STUDY ON EPIDEMIOLOGICAL FEATURES, APPLICATION OF DIAGNOSTIC KIT FOR DETECTION OF TRYPANOSOMIASIS CAUSED BY TRYPANOSOMA EVANSI IN CATTLE AND BUFFALOES IN A FEW NORTHERN MOUNTAINOUS PROVINCES AND RECOMMENDATION FOR PREVENTIVE AND TREATMENT MEASURES

MINISTRY OF EDUCATION AND TRAINING THAI NGUYEN UNIVERSITY DO THI VAN GIANG STUDY ON EPIDEMIOLOGICAL FEATURES, APPLICATION OF DIAGNOSTIC KIT FOR DETECTION OF TRYPANOSOMIASIS CAUSED BY T

MINISTRY OF EDUCATION AND TRAINING THAI NGUYEN UNIVERSITY

DO THI VAN GIANG

STUDY ON EPIDEMIOLOGICAL FEATURES, APPLICATION OF DIAGNOSTIC KIT FOR DETECTION OF TRYPANOSOMIASIS CAUSED BY TRYPANOSOMA EVANSI IN CATTLE AND BUFFALOES IN A FEW NORTHERN MOUNTAINOUS PROVINCES AND RECOMMENDATION FOR PREVENTIVE AND TREATMENT MEASURES

Speciality: Veterinary parasitology and microbiology Code: 62 64 01 04

SUMMARY OF PhD DISSERTATION IN VETERINARY MEDICINE

THAI NGUYEN - 2014

The dissertation has been completed at:

THAI NGUYEN UNIVERSITY OF AGRICULTURE AND

FORESTRY

Advisors:

1 PROFESSOR DOCTOR Nguyen Thi Kim Lan

2 DOCTOR Nguyen Quoc Doanh

Thesis PhD reviewer 1: ASSOCIATE PROFESSOR Dr Phan Dich Lan

Thesis PhD reviewer 2: Dr Nguyen Trong Kim

The dissertation was defended in front of a preliminary

examination commitee held at 8.30 on 21 - 9- 2013 in Thai Nguyen

The dissertation can be found in:

- National library inViet Nam

- The library of Thai nguyen university of agricultutre and forestry

DANH MỤC CÁC CÔNG TRÌNH CÓ LIÊN QUAN ĐẾN ĐỀ TÀI

1 Đỗ Thị Vân Giang, Nguyễn Thị Kịm Lan, Nguyễn Quốc Doanh,

Nguyễn Thị Bích Ngà (2013), “ Đặc điểm bệnh do T evansi gây

ra trên động vật thí nghiệm (chuột bạch)”, Tạp chí khoa học kỹ

thuật Thú y, tập 20, số 2, trang 49 - 55

2 Đỗ Thị Vân Giang, Nguyễn Thị Kim Lan, Nguyễn Thu Trang,

Trương Thị Tính (2013), “Đặc điểm bệnh lý do T evansi gây ra trên thỏ thí nghiệm”, Tạp chí khoa học kỹ thuật chăn nuôi, số

tháng 8, năm 2013, trang 83 - 90

3 Phạm Thị Tâm, Bùi Thị Hải Hòa, Nguyễn Thị Kim Lan, Đỗ Thị Vân Giang (2013), “Nghiên cứu thiết lập phản ứng ELISA chẩn đoán

bệnh tiên mao trùng cho trâu, bò Việt Nam”, Tạp chí Nông nghiệp và

phát triển nông thôn, kỳ 2, tháng 8, năm 2013, trang 41 - 45

4 Nguyễn Thị Kim Lan, Đỗ Thị Vân Giang, Nguyễn Văn Quang, Nguyễn Thị Ngân, Lê Minh, Phan Thị Hồng Phúc, Phạm Diệu

Thùy (2013), “Thử hiệu lực một số thuốc trị Trypanosoma evansi qua thử nghiệm in vitro và in vivo”, Tạp chí khoa học kỹ thuật

