KHẢO sát XOẮN KHUẨN LEPTOSPIRA và LEPTOSPIROSIS TRÊN CHÓ ở một số TỈNH ĐỒNG BẰNG SÔNG cửu LONG tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY

SUMMARY OF DOCTORAL THESIS
Major: PATHOLOGY AND TREATMENT OF ANIMALS
Major code: 62 64 01 02

NGUYEN THI BE MUOI

SURVEY OF LEPTOSPIRA AND LEPTOSPIROSIS
IN DOGS IN SOME PROVINCES
IN THE MEKONG DELTA

Can Tho-2019


THIS THESIS WAS COMPLETED AT CAN THO UNIVERSITY

Academic supervisor: Assoc. Prof. PhD. HO THI VIET THU

The thesis was defended at the university examination committee
At.………………………………………., Cantho University
At……….. hour ….…, on date……..month…..…. year……
Reviewer 1:
Reviewer 2:
Reviewer 3:

The dissertation is available in Libraries
1. Central library of Can Tho University.
2. National library of Vietnam.

Chapter 1: INTRODUCTION
1.1 The imperativeness of thesis
Leptospirosis is a common infectious disease to many species of
animals and it is caused by several species of pathogenic spirochetes
(pathogenic Leptospira species). The disease is prevalent throughout the
world, especially in hot, humid tropical areas, which are conducive to the
presence out of environments (Evangelista and Coburn, 2010). Pathogenic
germs exist in the renal tubules and they are excreted in the urine to
contaminate the environment (Adler and Moctezuma, 2010) and in naturals,
rats are used as a source of spirulina and a source of transmission for the
disease for dogs, humans, and other animals (Syke et al., 2011).
In Vietnam, there have been many studies on the Leptospira
infection in animals and the results show that the prevalence of Leptospira
infection is variable, depending on the survey’s animals, time and places by
some of the researchers (such as Vu Dinh Hung, 1995; Nguyen Dinh Ngan,
2000; Le Huynh Thanh Phuong, 2001; Hoang Manh Lam, 2002 and Hoang
Kim Loan, 2002). However, research on this disease in dogs is very
limited, especially in the Mekong Delta, where there is no study on
Leptospira and Leptospirosis in dogs. Thus, the thesis “Survey of
Leptospira and Leptospirosis in dogs in some provinces in the Mekong
Delta” was carried out.
1.2 The aim of thesis
- Survey of the spirochete Leptospira circulating in dogs and rats.
- To study the changes of pathogenic Leptospirosis in dogs.
- To study the effective treatments for Leptospirosis in dogs.
1.3 The new distribution of thesis
This is the first research discovered two species Leptospira
interrogans and Leptospira fainei presence in unvaccinated dogs
Leptospirosis in some provinces in the Mekong Delta.

1.4 Scientific significance of the study
This is the first research about the epidemiological of Leptospirosis
in dogs, as well as a scientific basis for the construction process and
treatment of the dog, limiting disease in humans and other animals.

1


Chapter III: CONTENTS AND METHODOLOGY
3.1 Content 1: Determination of Leptospira infection in dogs and rats
3.1.1 Research subjects
Survey of Leptospia circulation is done on dogs health and dogs
diseases, which are from 4 months old and above, unvaccinated
Leptospirosis dogs, from farms in 4 provinces (Can Tho, Vinh Long, An
Giang and Ca Mau) and three animal clinics (including healthy dogs and
dogs diseases) in Can Tho city. The survey of Leptospia circulation in rats
from being trapped in the dog sampled households surveyed.
3.1.2 Methodology
a. The method of blood sample
Serum samples of dogs: cross-sectional serological analysis was carried
out on 1.433 serum samples of healthy and dogs diseases, unvaccinated
Leptospirosis dogs according to breed, age, sex and farming methods The
intensity of infection was mainly concentrated at low antimicrobial
antibody titration values of 1: 200 to 1: 800 in the Mekong Delta.
Sample size: estimated sample size should be based on the Fosgate (2009)
epidemiological formula for each of the survey province.
Table 3.1 Quantity of dogs serum
P(1-P).Z2 0.2(1-0,2). 1.962
n=
=

= 245
d2
0.052

Quantity
(individual)
Can Tho
650
Vinh Long
256
The sample size which needs to be
An
Giang
263
collected in each province is 245 samples
Ca
Mau
264
of dogs serum or more and is shown in
Total
1.433
Table 3.1.
Serum samples of rats: cross-sectional serological analysis was carried
out on 647 serum samples rats in four provinces in the Mekong Delta.
Sample size: the estimated sample size should be based on the formula.

