Study on epidemiological characteristics of fasiolosis in buffaloes and bovines in thai nguyen, bac kan, tuyen quang province and preventive and treatment methods (2010 2013)

THAI NGUYEN UNIVERSITY --- PHAM DIEU THUY STUDY ON EPIDEMIOLOGICAL CHARACTERISTICS OF FASCIOLOSIS IN BUFFALOES AND BOVINES IN THAI NGUYEN, BAC KAN, TUYEN QUANG PROVINCE AND PREVENTI

THAI NGUYEN UNIVERSITY -

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)

SUMMARY OF PH.D DISSERTATION IN

VETERINARY MEDICINE

Thai Nguyen, 2014

THAI NGUYEN UNIVERSIY -

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)

Speciality: Parasites and Veterinary Microbiology

Code: 62.64.01.04

SUMMARY OF PH.D DISSERTATION IN

VETERINARY MEDICINE ADVISOR: Prof Nguyen Thi Kim Lan, Ph.D

Thai Nguyen, 2014

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

INTRODUCTION

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

- Laboratory of Veterinary and Animal Science faculty - College

of Agriculture and Forestry, Thai Nguyen University

- Institute of Ecology and Biological Resources

- 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

2.3.4 Studying prevention and control measures of fasioliasis

caused by F gigantica in buffaloes and bovines

2.4 Methods of study

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

2.4.3 Methodologies for studying epedemic charateristics of

Fascipla spp in buffaloes and bovines: use of Benedek sedimentation

method (1943) for examination of Fasciola eggs

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)

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

* 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

2.5 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

CHAPTER 3 RESULTS AND DISCUSSION

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)

Table 3.1 Results of the necropsy examination of buffaloes and

the collection of liver flukes

Place (province)

Number of necropsied buffaloes (bufalo)

Number of infected buffaloes (bufalo)

Percentage (%)

Infection intensity (liver fluke/bufalo)

Table 3.2 Results of the necropsy examination of bovines and the

collection of liver flukes

Place (province)

Number of necropsied bovines (bovine)

Number of infected bovines (bovine)

Percentage (%)

Infection intensity (liver fluke/(bovine)

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

Table 3.3 Indentification of parasitic liver fluke species in

buffaloes and bovines

Results of species determination

Fasciola gigantica species

Fasciola hepatica species

Intermidiate forms between the 2 species

Place

(province)

Number of liver flukes for determinat ion (fluke)

Number

of flukes

%

Number

of flukes

%

Number

of flukes

%

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

compared with the 2 samples mentioned above So the intermediate

forms were also F gigantica species

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

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

Infection intensity (eggs/g feces)

≤ 200 > 200 - 500 > 500

Place (province, district)

Number

of buffaloes examined (buffalo)

Number

of infected buffaloes (buffalo)

Percentage (%)

* Thai Nguyen 1800 851 47.28 b 447 52.52 262 30.79 142 16.69

χ 2

= 31.779; P = 0.000

* Bac Kan 1800 733 40.72 a 461 62.89 191 26.06 81 11.05

χ 2

= 63.055; P= 0.000

* Tuyen Quang 1800 934 51.89 c 568 60.81 289 30.94 77 8.25

Tuyen Quang

χ 2

= 13.784; P = 0.001

5400 2518 46.63 1.476 58.62 742 29.47 300 11.91

Total

χ 2 = 45.551; P = 0.000

* Notes: In vertical line, the numbers with superscriptions are different remarkably in stastistics

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%)

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%

Table 3.7 Infection rate and intensity of F gigantica in bovines in

the places investigated

Infection intensity eggs/g feces)

≤ 200 > 200 - 500 > 500

Places (province,

district)

Number of

bovines

examined

(bovine)

Number of infected bovines (bovine)

Percentage (%)

* Thai Nguyen 630 243 38.57 b 144 59.26 75 30.86 24 9.88

Dong Hy 210 110 52.38b 47 42.73 44 40.00 19 17.27

Vo Nhai 210 63 30.00a 45 71.43 15 23.81 3 4.76

Dai Tu 210 70 33.33a 52 74.29 16 22.86 2 2.85

χ 2

= 25.846; P = 0.000

* Bac Kan 630 188 29.84 a 123 65.43 51 27.13 14 7.44

ChoMoi 210 80 38.10b 48 60.00 27 33.75 5 6.25

Bach Thong 210 69 32.86b 52 75.36 12 17.39 5 7.25

Ngan Son 210 39 18.57a 23 58.97 12 30.77 4 10.26

χ 2

= 20.485; P= 0.000

Tuyen Quang 630 225 35.71 b 136 60.45 77 34.22 12 5.33

Yen Son 210 67 31.90 39 58.21 25 37.31 3 4.48

Ham Yen 210 75 35.71 46 61.33 23 30.67 6 8.00

Tuyen Quang

city 210 83 39.52 51 61.45 29 34.94 3 3.61

χ 2

= 2.655; P = 0.265

Total

χ 2

= 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

(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

3.2.2.3 Infection rate and intensity of F gigantica in buffaloes and bovines acording to seasons

Table 3.10 Infection rate and intensity of F gigantica in

buffaloes according to seasons

Infection intensity (eggs/g feces)

