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.
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 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: …………… 1st Opponent: …………… 2nd Opponent: ……………………… Reviewed Confirmation of Chairman ………… Thesis could be found at: Learning Resource Center, Can Tho University National Library of VietNam I PUBLISHED ARTICLES Published Articles in journals 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 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 Ha Huynh Hong Vu, Nguyen Ho Bao Tran, Pham Duc Phuc, Nguyen Huu Hung, 2016 Application of molecular marker-ITS-1 gene and PCRRFLP 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 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 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 II 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 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 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 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 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 age groups: under year old, 1-2 years old, and over 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 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 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 Annealing Primer sequence Gene Primer temperature Reference (5’ – 3’) o -Tm ( C) ITS1-F: TTG CGC ITS1TGA TTA CGT ITS1 F/ITS1- CCC TG 56 Itagaki T (2005) R ITS1-R: TTG GCT GCG CTC TTC ATC PCR products were incubated with restriction enzymes RsaI (5 IU) overnight at 37oC 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 Fasciola hepatica RsaI Fasciola gigantica Temperature, incubation time 370C, overnight incubation Predicted length of cutting (bp) Reference 367, 104 (68, 59, 5’ GT↓AC 3' 54, 28) 3' CA↑TG 5' 367, 172 (59, 54, 28) (Ichikawa et al, 2010) Location cuting Sequencing positive samples - Totally 12 liver fluke samples (Fasciola sp.) were collected from provinces in Mekong Delta, specifically 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 Number of Experiment Number of petri / trial eggs/petri Negative control Experiment 60 10 Experiment 60 10 Experiment 60 10 Experiment 1: Fasciola sp egg in petri disk with water levels of 0, cm, no illumination, pH from 6-8, the temperature from 26-290C Experiment 2: Fasciola sp egg in petri disk above 0, cm, lighting hours/day, pH from 6-8, the temperature from 26-290C Experiment 3: Fasciola sp egg in petri without water 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 species of snail Lymnaea Table 3.4 Identifying the longevity of miracidia in water Number of Number of petri disk/ Experiment miracidia/petri experiment 15 10 15 10 15 10 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 Infection dose (micracidium /snail) Lymnaea swinhoei Negative control Lymnaea viridis Negative control Lymnaea swinhoei Lymnaea viridis Number snail of experiment 80 80 3 160 160 3.4.3.5 Cattle infected by Fasciola larvae a Research objects In this experiment, cattle at the age of 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 different groups which were received 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 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 experiments and 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 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 albendazole: 10 mg/KgP 15 triclabendazole: 15 mg/KgP 15 mebendazole:10 mg/KgP 15 albendazole:15 mg/KgP 15 triclabendazole: 20 mg/KgP 15 mebendazole:15 mg/KgP 15 c Observing targets The efficacy of drugs afer 5, 10, 15 days post-treatment were observed the adverse effects of those drugs in treated cattle oral oral oral oral oral oral tested and 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 Intensity of infection Province Examined cattle Infected cattle Prevalence (%) + (%) An Giang Dong Thap Vinh Long Ben Tre Tra Vinh Soc Trang Total a ++ +++ (%) (%) a 1036 987 993 933 900 935 268 249 244 149 142 134 25.87 25.23a 24.57a 15.97b 15.78b 14.33b 73.13 70.28 71.31 81.21 83.80 85.07 20.15 20.88a 19.67a 14.77b 11.97b 11.94b 6.72a 8.84a 9.02a 4.03b 4.23b 2.99b 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+) 10 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