2

Location

Table 3.2 Quantity of rats serum
Locations
Quanlity
P(1-P). Z2
0.071(1-0,071). 1.962
(individual)
n=
=
= 101
Can Tho
195
d2
0.052
Vinh Long
140
An Giang
185
The sample size required for each
Ca Mau
127
province is 101 rats serum samples and is
Total
647
shown in Table 3.2.
Total 647 rats, in which rats (Rattus norvegicus) is 285, muskrats
(Suncus murinus) is 152 and small rats (Mus musculus) is 210.
b. Implementation methods
- The reaction MAT (microscopic Agglutination Test) is used to
detect specific antibodies against Leptospira in dogs and rats with

serogroup 18 by the Pasteur Institute in Ho Chi Minh City supply
(according to the process of the WHO, 2003).
- The reaction was considered positive when agglutination titer ≥ 1:
200 in dogs serum samples and ≥ 1: 20 in rats serum samples.
c. Survey criteria
- The prevalence of Leptospira in dogs and rats at survey sites.
- The prevalence of serogroup Leptospira in dogs and rats.
- The intensity of serogroup Leptospira infection in dogs and rats.
- The prevalence of Leptospira in dogs according to breed, age and
farming methods.
- The correlation coefficient (R2) between the positive rate of
Leptospira serogroup in rats and dogs, the linear regression equation y = a
+ bx.
3.2 Content 2: Detection of Leptospira from urine
The detection of Leptospira from urine is performed on the same 63
dogs examined the biochemistry physiology index of blood and urine from
111 dogs MAT ≥ 1: 400.
3.3.1 Detection of Leptospira from urine by PCR
Implementation methods: urine is collected midway through the urethra
or directly collected at eliminating the process with urine volume of 6-20
ml, divided into 3 parts for analysis.
- Part 1: 6 ml of urine taken no centrifuge to analyze the
physiological indicators of urine.

3


- Part 2: 3 ml of urine taken after centrifugation for DNA extraction.
- Part 3: 3 ml of urine taken after centrifugation to culture on the
environment Ellinghausen-McCullough-Johnson (EMJH).

The implementation of PCR
Sixty-three dog urine sample used for DNA extraction and PCR
reaction used on the basis of 16S rRNA gene amplification.
Table 3.3 Nucleotide sequence using primers 16S rRNA gene amplification
region.
Gene Total
Nucleotide sequence
bp
primers
M-27F
5’-AGA GTT TGA TCM TGG CTC AG-3’ 1500bp
16S
M-1492R 5’-TAC GGY TAC CTT GTT ACG ACT
T-3’
The total primers are based on the work of Weisburg et al. (1991).
Table 3.4 PCR reaction components
Components
Volume/1
Concentration/1
reaction
reaction
2X Master Mix
25X
1X
Primer M-27F (10 µM)
2 µL
0.4 µM
Primer M-1492R (10 µM)
2 µL
0.4 µM

DNA tách chiết
1-2 µL
50-100 ng
Bi.H20
Đủ 50 µL
Table 3.5 Heating process in PCR
Temperature
Time
Cycle
o
95 C
5 phút
1
95oC
30 giây
55oC
30 giây
30
72oC
1 phút 45 giây
72oC
5 phút
1
3.3.2 Detection of Leptospira from cultures
Method for culture Leptospira
- Leptospira cultured according to the WHO (2003) on environments
EMJH, observed under dark-field microscopic examination and identified
to species level by PCR and sequencing.
- PCR was performed as section 3.3.1

4


Sequence the 16S rRNA gene of Leptospira
- The PCR’s products were then purified and decoded in nucleotide
at company Macrogen (South Korea).
- The sample was sequenced 16S rRNA automatically on the ABI
3130 Sequencer with two primers 27F and 1492R.
- If the homogeneity of the nucleotide sequence in the 16S rRNA
gene is ≥ 95%, it can be determined to be a genus and ≥ 99%, which can be
determined by the species (based on the level of homology description of
La Scola et al., 2006).
- Phylogeny is based on nucleotide sequences using MEGA 7.0
software (Kumar et al., 2016). Looking up the results on BLAST SEARCH
and comparing a wide range of bacterias available in GeneBank and
database on NCBI are to identify the isolated species and two species of the
spirochete Leptospira reference used for comparison in the analysis of
species arising:
>Icterohaemorrhagiae NR_116542.1 Leptospira interrogans serovar
icterohaemorrhagiae strain RGA 16S ribosomal RNA gene, partial
sequence.
>Hurstbridge NR_043049.1 Leptospira fainei serovar hurstbridge strain
BUT 6 16S ribosomal RNA gene, partial sequence.
3.3 Content 3: Survey on the pathogenic changes of Leptospira in dogs
The pathological changes in dogs diseases surveyed base on
physiological biochemical index of blood and urine of 13 dogs positive for
Leptospira by PCR and MAT ≥ 1: 400.
a. Blood analysis
The testing of physiological blood was investigated by hematology
(Cell Dyn 3200, USA), including leukocyte, neutrophile%, lymphocytes%,

erythrocytes, hemoglobin, hematocrit, platelet and biological of blood was
investigated monitoring by blood chemistry analyzer (AU604, Japan): ure,
creatinine, AST, ALT and bilirubin and compared to standards of the
Merck (2010).
b. Urine analysis
The testing of the physiological index of urine surveyed by urine analyzer
urine (U120, USA) include: red blood cells, white blood cells, proteins, and
bilirubin and compared with Merck's standards (2010) and Khorami et
al. (2010).

