MINISTRY OF EDUCATION AND TRAINING
THAI NGUYEN UNIVERSITY
MINISTRY OF EDUCATION AND TRAINING
THAI NGUYEN UNIVERSIY
--------------------
--------------------
PHAM DIEU THUY
PHAM DIEU THUY
STUDY ON EPIDEMIOLOGICAL CHARACTERISTICS OF
FASCIOLOSIS IN BUFFALOES AND BOVINES IN THAI
NGUYEN, BAC KAN, TUYEN QUANG PROVINCE AND
PREVENTIVE AND TREATMENT METHODS
(2010 - 2013)
STUDY ON EPIDEMIOLOGICAL CHARACTERISTICS OF
FASCIOLOSIS IN BUFFALOES AND BOVINES IN THAI
NGUYEN, BAC KAN, TUYEN QUANG PROVINCE AND
PREVENTIVE AND TREATMENT METHODS
(2010 - 2013)
Speciality: Parasites and Veterinary Microbiology
Code: 62.64.01.04
SUMMARY OF PH.D. DISSERTATION IN
VETERINARY MEDICINE
SUMMARY OF PH.D. DISSERTATION IN
ADVISOR: Prof. Nguyen Thi Kim Lan, Ph.D.
VETERINARY MEDICINE
Thai Nguyen, 2014
Thai Nguyen, 2014
3
LIST OF WORKS RELATED TO THE DISSERTATION
1. Nguyen Thi Kim Lan, Pham Dieu Thuy, Pham Thi Trang
(2012), “Infection rate and intensity of Fasciola gigantica in
buffaloes and bovines in Thai Nguyen”, Animal husbandry magazine
July, pages 19 - 23.
2. Pham Dieu Thuy, Nguyen Thi Kim Lan, Hoang Thi Ngan
(2012), “Status of Fasciola infection in buffaloes and bovines in Bac
Kan province”, Animal husbandry magazine, August, pages, 26 - 31
3. Pham Dieu Thuy, Nguyen Thi Kim Lan, Tran Nhat Thang,
Tran Thi Phuong Thao (2014), “Study on egg viability and
development time of Fasciola gigantica larvae in the external
environment and in the intermediate host”, Journal of Agriculture
and Rural Development, June, pages 122 - 126.
4. Pham Dieu Thuy, Nguyen Thi Kim Lan, Tran Nhat Thang,
Tran Thi Phuong Thao (2014), “Study on the contamination of
Fasciola gigantica eggs and larvae in the environment”, Veterinary
sciences and Techniques, Vol. XXI, No6, pages 76 - 81.
1
2
INTRODUCTION
- Laboratory of Veterinary and Animal Science faculty - College
of Agriculture and Forestry, Thai Nguyen University
- Institute of Ecology and Biological Resources.
The Fascioliasis in buffaloes and bovines (Fassciolosis) caused
by two liver fluke species namely Fasciola hepatica and Fasciola
gigantica, is considered as a common parasitic disease which
causes great losses in the economy of buffalo and bovine
production in the world (Soulsby E. J. L., 1987). In recent years,
the Fascioliasis in buffaloes and bovines has occurred widely and
increased in the number due to the climate change and the
migration of animals between countries and/or regions (Kasib
Muhammad Khan et al., 2013).
There are some studies on the epidemiological characteristics of
fascioliasis in buffaloes and bovines and the effectiveness of the
treatment. But, only few works has been paid which considers the
issue in the mountainous area such as Thai Nguyen, Bac Kan and
Tuyen Quang province. Therefore, an effective measures of
prevention and the control of this diease in the mountainous area are
obviously needed.
CHAPTER 1
OVERVIEW
CHAPTER 2
MATERIALS, CONTENTS AND METHODS OF STUDY
2.1. Objects of study
- Buffaloes and bovines in three provinces: Thai Nguyen, Bac
Kan and Tuyen Quang
- Fasciolosis in buffaloes and bovines.
2.1.2. Study period: From 2010 - 2013
2.1.3. Places of study
2.1.3.1. Places of implementation: Thai Nguyen, Bac Kan and Tuyen
Quang province.
2.1.3.2. Places of samples
- Sperstructure Laboratory - National Institute of Hygiene and
Epidemiology.
2.2. Materials of study
2.2.1. Materials and samples of study
* Animals of study: buffaloes and bovines at diffenrent age, and
freshwater snails
* Samples of study: samples of fresh feces from Buffaloes and
bovines; samples of soil from animal house (sediment) floors, aquatic
grass samples, samples of snails without covers of the mouth,
Fasciola samples collected from necropsied buffaloes and bovines,
and bile samples, etc.
2.2.2. Instruments and chemicals: Light microscopy, counting
chamber Mc Master, cattle bedding, jars for specimen, surgical
instrument set, glass sinks, glass pots, plastic trays, enamel tray,
anthelmintic drugs against liver fluke, etc.
2.3. Contents of study
2.3.1. Determining parasitic liver flukes in buffaloes and bovines in
three Northern mountainous provinces
2.3.2. Epidemiological characteristics of fasciolosis caused by F.
gigantica in buffaloes and bovines
2.3.2.1. Investigating current status of animal husbandry and the
prevention of buffaloes and bovines from parasiolosis in three
studied provinces
2.3.2.2. Situation of F. gigantica infection in buffaloes and bovines
2.3.2.3. Study on eggs and larvae of F. gigantica in the external
environment and the intermediate host
2.3.3. Studying correlation between the number of F. gigantica
eggs in 1 gram of feces with the number of parasitic flukes
buffaloes and bovines
3
4
2.3.4. Studying prevention and control measures of fasioliasis
caused by F. gigantica in buffaloes and bovines
2.4.. Methods of study
* Determining the rate and intensity of infection F. gigantica of
bufalo in 2 months, 4 months after the trial
* Sugegesting the prevention and control procedures of
fascioliasis caused by F. gigantica in buffaloes and bovines.