Thú y, tập XX, số 6, trang 69 - 77

- Lesions in viscera of buffaloes experimentally infected with T

evansi have been found in heart, lungs, spleen, liver, kidneys (20 -

100 %) Microscopic lesions have been found in the internal organs

of the buffaloes experimentally infected with T evansi

1.3 Application of serological test kits for diagnosis of trypanosomiasis

- Detection rates of CATT kit and ELISA kit in positive serum

samples are 100 %

- CATT kit and ELISA kit have detected 15,50 % of serum

samples of buffaloes tested positive for T evansi

1.4 Susceptibility of T evansi to several drugs tested in white mice

- T evansi are highly susceptible to trypamidium samorin,

moderately to diminavet, and less susceptible to azidine and might

have been resistant to trypanosoma

- Diminavet or trypamidium samorin can be administered at dose

recommended by manufacturer or higher for the most effective

treatment of T.evansi

1.5 Preventive and control measures of trypanosomiasis in

cattle and buffaloes

- The treatment regimen consisting of trypamidium samorin ( at

dose 1 mg /kg B.W), saline solution (200 ml /animal), 20 % caffein

(15 ml /animal), 5 % vitamin C (20 ml /animal), 2.5 % vitamin B1 (20

ml /animal) have good efficacy in treating trypanosomiasis (100 %)

and safety to cattle and buffaloes

- First recommendation of preventive and control measures of

trypanosomiasis in cattle and buffaloes is thorough treatment of

trypanosomiasis in buffaloes and cattle; exterminating sucking flies

and gad flies that transmit trypanosomiasis to cattle and buffaloes by

using trypamidium samorin drug; supplying cattle and buffaloes with

feed actively in winter and spring, improvement of feeding and

caring, and management of cattle and buffaloes; culling buffaloes and

cattle over 8 years old

2 RECOMMENDATION

- Using treatment regime consisting of trypamidium samorin (at

dose 1 mg /kgB.W), saline solution (200 ml /animal), caffein (15 ml

/animal), 5 % vitamin C (20 ml animal), 2,5 % vitamin B1 (20 ml

/animal) for treatment of trypanosomiasis in buffaloes and cattle in

various places

- The preventive and control of trypanosomiasis caused by T

evansi in buffaloes and cattle should be allowed to be applied in

Thai Nguyen, Lang Son, Hoa Binh, Lai Chau and other provinces

INTRODUCTION

1 Urgency of the project

Trypanosomiasis or Trypanosomosis is a common disease in cattle and

buffaloes causing a great losses for cattle husbandry in our country and many

of other countries in the world

According to Phan Dich Lan (2004) cattle and buffaloes infected

with Trypanosoma evansi are often show emaciation, anemia, decrease

of disease resistance and death in winter and spring

In our country, many authors have studied trypanosomiasis (Phạm Sy Lang, 1982; Le Duc Quyet, 1995; Nguyen Quoc Doanh, 1996; Luong To Thu and Le Ngoc My, 1996; Vuong Thi Lan Phương, 2004; Phan Van Chinh, 2006…) However only few studies of trypanosomiasis have been performed in mountainous provinces

In order to make contribution to the control of trypanosomiasis in buffaloes and cattle in mountainous provinces we implement the project:

“Study on epidemiological features, application of diagnostic kit for detection of trypanosomiasis caused by Trypanosoma evansi in cattle and buffaloes in a few northern mountainous provinces and recommendation for preventive and treatment measures ”

2 Objective of the project Identification of Trypanosomes causing trypanosomiasis

epidemiological and pathological features of the disease; using Kit for detection of trypanosomes and recommendation of preventive and control measures of trypanosomiasis in cattle and buffaloes

3 Scientific and practical significance of the project

3.1.Scientific significance

The results of the project are scientific informations of epidemiological and pathological features and effects of detection kits for diagnosis of trypanosomiasis,preventive and control measures of this disease in cattle and buffaloes

3.2 Practical significance

The results of the study is a scientific basis to recommend animal producers apply preventive and control measures of trypanosomiasis in order to lower infection rates of trypanosomes and great losses caused

by T evansi, contribute to improving the productivity in animal

husbandry and promote development of cattle husbandry

4 New contribution of the project

- It is the first work, studying epidemiological and pathological features, diagnosis , prevention and treatment of trypanosomiasis in buffaloes and cattle systematically in 4 northern mountainous provinces

