Summary of doctoral thesis: Some epidemiological biology and of Fasciola sp. and the efficacy of anthelminthic treatments in cattle in the Mekong delta

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY

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

Ha Huynh Hong Vu

Some epidemiological biology and of Fasciola sp. and
the efficacy of anthelminthic treatments in cattle
in the Mekong delta

Can Tho- 2018


THIS THESIS WAS COMPLETED AT CAN THO
UNIVERSITY
Academic supervisor:

Assoc. Prof. DR. Nguyen Huu Hung

This thesis was defended against the Ph.D. dissertation council at the
university level.
Place: …………..
Time: ……………
1st Opponent: ……………..
2nd Opponent: ………………………

Reviewed Confirmation of Chairman
…………..

Thesis could be found at:
1. Learning Resource Center, Can Tho University.
2. National Library of VietNam.

I


PUBLISHED ARTICLES
Published Articles in journals
1. Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Nguyen Huu Hung, 2014.
Identification freshwater snail intermediate host of trematoda causing
animal disease in Vinh Long and Dong Thap Province. Journal of Science,
Can Tho University, Special issue agriculture, pp 8-12.
2. Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Nguyen Huu Hung, 2015.
Morphological and molecular characteristic of Fasciola sp infected in cattle
in Dong Thap province. Journal of Science-Technique of Veterinary
Medicine, 6: 63-69.
3. Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Pham Duc Phuc, Nguyen
Huu Hung, 2016. Application of molecular marker-ITS-1 gene and PCRRFLP technique for determining large liver flucke (Fasciola sp.) in cattle in
Mekong river Delta, 2016. Journal of Science-Technique of Veterinary
Medicine, 2: 85-92.
4. Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Nguyen Huu Hung, 2016.
Large liver fluke (Fasciola sp.) infection of cattle in the Mekong Delta and
results of treatment trials. Journal of Science, Can Tho University, Special
issue agriculture, pp 17-22.
5. Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Nguyen Huu Hung, 2018.
The surveillance on pathological characteristics of Fasciola gigantica
infected in Mekong delta. Journal of Science, Can Tho University, Special
issue agriculture, pp 12-17.

II


Chapter I: INTRODUCTION
1.1 Rationale
According to the World Health Organization (WHO), Fascioliasis is one of
the important diseases, which is found in humans and animals. More than
2.4 million people in 70 countries were affected by the disease (WHO,
2015; Amer, 1016). In Vietnam, Fascioliasis in humans tends to increase
gradually, from 2006 to 2010. In fact, 15,764 people and cases were
infected by Fasciola sp. in 2006 and those cases increased to over 20,000
people in 2011. The disease in 52 provinces from North to South and
pathogenic species is determined mainly Fasciola gigantica (Nair et al.
2012). Fasciolosis has been demonstrated and listed in zoonosis diseases.
The disease causes by the large liver flukes which require the intermediate
host (freshwater snail species) to complete its life cycle. The Mekong Delta
possesses the geographic features such as innumerable canals, rivers,
stream which is suitable to develop agriculture: paddy rice and vegetables
as well as provide the appropriate conditions for freshwater snail
development. Moreover, livestock husbandry also great develops because
famers take advantages the source of by-product from agricultural
processing. However, most of husbandry farms are small-scale farms where
people normally use by-products from agriculture and they do not have
well knowledge about applying the techniques in animal husbandry and
veterinary. As the results, their livestocks expose high prevalence of
helminthes infection. Therefore, it is crucial to research about fasciolosis
and how to manage the spreading of this disease in order to minimize the
damage from it. The study aimed to investigate “The epidemiological,
biological characteristics of Fasciola sp. and the efficacy of anthelmintic

treatments in cattle in Mekong Delta”
1.2 Objectives
- Identifying the species, distribution, biological characteristics and
influential factors to the liver flukes infection rate in cattle the Mekong
Delta.
- Suggesting the treatment methods for infected cattle in Mekong Delta.
1.3 Scientific significance
- This is a systematic research about liver flukes Fasciola gigantica in
cattle: determining the prevalence of infection and influential factors to the
pathogen. Species were identified by morphological and molecular
characteristics (PCR-RFLP, and sequencing)
- The life cycle of Fasciola gigantica in cattle in Mekong Delta were firstly
researched: identifying intermediate host (snails). Clinical symptoms and
3


anthelminthic testing would be useful for diagnosis and treatment.
- This thesis provides documentations about Fasciola sp. infected in cattle
(Mekong Delta), and supplies academic knowledge for veterinary
parasitology books to education and training purposes
1.4 Practical significance
- The thesis results are the scientific background for recommending farmers
in effectively diagnosis, treatment and prevention liver flukes that
minimizes the economic lose as well as contributes for the sustainable
development of livestock husbandry.
1.5 Innovative contributions of the thesis
This is the first research about Fasciola gigantica in infected cattle in
Mekong Delta which were identified by applying molecular biology
techniques.
This is also first research about the complete life cycle of Fasciola

gigantica.
Gross lesions and histopathological of Fasciolosis (causing by F.gigantica)
were completely described which were provided background for quickly
diagnosis and treatments
Chapter III: CONTENT AND RESEARCH METHODOLOGY
3.1 The research contents
3.1.1 Determining the prevalence of liver flukes of cattle in the Mekong
Delta provinces
- Determining the infection rate of liver flukes of cattle in the Mekong
Delta provinces by the fecal examination and necropsy methods.
3.1.2 Identifying the species of Fasciola sp. in the Mekong Delta
provinces
- Determining the species of Fasciola sp. by analyzing mophorlogical
molecular biology chacteristics and sequencing.
3.1.3 Researching about life cycle of Fasciola gigantica
- Observing the development of the Fasciola gigantica egg outside the
definite host.
- Observing the development of the larvaes of Fasciola gigantica in
intermediate host (Lymnaea swinhoei and Lymnaea viridis) to stage
cercaria infection.
- Analyzing and recording the every development stage of Fasciola
gigantica since embronated eggs to mature in definitive host.
4