2.4.1. Method of necropsy and examination are used to collect and
identify F. gigantica species in buffaloes and bovines in Thai
Nguyen, Bac Kan and Tuyen Quang province
2.4.2. Method of investigating veterinary hygiene and prevention of
fascioliasis caused by F. gigantica in cattle and bufaloes
2.5. Methods of data processing
The data acquired were analyzed and processed using the method
of Biostatistics (according to Nguyen Van Thien, 2008) based on
Excel 2003 and Minitab 14.0 software.
2.4.3. Methodologies for studying epedemic charateristics of
Fascipla spp. in buffaloes and bovines: use of Benedek sedimentation
method (1943) for examination of Fasciola eggs.
CHAPTER 3
RESULTS AND DISCUSSION
2.4.4. Studying eggs and larvae of F. gigantica in the external
environment and the intermediate host
2.4.5. Studying lifetime of F. gigantica eggs in the external
environment (when not released into water)
3.1. Results of the species determination of parasitic Fasciola spp.
in buffaloes and bovines in three provinces (Thai Nguyen, Bac
Kan and Tuyen Quang)
2.4.6. Study of time of miracidium released from eggs and survival in water
2.4.7. Study on the duration of F. gigantica larval in Lymnae viridis
snails - intermediate hosts
2.4.8. Method of determining correlation between the number of F.
gigantica eggs in 1 gram of feces and the number of parasitic liver
flukes in buffaloes and bovines.
2.4.9. Method of determining the effect of medicines against F.
gigantica
2.4.10. Trials on preventive measures of fascioliasis caused by F.
gigantica in buffaloes and bovines.
- Place of implementation: Dong Hy district - Thai Nguyen
Nguyen province.
- Trial object: buffalo at 2 - 4 years old.
Content:
* Arranging the test
Table 3.1. Results of the necropsy examination of buffaloes and
the collection of liver flukes
Place
(province)
Thai Nguyen
Bac Kan
Tuyen Quang
Total
Number of
necropsied
buffaloes
(bufalo)
Number of
infected
buffaloes
(bufalo)
Percentage
(%)
Infection intensity
(liver
fluke/bufalo)
150
150
150
450
89
78
97
264
59.33
52.00
64.67
58.67
5 - 89
3 - 72
6 - 78
3 - 89
Table 3.2. Results of the necropsy examination of bovines and the
collection of liver flukes
Place
(province)
Thai Nguyen
Bac Kan
Tuyen Quang
Total
Number of
necropsied
bovines
(bovine)
Number of
infected
bovines
(bovine)
Percentage
(%)
Infection intensity
(liver
fluke/(bovine)
20
20
20
60
8
6
8
22
40.00
30.00
40.00
36.67
7 - 52
1 - 45
4 - 64
1 - 64
5
6
The results in table 3.1 and 3.2 show that at post - mortem
examination of 450 buffaloes and 60 bovines in three provinces, the
infection rate in buffaloes and bovines was 58.67% and 36.67%,
respectively.
compared with the 2 samples mentioned above. So the intermediate
forms were also F. gigantica species.
Table 3.3. Indentification of parasitic liver fluke species in
buffaloes and bovines
Place
(province)
Thai Nguyen
Bac Kan
Tuyen Quang
Total
Number of
liver flukes
for
determinat
ion
(fluke)
262
356
538
1156
3.2. Epidemiological characteristics of fascioliasis caused by
Fasciola in buffaloes and bovines
3.2.2. Situation of F. gigantica infection in three Northern
mountainous provinces
Results of species determination
Fasciola
Intermidiate
Fasciola
hepatica
forms between
gigantica species
species
the 2 species
Number
of
flukes
%
Number
of
flukes
%
Number
of
flukes
%
258
347
517
1122
98.47
97.47
96.10
97.06
0
0
0
0
0
0
0
0
4
9
21
34
1.19
2.29
4.06
2.94
Table 3.3 shows that in the total of 1156 liver fluckes
classified, 97.06% of them was Fasciola gigantica, this was
from 96.10% - 98.47% between the provinces, and there was no
fluke belonging to Fasciola hepatica species. However, there
were 34 flukes (2.94%) in the intermediate forms between the
two species (F. gigantica and F. hepatica) (these flukes had
"shoulder" but not obvious). So we had redefined these samples
by biological molecular methods. The result of DNA sequencing
of three reprenstative samples showed that 99% of the samples
were homologous with the gen bank of F. gigantica. Thus, the
flukes in the intermediate forms were also F. gigantica species.
Comparing nucleotide sequence and amino acid sequence, it is
obvious that 2 samples of F. gigantica having the same sequences.