5


c. Survey criteria
- The change of biochemical and physiological index of blood and
urine in dogs having Leptospirosis.
- Clinical signs of suspected Leptospirosis in dogs: fever, reflux,
vomiting, hemorrhage,...
3.4 Content 4: Study on the treatment of Leptospirosis in dogs
Study subjects: healthy dogs and dogs diseases were brought to medical
examination and treatment at the veterinary clinic 3 in Can Tho city.
- Set up a collaborative regimen treatment with the owners.
- Sixty-three dogs in the content 2 titer antibodies against Leptospira
≥ 1: 400, with the presence of Leptospira in urine by dark-field microscopic
examination (is randomized with 32 dogs did not show clinical symptoms
and 31 dogs have some manifestation clinical symptoms suspected
Leptospirosis: vomiting, eating less, sad, sedentary, kidney failure, liver
failure, ascites, skin hemorrhages and jaundice).
Table 3.6 Experimental design
Treatment 1: Shoptapen

20 0.2 mg/kgP, Intramuscular or
7-14
(Penicillin
each three
Subcutaneous
days
G+Streptomycin)
days
injection
Treatment 2:
22 22 mg/kgP, Take medicine
7-14
Amoxicillin
twice a day
days
Treatment 3:
21 5 mg/kgP,
Take medicine
7-14
Doxycycline
twice a day
days
Monitoring criteria is to assess effectiveness of treatment based on
Goldstein, (2010); Sykes et al. (2011); Francey and Schweighauser, (2012)
and Schuller et al. (2015).
3.5 Data analysis and processing
- Data was processed for the statistics using Microsoft Excel 2013
software.
- The physiological, biochemical index of blood and urine are
presented as ±SE.

- The investigation of factors related to Leptospira infection
including breeds, age, sex, and farm method χ2 in Minitab 16.0
- The regression was used to analyze the correlation of Leptospira in
rats and dogs with the equation of y=a + bx, where a and b are constant, y
and x are positive rate in rats and dogs.

6


Chapter 4 RESULTS AND DISCUSSION
4.1 Survey results of Leptospira infection in dogs and rats
4.1.1 The prevalence of Leptospira in dogs in some provinces in
Mekong Delta
Table 4.1 The prevalence of Leptospira in dogs
Province
Number of serum
Number of
Positive rate
samples
positive serum
(%)
samples
Can Tho
650
159
24.46a
Vinh Long
256
69
26.95a

An Giang
263
53
20.15a
Ca Mau
264
50
18.94b
Total
1.433
331
23.10
Data in the same column with different caps are statistically different
(P<0.05).
A total of 1.433 dogs serum samples has 331 dogs positive of
Leptospira has the rate of 23.10% overall. In particular, the rate of
Leptospira infection in dogs is highest in Vinh Long (26.95%), followed by
Can Tho (24.46%), An Giang (20.15%) and Ca Mau (18.94%).
The comparison of Vu Dinh Hung study (1995), the surveys of dog
Leptospira infection rate in the southern province of Long An, Can Tho,
Dong Nai and the vicinity of Ho Chi Minh city was 44.44%. Le Huynh
Thanh Phuong (2001) survey in the northern provinces of Vietnam with
Leptospira infection rates average 25.27% in dogs; in DakLak in dogs
Leptospira infection rate 19.15% (Hoang Manh Lam, 2002) and the results
of Ly Thi Lien Khai (2012) in Can Tho province prevalence Leptospira in
dogs 40.47%. This result of research showed that the prevalence of
Leptospira in dogs between countries with different was due to differences
in climate, weather and geographical conditions of each country should
prevalence of various and the adaptation of each different dog so the
epidemiological situation in each locality will be different survey (Levett,

2001; Ellis, 2010).

7


Table 4.2 Prevalence of serogroup Leptospira in dogs
Total 1.433 serum samples
Serogroup Leptospira

Number infection

(%)

L. australis
L. autumnalis
L. bataviae
L. canicola
L. ballum
L. icterohaemorrhagiae
L. pyrogenes
L. cynopterie
L. gryppotyphosa
L. hebdomadis
L. javanica
L. panama
L. semaranga
L. pomona
L. tarassovi
L. sejroe
L. louisiana

L. hurstbridge

19
12
40
61
7
116
30
9
40
12
22
29
3
9
1
31
9
42

5.74
3.63
12.08
18.43
2.11
35.05
9.06
2.72
12.08

3.63
6.65
8.76
0.91
2.72
0.30
9.37
2.72
12.69

331
Total number of positive samples
By the Table 4.2 had demonstrated the circulation 18/18 Leptospira
serogroup in dogs in some provinces in the Mekong Delta. Serogroup
accounts for a high proportion of L. icterohaemorrhagiae (35.05%), L.
canicola (18.43%), L. bataviae (12.08%), L. pylotypes (12.08%) and L.
hurstbridge (69%). The compared with research of Vu Dat and Le Huynh
Thanh Phuong (1999) have demonstrated in dogs in Hanoi are infected
with L. serogroup bataviae, L. canicola, L. gryppotyphosa and L.

