Tóm tắt Luận án The epidemiological, biological characteristics of Fasciola sp. and the efficacy of anthelmintic treatments in cattle in Mekong Delta

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Major: PATHOLOGY AND TREATMENT OF ANIMALS Major code: 62 64 01 02 Ha Huynh Hong Vu Some epidemiological biology and of Fasciola sp. and the efficacy of anthelminthic treatments in cattle in the Mekong delta Can Tho- 2018 I THIS THESIS WAS COMPLETED AT CAN THO UNIVERSITY Academic supervisor: Assoc. Prof. DR. Nguyen Huu Hung This thesis was defended against the Ph.D. dissertation council at the university level. Place: .. Time: 1 st Opponent: .. 2 nd Opponent: Reviewed Confirmation of Chairman .. Thesis could be found at: 1. Learning Resource Center, Can Tho University. 2. National Library of VietNam. II PUBLISHED ARTICLES Published Articles in journals 1. Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Nguyen Huu Hung, 2014. Identification freshwater snail intermediate host of trematoda causing animal disease in Vinh Long and Dong Thap Province. Journal of Science, Can Tho University, Special issue agriculture, pp 8-12. 2. Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Nguyen Huu Hung, 2015. Morphological and molecular characteristic of Fasciola sp infected in cattle in Dong Thap province. Journal of Science-Technique of Veterinary Medicine, 6: 63-69. 3. Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Pham Duc Phuc, Nguyen Huu Hung, 2016. Application of molecular marker-ITS-1 gene and PCR- RFLP technique for determining large liver flucke (Fasciola sp.) in cattle in Mekong river Delta, 2016. Journal of Science-Technique of Veterinary Medicine, 2: 85-92. 4. Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Nguyen Huu Hung, 2016. Large liver fluke (Fasciola sp.) infection of cattle in the Mekong Delta and results of treatment trials. Journal of Science, Can Tho University, Special issue agriculture, pp 17-22. 5. Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Nguyen Huu Hung, 2018. The surveillance on pathological characteristics of Fasciola gigantica infected in Mekong delta. Journal of Science, Can Tho University, Special issue agriculture, pp 12-17. 3 Chapter I: INTRODUCTION 1.1 Rationale According to the World Health Organization (WHO), Fascioliasis is one of the important diseases, which is found in humans and animals. More than 2.4 million people in 70 countries were affected by the disease (WHO, 2015; Amer, 1016). In Vietnam, Fascioliasis in humans tends to increase gradually, from 2006 to 2010. In fact, 15,764 people and cases were infected by Fasciola sp. in 2006 and those cases increased to over 20,000 people in 2011. The disease in 52 provinces from North to South and pathogenic species is determined mainly Fasciola gigantica (Nair et al. 2012). Fasciolosis has been demonstrated and listed in zoonosis diseases. The disease causes by the large liver flukes which require the intermediate host (freshwater snail species) to complete its life cycle. The Mekong Delta possesses the geographic features such as innumerable canals, rivers, stream which is suitable to develop agriculture: paddy rice and vegetables as well as provide the appropriate conditions for freshwater snail development. Moreover, livestock husbandry also great develops because famers take advantages the source of by-product from agricultural processing. However, most of husbandry farms are small-scale farms where people normally use by-products from agriculture and they do not have well knowledge about applying the techniques in animal husbandry and veterinary. As the results, their livestocks expose high prevalence of helminthes infection. Therefore, it is crucial to research about fasciolosis and how to manage the spreading of this disease in order to minimize the damage from it. The study aimed to investigate “The epidemiological, biological characteristics of Fasciola sp. and the efficacy of anthelmintic treatments in cattle in Mekong Delta” 1.2 Objectives - Identifying the species, distribution, biological characteristics and influential factors to the liver flukes infection rate in cattle the Mekong Delta. - Suggesting the treatment methods for infected cattle in Mekong Delta. 1.3 Scientific significance - This is a systematic research about liver flukes Fasciola gigantica in cattle: determining the prevalence of infection and influential factors to the pathogen. Species were identified by morphological and molecular characteristics (PCR-RFLP, and sequencing) - The life cycle of Fasciola gigantica in cattle in Mekong Delta were firstly researched: identifying intermediate host (snails). Clinical symptoms and 4 anthelminthic testing would be useful for diagnosis and treatment. - This thesis provides documentations about Fasciola sp. infected in cattle (Mekong Delta), and supplies academic knowledge for veterinary parasitology books to education and training purposes 1.4 Practical significance - The thesis results are the scientific background for recommending farmers in effectively diagnosis, treatment and prevention liver flukes that minimizes the economic lose as well as contributes for the sustainable development of livestock husbandry. 