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

26

0
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

1

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
25

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.





24
- 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.
1
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.
2
- 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.
23
- 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 10
3
T. evansi /mouse), 1 - 2 days (at
dose 10
6
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.
22
+ 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%).
3

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
4
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 (10
3
and 10
6
T.
evansi /mouse, 10
7
T. evansi /rabbit, 2 x 10
8
and 3 x 10
8
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
0

C.
+ 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
21
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

30 30 100
Diminavet
3,5 mg/ kg
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

30 26 86,67
Adizine
4,0 mg/ kg
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

30 24 80,00
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.
20
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
MEASURES OF TRYPANOSOMIASIS
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.
5
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

Trypanosoma

Hanvet
(Viet Nam)
isometamidium
chloride chlorhydrate
0,8 1,0 1,2
I M
Azidin
Hanvet
(Viet Nam)
Diminazene aceturate 3,0 3,5 4,0
I M
Trypamidium

samorin
Merial
(France)
Isometamidium
chloride hydrochloride

0,8 1,0 1,2
I M
Diminavet
VMD
(Belgium)
Diaceturate diminazene
and antipyrine
3,0 3,5 4,0
I M
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.
6
Chapter 3
RESULTS OF STUDY

3.1. STUDYING EPIDEMIOLOGICAL FEATURES OF
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
T. evansi species Other species
Place
(province)
Number of
strains were
grouped into
species
Number of
strain
rate (%)

Number of
strain
rate (%)

Thai Nguyen

2 2 100 0 0.00
Lang Son 2 2 100 0 0.00
Hoa Binh 3 3 100 0 0.00
Lai Chau 4 4 100 0 0.00
Total 11 11 100 0 0.00

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
Bufaloes Cattle
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
(%)
Thai Nguyen

140 17 12,14 49 5 10,20
Hoa Binh 111 22 19,82 39 4 10,26
Lang Sơn 161 25 15,53 45 3 6,67
Lai Chau 161 24 14,91 0 0 0,00
Total 573 88 15,36 133 12 9,02
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
19
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
Group
Control Experimental
Drug dose (mg / kg/ body
weight)
0
2,5 3,5 4,5
Number of mice
10 10 10 10
Number of T. evansi
/microscopic field area
prior drug was used
(
X
mX 
)
91,10 ± 1,22 90,60 ± 1,79 86,20 ± 1,89 93,60 ± 1,80
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
24 hours
0 3/10 4/10 0 5/10 0 10/10 0
48 hours
0 8/10 6/10 0 10/10 0 10/10 0
72 hours
0 10/10

6/10 0 10/10 0 10/10 0

10 days
0 10/10

9/10 1/10

9/10 1/10

9/10 1/10

15 days
0 10/10

6/10 4/10

7/10 3/10

7/10 3/10

Number of mice in which
trypanosomes were still
surviving on day 15, 20 and
30 after drugs was used
- 2/6 0/7 0/7
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
18

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
Group
Control Experimental
Drug dose (mg / kg/ body
weight)
0
0,8 1,0 1,2
Number of mice
10 10 10 10
Number of T. evansi
/microscopic field area
prior drug was used

(
X
mX 
)
92.5 ± 1,92 93.6 ± 2,21 88.2 ± 1,60 92.2 ± 1.30
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
24 hours
0 1/10 0/10 3/10

0/10 0/10 3/10 0/10
48 hours
0 7/10 0/10 4/10

5/10 0/10 9/10 0/10
72 hours
0 10/10

3/10 7/10

10/10 0/10 10/10 0/10
10 days
0 10/10

3/10 7/10

10/10 0/10 10/10 0/10

15 days
0 10/10

3/10 7/10

10/10 0/10 10/10 0/10
Number of mice in which
trypanosomes were still
surviving on day 15, 20 and
30 after drugs was used
- 2/3 0/10 0/10
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).
7
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
8
Blood sucking fly and gadfly species
Provine District, city
S. calcitrans T. kiangsuensis T. rubidus
Dong Hy + + +
Phu Bình + + +
Thai Nguyen