8


icterohaemorrhagiae. Thus, it is depended on the epidemiological situation
of each locality differ on Leptospira serogroup in circulation.
Table 4.3The intensity of infection of serogroup Leptospira of in dogs
Serogroup
Leptospira
australis

1:200
Infecti
%
-on
15
78.95

1:400
Infet
%
i-on
4
21.05

1:800
Infect
%
i-on
0
0.00

1:1600
Infect
%
i-on
0
0.00

1:3200
Infect

%
i-on
0
0.00

autumnalis

11

91.67

1

8.33

0

0.00

0

0.00

0

0.00

bataviae

31

77.50

9

22.50

0

0.00

0

0.00

0

0.00

canicola

41

67.21

14

22.95

3

4.92

2

3.28

1

1.64

ballum

6

85.71

1

14.29

0

0.00

0

0.00

0

0.00

icterohaemorrh
agiae

86

74.14

22

18.97

6

5.17

2

1.72

0

0.00

pyrogenes

22

73.33

7

23.33

1

3.33

0

0.00

0

0.00

cynopterie

6

66.67

2

22.22

1

11.11

0

0.00

0

0.00

gryppotyphosa

30

75.00

7

17.50

2

5.00

1

2.50

0

0.00

hebdomadis

7

58.33

5

41.67

0

0.00

0

0.00

0

0.00

javanica

15

68.18

6

27.27

1

4.55

0

0.00

0

0.00

panama

17

58.62

9

31.03

3

10.34

0

0.00

0

0.00

semaranga

3

100

0

0.00

0

0.00

0

0.00

0

0.00

pomona

7

77.78

2

22.22

0

0.00

0

0.00

0

0.00

tarassovi

1

100

0

0.00

0

0.00

0

0.00

0

0.00

sejroe

26

83.87

5

16.13

0

0.00

0

0.00

0

0.00

louisiana

6

66.67

3

33.33

0

0.00

0

0.00

0

0.00

hurstbridge

33

78.57

9

21.43

0

0.00

0

0.00

0

0.00

Total 492

363

73.78

106

21.54

17

3.46

5

1.02

1

0.2

The intensity of infection was mainly concentrated at low
antimicrobial antibody titration values of 1: 200 to 1: 800, with a high rate
in two serogroups of L. canicola and L. icterohaemorrhagiae ranging from
1: 1600 to 1: 3200. While from the results of Vu Dinh Hung (1995), the
intensity of infestation was mainly concentrated in the agglutination titer of
1: 800 and in Le Huynh Thanh Phuong study (2001), it mainly
concentrated in the agglutination titer from 1: 800 to 1: 1600.
If the antibody titer higher prove Leptospira infection levels as high
as because antibody titres reflected antibody levels in the dogs’ blood. It is

9


depended on each area, each region, each country, dogs may be infected
with different serogroups of Leptospira at different intensities and
depending on the time of sampling, the health status of the animals when
collecting their serum and their nurturing conditions.
Table 4.4 Prevalence of Leptospira infection by breeds

Dog breeds
Number of
Number of
Rate (%)
serum samples
positive serum
samples
Domestic dog’s breeds
835
198
23.71a
Exotic dog’s breeds
598
133
22.24a
The prevalence of Leptospira in domestic dogs and exotic dog’s
breeds, which was 23.71% and 22.24%, had no statistical difference. This
might come from the fact that in the same habitat, the ability of being
exposed to the pathogen was the same for two dog breeds and the
prevalence of Leptospira infection did not differ between them, which is
contract with the results in Harland et al. (2013) and Maele et al. (2008)
studies, the factor of breeds did not affect the incidence of infection.
Table 4.5. Prevalence of Leptospira infection by age
Number of
Number of
Rate
Age
serum
positive serum
(%)

samples
samples
4 months < 12 months
21.51a
358
77
≤ 1-6 years old
22.52a
737
166
≥ 6 years old

a

26.04
338
88
The prevalence of positive Leptospira were older than 6 years old
had the highest rate of Leptospira infection (26.04%), followed by dogs
from 1 to 6 years old (22.52%) and dogs from 4 months to less than 12
months old had the lowest infection rate (21.51%), but statistical analysis
showed no significantly difference. That is to say, Leptospira can cause
disease in dogs of all ages and older dogs have higher rates of infection
(Harland et al., 2013).

10


Table 4.6 Prevalence of Leptospira infection by sex
Sex

Number of serum
Number of positive Rate (%)
samples
serum samples
Male dogs
715
173
24.20a
Female dogs
718
158
22.01a
Table 4.6 shows that, in the Mekong Delta, 24.20% of male dogs
were positive for Leptospira infections and it was 22.01% for female dogs,
and the difference was not statistically significant. This indicates that male
and female dogs are equally susceptible to Leptospira infection. According
to a survey done by Senthil et al. (2013), the rates of Leptospira infection
in male dogs (20.9%) and female dogs (21%) were the same, and the study
conducted by Dhliwayo et al. (2012) and Anabel et al. (2013) claimed that
the percentage of dogs positive for Leptospira does not depend on sex.
Table 4.7 Proportion of Leptospira-infected dogs by farming methods
Number of
Number of
Farming methods
positive serum
Rate (%)
serum samples
samples
Free-raising dogs
949

246
25.92a
Captive dogs
484
85
17.56b
Data in the same column with different caps are statistically different
(P<0.05).
Table 4.7 shows that free-raising dogs having positive rate for
Leptospira was 25.92%, which was higher than that in captive dogs at
17.65%, and there was statistically significant difference (P<0.01). This
may be explained that free-raising dogs had poor concern and poor hygiene
and they were more likely to be exposed to pathogens through daily food
seeking, urine expositing or drinking dirty water, eating food contaminated
with Leptospira more than captive dogs, which made free-raising dogs had
higher rate of Leptospira infection (Garde, 2013).