- Determining susceptibility of T evansi to several drugs against

trypanosomes

- Assessing efficacy of test kits for diagnosis of trypanosomes in

cattle and buffaloes

- Initially, recommending effective preventive and control measures

of trypanosomes in cattle and buffaloes and disseminating them to

various places

Chapter I OVERVIEW OF DOCUMENT Trypanosomiasis is a common disease in our country caused by

Trypanosoma evansi species

According to Pham Sy Lang (1982), Phan Dich Lan (2004), Phan Van

Chinh (2006), trypanosomiasis has appeared in many areas throughout the

country with high infection rates (13 - 30 % of buffaloes and 7 - 14 % of

cattle), mortality rate of affected animal was 6,3 - 20 %

Verma B B (1978), Payne R C (1992), Wuyts (1994), Nguyen

Dang Khai (1995), Da Silva A S (2010) indicate that clinical signs in

trypanosome infected buffaloes and cattle include falling and rising

fever, emaciation, anemia, edema, corneal inflammation, swelling of

the testes and testitis, swollen lympho nodes, limb paralysis, abortion

According to Damayanti R (1994), Reid S A (2001), Mekata H

(2013) gross lesions in T evansi infected buffaloes include hemorrhage in

pericardium membrance, pneumonia, hepatitis, swelling and edema of the

spleen, swollen lympho nodes, enlarged bone marrow

Many authors have studied trypanosomiasis in different animal

species (horses, dogs, cats, mice…): Hagos A (2010), Aquino L P

(2010), Tamarit A (2010), Habila N (2010), Gari F R (2010), Haridy

F M (2011), Desquesnes M (2011), Ramirez Iglesias J R (2011),

Tonin A A (2011), Dalla Rosa L (2012), Elshafie E I (2012), Sharma

P (2012), Takeet M I (2012), Kundu K (2013), Rosa L D (2013),

Nguyen Q D (2013), Faccio L (2013) …

Doan Van Phuc (1981), Nguyen Quoc Doanh and Pham Sy Lang

(1996 - 1997), Phan Van Chinh (2006) used several drugs for

treatment of trypanosomiasis in cattle and buffaloes in Viet Nam and

show that trypamidium, berenil, trypazen are high effective and safe

in treating trypanosomiasis in cattle and buffaloes

- There are 3 sucking fly and gadfly species transmitting trypanosomes to cattle and buffaloes

1.2 Ability of T evansi to cause disease in some animals experimentally

infected

* In white mice:

- T evansi appears in mouse blood 3 - 7 days after being

experimentally infected (at dose 103 T evansi /mouse), 1 - 2 days (at

dose 106 T evansi /mouse)

- Clinical signs appear in the mice on the last day before death, number of mice exhibiting symptoms make up 55 - 100 %; time to death

is 3 - 13 days after being experimentally infected

- The internal organs showing lesions make up 33,75 - 100 %

Microscopic lesions show apparently in organs of mice infected with

T evansi

- Internal organ weights of mice infected with T evansi are higher

than that of healthy mice

* In rabbits:

- Appearance time of T evansi in the blood of rabbit earliest on

day 4 and latest on day 14 after being experimentally infected

Appearance frequency of T evansi in the blood of mouse is 93,9 %

- Clinical signs appear in the mice experimentally infected with T

evansi on days 15 - 52 after being experimentally infected and 70 -

100 % of the rabbits having clinical manifestation Time to the rabbits killed is 37 - 82 after being experimentally infected

- Gross lesions in rabbits infected with T evansi have been

mainly found in heart, liver, spleen, lungs and kidneys accounting for

50 - 100 %

- The internal organs of the affected rabbits have shown microscopic lesions apparently

* In buffaloes:

- At high infective dose of T evansi, time to appear T evansi in

peripheral circulation is early and oppositely at low dose

- Buffaloes infected with T evansi manifest waves fever

Averagely, 3 - 8 days a fever appears

- 5 buffaloes infected with T evansi all appear clinical signs with

percentage of 20 - 100 %

- In trypanosome infected buffaloes there are apparently decreased amount of erythrocytes and increased amount and rates of granulocytes compared with the control buffaloes

+ Examination and treatment for trypanosomiasis in buffaloes and

cattle infected with T evansi in summer and Autumn in order to limit

outbreak of trypanosomiasis and mortality rates of buffaloes and cattle in

Winter and Spring

2 Exterminating sucking flies and gad flies that transmit

tripanosomiasis

- Exterminating flies and gad flies by changing their habitat,

clearing plants in each area, not letting water stagnate; composting

manure in order to kill eggs and larva of flies and gad flies; housing

animals with nets to prevent flies and gad flies from sucking

buffaloes and cattle These methods are effective ones, creating

unfavourite conditions for the life of flies and gad flies to prevent

them from growth and complete their life cycle

- Using chemiclals to exterminate sucking flies and gad flies: The

chemicals such as endosulfan, brophos,tetracloreinphos can be used…

3 Using chemicals for prevention of trypanosomiasis

Using trypamidium samorin at dose 0.5 mg /kg B.W twice a year

( in early summer and at the end of Autumn) in order to prevent

trypanosomiasis in the local places

4 Improvement of caring, feeding and management of buffaloes and

cattle (especially in Autumn and Winter, when the weather is

unfavourable and feed is rare ), supplying them with sufficient feed

in Winter and Spring to enhance disease resistance of cattle and

buffaloes in local places

5 Culling old cattle and buffaloes (over 8 years old) in order to

prevent infection from natural reservoir of T.evansi

CONCLUSION AND RECOMMENDATION

1.1 Epidemiological features of trypanosomiasis in cattle and

buffaloes in 4 northern mountainous provinces

- 11 parasitic trypanosome strains have been isolated, all of which

are T evansi species

- The infection rate of T evansi in buffaloes is 15,36 % and in

cattle is 9,02 %; These rates increases with aging of animal

- The Infection rate of T evansi in buffaloes and catle is the

highest in Autumn (26,16 %) and the lowest is in Spring (5,61 %)

The percentage of disease outbreaks is the highest in Winter

(66,67%) and the lowest is in Summer (9,09%)

Chapter 2 OBJECTS, MATERIALS, CONTENTS AND METHODS OFSTUDY 2.1 OBJECTS AND MATERIALS OF STUDY

2.1.1 Object of study

- Buffalo and cattle herds in a few districts of four of Northern mountainous provinces: Thai Nguyen, Lang Son, Lai Chau and Hoa Binh

- Trypanosomiasis caused by Trypanosoma evansi

2.1.2 Materials of study

- Blood samples collected from the places investigated

- Samples from animals experimentally infected with Trypanosomes

- T evansi is isolated from infected cattle and buffaloes

- Animals experimentally infected with Trypanosoma evansi:

rats, rabbits, buffaloes

- Recombinant antigen based-ELISA diagnostic kit and CATT kit using

antigens of T evansi species isolated from four of provinces of study

- Drugs for treatment of trypanosomiasis: azidine, trypamidium samorin, diminavet and suppotive drugs

- Auto hematology analyser Cellta - Mek - 6420K - Nihon Kohden (Japanese)

- Microtome

- Light microscope, chemicals and other experimental instruments 2.2 PLACES AND TIME PERIOD OF STUDY

* Places of study:

- places where samples were collected: Thai Nguyen, Lang Son, Lai Chau, Hoa Binh provinces

- Places where samples were tested: Thai Nguyen, university of agriculture and forestry, National veterinary institute, veterinary centre of disease diagnosis, biotechnological institute

* Time period of study: From 2011 to 2013

2.3 CONTENTS OF STUDY 2.3.1 Studying epidemiological features of trypanosomiasis in cattle and buffaloes in 4 of Northern mountainous provinces