Vo Nhai + + +
Hoa Binh Kim Boi + + +
Tam Duong + + +
Lai Chau
Than Uyen + + +
Lang Son city + + +
Chi Lang + + +

Lang Son
Van Lang + + +
Frequency of appearance (%)

100 100 100
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
(%)
Stomoxys calcitrans 374 47,77
Tabanus kiangsuensis 158 20,18
Thai
Nguyen

783
Tabanus rubidus 251 32,05
Stomoxys calcitrans 63 46,67
Tabanus kiangsuensis 40 29,63
Lai
Chau
135
Tabanus rubidus 32 23,70

Stomoxys calcitrans 147 50,87
Tabanus kiangsuensis 55 19,03
Hoa
Bình
289
Tabanus rubidus 87 30,10
Stomoxys calcitrans 231 38,89
Tabanus kiangsuensis 146 24,58
Lang
Son
594
Tabanus rubidus 217 36,53
Stomoxys calcitrans 815 45,25
Tabanus kiangsuensis

399 22,15
Total 1,801
Tabanus rubidus 587 32,59
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
17
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
Group
Control Experimental
Drug dose (mg / kg/ body
weight)
0 3,0 3,5 4,0
Number of mice
10 10 10 10
Number of T. evansi
/microscopic field area
prior drug was used
(
X

mX 
)
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
24 hours
0 2/10 0 2/10

3/10 1/10 5/10 1/10
48 hours
0 7/10 1/10 5/10

4/10 1/10 7/10 1/10
72 hours
0 10/10

2/10 5/10

6/10 3/10 9/10 1/10
10 days
0 10/10

1/10 7/10

6/10 4/10 9/10 1/10
15 days
0 10/10

1/10 9/10

6/10 4/10 7/10 3/10
Number of mice in which
trypanosomes were still
surviving on day 15, 20 and
30 after drugs was used
- 0/1 2/6 1/7
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 ).
16
CATTKit ELISA Kit
place
(district, city)
Number of
serum
samples
tested
Number of
positive
samples (+)
percentage
(%)
Number of
positive

samples (+)

percent
age
(%)
Thai Nguyen city

50 7 14,00 7 14,00
Dong Hy 50 6 12,00 6 12,00
Vo Nhai 50 10 20,00 10 20,00
Phu Binh
50 8 16,00 8 16,00
Total 200 31 15,50 31 15,50
Table 3.26 shows that when 2 types of kits were used for diagnosis
31/200 samples were detected to be positive with T. evansi; accounting
for 15.5 % (varied from 12 to 20 %). The results of mice inoculation
were similar. Thus, ability of these 2 kits to detect T. evansi infection in
buffaloes is equivalent to method of white mice inoculation.
3.4. TESTING SUSCEPTIBILITY OF T. EVANSI TO SOME
DRUGS TESTED IN WHITE MICE
3.4.1. Testing susceptibility of T. evansi to trypanosoma drug
Table 3.27. Time from using the drug to clearance of T. evansi from
white mice following trypanosoma drug was used
Group
Control Experimental
Drug dose (mg / kg/ body
weight)
0
0,8 1,0 1,2
Number of mice

10 10 10 10
Number of T. evansi
/microscopic field area
prior drug was used
(
X
mX 
)
89,4 ± 2,75 90,2 ± 1,71 88,1 ± 1,65 92,8 ± 2,31
Indic
ation
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
24 hours
0 3/10 0 2/10

0 4/10

0 1/10
48 hours
0 8/10 0 5/10

0 4/10

4/10 2/10
72 hours
0 10/10

0 9/10

3/10 6/10

5/10 5/10
10 days
0 10/10

0 10/10

3/10 7/10

5/10 5/10
15 days
0 10/10

0 10/10

1/10 9/10 3/10 7/10
Number of mice in which
trypanosomes were still
surviving on day 15, 20 and
30 after drugs was used
- - 0/1 2/3
Table 2.27 shows tthat trypanosoma drug at dose lower than dose
recommended by the manufacturer (0.8 mg /kg body weight) was not
9
Month of activities of flies and gad-flies Fly and gad fly
species
1 2 3 4 5 6 7 8 9 10 11