3.1.4 Fasciola gigantica Pathogen researching
-Determine the clinical symptoms, gross lesions and histopathology on
infected Fasciola gigantica cattle
3.1.5 Studying on treatments of Fasciola gigantica infected cattle
- Testing the efficacy of anthelminths: albendazole, mebendazole and

triclabendazole; and suggest the efficient treatment on Fasciola gigantica
infected cattle.
3.2 Subjects, timeline, and researching areas
3.2.1 Subjects: cattle in six provinces: Dong Thap, An Giang, Vinh Long,
Tra Vinh, Ben Tre and Soc trang; liver flukes-Fasciola sp.; the snail
Lymnaea spp.
3.2.2 Timeline: from 11/2013 to 06/2017
3.2.3 Rearching areas
The cattle in 6 provinces (Mekong Delta), slaughter houses, histologic
laboratory in the Department of Veterinary Medicine – College of
Agricultural and Applied Biology, Can Tho University; Department of
Clinical Veterinary Science and livestock - veterinary medicine – Nong
Lam University, Anatomical pathology laboratory - University of medicine
and pharmacy of Can tho.
3.3. Materials and Chemistry
Necessary materials and chemistry for diagnosis and molecular biology
techniques.
3.4 Research methology
3.4.1 Identifying the prevelance of infected cattle in Mekong Delta
Subjects:
- Domestic cattle, cross-breeding Sind, dairy cow were classified into 3 age
groups: under 1 year old, 1-2 years old, and over 2 years old. Slaughter
cattles were selected for this research basing on the original surveyed
provinces.
- Methods:
Fecal examination: modified sedimentation of Benedek (1943).
The autopsy methods: the operating SKRJABINE (1937)
-Observation targets
The overall infected rate of Fasciola sp. in cattle, the infection rate of this
disease according to seasons, husbandry methods, ages, and ecogeographic

areas; the intensity of the infection ( the number of species/ individual)
-Statistical analysis: Chi-Square test /Minitab program version to compare
infection rates.

5


3.4.2 Identification method
3.4.2.1 Identification method of trematode by morphology based on
documents written by David and Erasmus (1972), Soulsby (1980), Nguyen
Thi Le (2000).
3.4.2.2 Identification method of live flukes by molecular techniques
(PCR-RFLP) and sequence genes
Collecting and storing DNA extraction samples
Totally, 180 liver flukes were randomly collected from the liver and bile
ducts in slaughter cattle from 6 surveyed provinces. Specimens was stored
in physiological saline and brought to DNA laboratory.
DNA extraction
DNA concentration measurement.
PCR-RFPL techniques
PCR reaction:
Table 3.1 Primer sequence corresponding to the target gene
Annealing
Primer sequence
Gene Primer
temperature
Reference
(5’ – 3’)
o
-Tm ( C)

ITS1-F: TTG CGC
ITS1TGA TTA CGT
ITS1
F/ITS1- CCC TG
56
Itagaki T (2005)
R
ITS1-R: TTG GCT
GCG CTC TTC ATC
PCR products were incubated with restriction enzymes RsaI (5 IU)
overnight at 37oC. Then the products were run on 1.5% agarose gel with
Ethidium bromides in 80 votage in 30 minutes. The gels were visualized
under camera (Geldoc).
Table 3.2 Prediction the restriction patterns of the enzyme RsaI Enzyme
restriction patterns in the region of ITS1 Fasciola sp.
Species

Restriction
enzyme

Fasciola
hepatica
RsaI
Fasciola
gigantica

Temperature,
incubation
time

370C,
overnight
incubation

6

Predicted
length of
cutting
(bp)

Reference

367, 104
(68, 59,
5’..GT↓AC...3'
54, 28)
3'..CA↑TG...5'
367, 172
(59, 54,
28)

(Ichikawa
et al,
2010)

Location
cuting

Sequencing positive samples
- Totally 12 liver fluke samples (Fasciola sp.) were collected from 6
provinces in Mekong Delta, specifically 2 samples/one province.
PCR products were purified and sent to Macrogen (Korea) to
sequence (using Sanger sequence method)
Observation targets :
- Identification of Fasciola sp. were done by morphological and
molecular biology chacteristic.
- Comparasion the nucleotide sequence of target gene ITS-1 of
Fasciola sp among surveyed provinces and Fasciola sp. collected
worldwide in Genbank
Analyzing data :
- BLAST the ITS-1 sequence (in NCBI) was used to identify the
specific species, and comparing the level of similarity among
multisequence by CLUSTAL OMEGA, analyzing Pairwise
alignment/Calculate identity/Similarity for sequences (Bioedit).
3.4.3 Studies of life cycle of Fasciola gigantica.
3.4.3.1 Identification of freshwater snails
Basing on the classification system of the freshwater snails was
described by John (1982), Dang Ngoc Thanh and his colleagues (1980).
From this backgound knowledge, the snails Lymnaea were carefully
collected and feeded in the laboratory environment to produce the clean
snail generation. Miracidium from embronated eggs (Fasciola gigantica)
were infected to clean snail generation.
3.4.3.2 Observe the development of Fasciola gigantica egg in in vitro
a. Reseach objects:
Fasciola gigantica eggs, Lymnaea swinhoei and Lymnaea viridis snails is
"clean snail".
b. Experimental design
Table 3.3 Experiment designing for Fasciola gigantica eggs development

Number of
Experiment
Number of petri / trial
eggs/petri
Negative control
0
5
Experiment 1
60
10
Experiment 2
60
10
Experiment 3
60
10
Experiment 1: Fasciola sp. egg in petri disk with water levels of 0, 5 cm, no
illumination, pH from 6-8, the temperature from 26-290C. Experiment 2: Fasciola
sp. egg in petri disk above 0, 5 cm, lighting 4 hours/day, pH from 6-8, the
temperature from 26-290C. Experiment 3: Fasciola sp. egg in petri without water