1.5 Innovative contributions of the thesis This is the first research about Fasciola gigantica in infected cattle in Mekong Delta which were identified by applying molecular biology techniques. This is also first research about the complete life cycle of Fasciola gigantica. Gross lesions and histopathological of Fasciolosis (causing by F.gigantica) were completely described which were provided background for quickly diagnosis and treatments Chapter III: CONTENT AND RESEARCH METHODOLOGY 3.1 The research contents 3.1.1 Determining the prevalence of liver flukes of cattle in the Mekong Delta provinces - Determining the infection rate of liver flukes of cattle in the Mekong Delta provinces by the fecal examination and necropsy methods. 3.1.2 Identifying the species of Fasciola sp. in the Mekong Delta provinces - Determining the species of Fasciola sp. by analyzing mophorlogical molecular biology chacteristics and sequencing. 3.1.3 Researching about life cycle of Fasciola gigantica - Observing the development of the Fasciola gigantica egg outside the definite host. - Observing the development of the larvaes of Fasciola gigantica in intermediate host (Lymnaea swinhoei and Lymnaea viridis) to stage cercaria infection. - Analyzing and recording the every development stage of Fasciola gigantica since embronated eggs to mature in definitive host. 5 3.1.4 Fasciola gigantica Pathogen researching -Determine the clinical symptoms, gross lesions and histopathology on infected Fasciola gigantica cattle 3.1.5 Studying on treatments of Fasciola gigantica infected cattle - Testing the efficacy of anthelminths: albendazole, mebendazole and triclabendazole; and suggest the efficient treatment on Fasciola gigantica infected cattle. 3.2 Subjects, timeline, and researching areas 3.2.1 Subjects: cattle in six provinces: Dong Thap, An Giang, Vinh Long, Tra Vinh, Ben Tre and Soc trang; liver flukes-Fasciola sp.; the snail Lymnaea spp. 3.2.2 Timeline: from 11/2013 to 06/2017 3.2.3 Rearching areas The cattle in 6 provinces (Mekong Delta), slaughter houses, histologic laboratory in the Department of Veterinary Medicine – College of Agricultural and Applied Biology, Can Tho University; Department of Clinical Veterinary Science and livestock - veterinary medicine – Nong Lam University, Anatomical pathology laboratory - University of medicine and pharmacy of Can tho. 3.3. Materials and Chemistry Necessary materials and chemistry for diagnosis and molecular biology techniques. 3.4 Research methology 3.4.1 Identifying the prevelance of infected cattle in Mekong Delta Subjects: - Domestic cattle, cross-breeding Sind, dairy cow were classified into 3 age groups: under 1 year old, 1-2 years old, and over 2 years old. Slaughter cattles were selected for this research basing on the original surveyed provinces. - Methods: Fecal examination: modified sedimentation of Benedek (1943). The autopsy methods: the operating SKRJABINE (1937) -Observation targets The overall infected rate of Fasciola sp. in cattle, the infection rate of this disease according to seasons, husbandry methods, ages, and ecogeographic areas; the intensity of the infection ( the number of species/ individual) -Statistical analysis: Chi-Square test /Minitab program version to compare infection rates. 6 3.4.2 Identification method 3.4.2.1 Identification method of trematode by morphology based on documents written by David and Erasmus (1972), Soulsby (1980), Nguyen Thi Le (2000). 3.4.2.2 Identification method of live flukes by molecular techniques (PCR-RFLP) and sequence genes Collecting and storing DNA extraction samples Totally, 180 liver flukes were randomly collected from the liver and bile ducts in slaughter cattle from 6 surveyed provinces. Specimens was stored in physiological saline and brought to DNA laboratory. DNA extraction DNA concentration measurement. PCR-RFPL techniques PCR reaction: Table 3.1 Primer sequence corresponding to the target gene Gene Primer Primer sequence (5’ – 3’) Annealing temperature -Tm ( o C) Reference ITS1 ITS1- F/ITS1- R ITS1-F: TTG CGC TGA TTA CGT CCC TG ITS1-R: TTG GCT GCG CTC TTC ATC 56 Itagaki T (2005) PCR products were incubated with restriction enzymes RsaI (5 IU) overnight at 37 o C. Then the products were run on 1.5% agarose gel with Ethidium bromides in 80 votage in 30 minutes. The gels were visualized under camera (Geldoc). Table 