2.3.2 Ability of T evansi to cause disease in several experimentally

infected animals

2.3.3 Application of diagnostic kit to diagnosis of trypanosomiasis

in various places

2.3.4 Susceptibility testing of T evansi to several drugs in white mice

2.3.5 Establishing treatment regimens of trypanosomiasis and

recommendation of preventive and treatment measures of this disease

2.4 METHODS OF STUDY

- Samples were collected in 4 places of study by using method of

stratified random sampling

- Detecting trypanosomes in samples by direct observation under

microscope, giemsa staining, inoculation of laboratory animals

- Nomenclature of trypanosomes isolated from cattle and

buffaloes in northern mountainous provinces by using PCR method

(Polymerase Chain Reaction)

- Method of study of clinical pathological features:

+ Determination of number of trypanosomes/ml blood of white mice

by using neubauer counting chamber Based on the results obtained,

determining dose administered in experimental animals (103 and 106 T

evansi /mouse, 107 T evansi /rabbit, 2 x 108 and 3 x 108 T evansi /buffalo)

+ Monitoring animals experimentally infected with T evansi to

determine time period of the earliest appearing T evansi and the

latest in the blood, observing clinical signs and time to kill animals

post experimental infection

+ Determining frequency of T evansi appearance in blood of rabbits by

method of white mouse inoculation

+ Measuring body temperature of buffaloes by termometer 43 0C

+ Dissection and examination of internal organs of animals

experimentally infected which were dead (rabbits and mice) or were

still alive(buffaloes) Observing internal organs by naked eyes or by

using magnifier, taking pictures of sections in the body that manifested

typical gross lesions

+ Testing blood indices by using auto hematology analyzer-Cellta -

Mek - 6420K - Nihon Kohden (Japan)

+ Preparations were made based on Histology Technique of cutting

tissues: tissues are hardened by replacing water with paraffin The

Table 3.31 Efficacy of 3 treatment regimens of trypanosomiasis in

cattle and buffaloes

Treatment regimen

Treament drug and supportive drug

Dose

Number of buffaloes treated (buffalo)

Number of buffaloes from which Trypanosomes were cleared (buffalo)*

Percentage (%) Trypamidium

samorin

1,0 mg/ kg body weight Physiological

saline solution

200 ml/

animal 20% caffein 15 ml/ animal 5% vitamin C 20 ml/ animal

1

2.5%vitamin B1 20 ml/ animal

body weight Physiological

saline solution

200 ml/animal 20% caffein 15 ml/ animal 5% vitamin C 20 ml/ animal

2

2.5%vitamin B1 20 ml/ animal

body weight Physiological

saline solution

200 ml/

animal 20% caffein 15 ml/ animal 5% vitamin C 20 ml/ animal

3

2.5% vitamin B1 20 ml/ animal

Notes: * at examination, there were no T evansi 15 days and và 30 days after treatment

3.5.2 Recommendation of preventive and control measures of

trypanosomasis caused by T evansi in buffaloes and cattle in

mountainous provinces Combining results of our sutdy and results of study of other authors

in our country and in foreign countries with principles of prevention and control of trypanosomiasis, we recommend a procedure for prevention and control of trypanosomiasis including:

1 Killing trypanosomes in host body

- Using treatment regimen 1 (using trypamidium samorin) in treating trypanosomiasis in buffaloes and cattle During the treatment let the affected animals stay in their stable for 3 - 5 days and having good feeding ring and management