12

S. calcitrans - - + ++

+++

+++

+++

+++

+++

++

+ +
T. kiangsuensis - - - + +++

+++

+++

+++

+ + - -
T. rubidus - - - + +++

+++

+++

+++

++ + - -
Notes: +++: high activities
++ : average activities
+: less activities
- : no activites were found
* Rule of activities of sucking flies and gad flies in different time of the day
Table
3.9. Rule of activities of blood sucking flies and gad flies at various
time of the day
Time of the day (O’clock) Monitored
month
Fly and gadfly
species
6-8 8-10 10-12

12-14

14-16 16-18

18-20

S. calcitrans - - + + + - -
T. kiangsuensis - - - - - - -
March
T. rubidus - - - - - - -

S. calcitrans - + + + ++ + -
T. kiangsuensis - - + + + - - April
T. rubidus - - + ++ + + -
S. calcitrans + ++ +++ +++ +++ ++ +
T. kiangsuensis + + ++ +++ ++ + -
May -
August
T. rubidus + + ++ +++ ++ + -
S. calcitrans + + ++ ++ ++ + +
T. kiangsuensis + + ++ ++ + + -
September-

October
T. rubidus + + ++ ++ + + -
S. calcitrans - + + + ++ + -
T. kiangsuensis - - - - - - -
November
- December

T. rubidus - - - - - - -
Notes +++ : High activities
++ : average
+ :less activities
- :No activities have been seen
From the data obtained in table 3.8 and 3.9, we concluded that
sucking flies and gad flies were commonly found in the places
investigated. They were highly active from May to October of the
year ( in summer and autumn seasons), The highest active time was
from 10 to 16 o’clock of the day; This is time when animal producer
free buffaloes and cattle to graze in grazing land. S. Calcitrans flies

were active but weaker at nightfall (18 - 20 o’clock).
3.2. ABILITY OF T. EVANSI TO CAUSE DISEASE IN A FEW
EXPERIMENTALLY INFECTED ANIMALS
3.2.1. Ability of T. evansi to cause diseases in white mice
3.2.1.1. Time of T. evansi appearance in blood of white mice after
being experimentally infected
10
Table
3.10. Time of T. evansi appearance in blood of white mice after
being experimentally infected
Time of T. evansi appearance after
being experimentally intected
Time of
experimental
infection
group
Number
(mounse)
Infecting
dose
(T. evansi
/mounse)
earliest
(day)
(day)
average
(
X
mX 
) day

20 10
3
3 7 4,40 ± 0,32
Experimental

20 10
6
1 2 1,60 ± 0,12
1
Control 10 0 0 0 0,00
20 10
3
3 7 4,90 ± 0,31
Experimental

20 10
6
1 2 1,80 ± 0,09
2
Bontrol 10 0 0 0 0,00
The table 3.10 shows that at dose 10
3
T. evansi per mouse. It took
at least 3 days and longest 7 days for T. evansi to appear in blood
of mouse after being experimentally infected. At dose 10
6
T. evansi
per mouse, time of T. evansi to appear in blood of mouse was 1 - 2
days after being experimentally infected. .In the blood of the control
group of mice no T. evansi was found in peripheral blood.

3.2.1.4. Gross lesions in the affected white mice experimentally
infected
Table 3.13. Gross lesions and microscopic lesions in the white mice
experimentally infected with trypanosomes
Group Main gross lesion
Number of mice
at necropsy
(mouse)
Number of
mice showing
lesions (mouse)

Percentage

(%)
Flabby heart muscle 46 57,50
swollen and
hemorrhagic Liver
58 72,50
swollen and
hemorrhagic Lungs
60 75,00
swollen and
hemorrhagic spleen
80 100
Swollen kidneys 27 33,75
Subcutaneous tissues had
yellow víscous gel like
80
56 70,00