3.2 Prediction the restriction patterns of the enzyme RsaI Enzyme restriction patterns in the region of ITS1 Fasciola sp. Species Restriction enzyme Temperature, incubation time Location cuting Predicted length of cutting (bp) Reference Fasciola hepatica RsaI 37 0 C, overnight incubation 5’..GT↓AC...3' 3'..CA↑TG...5' 367, 104 (68, 59, 54, 28) (Ichikawa et al, 2010) Fasciola gigantica 367, 172 (59, 54, 28) 7 Sequencing positive samples - Totally 12 liver fluke samples (Fasciola sp.) were collected from 6 provinces in Mekong Delta, specifically 2 samples/one province. PCR products were purified and sent to Macrogen (Korea) to sequence (using Sanger sequence method) Observation targets : - Identification of Fasciola sp. were done by morphological and molecular biology chacteristic. - Comparasion the nucleotide sequence of target gene ITS-1 of Fasciola sp among surveyed provinces and Fasciola sp. collected worldwide in Genbank Analyzing data : - BLAST the ITS-1 sequence (in NCBI) was used to identify the specific species, and comparing the level of similarity among multisequence by CLUSTAL OMEGA, analyzing Pairwise alignment/Calculate identity/Similarity for sequences (Bioedit). 3.4.3 Studies of life cycle of Fasciola gigantica. 3.4.3.1 Identification of freshwater snails Basing on the classification system of the freshwater snails was described by John (1982), Dang Ngoc Thanh and his colleagues (1980). From this backgound knowledge, the snails Lymnaea were carefully collected and feeded in the laboratory environment to produce the clean snail generation. Miracidium from embronated eggs (Fasciola gigantica) were infected to clean snail generation. 3.4.3.2 Observe the development of Fasciola gigantica egg in in vitro a. Reseach objects: Fasciola gigantica eggs, Lymnaea swinhoei and Lymnaea viridis snails is "clean snail". b. Experimental design Table 3.3 Experiment designing for Fasciola gigantica eggs development Experiment 1: Fasciola sp. egg in petri disk with water levels of 0, 5 cm, no illumination, pH from 6-8, the temperature from 26-29 0 C. Experiment 2: Fasciola sp. egg in petri disk above 0, 5 cm, lighting 4 hours/day, pH from 6-8, the temperature from 26-29 0 C. Experiment 3: Fasciola sp. egg in petri without water Experiment Number of eggs/petri Number of petri / trial Negative control 0 5 Experiment 1 60 10 Experiment 2 60 10 Experiment 3 60 10 8 c. Observation targets - The length of time from eggs to develope to miracidium. - The length of time from the eggs hatching into miracidium to 50% eggs were hachted and miracidia liberating from the egg shells. 3.4.3.3 Observing the length of time of alive miracidia in water a. Research objects Fresh miracidia have just liberated from the egg shells. Those miracidia were observed to identify the their longevity in water. b. Experimental design After miracidium infected to the snails, infected snails were collected and necropsied at time points: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42 PI days in order to detect cercaria- free swimming stage in water and transforming to Adolescaria. For each time points, 10 of Lymnaea swinhoei and 10 Lymnaea viridis snails were surgery to the stage of development of the larva (redia and cercaria, sporocyst) in 2 species of snail Lymnaea. Table 3.4 Identifying the longevity of miracidia in water 3.4.3.4 Observing the development of larva stage of Fasciola in intermediate host Lymnaea spp snails. In this experiment, 960 miracidia were collected and infected to 240 Lymnaea swinhoei, and 240 Lymnaea viridis. Table 3.5 The invasion of Fasciola micracidia to Lymnaea snails Experiment Number of miracidia/petri Number of petri disk/ experiment 1 15 10 2 15 10 3 15 10 Experiment Infection dose (micracidium /snail) Number snail of experiment Lymnaea swinhoei Negative control 0 80 Lymnaea viridis Negative control 0 80 Lymnaea swinhoei 3 160 Lymnaea viridis 3 160 9 3.4.3.5 Cattle infected by Fasciola larvae a. Research objects In this experiment, 8 cattle at the age of 7 months-12 months old were bought from the local farmers in the surveyed areas. Before infecting, cattle were dewormed by albendazole and carefully tested the presence of liver fluke eggs as well as other helminths. b. The cercaria infection causing lab layout for experimental cattle. Six cattle were divided into 3 different groups which were received 3 doses 100, 150, and 200 cercaria. Those cattle were infected by ceraria through oral adminstration. Non-infected group was considered as negative control. Observing the presence of liver fluke eggs in feces of infected cattle The feces examination were conducted after 11weeks post infection and then feces samples were collect and check everyday until detecting the eggs of Fasciola sp. The sedimentation methods was applied to diagnose. The results were futher confirmed by necropsy method. Observation targets - Identification the timepoint of the presence of liver fluke eggs in cattle feces - The numember of liver flukes in experiment cattle as well as species identification by morphological and molecular biological features. 