7


c. Observation targets
- The length of time from eggs to develope to miracidium.
- The length of time from the eggs hatching into miracidium to 50% eggs
were hachted and miracidia liberating from the egg shells.
3.4.3.3 Observing the length of time of alive miracidia in water
a. Research objects

Fresh miracidia have just liberated from the egg shells. Those miracidia
were observed to identify the their longevity in water.
b. Experimental design
After miracidium infected to the snails, infected snails were collected and
necropsied at time points: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42 PI days in
order to detect cercaria- free swimming stage in water and transforming to
Adolescaria. For each time points, 10 of Lymnaea swinhoei and 10
Lymnaea viridis snails were surgery to the stage of development of the
larva (redia and cercaria, sporocyst) in 2 species of snail Lymnaea.
Table 3.4 Identifying the longevity of miracidia in water
Number of
Number of petri disk/
Experiment
miracidia/petri
experiment
1
15
10
2
15
10
3
15
10

3.4.3.4 Observing the development of larva stage of Fasciola in
intermediate host Lymnaea spp snails.
In this experiment, 960 miracidia were collected and infected to 240
Lymnaea swinhoei, and 240 Lymnaea viridis.
Table 3.5 The invasion of Fasciola micracidia to Lymnaea snails

Experiment

Infection dose
(micracidium /snail)

Lymnaea swinhoei
Negative control
Lymnaea viridis
Negative control
Lymnaea swinhoei
Lymnaea viridis

8

Number snail of
experiment

0

80

0

80

3
3

160
160

3.4.3.5 Cattle infected by Fasciola larvae
a. Research objects
In this experiment, 8 cattle at the age of 7 months-12 months old were
bought from the local farmers in the surveyed areas. Before infecting,
cattle were dewormed by albendazole and carefully tested the presence of
liver fluke eggs as well as other helminths.
b. The cercaria infection causing lab layout for experimental cattle.
Six cattle were divided into 3 different groups which were received 3 doses
100, 150, and 200 cercaria. Those cattle were infected by ceraria through
oral adminstration. Non-infected group was considered as negative control.
Observing the presence of liver fluke eggs in feces of infected cattle
The feces examination were conducted after 11weeks post infection and
then feces samples were collect and check everyday until detecting the eggs
of Fasciola sp. The sedimentation methods was applied to diagnose. The
results were futher confirmed by necropsy method.
Observation targets
- Identification the timepoint of the presence of liver fluke eggs in
cattle feces
- The numember of liver flukes in experiment cattle as well as
species identification by morphological and molecular biological
features.
3.4.4 Clinical symptoms and gross and histopathological changes on
cattle infected with Fasciola
3.4.4.1 Symptoms of cattle infected with Fasciola
- Physical and clinical examination were done in 60 infected cattle with
Fasciola and 6 infected cattle in infection experiment (3.4.3.5 )
3.4.4.2 Researching about the gross lesions and histopathology in liver
tissue causing by Fasciola gigantica infection

-Objects: livers from Fasciola gigantica infected cattle in this experiment
and 45 livers from Fasciola gigantica infected cattle in slaughter houses in
Mekong Delta.
3.4.5. Studying the prevention and treatment Fasciolosis in cattle
105 crossbred Sind cattle having in high infectious intensity from 2+ to 3+
were collected to test the efficacy of albendazole, mebendazole,
triclabendazol. The number of cattle were divided into 2 experiments and 5
cattles in control group.
Experiment 1: treatment dosage- following the manufacturer's instructions
Experiment 2: increasing the treatment dosage (higher dose the
manufacturer’s recommendation)
Control group: not use any treatment
9


Table 3.7 The efficiency of albendazole, mebendazole and triclabendazole
on Fasciola sp. infected cattle
Experiment

Doses
(mg/kg body weight)

Number
of cattle
treatment

Adminstration
Route

Control group

0
5
albendazole: 10 mg/KgP
15
1
triclabendazole: 15 mg/KgP
15
mebendazole:10 mg/KgP
15
albendazole:15 mg/KgP
15
2
triclabendazole: 20 mg/KgP
15
mebendazole:15 mg/KgP
15
c. Observing targets
The efficacy of drugs afer 5, 10, 15 days post-treatment were
observed the adverse effects of those drugs in treated cattle.

oral
oral
oral
oral
oral
oral
tested and

CHAPTER IV RESULTS AND DISCUSION

4.1 The prevalence of liver flukes infected cattle in Mekong Delta
4.1.1 The results of fecal examination of Fasciola sp. infected cattle in
Mekong Delta
Table 4.1 The prevalence of liver flukes infected cattle in Mekong Delta
Intensity of infection
Province

Examined
cattle

Infected
cattle

Prevalence
(%)

+
(%)

An Giang
Dong Thap
Vinh Long
Ben Tre
Tra Vinh
Soc Trang
Total

a

++

+++

(%)