Experimental
infected
group 1 + 2

Atrophy of testes
(male mice)
40 39 97,50
Control
group
Lesions mentioned
above
20 0 0,00
Table 3.13 shows that mice experimentally infected with T.evansi all
had lesions in internal organs, making up 33,75 - 100 %. At necropsy of
20 mice in the control group, no lesions were found in the internal
organs and subcutaneous tissues of the mice.
15
3.3.1.1. Detection rate of all positive samples performed by CATTT kit
The results were illustrated in table 3.24.
Table 3.24. Rate of positive samples of all positive samples
detected by CATT kit (+)
Serum of buffaloes infected with
trypanosomes
Serum of cattle infected with
trypanosomes
Positive
reaction ( +)
Negative
reaction (-)
Positive

reaction ( +)

negative
reaction (-)
Place
Number of
samples
used kit
n % n %
Number of
samples
used kit
n % n %
Thai Nguyen

17 17 100 0 0 5 5 100

0 0
Hoa Binh 22 22 100 0 0 4 4 100

0 0
Lang Son 25 25 100 0 0 3 3 100

0 0
Lai Chau 24 24 100 0 0 0 0 0 0 0
88 88 100 0 0 12 12 100

0 0
Total
Total detection rate: 100/100 (+) = 100 %

Table 3.24 shows that 100 serum samples from cattle and
buffaloes experimentally infected with T. evansi, were all positive
when CATT kit was used (making up 100 %), of which 88/88 of
serum samples from the buffaloes and 12/12 of serum samples from
the cattle were positive (accounting for 100 %).
3.3.1.2. . Rate of positive samples detected by ELISA kit from all of
positive serum samples (+)
Table 3.25. Rate of positive samples detected by ELISA kit from all of
positive serum samples (+)
Serum from the experimentally
trypanosome infected buffaloes
Serum from cattle infected
with trypanosomes
reaction
( +)
reaction
(-)
reaction
( +)
reaction
(-)
Place
Number
of samples
used kit
n % n
%
Number of
samples
used kit

n
% n %
Thai Nguyen

17 17 100 0 0 5 5 100

0 0
Hoa Binh 22 22 100 0 0 4 4 100

0 0
Lang Son 25 25 100 0 0 3 3 100

0 0
Lai Chau 24 24 100 0 0 0 0 0 0 0
88 88 100 0 0 12 12 100

0 0
Total
Total detection rate: 100/100 (+) = 100 %
Table 3.25 reveals that ability of ELISA kit to detect antibody
against T. evansi in positive serum samples was very sensitive (100 %).
3.3.2. Application of the kit to diagnose trypanosomiasis
200 serum samples were randomly collected from buffaloes in
Thai Nguyen. The results were illustrated in table 3.26.
Table 3.26. Infection rate of trypanosomes in buffaloes in Thai
Nguyen obtained from diagnosis kit
14
thrombocytes and decreased number of erythrocytes compared with
the control buffaloes (table 3.21).
Amount and proportion of various types of leucocytes were

illustrated in table 3.22.
Table 3.22. Amount and proportion of various types of leucocytes
in the buffaloes experimentally infected compared with the control
buffaloes
Group Control buffaloes
Experimentally infected
buffaloes
Number of buffaloes
(buffalo)
3 5
Type of leucocytes

Amount
(
x
X± m
)
Percentage
(%)
Amount
(
x
X ± m
)
Percentage
(%)
Contrast the
control group
and
experimentally

infected group
Lymphocytes
thousand /ml)
10.333 ±
1.782,48
75,98 7.040 ± 182,35

40,79
χ
2
= 2182,22
P = 0,000
Monocytes
(thousand/ml)
2.333 ± 47,14 17,16 1.180 ± 74,16 6,84
χ
2
= 60,46
P = 0,000
Granulocytes
(thousand /ml)*
934 ± 232,14 6,86 9.040 ± 1.295,96

52,37
χ
2
= 1115,86
P = 0,000
Notes: *
Granulocytes include eosinophils, basophils and neutrophils