The other one had 5 nucleotides and 3 amino acids different
3.2.2.1. Infection rate and intensity of F. gigantica in buffaloes and
bovines in Thai Nguyen, Bac Kan and Tuyen Quang province
Table 3.6. Infection rate and intensity of F. gigantica in buffaloes
in the places studied
Place
(province,
district)
* Thai Nguyen
Dong Hy
Vo Nhai
Dai Tu
Number
of
buffaloes
examined
(buffalo)
Number
of
infected
buffaloes
(buffalo)
Percentage
(%)
n
%
n
%
n
%
1800
600
600
600
851
335
238
278
47.28b
55.83c
39.67a
46.33b
447
162
151
134
52.52
48.36
63.45
48.20
262
111
58
93
30.79
33.13
2437
33.45
142
62
29
51
16.69
18.51
12.18
18.35
40.72a
53.33b
31.67a
37.17a
461
186
141
134
62.89
58.13
74.21
60.09
191
102
30
59
26.06
31.87
15.79
26.46
81
32
19
30
11.05
10.00
10.00
13.45
51.89c
56.00b
45.83a
568
228
166
60.81
67,86
60.36
289
89
82
30.94
26.49
29.82
77
19
27
8.25
5.65
9.82
3.83b
174
53.87
118
36.53
31
9.60
46.63
1.476
58.62
≤
200
Infection intensity
(eggs/g feces)
> 200 - 500
> 500
χ2= 31.779; P = 0.000
1800
733
600
320
600
190
600
223
χ2= 63.055; P= 0.000
* Tuyen Quang
1800
934
Yen Son
600
336
Ham Yen
600
275
* Bac Kan
Cho Moi
Bach Thong
Ngan Son
Tuyen Quang
city
600
323
2
Total
χ = 13.784; P = 0.001
5400
2518
742
29.47
300
χ2= 45.551; P = 0.000
* Notes: In vertical line, the numbers with superscriptions are
different remarkably in stastistics.
11.91
7
8
Table 3.6 shows that in three provinces the infection rate of F.
gigantica in buffaloes was 46.63%, ranging from 40.72% to 51.89%.
The difference between the three provinces was significant (P <
0.001). Particularly, in Tuyen Quang, the infection rate in buffaloes
was highest (51.89%), followed by Thai Nguyen (47.28%), the
lowest was in Bac Kan proince (40.72%).
(38.57%), and the lowest rate was in Bac Kan province (29.84%). However,
the differences in prevalence of bovines Fasciola in Thai Nguyen and Tuyen
Quang were not significant (P > 0.05). In different districts the prevalence was
apparent (P < 0.001), but in Tuyen Quang province, the differences in
prevalence between districts were not clear (P > 0.05). In term of infection
intensity, there was lighter and more moderate intensity in bovines compared
with buffaloes, but the severe infection intensity was less.
In general, the infection intensity in buffaloes was mild. However,
buffaloes infected intensity was accounted for 58.62%; the average intensity
made up 29.47%; while the severe intensity accounted for 11.91%.
3.2.2.3. Infection rate and intensity of F. gigantica in buffaloes and
bovines acording to seasons
Table 3.7. Infection rate and intensity of F. gigantica in bovines in
the places investigated
Number of Number of
infected Percentage
Places (province, bovines
district)
examined
bovines
(%)
(bovine)
(bovine)
* Thai Nguyen
Dong Hy
Vo Nhai
Dai Tu
630
243
≤
n
200
%
Infection intensity
eggs/g feces)
> 200 - 500
n
%
n
> 500
%
38.57b
144
59.26
75
30.86
24
9.88
b
Table 3.10. Infection rate and intensity of F. gigantica in
buffaloes according to seasons
Number of Number
Places
buffaloes of infected Percentage
Season
(province)
examined buffaloes
(%)
(buffalo (buffalo)
210
110
210
63
210
70
χ2= 25.846; P = 0.000
* Bac Kan
630
188
ChoMoi
210
80
Bach Thong
210
69
Ngan Son
210
39
χ2= 20.485; P= 0.000
Tuyen Quang
630
225
Yen Son
210
67
Ham Yen
210
75
52.38
30.00a
33.33a
47
45
52
42.73
71.43
74.29
44
15
16
40.00
23.81
22.86
19
3
2
17.27
4.76
2.85
Sping
Summer
Thai
Nguyen Autumn
Winter
29.84a
38.10b
32.86b
18.57a
123
48
52
23
65.43
60.00
75.36
58.97
51
27
12
12
27.13
33.75
17.39
30.77
14
5
5
4
7.44
6.25
7.25
10.26
Sping
Summer
Bac Kan Autumn
Winter
35.71b
31.90
35.71
136
39
46
60.45
58.21
61.33
77
25
23
34.22
37.31
30.67
12
3
6
5.33
4.48
8.00
Tuyen Quang
city
39.52
51
61.45
29
34.94
3
3.61
Sping
Summer
Autumn
Winter
34,71
403
61.43
203
30.95
50
7.62
210
83
Tuyen
Quang
2
Total
χ = 2.655; P = 0.265
1.890
656
2
χ = 11.015; P = 0.004
* Notes:I vertical line, the numbers with superscription are different
remarkably in statistics.