(%)
a

1036
987
993
933
900
935

268
249
244
149
142
134

25.87
25.23a
24.57a
15.97b
15.78b
14.33b

73.13

70.28
71.31
81.21
83.80
85.07

20.15
20.88a
19.67a
14.77b
11.97b
11.94b

6.72a
8.84a
9.02a
4.03b
4.23b
2.99b

5784

1186

20.50

75.80

17.62

8.68

a,b in the same row showed the statistically significant difference at P< 0.05

Table 4.1 showed that the overall infected cattle by Fasciola sp. 20.50%. In
particular, cattle in An Giang province had the highest infectious rate of
Fasciola sp. 25.87%, following by cattle in Dong Thap (25.23%) and Vinh
Long with 24.57%. The infectious rate of Fasciola sp. in cattle in Ben Tre,
Tra Vinh and Soc Trang was 15.97%, 15.78% and 14.33%; respectively.
Most of infected cattle had the low intensity of infection (1+) which
oocupied of 78.80%, following by the (2+) intensity with 17.62%, and (3+)
10


with 8.68%. The infectious rate of Fasciola sp. in cattle in An Giang, Dong
Thap and Vinh Long had statistically significant higher than those in Tra
Vinh, Ben Tre and Soc Trang (p<0.05). The above results were explained
that An Giang and Dong Thap province is located at the upstream of the
Tien and the Hau River, Vinh Long is by the sides of the rivers. Due to the
geographic features such as innumerale canals and waterways, it is the
appropriate conditions for the development of intermediate hosts (snail) of
Fasciolosis. Therefore, the infectious rate of Fasciolosis in cattle in An
Giang, Dong Thap ang Vinh Long had the higher than those in Tra Vinh,
Ben Tre and Soc Trang (the latter provinces locate near the sea and have
the saltwater and blrackish water areas – not appropriate for intermediate
hosts (snails) development. This result confirmed that the geographic
climate and the ecological conditions had great influence on the growth of
intermediate hosts of Fasciolosis and by the way influence on the infectious
rate of Fasciolosis in cattle. This studying results is compatible with the
researches of Pham Van Khue and Phan Luc1996); Phan Dich Lan et al.

(2002); Nguyen Thi Kim Lan et al. (2008).
Table 4.2 The infection rate of liver fluke egg in cattle according to the
breed
Provinces

Gender
Crossbred Sind
Prevalence (%)

Dairycow
Prevalence (%)

Local cattle
Prevalence (%)

24.91b
23.48b
a
12.31
25.58b
14.99b
15.43b
a
7.07
17.07b
a
9.15
20.86b
a,b in the same row showed the statistically significant difference at P< 0.05

An Giang
Dong Thap
Vinh Long
Ben Tre
Tra Vinh
Soc Trang
Total

26.99b
24.53b
27.25b
17.04b
16.16b
15.47b
21.55b

The results in table 4.2 showed that the infectious rate of Fasciolosis in
local cattle were 21.55%, that was higher than crossbred Sind 20.86% and
the lowest infection rate in dairy cows (9.15%). Through statistical analysis
we found that the infection rates of dairy cows and crossbred Sind as well
as local cattle had statistical significant difference. Meanwhile, local cattle
and crossbred Sind had with no difference. According to breed factor, the
infectious rate of Fasciolosis in cattle among provinces also provided the
same patterns.
Table 4.3 showed that the prevalence of Fasciola infectious rate in cattle
increased according to ages; namely under 1 year old cattle (8.24%);
closely by from 1 to 2 year-old-cattle (19.06%); and over 2 year-old cattle
11

(31.38%).Through statistical analysis showed that there was significant
difference among 3 various age groups. Over 2 year-old cattle had longterm environmental exposure to environmental conditions which especially
had more opportunities to be infected intermediate hosts for Fasciolosis.
Moreover, the immunune system of old animals decreased so it was easy to
be infected by other pathogens. Young cattle (<1 year old) were still in
breast feeding period so the opportunity for them to contact to larvae stages
of Fasciola sp. was lowest among 3 surveyed groups. In conclusion, the
liver fluke infection rate in cattle depended on age of the host which were
similar to finding of authors Do Duong Thai (1978), Ho Thi Thuan (1986),
Phan Dich Lan (2000), Le Huu Khuong et al.. (2001), Nguyen Duc Tan et
al. (2010), Nguyen Khac Luc et al. (2010), Vo Thi Hai Yen et al. (2010),
Nguyen Huu Hung (2011), Nguyen Thu Huong et al. (2012), Nguyen Thi
Sam et al. (2012)
Table 4.3 The infection rate of liver fluke egg in cattle according to ages
group
Infected rate to age (%)
<1 year
1-2 years old
>2 years old
10.53a
23.97b
41.12c
An Giang
a
b
9.23
21.79
40.79c
Dong thap
a

b
10.14
22.93
39.74c
Vinh Long
a
b
6.91
14.79
24.21c
Ben Tre
a
b
6.51
14.07
22.91c
Tra Vinh
a
b
5.37
12.69
21.82c
Soc Trang
a
b
Total
8.24
19.06
31.38c
a,b,c in the same row showed the statistically significant difference at P< 0.05

Provinces

Table 4.4 The infection rate of liver fluke egg in cattle according to the
raising methods
Provinces

An Giang
Dong thap
Vinh Long
Ben Tre
Tra Vinh
Soc Trang
Total

Infected rate to raising method (%)
Confined
Partial- free range

12.41a
11.95a
11.67a
8.11a
9.83a
8.53a
10.55a

34.17b
31.67b
34.03b
19.62b

19.67b
18.21b
26.74b

a,b in the same row showed the statistically significant difference at P< 0.05

Table 4.4 showed that the liver fluke infection rate of partial free-range
cattle in Mekong Delta (26.74%) was statistically higher than the confined
cattle (10.55%) with p value <0.05. Similarly, the infectious rate of kept
12


cattle and grazing cattle in every province such as An Giang, Dong Thap,
Vinh Long, Tra Vinh, Ben Tre and Soc Trang also followed the same
pattern.
Table 4.5 The infection rate of liver fluke egg in cattle according to the
seasonal
Provinces

