and by Le Van Dung et al.. (2013) , which was 55.10%, but
higher than finding of Nguyen Thi Kim Lan and others (2016, 2017)
when isolating bacteria causing pneumonia in pigs in Bac Ninh
province from lungs and retropharyngeal lymph nodes of pigs found
that the prevalence of S. suis was 38.67% and in pigs raised in Tuyen
Quang province was 13.0%. As a result, isolation of S. suis in pigs in
Thai Nguyen showed that this bacterium had played an important role
in pneumonia and arthritis in pigs for the past years in Thai Nguyen.
Table 3.4: Results of S. suis isolated from pigs at different age
suffering pneumonia and arthritis
Orde
r
1

2

3

4

Age
(month old)
Piglets
( ≤ 1 month
old)
Post weaned
piglets

(>1 - 3
months old)
Porker
(> 3 - 6
months old)
Gilts, sows
(≥ 7 months
old)
Total

15

Sample

Number of
samples
exanimated

Number
of positive
samples
(+)

Percen
t
(%)

37

16

43.24

35
35

18
15

51.42
42.85

44

25

56.81

43
42

31
20

72.09
47.61

37

18

48.64

36
35

25
14

69.44
40.00

23

11

47.82

20
16

13
6

65.00
37.50
52.60

Retropharyngea
l fluid

Lung
Synovial fluid
Retropharyngea
l fluid
Lung
Synovial fluid
Retropharyngea
l fluid
Lung
Synovial fluid
Retropharyngea
l fluid
Lung
Synovial fluid
403

212

Total
(%)

45.79

58.91

52.77

50.84

16
3.2.2. Results of investigating some biological and chemical
characteristics of isolated Streptococcus suis strains
Results of investigating some biological and chemical
characteristics of S. suis bacteria isolated from pigs's specimens with
pneumonia and arthritis in Thai Nguyen province found that all strains
had typical biological and chemical characteristics of S. suis bacteria as
described by scientific documents.
3.2.3. Results of investigating some biological and chemical
characteristic of S. suis bacteria isolated by API 20 Strep kit system
Results of investigating some biological and chemical
characteristics of S. suis bacteria isolated by API 20 Strep kit system, of
which 20 reactions prepared in plastic tray produced by BioMrieux
company, including: VP, HIP. ESC, PYRA, αGAL, βGUR, βGAL, PAL,
LAP, ADH, RIB, ARA, MAN, SOR, LAC, TRE, INU, RAF, AMD,
GLYG. The results showed that S. suis strains isolated from pigs in Thai
Nguyen had all the typical biological and chemical characteristics of S.
suis bacteria.
3.2.4. Result of antibiotic susceptibility test of isolated S. suis strains.
Table 3.5: Result of antibiotic susceptibility test of
isolated S. suis strains
Orde
r

Antibiotic

Number
of
strains
tested

High
(+)

1

Ceftiofur

168

142

2

Florfenicol

168

137

3

Amoxicillin

168

135

4

Ampicillin

168

122

5

Ofloxaxin

168

90

6

Tetracyclin

168

71

7

Gentamicin

168

43

8

Lincomycin

168

21

9

Penicillin G

168

17

16

Evaluation of susceptibility
Average
Resistant
(%)

84.5
2
81.5
4
80.3
5
72.6

1
53.5
7
42.2
6
25.5
9
12.5
0
10.1

(+)

(%)

(+)

(%)

15

8.92

11

6.54

17

10.1

1

14

8.33

15

8.92

18

10.71

13

7.73

33

19.64

60

35.71

72

42.85

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

1
10

Neomycin

168

9

5.35

38

11

Colistin

168

4

2.38

32

12

Erythromycin

168

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

erythromycin (82.73%), colistin (78.57%), neomycin (72.02%) and
penicillin G (58.33%). Our results were in agreement with those of
Nguyen Thi Kim Lan and others. (2017), when testing the susceptibility
of S. suis strains isolated in pigs in Bac Ninh province to some
antibiotics, it showed high susceptibility to ceftiofur (81.82%),
amoxicillin (79.55%), florfenicol (75.00%) and ampicillin (70.45%).
The above results were the basis for us to select antibiotics for treatment
of streptococcal disease associated with S. suis in pigs in Thai Nguyen
to reach high effectiveness.
3.2.5. Results of serotyping isolated S.suis strains
Table 3.6 showed that number of S. suis serotype 2 strains was the
highest (58.17%), followed by serotype 9 (16.34%), serotype 7 (7.19%),
the lowest was serotype 21 and 29 (1.96 and 2.61% respectively); S. suis
serotype was not determinated in the rest 21 strains, accounting for
13.72%. For serotype 2, the highest was in post-weaning pigs (64.00%),
followed by piglets <1 month old (58.97%) and the lowest was in porkers
(51.21%). Results of other S. suis serotypes of S. suis isolated in Thai
Nguyen were not determinated.
Table 3.6: Results of serotyping isolated S. suis strains
Different ages
of pigs
(months old)