,
Table 3.22 shows that amount and rates of granulocytes in the
buffaloes experimentally infected with trypanosomes were higher
than that in the control ones. In contrast, amount and rates of
lymphocytes and monocytes in the buffaloes experimentally infected
with trypanosomes were much lower than that in the control ones.
The differences were significant (P < 0,001).
3.2.3.5. Main gross lesions in buffaloes experimentally with Trypanosomes
Gross lesions in buffaloes experimentally with Trypanosomes
were similar to the lesions found in white mice and rabbits, varying
20 - 100 %.
3.2.3.6. Microscopic lesions in some internal organs of affected
buffaloes due to being experimentally infected with trypanosomes
Microscopic lesions in the internal organs of buffaloes
experimentally infected with trypanosome consisted of dilated
myocardium, enlargement of cadiac muscle fibers; dilated hepatic
sinusoid, hepatic vein like spokes being dilated, degeneration of liver
cells; congested lungs, accumulated with edema fluid; hemorrhage of
splenic tissues filtrated inflamatory cells and macrophages;
hemorrhagic kidney tissues, dilated and hemorrhagic renal ducts.
3.3. APPLICATION OF SEROLOGICAL TEST KIT FOR DIAGNOSIS
OF TRYPANOSOMIASIS IN CATTLE AND BUFFALOES
3.3.1. Rate of positive serum samples detected by the kit
11
3.2.2. Ability of T. evansi to cause diseases in rabbits
3.2.2.2. Time of T. evansi appearance in blood of rabbits experimentally
infected with T. evansi and time period of killing rabbits
Table 3.16. Frequency of T. evansi appearance in blood of rabbits
experimentally infected with T. evansi and time period of killing rabbits
Time of

infecting

Group
Experimental
infected
rabbit

Number of
times for
examining

Number of
times of
appearance

Percentage
(%)
Time period of
killed rabbits
after being
infected (day)

1 10 9 90,00 82
2 10 9 90,00 54
3 10 8 80,00 64
4 10 10 100 58
Experimentally
infected
5 9 9 100 37
1

CONTROL 5 rabbits 50 0 0,00 Not killed
1 10 10 100 40
2 10 9 90,00 48
3 10 10 100 38
4 10 10 100 61
Experimental
infected
5 10 9 90,00 75
2
CONTROL 5 rabbits 50 0 0,00 Not dead
Experimental
infected
10 rabbits 99 93 93,90 55,7 ± 5,14
Tính
chung
CONTROL 10 rabbits 100 0 0,00 Not killed
Table 3.16 shows that: there were many times that T. evansi was
detected in blood of rabbit. 93 of 99 times for being tested. in blood
of 10 rabbits T. evansi was found making up 93,90 %. Rabbits
experimentally infected with T. evansi were all killed on day 37 to
day 82 after being experimentally infected.
* In blood of rabbits in the control group in twice of experimental
infections T. evansi was nof found. At the end of the experiment no
rabbits were killed.
3.2.2.3. Time to appear clinical signs and develop symptoms
Table 3.17 shows that all of 10 rabbits experimentally infected with T.
evansi in twice of experiments manifested clinical signs accounting for 70
- 100 %. Time to appear the earliest clinical signs on day 15 and the latest
was on day 52 after being experimentally infected, generally 25 - 40 days
after being experimentally infected rabits presented clinical signs.

Table 3.17.Time of appearance of main clinical signs in rabbits after
being experimentally infected
12
Time of appearance of clinical signs
after being experimentally infected

Group Clinical signs
Number
of rabbits
moni-
tored
(rabbit)
Number of
rabbits
showing
clinical signs
(rabbit)
Percen-

tage
(%)
earliest
(day)
Latest

(day)

Average
(
X

mX 
)(day)

Edematous ear
(ear was prone to
the back side)
10 100 15 49 27,5 ± 3,60
Respiration
disorder
10 100 16 44 25,2 ± 2,87
Swollen eyes with
rheum
10 100 21 41 33,3 ± 2,17
Swollen and
edematous lips
7 70,0 26 46 38,6 ± 2,42
Experimen-
tally
infected
Slow moving
10
8 80,0 31 52 40,9 ± 2,21
Control
Symptoms
mentioned above