An Giang
Dong thap
Vinh Long
Ben Tre
Tra Vinh
Soc Trang
Total

Infected rate to seasonal (%)
Rainy season
Dry season

31.42a
30.38a
29.52a
22.02a
20.56a
20.43a
26.07a

20.23b
18.66b
19.60b
10.66b
10.73b
8.42b
14.79b

a,b in the same row showed the statistically significant difference at P< 0.05

Table 4.5 illustrated that the Fasciola infection rate in cattle in the Mekong
Delta was influenced by season factor. In fact, the infectious rate in dry
season was higher than in the rainy season (14.79%). Through statistical
analysis of Fasciola infection rates between the dry season and the rainy
season in the Mekong Delta have the discrepancy is significant statistically
(p < 0.05). Similarly, in An Giang, Dong Thap, Vinh Long, Tra Vinh, Ben
Tre and Soc Trang province, the Fasciola infection rates was statistically
higher in the dry season than in rainy season.
Table 4.6 Fasciola infection rate on cattle by eco-region
Provinces

An Giang
Dong thap
Vinh Long
Ben Tre
Tra Vinh
Soc Trang
Total

Infected rate to eco-region(%)

Saltwater and brackish
-

9.38a
9.47a
8.33a
9.01a

Fresh water
25.87
25.23
24.57
22.06b
21.11b
22.11b
24.14b

a,b in the same row showed the statistically significant difference at p < 0.05

Table 4.6 showed that the Fasciola infection rate in cattle in the Mekong

Delta had changed according to the ecological regions. In fact, the
percentage of infected cattle in freshwater areas was the highest one
(24.14%), following by the cattle in saltwater and brackish ecological
region 9.1%. The results of statistically analyzing also pointed out that this
difference was statistical discrepancy (p<00.5)
13


4.1.2 The results of autopsy to determine the prevalence of Fasciola
infection in cattle in Mekong Delta
Table 4.7 Prevalence of Fasciola infection in cattle in the Mekong Delta
provinces (by autopsy)
Provines

AG
ĐDT
VL
BT
TV
ST
Total

Examined cattle

279
257
263
270
257
246

1,572

Infection Cattle

75
70
68
48
45
40
346

Prevalence
(%)

Intensity
(X±SE)

26.88a
27.24a
25.86a
17.78b
17.51b
16.26b
22.01

12.88±0.79
12.37±0.88
11.91±0.83
7.02±0.48

6.71±0.56
6.40±0.61
9.93±0.34

a,b in the same column showed the statistically significant difference at P< 0.05

Table 4.7 showed that cattle in 6 provinces had the Fasciola infection rate
of 22.01% with infection intensity: 0.34 ± 9.93. Cattle in An Giang, Dong
Thap and Vinh Long province were high infectious rate group. Specifically,
the cattle in An Giang had the highest infection rate of 26.88% with the
intensity of infection (IF) 12.88±0.79, following closely by Dong Thap
with 27.24%, IF 12.37±0.88, and the lowest one in this group with 25.86%
and IF 11,91±0,83. Cattle in Ben Tre, Tra Vinh and Soc Trang province
belonged to low Fasciola infection group. In this group, the infectious rate
of cattle in Ben Tre, Tra Vinh and Soc Trang were 17,78% with IF
7.02±0.48; 17.51% with IF 6.71±0.56; 16.26% with IF 6.40±0.61;
respectively.
4.2 Results of liver fluke identification
4.2.1 Liver fluke identification by morphological method
Totally 2253 liver flukes were collected from 6 provinces in Mekong Delta,
then were measure the length and analyzed the morphological
characteristics. The results found that the size of those liver flukes had the
length: 33.21±0.48mm, width: 9.24±0.09mm, oral sucker 1.056 ± 0,
008mm, ventral sucker 1.468 ± 0, 011mm and lengh/width rates/3.49 ±
0.04 (the size fluctuating from 28.64 ± 0, 16 mm to 38.43 ± 0, 61mm for
length and from 6.44 ± 0, 29mm to 10.77 ± 0, 14 mm for width,
length/width ratio ranged from 2.83 ± 0.043 to 4.22 ± 0.105). Liver flukes
are leaf-shaped, broader anterior than posteriorly, intestine branching and
has distributed the vitelline glands throughout the body. Ventral sucker is
usually bigger than oral sucker. The testes and ovaries are located just

behind the ventral sucker and the feeling in the middle of the body expels
the leaves. According to Phan The Viet et al. (1977) and Nguyen Thi Le
14


(2000), 2,259 Fasciola obtained in 6 provinces of the Mekong Delta are
species of Fasciola gigantica.
4.2.2 The results of liver fluke identification by molecular biology
4.2.2.1 Analyzing PCR-RFLP results in liver fluke identification on
cattle in the Mekong Delta

360b
p170b
p60bp
Figure 4.2: The PCR products was digested by using restriction enzyme RsaI
From left to right, M: Marker 100bp, Wells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10: samples: 11:
negative control, 12: positive control- Fasciola gigantica.

Totally 180 liver fluke samples collecting from 6 provinces were analyzed
using PCR to multiply the ITS1 gene fragment by specific primer ITS1-F
and-R ITS1 (Itagaki et al., 2005) designed to Fasciola spp. detection with
the size of PCR products around 680 bp (Ichikawa and Itagaki , 2010).
Through the results of electrophoresis PCR-RFLP products, 180/180
samples had the cutting pattern of Fasciola gigantica which had 3 different
bands with size 360, 170, 60 bp. The results in this study strongly
confirmed that liver flukes in Mekong Delta was Fasciola gigantica
4.2.2.2 Analysing the ITS1 gene sequencing of Fasciola gigantica
The PCR products from 12 liver flukes in 6 provinces were purified and
sequenced and BLAST with the nucleotide sequence of ITS-1 in Genbank.
The sequencing results showed that 12/12 samples were Fasciola

gigantica. This result pointed out that PCR-RFLP is the effective methods
to distinguish the differences between F.gigantica and F.hepatica. The
ITS-1 sequence of F.gigantica (this study) 660 bp had high level of
similarity, just different from 1-2 nucletotide in the gene fragment was
assigned area of. Among them, 8/12 samples had sequence perfectly match
with samples of Fasciola gigantica in Thailand (F-T), Vietnam (F-VN)up
to 99.8% to 100% (Ichikawa & Itagaki, 2010). This demonstrates that this
gene has a very high similarity conservation among collected F.gigantica,
which does not depend on the location of sampling. Fasciola gigantica
expels patterns in the Mekong Delta provinces have similar proportions
15


with the samples of Thailand, SLGL China, Japan, South Korea, Australia
and Vietnam range from 99.0% to 98.9%. Therefore, large liver fluke
circulation in cattle in the Mekong Delta region was F.gigantica.
Table 4.8 Comparison the ITS1 nucleotide sequence of Fasciola sp. in the
study and other strains of Fasciola worldwide
Position of Nucleotide variation
No