11


4.1.2 Prevalence of Leptospira in rats and positive correlation between
serogroups Leptospira in rats and dogs
4.1.2.1 Prevalence of Leptospira in rats
Table 4.8 Prevalence of Leptospira in rats in some Mekong Delta provinces
Number
Number of
Rate
Types of rats
of serum
positive serum

(%)
samples
samples
Rats (Rattus norvegicus)
285
132
46.32 a
Muskrats (Suncus murinus)
152
40
26.32b
Small rats (Mus musculus)
210
57
27.14b
Total
647
229
35.40
Data in the same column with different caps are statistically different
(P<0.05).
Table 4.8 shows that 35.40% of rats are positive for Leptospira, in
which Rattus norvegicus had the highest infection (46.32%), followed by
Suncus murinus (27.14%) and the lowest incidence was Mus musculus
(26.32%) and the difference was statistically significant (P<0.05).
Compared with the survey on the prevalence of Leptospira in rats
between these authors have different Leptospira infection rates as study of
Vu Dinh Hung (1995) the prevalence of Leptospira in rats was 34.01% in
Hanoi, Nguyen Thi Ngan (2000) and Le Huynh Thanh Phuong (2001)
examined the prevalence of Leptospira in rats in the North Vietnam 2 years

(2000-2001) prevalence was 63%, and Ly Thi Lien Khai (2012) in Can Tho
was 55.55%. The difference may be due too various for geographic
location, natural conditions and rats are rodents often live in sewers, areas
of waste, the proportion positive for Leptospira in rats between the studies
also differ together (Suepaul et al., 2014).
Table 4.9 Prevalence of serogroup Leptospira in dogs
Total 647 serum samples
Serogroup Leptospira
L. australis
L. autumnalis
L. bataviae
L. canicola

12

Number infection

(%)

12
9
32
47

5.24
3.93
13.97
20.52

Total 647 serum samples
Serogroup Leptospira

Number infection

(%)

L. ballum
L. icterohaemorrhagiae
L. pyrogenes
L. cynopterie
L. gryppotyphosa
L. hebdomadis
L. javanica
L. panama
L. semaranga
L. pomona
L. tarassovi
L. sejroe
L. louisiana
L. hurstbridge

4
93
19
10
18
6
13
25

1
17
7
22
10
25

1.75
40.61
8.30
4.37
7.86
2.62
5.68
10.92
0.44
7.42
3.06
9.61
4.37
10.92

229
Total number of positive samples
Table 4.9 show that had demonstrate the circulation 18/18 Leptospira
serogroup in dogs in some provinces in the Mekong Delta. The serogroup
was high in L. icterohaemorrhagiae (40.61%), L. canicola (20.52%), L.
bataviae (13.97%), L. panama (10.92%) and serogroup L. hurstbridge
(10.92%).
The comparison with the results of Vu Dinh Hung study (1995) rats

infected with L. bataviae and L. pomona or research of Hoang Kim Loan et
al. (2013) of the prevalence of Leptospira in rodents in southern Vietnam
have two serogroups high percentage in rats including L. bataviae and L.
hurstbridge.

13


Table 4.10 The intensity of infection of serogroup Leptospira of in rats
Serogroup
Leptospira
australis
autumnalis
bataviae