Table 3.7 shows that the infection rate of F. gigantica in bovines was
34.71% (much lower than buffaloes), the highest rate found in Thai Nguyen
Total
Sping
Summer
Autumn
Winter
450
173
486
291
414
218
450
169
χ2= 66.906; P= 0.000
378
115
468
245
450
202
504
171
χ2= 55..697; P = 0.000
503
254
453
271
412
220
432
189
χ2= 23.653; P = 0.000
1.331
542
1.407
807
1.276
640
1.386
529
χ2= 129.022; P = 0.000
Infection intensity
(eggs/g feces)
> 500
≤ 200 > 200 - 500
n
%
n
%
n
%
38.44a
59.88c
52.66b
37.56a
78
165
117
87
45.09
56.70
53.67
51.48
63
80
69
50
36.42
27.49
31.65
29.59
30.42a
52.35c
44.89b
33.93a
80
146
119
116
69.57
59.59
58.91
67.84
31
66
54
40
26.96 4 3.47
26.94 33 13.47
26.73 29 14.36
23.39 15 8.77
50.50b
59.82c
53.40bc
43.75a
136
187
138
107
53.54
69.00
62.73
56.61
93
63
67
66
36.61
23.25
30.45
34.92
25
21
15
16
9.85
7.75
6.82
8.47
40.72a
57.36c
50.16b
38.17a
294
498
374
310
54.24
61.71
58.44
58.60
187
209
190
156
34.5
25.9
29.69
29.49
61
100
74
65
11.26
12.39
11.87
11.91
32
46
32
32
18.49
15.81
14.68
18.93
* Notes: in vertical line, the numbers that carry different letters are
different in stastistic signinficance.
9
10
The results in table 3.10 shows that regarding infection rate:
overall, the infection rates of liver flukes in buffaloes in three
provinces were different with seasons. The highest rate was in
Summer (57.36%), followed by Autumn (50.16%); Spring (40.72%)
and the lowest was in Winter (38.17%). However the infection rate of
liver flukes in buffaloes between Winter and Spring was not
markedly different (P > 0.05).
comparing shows that the prevalence between bulls and cows was not
markedly different (P > 0.05). Statistically there were also no
significant differences of infection intensity between bulls and cows.
This result was consistent with study findings on cattle by Pham
Ngoc Vinh, Nguyen Trong Kim (1997). The study results on goats by
Nguyen Thi Kim Lan et al. (1999). Khan M. K. et al. (2009).
* Regarding the intensity of infection:
Buffaloes infected mainly in mild and moderate intensity in all four
seasons of the year. In severe intensity the infection rates of liver flukes in
buffaloes and bovines in summer and were the lowest in Winter.
3.2.2.4. Infection rate and intensity of F. gigantica in buffaloes and
bovines with different sex.
Table 3.13. Infection rate and intensity of F. gigantica in
buffaloes with sex of cattle
Places
(province)
Sex
Number of
Number of
bovine
Percentage
infected
(%)
examined
cattle(cattle)
(cattle)
Infection intensity
(eggs/g feces)
≤ 200
n
%
> 200 - 500
n
n
> 500
%
n
Bull
239
85
35.56
51 60.00 26 30.59 8
9.41
Cow
391
158
40.41
93 58.86 49 31.01 16 10.13
Bull
284
86
30.28
53 61.63 28 32.56 5
5.81
Cow
346
102
29.48
70 68.63 23 22.55 9
8.82
Bull
164
58
35.37
34 58.62 21 36.21 4
5.17
Cow
466
167
35.84
102 61.08 56 33.53 8
5.39
Bull
687
229
33.33
138 60.26 75 32.75 17
6.99
Cow
1203
427
35.49
265 62.06 128 29.98 33
7.96
Thai Nguyen
Bac Kan
Tuyen
Quang
Total
χ2= 0.901; P = 0.342
Table 3.13 shows that the infection rates of F. gigantica in bulls
and cows were 33.33% and 35.49% respectively. Statistical
3.2.3. Study on eggs and larvae of F. gigantica in the external
environment and in intermediate hosts
3.2.3.2. Contamination of F. gigantica eggs on grazing land
Table 3.16. Contamination of F. gigantica eggs in grazing land of
buffaloes and bovines
Top soil from grazing land
Pool
Place
Number of Number of
Number of Number of
Percentage
percentage
(province, district) samples positive
samples positive
(%)
(%)
examined samples (+)
examined samples (+)
* Thai Nguyen
450
33
7.33
450
61
13.56
Dong Hỷ
150
11
7.33
150
20
13.33
Vo Nhai
150
10
6.67
150
16
10.67
Dai Tu
150
12
8.00
150
25
16.67
* Bac Kan
450
38
8.44
450
63
14.00
Cho Moi
150
14
9.06
150
23
15.33
Bach Thong
150
13
8.84
150
25
16.67
Ngan Son
150
11
7.56
150
15
10.00
* TuyenQuang
450
42
9.33
450
78
17.33
Yen Son
150
16
10.67
150
32
21.33
Ham Yen
150
14
9.33
150
24
16.00
Tuyen
150
12
8.00
150
22
14.67
Quang city
Total
1.350
113
8.37
1.350
202
14.96
χ2= 1.178; P = 0.555
χ2= 3.016; P = 0.221
Table 3.16 shows that overall 8.37% of the samples of grazing
land surface and 14.96% of water sampes from the hollows on
grazing land in the places of the three provinces under study were
contaminated with liver fluke germs.