13

48

149

173

265

359

437

457

F-A

A

T

G

T

T

C

T

C

F-C

A

T

G

T

T

C

T

C

3

F-J

A

Y

G

Y

W

Y

W

Y

4

F-K

A

Y

G

Y

W

Y

W

Y

5

BT2

G

C

G

T

T

T

A

T

6

TV2

G

C

G

T

T

T

A

T

7

AG1

C

C

G

T

T

T

A

T

8

DT1

A

C

A

T

T

T

A

T

9

ST2

A

C

G

T

T

T

A

T

10

ST1

A

C

G

T

T

T

A

T

11

VL2

A

C

G

T

T

T

A

T

12

BT1

A

C

G

T

T

T

A

T

13

AG2

A

C

G

T

T

T

A

T

14

F-T

A

C

G

T

T

T

A

T

15

F-VN

A

C

G

T

T

T

A

T

16

DT2

A

C

G

T

T

T

A

T

17

VL1

1
2

A
C
G
T
T
T
A
T
TV1
A
C
G
T
T
T
A
T
Annotation: AG1 AG2 liver flukes, collected in An Giang Province; BT1, BT2
samples in Ben Tre province; ĐT1, ĐT2: samples in Dong Thap province; ST1,
ST2: samples collected in the province; TV1 TV2: samples in Tra Vinh province;
VL1 VL2 samples in Vinh Long province; F-T: Fasciola gigantica in Thailand; FVN: Fasciola gigantica in Vietnam; F-A: Fasciola hepatica in Australia; F-C:
Fasciola hepatica; F-J: hybrid Fasciola in Japan; F-K:. hybrid Fasciola in South
Korea
18

4.3 Life cycle of Fasciola
4.3.1 The distribution of freshwater snail species-intermediate host of
F. gigantica
Totally 15,848 snails were collected in the Mekong Delta provinces.
Through the identification of classification based on the classification

16


system of the freshwater snails by Dang Ngoc Thanh et al. (1980) and John
(1982), the results showed that collected snails in the Mekong Delta
provinces were Lymnaea swinhoei (15.61%), followed by species of
Lymnaea viridis (9.71%).
Table 4.9 The development of Fasciola sp. egg in water environment
Experiment
STKS
TGTBĐN (day)
TGTN (50%) (day)
1

600

15.18±0.16

19.05±0.129

2

600

10.57±0.14

15.88±0.10

3

600

-

-

Notes: Experiment 1: Fasciola sp. Eggs in petri disk with water levels of 0, 5 cm, no
illumination, pH from 6-8, the temperature from 26-290C. Experiment 2: Fasciola sp. egg in
petri disk above 0, 5 cm, lighting 4 hours/day, pH from 6-8, the temperature from 26-290C.
Experiment 3: Fasciola sp. egg in petri without water. STKS: survey number of eggs;
TGTBĐN: the time the eggs begin to hatch, TGTN (50%): the time the eggs hatch 50% rate.

Table 4.9 showed that Fasciola eggs cultivated in petri disk in experiment
1, the length of time for egg hatching was 15.18 ± 0.16 days. Fasciola sp.
egg hatching time reached 50% rate is 19.05 ± 0.129 days. In experiment 2,
the eggs started to hatch at 10.57 ± 0.14 days, the 50% of eggs hatching
was after 15.88 ± 0.10 days. In experiment 3, there is no egg hatching. So,
in the same condition as when illuminated, the time the eggs start to hatch
the egg hatching time and reaches 50% earlier than when those were not
illuminated. Thus, the water and light were identify as crucial element
which has a great influence to the development of Fasciola sp. egg and
miracidium shell escape. The light is an important factor in facilitating the
miracidium escaping from shell eggs. Through the observation, we found
that the development of Fasciola sp. eggs  miracidium were not
happened at the same time.
A

B

C

D

Figure 4.3: The development stages of Fasciola sp. egg form miracidium
A. Eggs Fasciola sp.; B. the miracidium in eggs;
C. the Miracidium in eggs are about escaping the shell; D. Miracidium

17


4.3.3 Time of miracidium in water environment
Tracking time of miracidium in conditions 26-290C showed that the
miracidium lived from 10-12days in aquatic environment. In first 10 hours
after hatching miracidium, no death miracidium were recorded. The death
after 10 hours, 10-11 hours, after 11-12 hours hatching were 8.22%,
41.56% and 52.89%; respectively. The research results of longevity of
miracidium in water was shorter than other research results of foreign
studies, but still longer than some documents of of Pham Van Khue and
Phan Luc (1996); Pham Dieu Thuy et al. (2014).
4.3.4 The development of Fasciola gigantica larvae in the intermediate
host Lymnaea swinhoei and Lymnaea viridis.
Through experimental design, the larvae of Fasciola gigantica were
infected on Lymnaea swinhoei and Lymnaea viridis. The results showed
that the Lymnaea swinhoei and Lymnaea viridis larvae were infected by
sporocyst after 10 days, redia found in snails from 14 to 38 days after
infecting miracidium, the cercaria found in snails from 26-42 days after
infecting miracidium. At the same time, observing the miracidium culture
water, we detected the presence of cercaria-swimming in water using very
strong activity tail, after 1-2 hours then the cercaria tail loss to form
adolescaria. So the length of time for development from miracidium

invasion to Lymnaea swinhoei and Lymnaea viridis to cercaria was from
26 to 42 days.
The results showed that both Lymnaea swinhoei and Lymnaea viridis snails
were infected by Fasciola sp. larvae as conducting experimental infection.
Thus, the intermediate host of Fasciola gigantica in Mekong Delta region
is Lymnaea swinhoei and Lymnae viridis.

