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 ). [...]... 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... the same clinical 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 Number of Number of Percenbuffaloes buffaloes showing tage monitored clinical... 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 15 Table 3.10 Time of T evansi appearance in blood of white mice after 3.3.1.1 Detection rate of all positive samples performed... 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. .. 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... 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 3.2.2 Ability of T evansi to cause diseases in rabbits 3.2.2.2 Time of T evansi appearance... 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. .. 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 Place Thai Nguyen Hoa Binh Lang Son Lai Chau Total Serum of buffaloes infected with Serum of cattle infected with trypanosomes trypanosomes Number of Positive Negative Number of Positive negative... 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. .. 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 Group Number of buffaloes (buffalo) Type of leucocytes Control buffaloes Experimentally infected buffaloes 3 5 Amount (X± m ) x Percentage (%) Amount (X ± m ) x Percentage . 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. 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. (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),