- Notes

+ Treating trypanosomiasis in over five year old buffaloes and cattle thorougly

cleared 15, 20 and 30 days after the experiment there were no mice

in which T evansi reappeared

10 mice in the control group were all killed 4 - 6 days after being

experimentally infected

Thus, diminavet drug was high effective against T evansi at

recommended dose or higher

The results of study shows that T evansi were very susceptible

to trypamidium samorin, moderately susceptible to diminavet, and

less susceptible to adizine whereas they might have been resistant to

trypanosoma drug Despite having been used for treatment of

trypanosomiasis for decades, trypamidium is still the drug of choice

in order to establish treatment regimens in treating trypanosomasis in

buffaloes and cattle effectively

3.5 ESTABLISHMENT OF TREATMENT REGIMENS AND

RECOMMENDATION OF PREVENTIVE AND CONTROL

3.5.1 Design of treatment regimens for high effective treatment

of trypanosomiasis

After the trial of susceptibility of T evansi to 4 drugs in

laboratory we found that 3 drugs, trypamidium samorin, diminavet

and azidine were effective to treat trypanosomiasis caused by T

evansi with different extents (trypamidium samorin and diminavet

were more effective to kill T evansi at the recommended dose,

azidine was more effective at dose higher than the recommended

dose) Therefore we designed 3 treatment regimens (each regimen

including drug against trypanosomes at effective dose that had been

determined added with supportive drugs) to treat T evansi infection

in several T evansi infected buffaloes and cattle 15 and 30 days

post treatment, efficacy of treatment regimen 1 was examined by

taking blood from buffaloes to inoculate white mice The results of

efficacy in 3 treatment regimens were illustrated in table 3.31

Table 3.31 shows that 3 regimens were all effective for treatment

of trypanosomiasis in buffaloes and cattle Clearance rates of T.evansi

post treatment were 80 - 100 %, of which regimen 1 had the highest

efficacy of treatment, regimen 3 had the lowest efficacy That was why

we chose regimen 1 for recommendation of using in treating

trypanosomiasis in cattle and buffaloes in various areas

tissue is then cut in the microtome From there the tissue can be mounted on a microscope slide stained with Hematoxylin - Eosin and examined under light microscope magnification of 200-400 times to observe microscopic changes in the slide

- Flies and gad-flies were classified based on classification key by Stekhoven Ricardo (1959)

- Using CATT kit and ELISA kit in order to detect antibody

against T evansi in the positive samples, then using these two kits to determine infection rate of T evansi in buffaloes in Thai Nguyen

- Testing susceptibility of T evansi to 4 drugs by injecting them in

white mice (each drug used at three different dose levels):

Dose indicated by the manufacturer (mg /kg body weight) Drug name Manufacturer Active ingredient

lower equal higher

Route of infection

(Viet Nam)

isometamidium

Trypamidium samorin

Merial (France)

Isometamidium

(Belgium)

Diaceturate diminazene

Monitoring in order to identify time from experimal treatment to clearance of trypanosomes in mouse body in both the control group and the experimental group Based on results obtained identify susceptibility of trypanosomes to 4 drugs used

- Establishment of trypanosome treatment regimens for cattle and buffaloes in the investigated places, each regimen consists of drugs against trypanosomes (at effective dose to kill trypanosomes) added supportive drugs

2.5.TREATMENT OF DATA Data collected is treated by methods of biostatistics (According to document of Nguyễn Van Thien, 2008), on Excel software 2003 and Minitab software 14.0

Chapter 3 RESULTS OF STUDY

TRYPANOSOMIASIS IN CATTLE AND BUFFALOES IN 4 OF

NORTHERN MOUNTAINOUS PROVINCES

3.1.1 Nomenclature of Trypanosoma sp isolated from cattle and

buffaloes in four of Northern mountainous provinces

Table 3.1 Result of nomenclature of Trypanosoma species in four

of Northern mountainous provinces

Result

Place

(province)

Number of

strains were

grouped into

species

Number of strain rate (%)

Number of

Table 3.1 shows that 11/11 trypanosomes isolated from infected cattle

and buffaloes in four of northern mountainous provinces all belonged to

T evansi, no other trypanosome species were found Thus, T evansi was

the only parasite species of trypanosomes causing trypanosomiasis in

cattle and buffaloes in northern mountainous provinces

3.1.2 Trypanosoma infection in cattle and buffaloes in four of

Northern mountainous provinces

3.1.2.1 Infection rates of Trypanosoma in cattle and buffaloes in

various places

Table 3.2 Infection rate of Trypanosoma in cattle and buffaloes in

four of northern mountainous provinces

Place

(province)

Number of

buffaloes

tested

(buffalo)

Number of buffaloes infected (buffalo)

Infection rate (%)

Number

of cattle tested (buffalo)

Number

of cattle infected (cattle )

Infection rate (%)