A

B

C

Figure 4.4 The larval stages of Fasciola developed in Lymnaea
swinhoei and Lymnaea viridis
A. Sporocyst; B. The redia; C. Cercaria
4.3.5 Results of infection cause larvae Fasciola for cattle
Totally 6 cattle were chose and infected by cercaria to observe the
presence of Fasciola sp. eggs in cattle feces. In the experiment 1 (100
18


cercaria/ 1 cattle), Fasciola sp. eggs were detected from 114 to 115 days
after infection with the intensity of infection (IF) at 1+ and 2+. In the
experiment 2 (150 cercaria/1 cattle), the eggs were also detected at the
same time with experiment 1, and the IF were 2+. In the experiment 3 (200
cercaria/1 cattle), the eggs were somehow detected earlier from 111 to 114
days with IF 3+. Therefore, the higher number of cercaria invaded to host,
the higher intensity infection. Cercaria continued developing in cattle
(definite host) and developed to mature live flukes and laid eggs in 111-115

days after infection.
Table 4.10 Operating results Fasciola infection in experimental cattle
Experiment

Cattle

ĐC

1
2

1
Total

1
2

2
Total
3

1
2

Number of
cercaria
infection

Number of liver fluke
collect

Prevalence of liver
fluke collect (%)

0
100
100

0
29
30

0
29.0
30.0

200

59

29.50a

150
150
300
200
200

47
46

93
63
65

31.33
30.67
31.0a
31.5
32.5

Total
400
128
32.0a
In the experiment 1, the number of adult liver flukes/ number of cercaria
infected were 29.50% with the intensity infection 29-30 liver flukes. In the
experiment 2, the proportion between adult liver flukes and cercaria
infected were 31% with higher intensity 46-47 liver flukes. Similarity, in
the experiment 3, this proportion rised upto 32% and the intensity 63-65
liver flukes. However, the statistically analyzing showed that there was no
signicant difference among 3 experiment. The results of intensity of
infection in 3 groups were also tested by fecal examination which also
provided the same pattern.
4.3.6 Species of Fasciola determination results of infecting cattle
4.3.6.1 Results determine the species of liver fluke by using traditional
methods
Through the analysis of morphological characteristics and the structures of
the 280 liver fluke infection-causing cattle obtained experimentally. All of
liver flukes were Fasciola gigantica (basing on the features described in
19

speices identification of Phan The Viet et al. (1977) and Nguyen Thi Le
(2000). To determine the exact species liver fluke obtained from
experimental infections in this study, PCR-RFLP method was applied to
speices identification. The results from PCR-RFLP were also F.gigantica.
Therefore, the liver flukes in Mekong Delta provinces were Fasciola
gigantica .
4.3.6.3 Summary of life cycle of Fasciola gigantica

Figure 4.5. Summary of life cycle of Fasciola gigantica in cattle.
Life cycle of liver fluke: from embryonated egg stage in the water
environment then developing to miracidium 10-19 days. Excysted
miracidium freely moved in water from 10 hours to 12 hours. In Lymnaea
swinhoei and Lymnaea viridis snails. miracidium  cercaria took for 42
days, after 1-2 hours cercaria lost their tails and encysted as metacercaria
(infective larva). Fasciola gigantica become mature in definite host after
111-115 days infection.
4.4 Results of the investigation of symptoms of Fasciola gigantica
infected cattle
The study investigated 60 infected with Fasciola gigantica in nature and 6
infected cattle with Fasciola gigantica from experimental infection. The
results showed that Fasciola gigantica infected cattle had some clinical
20


symptoms such as emaciation, reduced rumination, diarrhea alternating
with constipation, anorexia, pale mucous membrane
4.5. Results of investigating pathological changes in liver of cattle
infected with Fascioliasis

4.5.1 Gross lesions on Fasciola gigantica infected liver
A

B

C

t

F

E

D

Figure 4.6 Gross lesion on Fasciola gigantica infected liver
A. Hemorrhages on the liver; B. Fasciola gigantica in bile ducts; C. Liver swelling
and fibrosis; D. Gallbladder walls thicken and accumulate calcium in the bile
ducts; E. white bile ductus; F. Fatty liver, swollen gall bladder.

A survey of 45 Fasciola gigantica infected bovine liver in nature and 6
infected cattle in experimental infection were collected to investigate gross
lesions. The observation of pathology in liver fluke infected livers showed
that macro pathology consisted of the marked thickness of bile ducts in
liver, yellowis-white lesions and hemorrhages due to the destruction of
tissues, and the surface of the liver. The lesions on liver surface revealed
the particular cirrhosis.
4.5.2 Histopathology of liver infection with Fasciola
Recognizing the generalized lesions on 15 infected cattle at infection
intensity of over 20 flukes in nature and 6 bovine liver in experimental

Fasciola gigantica infection for microscopic examination. Histopathology
of liver infection of fluke resulted from the intensive destruction of liver
parenchyma, marked hemorrhage and necrosis. The walls of the bile ducts
are commonly calcified fibrosis. Cirrhosis tissues were the signs of liver
parenchyma

21


A

B

C

F

E

D

Fig. 4.7 Microscopic examination of Fasciola gigantica infected liver
A. Liver faintly between the lobe; B. Blood vessels in the interstitial lobe; C.
thick fibrosis of the liver lobe; D. fibrous sheet penetration wall; E. bovine
proliferation and fibrosis; calcium; F. Juice resides in the liver parenchyma