11
12
3.2.3.3. The distribution of freshwater snails - intermediate hosts of
F. gigantica
3.2.3.4. The prevalence of liver fluke larvae in freshwater snails intermediate hosts
Table 3.17. Results of freshwater snail classification
Number of Lymnaea viridis
Lymnaea
Places
snails
species
swinhoei species
(province, district) classified
n
(%)
n
(%)
(snail)
* Thai Nguyen
2160
768
35.56
621
28.75
Dong Hỷ
720
224
31.11
195
27.08
Vo Nhai
720
293
40.69
235
32.64
Dai Tu
720
251
34.86
191
26.53
* Bac Kan
2160
663
30.70
437
20.23
Cho Moi
720
221
30.69
174
24.17
Bach Thong
720
230
31.94
147
20.42
Ngan Son
720
212
29.45
116
16.11
*Tuyen Quang
2160
520
24.07
978
45.28
Yen Son
720
234
32.50
286
39.72
Ham Yen
720
120
16.67
365
50.69
Tuyen Quang city
720
166
23.05
327
45.42
Total
6480
1951 30.11 2036 31.42
Other species*
n
(%)
Table 3.19. The prevalence of F. gigantica larvae in freshwater
snails
Place (province)
Description
Snail species
771
301
192
278
1.060
325
343
392
662
200
235
227
2493
35.69
41.81
26.67
38.61
49.07
45.14
47.64
54.44
30.65
27.78
32.64
31.53
38.47
Table 3.17 shows that in 6480 snails collected 30.11% belonged
to the L. viridis species; 31.42% belonged to L. swinhoei species
38.47% were the other snail species (not the intermediate host of
liver fluke). Thus more than 60% of the snails collected belonged to
two species L. viridis and L. swinhoei. According to Dang Ngoc
Thanh et al. (1980), Nguyen Trong Kim and Pham Ngoc Vinh
(1997), intermediate hosts of Fasciola in Northern part of Viet Nam
are two snails species: L. viridis and L. swinhoei. Results of snail
classification in Thai Nguyen, Bac Kan and Tuyen Quang showed
that two snail species accounted for a high proportion of snails
collected. Common distribution with large numbers of two snail
species creates favorable conditions for F. gigantica complete their
life cycle.
Number of snails
examined (snail)
Number of snails
infected with
F. gigantica
larvae(snail)
Percentage (%)
n (snail)
Sporocyst
(%)
n (snail)
Redia
%
n (con)
Cercaria
(%)
Thai Nguyen
Bac Kan
Tuyen Quang
Lymnaea
viridis
Lymnaea
swinhoei
Lymnaea
viridis
Lymnaea
swinhoei
Lymnaea
viridis
Lymnaea
swinhoei
768
621
663
437
520
978
108
40
173
41
208
318
14.06
55
50.93
63
58.33
66
61.11
6.44
13
32.50
19
47.50
17
42.50
26.09
95
54.91
97
56.07
104
60.12
9.38
18
43.90
25
60.98
22
53.66
40.00
76
36.54
96
46.15
103
49.52
32.52
124
38.99
131
41.19
141
44.34
Table 3:19 shows that on the prevalence of larvae in snails there were
14.06% of L. viridis and 6.44% of L. swinhoei in Thai Nguyen; 26.09% of
L. viridis 9.38% L. swinhoei in Bac Kan; 40% of L. viridis and 32.52% of
L. swinhoei in Tuyen Quang being infected with F. gigantica larvae. Thus,
both species of snails were infected with liver fluke larvae ranging 20% 70%. In which L. viridis was infected with liver flukes with significantly
higher rate compared with L. swinhoei (this shows in all three provinces
studied). It is believed that L. viridis species was more susceptible to F.
gigantica compared with L. swinhoei.
- Regarding the prevalence of larval forms of F. gigantica in snail:
L. viridis was infected with sporocyst larvae from 36.54% to 54.91%; Redia
larvae from 46.15% to 58.33% and Cercaria larvae 49.52% to 61.11%. L.
swinhoei infected with Sporocyst larvae from 32.50% to 43.90%; Redia larvae
from 41.19% to 47.50% and Cercaria larvae from 42.50% to 53.66%.
13
14
3.2.3.8. Study on the time of Miracidium hatched released and
surviving in water
3.2.3.9. Study of time for development of F. gigantica larvae in snail
- intermediate hosts
Table 3.26. Time to complete the larval stages of F. gigantica
(from the time the eggs fall into the water)
Table 3.24. Time of Miracidium hatched and released into
water (from releasing eggs in water)
Initial time of
Time for
Miracidium
all
Miracidium
Number Temperature releaseed from
released
from eggs
Season Experiment of
and pH
eggs
samples
water
X ± mX
total ( X ± mX ) total
(day)
(day)
(day)
(day)
15.40 ± 0.45
14.40 ± 0.36
I
10
22 - 23oC,
Spring
14.60 ± 0.41 14 16.80 ± 0.25 16
II
10
6-7
15.20 ± 0.25
15.20 ± 0.62
III
10
(
Summer
Autumn
Winter
I
10
II
10
III
10
I
10
II
10
III
10
I
10
II
10
III
10
o
26 - 27 C,
6-7
)
8.80 ± 0.33
8.90 ± 0.31
Season
Number of
samples
Spring
5
Summer
5
Atumn
5
Winter
5
9.70 ± 1.58
8
9.50 ± 0.23
24 - 25oC, 12.00 ± 0.58
6-7
13.70 ± 0.30 11
11.40 ± 0.21
20.00 ± 0.47
18 - 19oC,
19.30 ± 0.74 19
6-7
19.90 ± 0.42
8.30 ± 1.78
10
10.10 ± 2.18
14.10 ± 2.79
12.20 ± 2.81
15
15.40 ± 2.92
19.70 ± 0.63
21.10 ± 0.79
21
20.30 ± 1.57
Table 3.24 shows that in Spring Miracidium hached and released
from egg earliest was 14 days, no later than 16 days; In Summer the
earliest was 8 days and no later than 10 days; In Autumn, the earliest
time was 11 days and no later than 15 days; In Winter the earliest
was 19 days and no later than 21 days. Thus, the time required for
development from the egg into the water to hatch into miracidium
varied from 8 to 21 days. In winter, due to cold weather the
development of eggs in water lasted longer. Whereas, in Summer the
weather was hot, the development of eggs was markedly shorter.