Piglets
( ≤ 1 month old)
Post weaned
piglets (>1- 3
months old)
Porkers (>3 - 6
months old)

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
(%)
(+)

Serotyping results
Serotype 9
Serotype 21
Numbe
Numbe
r of
Percen
r of
Percen
positive
t
positive
t
strains
(%)
strains
(%)
(+)
(+)

Serotype 29
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

2.56

7

17.94

50

32

64.00

3

6.00

7

14.00

1

2.00

1

2.00

6

12.00

41

21

51.21

3

7.32

9

21.96

1

2.44

1

2.44

6

14.64

23

13

56.52

2

8.69

3

13.02

1

4.34

1

4.34

2

8.69

153

89

58.17

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

isolated S. suis
The thesis results showed that among the genes encoding the
virulence factors, arcA gene carried by S. suis strains accounted the highest
percentage (75.5%), followed by mrp gene (71.1%), sly gene (62.2%) and
epf gene (57.7%). 5 types of gene combinations encoding virulence factors
were identified including: arc/sly, arcA/mrp, arcA/ epf, arcA/mrp/sly,
arcA/mrp/sly and arcA/mrp/sly/epf in 45 S. suis strains studied. Our thesis
results were similar to those of some authors such as Do Ngoc Thuy and et
al. (2009) from healthy pigs and pigs with pneumonia symptoms, PRRS
arthritis symptoms, or encephalitis from which S. suis were isolated at
different rates. In 211 S. suis strains studied 18 serotypes of which were
identified, most commonly were serotype 2, 7, 9 and 7 combinations of
virulence genes. Investigation of Cu Huu Phu et al. (2013) showed that
serotype 2 strains carried the most gene combinations which were
scatteredly distributed in 5 combinations: arcA, arcA/sly; arcA/mrp;
arcA/mrp/sly and arcA/mrp/sly/ epf.
Results of PCR used for determining genes encoding virulence of S.
suis strains isolated were shown in Figure 3.2.

Notes:
Marker: 100 bp

18

Well 1: arcA Gene

Well 2: mrp Gene


19

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

Strain letter

S-TN2-1

19

Numbe
Injectio
r of
Serotyp
n dose
mice
e
(ml/
injected
mouse)
(mouse)
2

2

0.5

Results
Numbe
Time
r of
period Percent
dead
for death (%)

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

2
2
2
2
2
2
2
2

0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5

2/2
2/2
1/2
2/2
1/2
2/2
2/2
2/2

12 - 24
12 - 24

< 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.

Thus, it can be seen that serotype 2, 7 and 9 strains were the three
serotype types that dominated among S. suis strains isolated from pigs
with pneumonia and arthritis in Thai Nguyen that were high virulent
strains. These strains can cause death of mice shortly after 5 passage
cultures. This was a very important feature for screening and selection
of bacterial strains to produce Autovaccine.
Based on the formula of LD50 calculation of Reed L.J. &
Muench H. (1938) we determined LD50 of S. suis isolated as follows:
LD50 of serotype 2 = 4.5 x 10 7; LD50 of serotype 7 = 4.9 x 10 7 and
LD50 of serotype 9 = 4.8 x 107.
3.3. Study results of experimental manufacturing Autovaccine from
S. suis strains isolated
3.3.1. Selection of S. suis strains to produce experimental Autovaccine
From the thesis results on biochemical characteristics, S. suis
serotyping and virulence of S. suis strains isolated in pigs in Thai
Nguyen were determined, we selected 3 serotypes 2, 7 and 9 to produce
inactivated Autovaccine with aluminum adjuvant. Selected serotypes
accounted for high percentage of all serotypes isolated that expressed
gene encoding the virulent extracellular factor (EF-epf), the protein that
was muramidase releasing protein (MRP), hemolytic factor Suilysin
(SLY-sly) and arginine deiminase enzyme (ARC-arcA) that were genes
producing toxins that play an important role in pig disease. In addition,

20


21
the serotypes above were all highl virulent ones, causing death of
laboratory mice within 12-36 hours.
3.3.2. Autovaccine manufacturing for preventing pigs from S.suis

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

broth
1.65 x 108
1.63 x 108
1.64 x 108
1.64 x 108
1.71 x 108
1.68 x 108
1.69 x 108
1.69 x 108
1.72 x 108
1.70 x 108
1.71 x 108
1.71 x 108