1:200
Infecti
%
on
9
75.00
6
66.67
18
56.25

Infec
tion
2
2

1:400
%
16.67
22.22

1:800
Infecti
%
on
1
8.33
0
0.00

1:1600
Infec
%
tion
0
0.00
1
11.11

8

25.00

4

12.50

2

6.25

1:3200
Infecti
%
o
0
0.00
0
0.00
0.00
0

canicola

36

76.60

6

12.77

4

8.51

1

2.13

0

0.00

ballum

4

100

0

0.00

0

0.00

0

0.00

0

0.00

icterohaemorrh
agiae

54

58.06

23

24.73

13

13.98

2

2.15

1

1.08

pyrogenes

11

57.89

5

26.32

2

10.53

1

5.26

0

0.00

cynopterie

9

90.00

1

10.00

0

0.00

0

0.00

0

0.00

gryppotyphosa

13

72.22

4

22.22

1

5.56

0

0.00

0

0.00

hebdomadis

4

66.67

3

50.00

0

0.00

0

0.00

0

0.00

javanica

10

76.92

2

15.38

0

0.00

0

0.00

0

0.00

panama

17

68.00

6

24.00

1

4.00

1

4.00

0

0.00

semaranga

1

100

0

0.00

0

0.00

0

0.00

0

0.00

pomona

10

58.82

5

29.41

2

11.76

0

0.00

0

0.00

tarassovi

7

100

0

0.00

0

0.00

0

0.00

0

0.00

sejroe

16

72.73

4

18.18

2

9.09

0

0.00

0

0.00

louisiana

8

80.00

2

20.00

0

0.00

0

0.00

0

0.00

hurstbridge

14

56.00

6

24.00

3

12.00

2

8.00

0

0.00

Total 370

247

66.76

79

21.35

33

8.92

10

2.70

1

0.27

14


Table 4.10 show that the intensity of Leptospira infection in rats in
four provinces was concentrated at low antimicrobial antibody titration
values of 1: 20 to 1: 40. This indicates that the rats in the study locations
had only low levels of Leptospira, which means these rats are at stages of
uplift carries the disease but they can transmit the disease to other animals,
including humans (Levett, 2001).
4.1.2.2. Positive correlation between serogroup Leptospira in rats and
dogs
Table 4.11 Positive rates of serogroup Leptospira in rats and dogs
Positive rate in Positive rate in
rats
dogs
The serogroup
No
Leptospira
(%)
(%)

1
L. australis
5.24
5.74
2
L. autumnalis
3.93
3.63
3
L. bataviae
13.97
12.08
4
L. canicola
20.52
18.43
5
L. ballum
1.75
2.11
6
L. icterohaemorrhagiae
40.61
35.05
7
L. pyrogenes
8.30
9.06
8
L. cynopterie

4.37
2.72
9
L. gryppotyphosa
7.86
12.08
10
L. hebdomadis
2.62
3.63
11
L. javanica
5.68
6.65
12
L. panama
10.92
8.76
13
L. semaranga
0.44
0.91
14
L. pomona
7.42
2.72
15
L. tarassovi
3.06
0.30

16
L. sejroe
9.61
9.37
17
L. louisiana
4.37
2.72
18
L. hurstbridge
10.92
12.69
2
R = 0.90; P<0.01

15


Table 4.11 shows that 18/18 serogroup Leptospira were detected
simultaneously in rats and dogs in the Mekong Delta. Four serogroups
having high infections were found in rats and dogs including serogroup L.
bataviae (13.97% and 12.08%), serogoup L. canicola (20.52% and
18.43%), serogroup L. icterohaemorrhagiae (40.61% and 35.05%) and L.
hurstbridge serogroup (10.92% and 12.69%). Comparing with the results of
research by Hoang Manh Lam (2002) when he did a study on the situation
of Leptospira infection in cattle and people in Daklak province, the same
serogroup was detected in rats and dogs as serogroup L.
icterohaemorrhagiae or in Le Huynh Thanh Phuong (2001) study,
serotypes with high infections in rats and dogs including serogroup L.
bataviae, L. canicola, L. gryppotyphosa, L. icterohaemorrhagiae and L.

pomona.
An analysis of the regression equation shows that the relationship
between the prevalence of Leptospira in rats and dogs ratios was quite
close, with correlation coefficients R2 = 0.90 indicating high correlation
and regression equation: y = 0.466 + 0.868x.

Prevalence of positive
Leptospira in dogs (%)

40

y = 0.466 + 0.868x
R² =0.90

35
30
25
20
15
10
5
0

0

10

20

30

40

Figure 4.1 Linear relationship
between
rate in rats (x) and
Prevalence of
positiveLeptospira
Leptospirapositive
in rats (%)
dogs (y)

16


4.2 Detection of Leptospira from urine
4.2.1 Results of detection of Leptospira from urine by PCR
Table 4.12 Proportion of detected Leptospira directly from urine
Number of
Number of
Rate
Clinical symptoms
urine samples
positive urine
(%)
samples
Dogs without clinical
32
6
18.75a

symptoms
Dogs with clinical
31
7
22.58a
symptoms of Leptospira
Total
63
13
20.63
A total of 63 urine samples was tested of Leptospira in urine by
dark-field microscopic examination; 13 (20.63%) samples were positive for
Leptospira by PCR. Including 18.75% (6/32) positive samples from dogs
without clinical symtomps and 22.58% (7/31) positive samples from dogs
with clinical symptoms suspected infection of Leptospira and not
difference statistical significance. When dogs were infected, Leptospira
enter the bloodstream, remain in dogs’ kidneys and was released through
the urine for a long time without clinical symptoms in dogs. If there were
clinical symptoms in dogs, it would be very difficult to treat (Levvet,
2001).
4.2.2 Results of detection of Leptospira from culture samples
Table 4.13 Proportion of detected Leptospira according to the sampling
locations
Number of
Number of
Number of
Locations
urine
suspected samples detected
samples

of Leptospira
Leptospira
infection
Veterinary clinic –
28
14 (50%)
0 (0%)
Can Tho University
Veterinary clinic
11
2 (18.18%)
0 (0%)
inter Ninh Kieu
District - Binh Thuy
Veterinary clinic,
72
47 (65.27%)
3 (6.38%)
50 Vo Van Kiet
Street, Can Tho city
Total
111
63 (56.76%)
3 (4.76%)
17


A total of 111 urine samples from dogs with antibody titers of 18
serogroups Leptospira MAT≥ 1: 400, the results showed that 63 (56.76%)
suspected infection of Leptospira by dark-field microscopic examination.