Stage of larvae
Egg à Miracidium
Miracidium à Sporocyst
Sporocyst à Redia
Redia à Cercaria
Cercaria à Adolescaria
* Egg à Adolescaria
Egg à Miracidium
Miracidium à Sporocyst
Sporocyst à Redia
Redia à Cercaria
Cercaria à Adolescaria
* Egg à Adolescaria
Egg à Miracidium
Miracidium à Sporocyst
Sporocyst à Redia
Redia à Cercaria
Cercaria à Adolescaria
* Egg à Adolescaria
egg à Miracidium
Miracidium à Sporocyst
Sporocyst à Redia
Redia à Cercaria
Cercaria à Adolescaria
* Egg à Adolescaria
Time to
complete (day)
14 - 16
3-4
6-8
18 - 20
2 - 4*
41 - 48
8 - 10
2-3
4 -7
15 - 17
2 – 4*
29 - 37
11 - 15
3–4
5–8
17 - 18
2 - 4*
36 - 45
19 - 21
4-5
8-9
20 - 21
3 - 5*
51 - 56
The results in table 3.26 shows that in winter, the time of
developing egg and larval forms in the snail were the longest. The
rule of the development time of larvae in intermediate hosts with
seasons reveals that the temperature is an important factor and a
major influence on the development of eggs and larvae of F.
gigantica in the external environment and in intermediate hosts.
15
16
3.3. Studies of correlation between number of parasitic F. gigantica in
buffaloes and bovines with number of eggs in 1 g of feces.
number of parasitic Fasciola / buffalo and number of eggs /g feces
was correlated.
The correlation coefficient of r = 0.96 indicates a very close
correlation.
* Correlation between number of eggs /g faeces and number of
parasitic Fasciola / bovine was as follows:
The linear regression equation y = a + bx
(y: number of eggs/g faeces. x: number of parasitic Fasciola /bovine)
Among them:
a = 4.145
b = 8.094
→ the linear regression equation: y = 4.145 + 8.094x
The correlation coefficient: r = 0.969
* The correlation between the number of parasitic Fasciola
/buffalo with number of eggs in 1 gram of feces was determined on
Minitab 14.0 software. The results were as follows:
Linear regression equation: y = a + bx
(y: number of eggs / g of feces. x: number of parasitic Fasciola / buffalo)
Among them:
a = 0.194
b = 8.101
→ linear regression equation: y = 0.194 + 8.101 x
Correlation coefficient: r = 0.96
The correlation between the number of parasitic Fasciola / buffalo
and number of eggs /g feces is illustrated in Figure 3.10
Correlation between number of F. gigantica eggs /g faeces and
number of parasitic Fasciola /bovine is illustrated in figure 3.11.
8 00
5 00
7 00
6 00
4 00
3 00
4 00
y
y
5 00
3 00
2 00
2 00
1 00
1 00
0
0
10
20
30
40
50
60
70
80
90
x
0
0
10
20
30
40
50
60
70
x
Figure 3.10. The graph illustrating the equation y = a + bx of the
relationship between the number of parasitic Fasciola /buffalo
with number of eggs /g faeces.
The graph 3.10 shows that number of corresponding points
between the parasite count through buffaloes at postmortern
examination with number of eggs in 1 gram of feces were mostly
located around the curvature of the linear regression equation y = a +
bx, away from lower left to upper right, there were no points lying far
from this curvature. That means that the correlation between the
Figure 3.11. The graph illustrating the equation y = a +
bx of the relationship between number of parasitic Fasciola
/bovine with number of eggs /g faeces.
The graph 3.11 shows that number of corresponding points
between the parasite count through bovines at necropsy with number
of eggs in 1 gram of feces were mostly located around the curvature
of the linear regression equation y = a + bx, from lower left to upper
right, there was only one point lying a bit far from this curvature.
17
18
That means that the correlation between the number of parasitic
fasciola /bovines and number of eggs /g of feces was direct
correlated.
The correlation coefficient r = 0.969 shows that this is closely
corelated.
3.4. Study of prevention and treatment measures of fascioliasis
caused by F. gigantica in buffaloes and bovines
3.4.1. Identifying drug for prevention and treatment of fascioliasis
caused by F. gigantica that is highly effective and safe
3.4.1.2. Trial on efficacy and safety of some drugs against F. gigantica in
buffaloes and bovines
* Testing on a small scale
Table 3.30. Efficacy of three drugs against F. gigantica in
experimental bovine
Table 3.30 shows that three drugs used for treatment of cattle
infected with F. gigantica at dose above, efficacy achieved similar to
the test on buffaloes (15/15 removed parasite eggs in feces after 10
days of dosing, 3/15 of cattle that were necropsied to examine liver
flukes in bile ducts after 25-35 days using dewormers). At the same
time, all 15 cows were no signs of any unusual after dosing. Thus,
doses of three drugs used for cattle were safe.
* Trial on a large scale
We tested the efficacy of triclabendazole treatment at dose 15 mg
/kg B.W albendazole and nitroxinil - 25 at dose 12mg /kg B.W in 450
buffaloes and 270 bovines in the studied places. The results are
illustrated in table 3.31 and 3.32.