Table 3.8 showed that after culture period, there were average
bacteria concentration of 1.64 x 10 8 to 1.71 x 108 bacteria/ml in broth
from 3 Autovaccine batches which were produced experimentally from
the S. suis strains isolated. According to QCVN-01-187: 2018, number
of bacteria that present in 1 ml of vaccine to produce the vaccine must
reach at least 1.5 x 108 bacteria/ml, so the above batches reached the
criteria. Based on the thesis results of Torremorell M. (1997), we
calculated the Autovaccine injection dose for pigs to be 2.5 ml / pig,
equivalent to the average bacterial density of about 4.2 x 108 CFU/ml.
3.3.2.2. Results of testing culture purity of S. suis broth used to produce
Autovaccine
Testing culture purity was carried out simultaneously with the
individual culture steps of each S. suis strain from master seeds to
working seeds. The results showed that the broth used to produce 3

21

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

days, and protection reached 100% whereas in the control group,
mortality was 100% 18 to 24 hours after challenge
- Challenged with S. suis serotype 7:
21 days after the first Autovaccine injection, all experimental and
control mice was challenged with S. suis serotype 7 at dose of 10LD50
(LD50 = 4.9 x 107 CFU). The results showed that after 10 days there
were 9/10 mice surviving in 2 groups immunized with Autovaccine
(group I and II), reaching the protection rate of 90%, in the third group,
10/10 mice survived reaching protection rate of 100%. In control
groups, the mortality was 100% during 18-24 hours.

22


23
- Challenged with S. suis serotype 9:
The experiment was conducted in the same way as the serotype
above, 21 days after the first Autovaccine injection, both experimental
and control mice were challenged with S. suis serotype 9 at dose of
10LD50 (LD50 = 4.8 x 107 CFU). The results showed that after 10
days, there were 10/10 mice surviving in 2 groups immunized with
Autovaccine (group I and III), reaching the protection rate of 100%, in
group II there were 9 of 10 mice surviving reaching protection rate of
90 % In control group, the mortality was 100% during 18-24 hours.
Thus, all 3 batches of experimental Autovaccine manufacture
were sterile, safe and efficacious for laboratory mice.
3.3.3. Results of determination of experimental Autovaccine immune
response for preventing pigs from S. suis
3.3.3.1. Results of antibody titre determination of the experimental pigs
30 days after autovaccination

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

Number
of
serum
samples
tested
35
5
35
5
35
5

1/16
Number
of
positive
samples
(+)
35
0
35
0
35
0

Percen
t
(%)
100

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

of
serum
samples
tested
35
5
35
5
45
5

Number
of
serum
samples
tested
35
5
35
5
35
5

1/64
Number
of
positive
samples
(+)
8

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

24
of pigs tested , at dilution of 1/32, in 77.1% and at at dilution of 1/64
in 20.0%.
3.3.3.2. Results of antibody titre determination of the experimental pigs
60 days after autovaccination
Table 3.10: Results of antibody titre determination of the
experimental pigs 60 days after autovaccination
Antigen
used for
testing

Number of pigs
tested by serums
(pig)

S. suis
serotype 2

Exprimental

S. suis
serotype 7

Exprimental

S. suis
serotype 9

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

(%)

Antibody titre
1/32
Number Number
of
of
Percen
serum
positive
t
samples samples
(%)
tested
(+)

1/64
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

34.2
0

35

35

100

35

30

85.7

35

9

25.7

5

0

0

5

0

0

5

0

0

35

35

100

35

31

88.5

35

10

28.5

5

0

0

5

0

0

5

0

0

Table 3.10 showed that the Autovaccine stimulated pigs to
produce good immunity at the time of 60 days after immunization with
antigens from 3 bacterial strains including S. suis serotype 2, 7 and 9,
which agglutination occurred in 100% of serum samples of pigs at
serum dilution of 1/16 and aglutination reaction increased in serum
samples of most pigs at dilution of 1/32 and 1/64 compared to the first
test after 30 days. Specifically: With serotype 2 antigen: At dilution of
1/16, agglutinating antibody titre was determinated in 100% of pigs; at
dilution of 1/32, in 91.4%, increasing by more than 14.3%; At dilution
of 1/64 in 34.2% increasing by more than 14.4%. With the serotype 7
antigen: At dilution of 1/16 agglutinating antibody titre was
determinated in 100% of serum samples; at dilution of 1/32, in 85.7%
increasing more than 11.2%; at dilution of 1/64, in 25.7% increasing by

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

Exprimental
Control
Exprimental
Control

35
5
35
5
35
5

Number
of
positive
samples
(+)
35
0
35
0
35
0

Percent
(%)
100
0
100
0

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

31
88.5
19
54.2
0
0
0
0
32
91.4
20
57.1
0
0
0
0

1/64
Number
of
Percent
positive
(%)
samples
(+)
6
17.1
0
0
4

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

dilution of 1/16 to 1/64 aglutination rate of serum antibody in the
experimental pigs decreased gradually compared to the third test 90
days after immunization with Autovaccine. Specifically: With serotype
2 antigen: At serum dilution of 1/8, agglutinating antibody titre was
determinated in 100% of pigs; at the dilution of 1/16, in 68.5%,

25