Table 3.2 revealed that examination of 573 buffaloes and 133 cattle,

infection rate of T evansi was 15,36 % and 9,02 % respectively The

At recommended dose (1,0 mg /kg B.W), the drug was high

effective to kill T evansi in mouse body (10/10 from which T evansi

was cleared)

At dose higher than recommended one (1,2 mg /kgB.W), 10 days

after the experiment 100% of mice from which T evansi was removed

15, 20 and 30 days after the experiment T evansi was not found to

reappear in the blood of mice

10 mice in the control group were all killed 4 - 6 days after being

experimentally infected with T evansi Thus, the efficacy of trypamidium samorin to kill T evansi was

very high at the dose recommended by manufacturer or higher

3.4.4 Determining susceptibility of T evansi to diminavet drug in white mice

Table 3.30 Time period to clear T evansi from mouse body when

diminavet drug was used

Drug dose (mg / kg/ body

Number of T evansi

/microscopic field area prior drug was used (

X

m

Indication Results

of monitoring afterthe drug was administered

Number of mice cleared of

trypanosomes

Number

of mice

killed

Number of mice cleared of

trypanosomes

Numbe

r of mice

killed

Number of mice cleared

of

trypanosomes

Number

of mice

killed

Number of mice cleared of

trypanosomes

Number

of mice

killed

Number of mice in which trypanosomes were still surviving on day 15, 20 and

30 after drugs was used

Table 3.30 shows that at dose lower than recommended dose (3.0 mg /kg B.W) the drug was able to kill trypanosome (6/10 of

mice from which T evansi were cleared), but in 2/6 of mice T evansi

reappeared 20 days after the drug was used At recommended dose (3.5 mg /kg B.W) or higher (4.0 mg /kgB.W), the drug had good

efficacy to kill T evansi (7/10 of mice from which T evansi were

When being used at recommended dose (3.5 mg /kg B.W) the

drug killed T evansi in mouse body quite well (6/10 of experimental

mice from which T evansi were cleared) However T evansi

reappeared in 2/6 mice 20 days after the drug being used

At dose higher than the recommended dose (4.0 mg /kg B.W), the

drug was the most effective to kill T evansi (7/10 of mice from which

T evansi were cleared but T evansi reappeared in 1/7 of mice 20 days

after the drug was administered The experiment above showed that

azidine was more effective to kill T evansi in mouse body than

trypanosoma drug, however this effect was not high enough 10 mice

in the control group were all killed because the drugs were not used

Our results of study were similar to that of Lun Z R et al (1991)

[106] (The author had treated 8 buffaloes infected with T evansi by

using 3.5 mg /kg diminazene aceturate in southern part of China, 2

months after treatment the disease relapsed into 2 buffaloes)

3.4.3 Determining susceptibility of T evansi to trypamidium

samorin drug in white mice

Table 3.29 Time to clearance of T evansi from mice after

Trypamidium samorin was used

Drug dose (mg / kg/ body

Number of T evansi

/microscopic field area

prior drug was used

(

X

m

Indication

Results

of monitoring

afterthe drug

was administered

Number of mice cleared of

trypanosomes

Number

of mice

killed

Number of mice cleared of

trypanosomes

Numbe

r of mice

killed

Number of mice cleared

of

trypanosomes

Number

of mice

killed

Number of mice cleared of

trypanosomes

Number

of mice

killed

Number of mice in which

trypanosomes were still

surviving on day 15, 20 and

30 after drugs was used

Table 3.29 shows that at dose lower than recommended dose:

(0,8 mg /kg B.W), trypamidium samorin was less effective to kill T

evansi in mouse body (3/10 of mice from which T evansi was removed,

2/3 of mice in which T evansi reappeared 20 days after being infected)

highest infection rate of trypanosomes was in buffaloes in Hoa Binh (19,82 %), the second highest was in Lang Son (15,53 %) and Lai Chau (14,91 %); the lowest was in Thai Nguyen province (12,14 %) The highest infection rate of trypanosomes in cattle was in Hoa Binh province (10,26 %), the second highest was in Thai Nguyen (10,20 %) and the lowest was in Lang Son (6,67 %)