Table 3.31. Efficacy of three dewormers angainst F. gigantica
in cattle on a large scale
Day after
treatment
Necropsy after treatment
Prior to
(eggs / g feces)
Order
treatment
Drugs and dosage number
Day
(eggs / g
Comparison
of bovine
at necropsy
feces)
5
10 15
sluke/cattle
after
(fluke)
treatment
Albendazol
(12mg/kg B.W)
Triclabendazole
(15 mg/kg kg B.W)
Nitroxinil - 25
(12mg/kg kg B.W)
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
285
315
375
405
255
450
410
380
295
320
260
350
300
430
330
30
30
45
60
15
55
60
40
30
35
25
35
30
45
40
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
35
25
30
-
Before dewworming
Time
0
12
50
465.00 ± 56.00
3
45.60 ±10.30
47
94.00
Triclabendazole
15
50
418.30 ± 48.65
0
0
50
100
Nitroxinil - 25
12
50
445.28 ± 30.50
3
35.20 ±8.70
47
94.00
Albendazol
12
50
520.80 ± 53.10
5
60.80 ±14.40
45
90.00
II Triclabendazole
15
50
560.00 ± 46.50
0
0
50
100
Nitroxinil - 25
12
50
490.35 ± 28.50
4
50.00 ±13.40
46
92.00
Albendazol
12
50
420.30 ± 46.00
5
25.80 ±6.72
45
90.00
III Triclabendazole
15
50
380.50 ± 60.78
0
0
50
100
12
50
415.27 ± 23.60
5
40.50 ± 9.86
45
90.00
Nitroxinil - 25
0
Dewworming eficacy
Number of
Number of Infection intensty Number of
Intensity
Dose (mg/
buffaloes
infected
infected
( X ± mx )
( X ± m x ) cleared from Efficacyy
kg B.W)
buffaloes
buffaloes
(%)
eggs
(buffaloes) (eggs/ feces g ) (buffaloes (eggs/ feces g )
(buffalo)
Albendazol
I
0
Type of drug
15 days after deworming
Effectiveness of albendazole was quite high, reaching 90 - 94%.
the infection intensity decreased from 420-465 eggs /g feces to 2560 eggs /g faeces. Nitroxinil - 25 also had althelmintic effectiveness
reaching 90 - 94%, reducing infection intensity from 415 - 490 eggs
19
20
The results in table 3:31 shows that in all three treatments.
triclabendazole has the highest althelmintic efficacy (100%). 15 days
after dosing, examining feces, eggs were not detected. The
althelmintic /g of feces to 35-50 eggs /g of feces 15 days after
deworming. Observing buffaloes before and after dosing, we found
that the activities of the whole buffaloes were normal, eating and
ruminating normally, no buffaloes had any side effects after receiving
the drug. So we evaluated: dose levels of three drugs used were
100% safe for cattle.
Table 3.35. Infection rate and intensity of F. gigantica after 4
months of the experiment
3.4.2. Trial on therapeutic measures in treating fascioliasis
caused by F. gigantica in buffaloes
Infection rate and intensity in buffaloes of the experimental group
and the control prior to the experiment are illustrated in table 3.33.
Table 3.33. Infection rate and intensity of F. gigantica in
buffaloes prior to the experiment
Group
Description
Number of buffaloes examined
(buffalo)
Number of infected buffaloes
((buffalo)
Percentage (%)
n
≤ 200
%
Infection
n
intensity
>200
(number of eggs/ - 500
%
gram of feces)
n
>
500
%
Experime
ntal group
Control
Statistical
significance
(P)
80
80
-
31
30
-
38.75
18
58.06
11
35.48
2
6.45
37.50
17
56.67
11
36.67
2
6.67
> 0.05
> 0.05
> 0.05
> 0.05
Table 3.33 shows that infection rate and intensity of F. gigantica
in the experimental group and control group prior the experiment
were equivalent
3.4.2.2. Infection rate and intensity of F. gigantica after 4 months
of the experiment
Group
Description
Number of buffaloes examined
(buffalo)
Experimental
group
Control
Statistical
significance (P)
80
80
-
14
36
-
17.50
13
92.86
1
7.14
0
0.00
45.00
24
66.67
10
27.78
2
5.56
< 0.001
< 0.001
< 0.001
< 0.001
Number of infected buffaloes
(buffalo)
Percentage (%)
Infection
intensity
(number of
eggs/ gram
of feces)
≤ 200
>200 -500
> 500
n
%
n
%
n
%
Table 3.35 shows that the infection rate after 4 months of
experiment, the prevalence of F. gigantica in buffaloes in the
experimental group was 17.50%, while the prevalence in the control
group was 45.00%. This difference was very clear (P <0.001).
The intensity of infection:
+ Mild infection intensity: 92.86% of buffaloes were infected with
liver fluke in experimental groups in light intensity, whereas in the
control group, mild prevalence was 66.67%, this difference was very
clear (P <0.001).
+ Average intensity of infection: In experimental group mean intensity of
infection was 7.14%, while that in the control group was 27.78%.
+ Heavy infection intensity: There were no buffaloes in
experimental group being infected in severe intensity, while the
proportion of severe infections in the control group was 5.56%. The
difference had statistical significance (P <0.001).
21
22
3.4.3. Recommendation of combining preventive and control
procedures to prevent fascioliasis caused by F. gigantica in
buffaloes.
5. Killing intermediate hosts of Fasciola, drainage of water,
drying muddy, wet pastures and grazing land are effective measures
to kill Lymnaea spp... Fresh snails can be killed by using lime poder,
copper sulffate... or rearing waterfowls (ducks, muscovy ducks, geese)
and black carps (mylopharyngodon piceus). However it depends on
place to apply appropriate measures in order to kill intermediate hosts
of Fasciola effectively.