≤ 200 > 200 - 500 > 500

Places (province) Season

Number of buffaloes examined (buffalo

Number

of infected buffaloes (buffalo)

Percentage (%)

Thai Nguyen

χ 2

= 66.906; P= 0.000

Bac Kan

χ 2

= 55 697; P = 0.000

Tuyen Quang

χ 2

= 23.653; P = 0.000

Total

χ 2

= 129.022; P = 0.000

* Notes: in vertical line, the numbers that carry different letters are different in stastistic signinficance

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)

* 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

Infection intensity (eggs/g feces)

≤ 200 > 200 - 500 > 500

Places

(province) Sex

Number of bovine examined (cattle)

Number of infected cattle(cattle)

Percentage (%)

Bull 239 85 35.56 51 60.00 26 30.59 8 9.41

Thai Nguyen

Bull 284 86 30.28 53 61.63 28 32.56 5 5.81

Bac Kan

Bull 164 58 35.37 34 58.62 21 36.21 4 5.17

Tuyen

Quang 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

Total

χ 2 = 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

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)

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

(province, district)

Number of samples examined

Number of positive samples (+)

Percentage (%)

Number of samples examined

Number of positive samples (+)

percentage (%)

Tuyen

1.350 113 8.37 1.350 202 14.96

Total

χ 2

= 1.178; P = 0.555 χ 2

= 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

3.2.3.3 The distribution of freshwater snails - intermediate hosts of

F gigantica

Table 3.17 Results of freshwater snail classification

Lymnaea viridis species

Lymnaea swinhoei species Other species*

Places

(province, district)

Number of snails classified

* Thai Nguyen 2160 768 35.56 621 28.75 771 35.69

* Bac Kan 2160 663 30.70 437 20.23 1.060 49.07

*Tuyen Quang 2160 520 24.07 978 45.28 662 30.65

Total 6480 1951 30.11 2036 31.42 2493 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

3.2.3.4 The prevalence of liver fluke larvae in freshwater snails - intermediate hosts

Table 3.19 The prevalence of F gigantica larvae in freshwater

snails

Place (province)

viridis

Lymnaea swinhoei

Lymnaea viridis

Lymnaea swinhoei

Lymnaea viridis

Lymnaea swinhoei

Number of snails

Number of snails infected with

F gigantica larvae(snail)

Sporocyst

Redia

Cercaria

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%

3.2.3.8 Study on the time of Miracidium hatched released and

surviving in water

Table 3.24 Time of Miracidium hatched and released into

water (from releasing eggs in water)

Initial time of

Miracidium

releaseed from eggs

Time for

all Miracidium

released from eggs Season Experiment

Number

of samples

Temperature and pH water (X ±m X)

(day)

total (day) (X ±m X)

(day)

total (day)

Spring

22 - 23oC,

6 - 7

15.20 ± 0.25

14

15.20 ± 0.62

16

Summer

26 - 27oC,

6 - 7

9.50 ± 0.23

8

10.10 ± 2.18

10

Autumn

24 - 25oC,

6 - 7

11.40 ± 0.21

11

15.40 ± 2.92

15

Winter

18 - 19oC,

6 - 7

19.90 ± 0.42

19

20.30 ± 1.57

21

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

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) Season Number of

samples Stage of larvae

Time to complete (day)

* Egg à Adolescaria 41 - 48

* Egg à Adolescaria 29 - 37

* Egg à Adolescaria 36 - 45

* Egg à Adolescaria 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

90 80 70 60 50 40 30 20 10 0

800

700

600

500

400

300

200

100

0

3.3 Studies of correlation between number of parasitic F gigantica in

buffaloes and bovines with number of eggs in 1 g of feces

* 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

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

x

7 0

6 0

5 0

4 0

3 0

2 0

1 0 0

5 0 0

4 0 0

3 0 0

2 0 0

1 0 0 0

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

Correlation between number of F gigantica eggs /g faeces and number of parasitic Fasciola /bovine is illustrated in figure 3.11

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