3.4.4 Clinical symptoms and gross and histopathological changes on cattle infected with Fasciola 3.4.4.1 Symptoms of cattle infected with Fasciola - Physical and clinical examination were done in 60 infected cattle with Fasciola and 6 infected cattle in infection experiment (3.4.3.5 ) 3.4.4.2 Researching about the gross lesions and histopathology in liver tissue causing by Fasciola gigantica infection -Objects: livers from Fasciola gigantica infected cattle in this experiment and 45 livers from Fasciola gigantica infected cattle in slaughter houses in Mekong Delta. 3.4.5. Studying the prevention and treatment Fasciolosis in cattle 105 crossbred Sind cattle having in high infectious intensity from 2+ to 3+ were collected to test the efficacy of albendazole, mebendazole, triclabendazol. The number of cattle were divided into 2 experiments and 5 cattles in control group. Experiment 1: treatment dosage- following the manufacturer's instructions Experiment 2: increasing the treatment dosage (higher dose the manufacturer’s recommendation) Control group: not use any treatment 10 Table 3.7 The efficiency of albendazole, mebendazole and triclabendazole on Fasciola sp. infected cattle Experiment Doses (mg/kg body weight) Number of cattle treatment Adminstration Route Control group 0 5 - 1 albendazole: 10 mg/KgP 15 oral triclabendazole: 15 mg/KgP 15 oral mebendazole:10 mg/KgP 15 oral 2 albendazole:15 mg/KgP 15 oral triclabendazole: 20 mg/KgP 15 oral mebendazole:15 mg/KgP 15 oral c. Observing targets The efficacy of drugs afer 5, 10, 15 days post-treatment were tested and observed the adverse effects of those drugs in treated cattle. CHAPTER IV RESULTS AND DISCUSION 4.1 The prevalence of liver flukes infected cattle in Mekong Delta 4.1.1 The results of fecal examination of Fasciola sp. infected cattle in Mekong Delta Table 4.1 The prevalence of liver flukes infected cattle in Mekong Delta Province Examined cattle Infected cattle Prevalence (%) Intensity of infection + ++ +++ (%) (%) (%) An Giang 1036 268 25.87 a 73.13 20.15 a 6.72 a Dong Thap 987 249 25.23 a 70.28 20.88 a 8.84 a Vinh Long 993 244 24.57 a 71.31 19.67 a 9.02 a Ben Tre 933 149 15.97 b 81.21 14.77 b 4.03 b Tra Vinh 900 142 15.78 b 83.80 11.97 b 4.23 b Soc Trang 935 134 14.33 b 85.07 11.94 b 2.99 b Total 5784 1186 20.50 75.80 17.62 8.68 a,b in the same row showed the statistically significant difference at P< 0.05 Table 4.1 showed that the overall infected cattle by Fasciola sp. 20.50%. In particular, cattle in An Giang province had the highest infectious rate of Fasciola sp. 25.87%, following by cattle in Dong Thap (25.23%) and Vinh Long with 24.57%. The infectious rate of Fasciola sp. in cattle in Ben Tre, Tra Vinh and Soc Trang was 15.97%, 15.78% and 14.33%; respectively. Most of infected cattle had the low intensity of infection (1+) which oocupied of 78.80%, following by the (2+) intensity with 17.62%, and (3+) 11 with 8.68%. The infectious rate of Fasciola sp. in cattle in An Giang, Dong Thap and Vinh Long had statistically significant higher than those in Tra Vinh, Ben Tre and Soc Trang (p<0.05). The above results were explained that An Giang and Dong Thap province is located at the upstream of the Tien and the Hau River, Vinh Long is by the sides of the rivers. Due to the geographic features such as innumerale canals and waterways, it is the appropriate conditions for the development of intermediate hosts (snail) of Fasciolosis. Therefore, the infectious rate of Fasciolosis in cattle in An Giang, Dong Thap ang Vinh Long had the higher than those in Tra Vinh, Ben Tre and Soc Trang (the latter provinces locate near the sea and have the saltwater and blrackish water areas – not appropriate for intermediate hosts (snails) development. This result confirmed that the geographic climate and the ecological conditions had great influence on the growth of intermediate hosts of Fasciolosis and by the way influence on the infectious rate of Fasciolosis in cattle. This studying results is compatible with the researches of Pham Van Khue and Phan Luc1996); Phan Dich Lan et al. (2002); Nguyen Thi Kim Lan et al. (2008). Table 4.2 The infection rate of liver fluke egg in cattle according to the breed Provinces Gender Dairycow Prevalence (%) Crossbred Sind Prevalence (%) Local cattle Prevalence (%) An Giang - 24.91b 26.99b Dong Thap - 23.48b 24.53b Vinh Long 12.31a 25.58b 27.25b Ben Tre - 14.99b 17.04b Tra Vinh - 15.43b 16.16b Soc Trang 7.07a 17.07b 15.47b Total 9