From the results of the study we recommend combining
preventive and control procedures against fascioliasis in buffaloes
and bovines follows:
1 Deworming F. gigantica for buffaloes and bovines: Deworm
periodically liver fluke twice/ year (phase 1 in April- May; phase 2 in
September - October) for the whole herd of buffaloes and bovine
once examining feces shows that 20-30% of these animals are
infected with liver fluke.
Three drugs at dose levels having been tested (triclabendazole, at
dose 15 mg /kg B.W, albendazol and nitroxinil - 25, at dose of 12
mg /kg) all were effective in removing liver fluke. Depending on the
locale and on the specific conditions, one of those drugs to remove
liver fluke from buffaloes can be selected. However, the use of
triclabendazole is the best effective.
2 Treatment of cattle manure to kill eggs of F. gigantica: daily
collect manure from stable and put in pitches of manure composting,
or gather them in one place as a pile and cover with thick mud
contained 5 -10 cm
3 Cleaning cattle stables every 2 months, using disinfectants to
spray in order to kill F. gigantica eggs in the floor of cattle houses
and their surrounding areas.
4. Improving pastures, grazing land for buffaloes and bovines:
Removing stagnant water on pastures, collecting manure from
grazing land for composting are needed in order to limit the spread
and development of eggs and larvae of trematodes in the
environment.
6. Strengthening the care and feeding animals: For buffaloes and
bovines at different ages (especially calving cows), it is necessary to
ensure adequate rations in terms of quantity and quality. Cattle over 8
years old must be culled so that they will not shed F. gigantica eggs
into the environment.
CONCLUSIONS AND RECOMMENDATIONS
1. Conclusion
1- 450 buffaloes and 60 cattle in 3 provinces were necropsied, the
infection rate in buffaloes was 58.67% and in cattle was 36.67%.
F. gigantica is the only species of trematode parasitising liver and
bile ducts of buffaloes and bovines the places invstigated, common
prevalence is 100%
2 - Epidemiological characteristics:
+ Status of animal husbandry and prevention of parasitosis in
buffaloes and bovines in 3 provinces is not good, especially
preventive measures of parasitosis in buffaloes and cattle.
+ The prevalence of F. gigantica in buffaloes and bovinein three
provinces is 46.63%; in cattle is 34.71%. The rate and intensity of
infection increases with aging.
Buffaloes and bovines are infected with F. gigantica in larger
number and more severe in summer. The rate and intensity of
infection in the other seasons are lower and lighter.
23
24
Variation in the rate and intensity of infection of cows and bulls
are markedly irregular.
3- Correlation between the number of eggs /g of feces with
number of parasitic liver flukes / animal is correlated quite closely, in
accordance with the linear regression equation y = a + bx.
+ The areas around stables and barns of buffaloes and bovines are
contaminated with F. gigantica eggs at the rate of 20 - 43%.
8.37% of the soil samples from the surface of grazing land and
14.96% of water samples from the hollows on grazing land were
contaminated with F. gigantica eggs.
+ L. viridis and L. swinhoei - intermediate hosts of F. gigantica
are commonly distributed in all investigated places.
The infection rate of liver fluke larvae in snails varies 20 - 70%.
Infection rate of L. viridis is 61%.
+ Samples of aquatic grasses in Thai Nguyen. Bac Kan and Tuyen
Quang are contaminated with Adolescaria at the rate of 14 - 23%.
+ In Spring, survival of F. gigantica eggs lasts 12 - 48 days (in
dry feces), 43 - 125 days (in wet feces). Survival of eggs is shorter
than that in Summer, longer in Autumn and the longest in winter (19
- 80 days in dry feces, 75 - 160 days in wet feces).
The survival time of eggs is short in dry soil, longer in wet soil. In
Summer, the survival time of eggs in the ground is shorter than in
other seasons.
+ In Spring, Miracidium hatches and is releases from the egg
earliest in 14 days, no later than 16 days; in Summer time this is 8
and 10 days; in Autumn 11 and 15 days; in Winter 19 and 21 days.
After hatching from the egg, Miracidium in the water is not more
than 10 hours (in Spring), 11 hours (in Summer), 14 hours (in
autumn) and 9 hours (in Winter).
The time from releasing F. gigantica eggs in water until forming
Adolescaria is the longest in winter (51 - 56 days), followed by
Spring (41 - 48 days). Autumn (36 - 45 days) and the shortest is in
Summer (29 - 37 days).
4- At the recommeded doses, efficacy of han - dertin B and
fasciolid against F. gigantica is not high (78 - 86%).
Efficacy of triclabendazole against F. gigantica at dose of 15 mg
/kg B.W is 100%; Efficacy of albendazole and nitroxinil - 25 at the
same dose (12mg /kg B.W) reaches 90 - 96%. All of 3 drugs are safe
for cattle and buffaloes.
5- Prevention and control of F. gigantica in buffaloes and cattle
should be performed by using five synchronized key measures
(deworming liver flukes with triclabendazole, composting manure.
killing snails, intermediate hosts, sanitation of feed, water, stables
and grazing land), culling the animals older than 8 years old.
2. Recommendation
In the Northern mountainous provinces, cattle are raised under
grazing methods, utilizing the existence of natural conditions.
Therefore, it is needed to implement the combined prevention and
control of Fascioliasis gigantica for buffaloes and bovines as the six
contents of the above conclusion. This is believed to help reducing
the economic losses caused by the parasiolois disease, improving
livestock productivity, and promoting the industry of animal
husbandry.
25
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