10 0 0,0 0 0 0
All of 10 rabbits in the control group didn’t show any clinical
signs during the experiments.
3.2.2.4. Main gross lesions in rabbits experimentally infected with

trypanosomes
All of 10 rabbits showed lesions in the internal organs with various
extents. Gross lesions were similar to that in the white mice.
3.2.2.5. Microscopic lesions in the internal organs of the affected rabbits
experimentally infected
The internal organs of rabbits infected with trypanosomes showed
microscopic lesions clearly including pericardial cavity accumulated with
a lot of fluid, dilation of various layers of heart muscle; dilated hepatic
portal vein, degenerative hepatocytes; congested lungs; hemorrhagic and
highly necrotic splenic tissues; hemorrhagic and enlarged renal ducts.
3.2.3. Ability of T. evansi to cause disease in buffaloes
3.2.3.2. Body temperature after being experimentally infected
- In the control group; the body temperature curve in all of 3
buffaloes in the control group varied from 37,8 to 38,5
0
C. Fever was
not found during the experiments.
- In the experimental group, fever was found in 5 buffaloes experimentally
infected with T. evansi (over 38,5
0
C). There were 3 - 3 times of fever in the 5
buffaloes corresponding to 3 - 4 waves fevers of trypanosomes.
3.2.3.3. Main clinical signs of buffaloes after being experimentally
infected
Table 3.20 indicates that 5 buffaloes being experimentally infected
with 2 different doses of trypanosomes manifested the same clinical
13
signs: in some early days their appetíte was normal, not manifesting any
clinical signs of trypanosomiasis, later their symptoms appeared (rate of
animals manifesting clinical signs varied 20 - 100 %).

Table 3.20. Main clinical signs of buffaloes experimentally infected
with trypanosomes
Experimental
groups
Main clinical signs
Number of
buffaloes
monitored
(buffalo)
Number of
buffaloes showing
clinical signs
(buffalo)
Percen-
tage
(%)
Undulant fever 5 100
Swollen eyes with mucus
running out of eyes
5 100
Dry,rough hair coat 5 100
Edema in jaws 4 80,00
Edema in chest and abdomen

3 60,00
Trembling 5 100
The group was
experimentally
infected with
T.

evansi
Paralysis of the hindlegs
5
1 20,00
The control
group
Clinical signs mentioned
above
3 0 0,00
3.2.3.4. Changes of some blood cell indices, number of leucocytes and
proportion of various types of leucocytes in buffaloes experimentally
infected with trypanosomes compared with that in control buffaloes
Table 3.21. Changes of some blood cell indices of buffaloes after
being infected
Blood cell indices
Control bufaloes
(
x
X ± m
)
Bufaloes experimentally
infected with
trypanosomes
(
x
X ± m
)
Number of buffaloes (buffalo)

3 5

Comparison
between
control and
exprimental
groups
Number of erythrocytes
(million/ml of blood)
8,470 ± 142,11 6.936 ± 943,00
χ
2
= 290.353
P = 0.000
Number of leucocytes
(thousand/ml of blood)
13,600 ± 2250.53

17.260 ± 1344.70
χ
2
= 328.92
P = 0,000
Number of thrombocytes
(thousand/ml of blood)
213.,67 ± 89,88 342.80 ± 33.75
χ
2
= 175.43
P = 0.000
Hemaglobin content
(g/l)

125.33 ± 0.33 114.00 ± 1.46
χ
2
= 0.11
P = 0.999
Average volume of
erythrocytes (fl)
47.20 ± 3.16 51.58 ± 0.17
χ
2
= 0.65
P = 0.957
Mean corpuscular hemoglobin
concentration (g/l)
302.33 ± 2.,40 300.4 ± 1.25
χ
2
= 0.03
P = 1.000
Results of testing 5 T. evansi infected buffaloes: showed that T.
evansi infection led to increased number of leucocytes and