3.1.2.2 Infection rates of trypanosomes with aging in cattle and buffaloes

Trypanosome infection rates with aging in cattle and buffaloes

was illustrated in figure 3.2

Figure 3.2 Infection rates of trypanosomes in buffaloes and cattle

were illustrated by the line graph in figure 3.2

Infection rates of trypanosomes in cattle and buffaloes in mountainous provinces tended to increase gradually with aging of animal Our results of study were similar to study results obtained by Phan Luc et al (1996) ,buffaloes and cattle at any age were infected

with Trypanosomes, but the infection rate increased with aging of

animal, the older the animal the higher the infection rates were

3.1.3 Study of blood sucking flies and gad flies which transmitted trypanosomes

3.1.3.1 Nomenclature of blood sucking fly and gad fly species in places investigated

The table 3.6 shows that there were 3 blood sucking fly species being vectors to transmit trypanosomes to buffaloes and cattle in the places

investigated: These flies were Stomoxys calcitrans, Tabanus kiangsuensis flies and Tabanus rubidus gadfly The fly species all appeared in all places

investigated and frequency of appearance was 100 %

Table 3.6 Result of nomenclature, distribution and frequency of

appearance of blood sucking flies and gad flies

Blood sucking fly and gadfly species

Provine District, city

S calcitrans T kiangsuensis T rubidus

Thai Nguyen

Lai Chau

Lang Son

3.1.3.2 Rates of blood sucking flies and gad flies in collected samples

Table 3.7 Rates of blood sucking flies and gad flies in collected

samples in the investigated places

Province Number of flies and gad-

flies collected (fly) Fly and gad-fly species

Number (fly)

Percentage (%)

Thai

Lai

Hoa

Lang

The table 3.7 shows that Stomoxys calcitrans flies made up 45,25

% of total 1.801 flies and gad flies collected, T kiangsuensis gad flies

accounted for 22,15 % and T Rubidus 32,59 %

According to Phan Dich Lan (1983) climate and ecological

conditions are suitable for Tabanidea gadfly and Stomoxy fly vectors

transmitting trypanosomiasis to develop

3.1.3.3 Rule of activities of blood sucking fly and gadfly species in the

investigated places

* Rule of their activities in the months of the year:

Table 3.8 Rule of activities of blood sucking fly and gadfly species in

various months

effective to kill T evansi in mouse body 10/10 mice were all killed

by T evansi

At dose recommended by the manufecturer (1.0 mg /kg body

weight), the drug was less active to kill T evansi (only 1/10 mice from which T evansi were cleared ( number of mice killed was 9/10 )

- At higher dose (1.2 mg //kg body weight), the drug killed T

evansi better than recomended dose (3 mice from which T evansi

was cleared, 7 mice were killed) However, after 15 days T evansi

reappeared in 2/3 mice

As the result Trypanosoma drug had low efficacy to kill T evansi Thus, T evansi might have been resistant to Trypanosoma drug

3.4.2 Determination of susceptibility of T evansi to azidine tested

in white mice Table 3.28 Time from using the drug to clearance of T evansi from

white mice after azidine was used

Drug dose (mg / kg/ body

Number of T evansi

/microscopic field area prior drug was used (

X

m

89,9 ± 1,41 91,4 ± 1,81 89,4 ± 2,0 90,3 ± 1,69

Indication Results

of monitoring afterthe drug was administered

Number of mice cleared of

trypanosomes

Number

of mice

killed

Number of mice cleared of

trypanosomes

Numbe

r of mice

killed

Number of mice cleared

of

trypanosomes

Number

of mice

killed

Number of mice cleared of

trypanosomes

Number

of mice

killed

Number of mice in which trypanosomes were still surviving on day 15, 20 and

30 after drugs was used

Table 3.28 shows that at dose lower than the recommended dose (3.0 mg /kg body weight) azidine was less effective to kill T evansi in

mouse body

At dose lower than the recommended dose (3.0 mg /kg B.W) azidine

was less effective to kill T evansi in mouse body (after the experiment only 1/10 mice from which T evansi were cleared )