These sixty-three samples were implanted into the EMJH medium and were
monitored for up to 3 months, the results of three samples (4.76%) had
developing Leptospira in 17 days. Due to Leptospira is characterized by its
difficult culture and long culturing time, it requires a lot of experience and
skills, and it is easy to contaminate with others bacteria.

Figure 4.3 Results of electrophoresis of
Figure 4.2 Leptospira under darkPCR’s products on agarose gel 1.5%, 90V,
field microscopic examination
45 minute. Sample 1, 2 are negative.
(X40) at being cultured for 17 days Samples 3, 4 and 5 are positive.
Combining the results from identification species of spirochetes with
the results of assessment, serological method of fixation was performed
simultaneously on the same sample of dog, and it shows sample No. 3 with
the total number of nucleotides (nt) is 1374nt and sample No. 5 with the
total of number of 1369nt nucleotides that are homologous to the reference
strain Leptospira interrogans serovar icterohaemorrhagiae strain RGA
with a gene pool number of NR 116542.1 at ≥ 99%. Sample No. 4 with the
total number of nucleotides sequenced is 1393nt, when compared with
strain spirochetes reference Leptospira fainei serovar hurstbridge strain
BUT6 codes on GeneBank is NR 043049.1 with the degree of similarity ≥
99% and may determine Leptospira fainei (intermediate pathogenic
Leptospira).

18


Table 4.14 Identification of species Leptospira based on the similarity of
the 16S rRNA gene
Sample

Number of nucleotide
Degree of Species of detected
No
and gene code
homology Leptospira
spirochetes
1
3
1374 nt, NR 116542.1
99%
2
5
1369 nt, NR 116542.1
99%
3
97
1385 nt, NR 116542.1
93%
4
181
1392 nt, NR 116542.1
99%
5
204
1412 nt, NR 116542.1
98%
6
307
1391 nt, NR 116542.1
99%

Leptospira
7
308
1408 nt, NR 116542.1
99%
interrogans
8
310
1390 nt, NR 116542.1
99%
9
312
1394 nt, NR 116542.1
99%
10
313
1390 nt, NR 116542.1
99%
11
490
1393 nt, NR 116542.1
99%
12
493
1390 nt, NR 116542.1
99%
13
494
1396 nt, NR 116542.1
98%

14
4
1393 nt, NR 043049.1
99%
15
328
1391 nt, NR 043049.1
99%
Leptospira fainei
16
330
1413 nt, NR 043049.1
99%
A total of 16 samples with DNA Leptospira in Table 4.14 were
sequenced, 13 samples belonged to the Leptospira interrogans group (3, 5,
97, 181, 204, 307, 308, 310, 312, 313, 490, 493, and 494) and three
samples belong to the Leptospira fainei group (4, 328 and 330). The results
of this study demonstrate that the presence of Leptospira DNA in dog urine
of the Leptospira interrogans group is similar to that of Lofflera et al.
(2014) in Argentina and a study in Thailand, Alongkorn et al. (2017) and
serogroup Leptospira fainei (serovar hurstbridge) has been detected from
human with Weil's syndrome (Petersen et al., 2001).
From the tree diagram (Fig. 4.4), 13 isolated samples were found in
the first branch of L. interrogans, sample No. 308 had close relation to
Leptospira interrogans, followed by samples 307, 5, 313 and the farthest
samples No. 97 and No. 3 samples belong to the second branch of
Leptospira fainei, sample No. 4 has close relation with Leptospira fainei,
followed by sample No. 330 and sample No. 328.
19

The analysis of the degree of homology has been identified
Leptospira interrogans in pathogenic Leptospira groups and Leptospira
fainei belongs to the intermediate pathogenic Leptospira of opportunistic
pathogens present in dogs urine in Can Tho city. This is the first study to
use the 16S rRNA genome to detect Leptospira in dogs in Mekong Delta.

Figure 4.4 The phylogenetic tree of the Leptospira serogroups
4.3 Survey of changes pathological Leptospirosis in dogs
4.3.1 The change of hematological index in dogs of Leptospira infection
Table 4.15 Physiological index of blood in dogs infected with Leptospira
Index monitoring
Total number of
Number of samples
blood physiological with abnormal
samples
blood physiology
(%)
(n=13)( ±SE)
White blood cell (109 /L)
33.50±3.29
13/13 (100)
(6-17)
Neutrophil % (60-70)
74.52 ±2.34
10/13 (76.92)
Lymphocyte % (8-21)
36.43±6.78
7/13 (53.85)
Red blood cell (1012/L)