4.6 Results of treatment of liver fluke in cattle
Table 4.11 Efficacy of albendazole in treatment of liver fluke in cattle
Before

No of
cattle
without
liver
fluke
eggs

Non-treat
ment
Experiment
1
Experiment
2

5

Efficacy of drug
After 10 days

Prevalence
(%)

No of
cattle
without
liver
fluke
eggs

0

0

15

5

15

13

o

Experiment

After 5 days

N of
infected
cattle

After 15 days

Prevalence
(%)

No of
cattle
without
liver

fluke
eggs

Prevalence
(%)

0

0

0

0

33.33

10

66.67

12

80

86.67

15

15

100

100

Experiment1: 10 mg/kgbodyweight; Experiment 2: 15 mg/kgbodyweight;

Table 4.11 showed that the experiment 2, all of cattle (15/15) were treated
with albendazole at the dose of 15 mg / kg body weight that achieved 100%
efficacy after 10 days of treatment. With the dosage of 10 mg/kg, 12/15
(80%) cattle were erradiated from Fasciola sp. Therefore, using
albendazole with dosage 15 mg/k.g body weight provided the highest
efficacy 100% with no side effects
Table 4.12 showed that the experiment 2, all of cattle (15/15) were treated
with triclabendazole at the dose of 20 mg / kg body weight that reached to
100% efficacy after 10 days of treatment. At the dosage of 15 mg/kg,
13/15 (88.67%) cattle were erradiated from Fasciola sp. Therefore, using
22


albendazole with dosage 15 mg/k.g body weight provided the highest
efficacy 100% with no side effects.
Bảng 4.12 Efficacy of triclabendazole in treatment of liver fluke in cattle
Efficacy of drug
Before

After 5 days

After 10 days

o

Experiment

o

N of
infected
cattle

N of
cattle
without
liver
fluke
eggs

o

Prevalence
(%)

No of
cattle
without
liver
fluke
eggs

Prevalence
(%)

0

0

0

0

40

12

80

13

86.67

80

15

100

15

100

N of

cattle
without
liver
fluke
eggs

0

Prevalence
(%)

Non-treat
5
0
ment
Experiment
15
6
1
Experiment
15
12
2
Experiment1: 15 mg/kgbodyweight;

After 15 days

Experiment2: 20 mg/kgbodyweight;

Table 4.13 Efficacy of mebendazole in treatment of liver fluke in cattle

Effect of on the treatment of fascioliasis in cattle
Before
Experiment

Non-treat
ment
Experimen
t1
Experimen
t2

No of
infected
cattle

Efficacy of drug
After 10 days

After 5 days
No of
cattle
without
liver
fluke
eggs

Prevalence
(%)

0

No of
cattle
without
liver
fluke
eggs

After 15 days

Prevalence
(%)

No of
cattle
without
liver
fluke
eggs

Prevalence
(%)

0

0

0

0

60

5

0

15

4

26.67

6

40

9

15

5

33.33

8

53.33

11

73.33

Experiment1:10 mg/kgbodyweight; Experiment2: 15 mg/kgbodyweight

Table 4.13 showed that in two experiments:1 and 2 of mebendazole in liver fluke
treatment in cattle at 10 mg / kg body weight and 15 mg / kg body weight
provided the low efficacy with only 60% and 73%; respecitvely, no adverse
effects during experiments.
The results presenting in table 4.11, 4.12 and 4.13 found that using the
recommendation dose from manufacturer instruction could not bring the highest
efficacy in eliminate liver flukes. However, using the higher dose than
23


recommendation dose of albendazole and triclabendazole had 100%
effectiveness.
Chapter V: CONCLUSION AND RECOMENDATION
5.1 Conclusion
From the results of the thesis, we had some conclusions as follows:
1. The overall prevalence rate of liver fluke infection in cattle in 6 surveyed
provinces in Mekong Delta was 20.50%. The Fasciola sp. infection rate
was influenced by animal breed, animal age, animal husbandry methods
and ecological areas
2. Analyzing morphological characteristics and molecular biology
including PCR-RFLP and sequencing were applied in Fasciola sp.
identification. The results from 3 above methods have confirmed that liver
flukes in cattle in Mekong Delta were Fasciola gigantica.
3. Life cycle of liver fluke: from embryonated egg stage in the water
environment then developing to miracidium 10-19 days. Excysted
miracidium freely moved in water from 10 hours to 12 hours. In Lymnaea

swinhoei and Lymnaea viridis snails. miracidium  cercaria took for 42
days, after 1-2 hours cercaria lost their tails and encysted as metacercaria
(infective larva). Fasciola gigantica developed to adult liver fluke 111-115
days infection.
4. Fasciola gigantica infected cattle had some clinical symptoms such as
emaciation, reduced rumination, diarrhea alternating with constipation,
anorexia, pale mucous membrane, bad hair condition. The gross lesions:
liver inflammation and cirrhosis, hemorrhage, brown round neoplasm in
liver surface, the infected livers having ivory-white yellow or clay-colored.
Histopathology of liver infection of fluke resulted from the intensive
destruction of liver parenchyma, marked hemorrhage and necrosis. The
walls of the bile ducts are commonly calcified fibrosis. Cirrhosis tissues
were the signs of liver parenchyma.
5.Albendazole (15 mg/kg) and triclabendazole (20 mg/kg) with an oral
single dose were both effective up to 100% in eliminating Fasciola. These
drugs were safety and no adverse effects noticed during treatment.
5.2 Suggestions
- It is necessary to propagate and disseminate widely about the harm and
transmission route of fascioliasis caused by Fasciola gigantica parasite in
cattle in Mekong Delta provinces.
- It is recommended that cattle producers in the Mekong Delta should use
albendazole 15mg / kg body weight or triclabendazole 20mg / kg body
weight to prevent periodic cure twice a year.
24