5.44±0.82
9/13 (69.23)
(5.5-8.5)
Hemoglobin (g/dL) (12-18)
11.98±1.34
7/13 (53.85)
Hematocrit % (35-57)
35.75±3.92
7/13 (53.85)
9
Platelet (10 /L) (170-400)
106.4±19.2
9/13 (69.23)
The dogs of positive Leptospira by PCR have the mean values of
white blood cell count, the percentage of neutrophil and lymphocyte were
higher than normal. The average value of erythrocytes, hemoglobin
20


concentration, hematocrit and platelets in this study is lower than the
normal. The cause of anemia may be due to the entry of Leptospira into the
body to produce toxin that disrupts red blood cells, or the anemia may also
be caused through the gastrointestinal tract or hemolysis. Leptospira secrete
toxins that break down into capillaries and connective tissue under the skin
causing hemorrhage, edema and jaundice (Lee et al., 2000) or acute
vasculitis at acute infection and because of disruption of platelets in
peripheral blood or bone marrow reduces platelet production (Geisen et al.,
2007).
Table 4.16 Biochemical index of blood in dogs infected with Leptospira
Total blood

Number of
Index monitoring
biochemistry samples
samples with
abnormal blood
(n=13) ( ±SE)
biochemictry (%)
Ure (µmol/L) (2.9-10)
33.37±4.91
12/13 (92.31)
Creatinin (µmol/L) (44-150)
458.2±92.7
11/13 (84.62)
AST (U/L) (8.9-48.5)
299±109
10/13 (76.92)
ALT (U/L) (8.2-57.3)
183.2 ±50.9
10/13 (76.92)
Biirubin TP (U/L) (0-17)
390±170
6/13 (46.15)
Based on tests for biochemical criteria of blood, 13 dogs with
Leptospira infection (MAT≥ 1: 400) showed higher levels of uremic blood,
creatine, AST, ALT and total bilirubin than normal. Because Leptospirosis
is a multi-system disease, it affects many organs, especially the liver and
kidneys. In the acute phase of Leptospira disease, it causes renal cell
damage, acute interstitial nephritis, decreasing of blood volume to the
kidneys leading to renal tubular necrosis, decreasing kidney filtration
function (Levett, 2001; Geisen et al., 2007). The liver is also a large organ

that is affected, after entering into the body, Leptospira will go to the liver,
causing hepatitis to whole liver or liver part, necrotizing hepatocellular and
edema can lead to acute hepatitis or chronic. On the other hand, when total
bilirubin in the blood rises, there may be hepatic nephropathy (Figure 4.5),
jaundice (Figure 4.6) and dark urine like Nally et al. (2004); Brito et al.
(2006) and Sykes et al. (2011) had mentioned.

21


Figure 4.5 Liver necrosis caused by Leptospirosis in dogs

Figure 4.6 Jaundice caused by Leptospirosis in dogs
Table 4.17 Urine physiology in dogs infected with Leptospira
Total urine
Number of
Index monitoring
physiology
samples with
samples (n=13)
abnormal urine
physiology
(%)
( ±SE)
Red blood cells (cell/µL) (0-5)
8.54±2.3
8/13 (61.54)
White blood cells (cell/µL) (517.15±5.01
2/13 (15.38)
40)

Protein (g/L Albumin) (0.1-0.3)
1.24±0.3
8/13 (61.54)
Bilirubin (µmol/L) (8.6-17)
14±4.33
4/13 (30.77)
From the results tests of urine physiology showed that 61.54% of red
blood cells and protein in urine from urine, this is explained that dogs had
renal dysfunction, it demonstrates acute kidney disease, glomerular injury,
acute interstitial nephritis, and renal tubular dysfunction (Katz et al., 2001;
Goldstein et al., 2006; Sykes et al., 2011) and when toxins disrupt
erythrocytes release of bilirubin in the blood and excreted through the urine
(Geisen et al., 2007).
22


4.3.2. The clinical symptoms in dogs positive for Leptospira by PCR
Table 4.18 Clinical symptoms in dogs positive for Leptospira
Clinical symptoms
Dogs’ PCR positive
Rate (%)
for Leptospira
Lethargy, sedentary
100
7
Reluctant to eat and anorexia
100
7
Skin and mucous membranes
5

71.43
jaundice
Color dark urine
5
71.43
Vomiting
5
71.43
Fever
4
57.14
Colorless mucus
6
85.71
Absorb abdominal water
3
42.85
Skin hemorrhage
2
28.57
Diarrhea
1
14.28
Clinical symptoms of 7 dogs were positive for Leptospira by PCR
such as lethargy, reluctant to eat and anorexia (100%), followed by
colorless mucus (85.71%), skin and mucosa jaundice, color dark urine,
vomiting (71.43%), and the lowest was diarrhea (14.28%). The symptoms
of lethargy, sedentary, reluctant to eat and anorexia is common
manifestations of many diseases (Goldstein et al., 2006; and Naiari et al.,
2011), but the symptoms of jaundice and urine color dark are most apparent

for Leptospirosis (Geisen et al., 2007).
4.4 Study on the treatment of Leptospirosis in dogs
Table 4.19 Rate of cure
Used medicine
Number of
Cure results
treated
Recovery dogs
Rate
dogs
(%)
Treatment 1 Shoptapen
20
8
40a
(Penicillin G +
Streptomycin)
Treatment 2 Amoxicillin
22
11
50a
Treatment 3
21
13
61.90a
Doxycycline
Total

63
32

50.80
Table 4.19 shows that the rate of recovery of the first treatment is
40%, the second treatment rate is 50% and the third treatment rate is
23