Summary of doctoral thesis: Some epidemiological biology and of Fasciola sp. and the efficacy of anthelminthic treatments in cattle in the Mekong delta

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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.

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Nội dung Text: Summary of doctoral thesis: Some epidemiological biology and of Fasciola sp. and the efficacy of anthelminthic treatments in cattle in the 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: 1. Learning Resource Center, Can Tho University. 2. National Library of VietNam. I
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. 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 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 3
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. 4
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. 5
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 Annealing Primer sequence Gene Primer temperature Reference (5’ – 3’) o -Tm ( C) ITS1-F: TTG CGC ITS1- TGA 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. Predicted Temperature, Restriction Location length of Species incubation Reference enzyme cuting cutting time (bp) 367, 104 Fasciola (68, 59, hepatica 370C, 5’..GT↓AC...3' (Ichikawa 54, 28) RsaI overnight 3'..CA↑TG...5' et al, 367, 172 2010) Fasciola incubation (59, 54, gigantica 28) 6
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 Number of Experiment Number of petri / trial eggs/petri Negative control 0 5 Experiment 1 60 10 Experiment 2 60 10 Experiment 3 60 10 Experiment 1: Fasciola sp. egg in petri disk with water levels of 0, 5 cm, no illumination, pH from 6-8, the temperature from 26-290C. Experiment 2: Fasciola sp. egg in petri disk above 0, 5 cm, lighting 4 hours/day, pH from 6-8, the temperature from 26-290C. Experiment 3: Fasciola sp. egg in petri without water 7
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 Number of Number of petri disk/ Experiment miracidia/petri experiment 1 15 10 2 15 10 3 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 Infection dose Number snail of Experiment (micracidium /snail) experiment Lymnaea swinhoei 0 80 Negative control Lymnaea viridis 0 80 Negative control Lymnaea swinhoei 3 160 Lymnaea viridis 3 160 8
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 9
Table 3.7 The efficiency of albendazole, mebendazole and triclabendazole on Fasciola sp. infected cattle Experiment Doses Number Adminstration (mg/kg body weight) of cattle Route treatment Control group 0 5 - albendazole: 10 mg/KgP 15 oral 1 triclabendazole: 15 mg/KgP 15 oral mebendazole:10 mg/KgP 15 oral albendazole:15 mg/KgP 15 oral 2 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 Intensity of infection Examined Infected Prevalence Province cattle cattle (%) + ++ +++ (%) (%) (%) a a An Giang 1036 268 25.87 73.13 20.15 6.72a Dong Thap 987 249 25.23a 70.28 20.88a 8.84a Vinh Long 993 244 24.57a 71.31 19.67a 9.02a Ben Tre 933 149 15.97b 81.21 14.77b 4.03b Tra Vinh 900 142 15.78b 83.80 11.97b 4.23b Soc Trang 935 134 14.33b 85.07 11.94b 2.99b 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+) 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
(31.38%).Through statistical analysis showed that there was significant difference among 3 various age groups. Over 2 year-old cattle had long- term environmental exposure to environmental conditions which especially had more opportunities to be infected intermediate hosts for Fasciolosis. Moreover, the immunune system of old animals decreased so it was easy to be infected by other pathogens. Young cattle (
cattle and grazing cattle in every province such as An Giang, Dong Thap, Vinh Long, Tra Vinh, Ben Tre and Soc Trang also followed the same pattern. Table 4.5 The infection rate of liver fluke egg in cattle according to the seasonal Infected rate to seasonal (%) Provinces Dry season Rainy season An Giang 31.42a 20.23b Dong thap 30.38a 18.66b Vinh Long 29.52a 19.60b Ben Tre 22.02a 10.66b Tra Vinh 20.56a 10.73b Soc Trang 20.43a 8.42b Total 26.07a 14.79b a,b in the same row showed the statistically significant difference at P< 0.05 Table 4.5 illustrated that the Fasciola infection rate in cattle in the Mekong Delta was influenced by season factor. In fact, the infectious rate in dry season was higher than in the rainy season (14.79%). Through statistical analysis of Fasciola infection rates between the dry season and the rainy season in the Mekong Delta have the discrepancy is significant statistically (p < 0.05). Similarly, in An Giang, Dong Thap, Vinh Long, Tra Vinh, Ben Tre and Soc Trang province, the Fasciola infection rates was statistically higher in the dry season than in rainy season. Table 4.6 Fasciola infection rate on cattle by eco-region Infected rate to eco-region(%) Provinces Saltwater and brackish Fresh water - An Giang 25.87 - Dong thap 25.23 - Vinh Long 24.57 Ben Tre 9.38a 22.06b Tra Vinh 9.47a 21.11b Soc Trang 8.33a 22.11b Total 9.01a 24.14b a,b in the same row showed the statistically significant difference at p < 0.05 Table 4.6 showed that the Fasciola infection rate in cattle in the Mekong Delta had changed according to the ecological regions. In fact, the percentage of infected cattle in freshwater areas was the highest one (24.14%), following by the cattle in saltwater and brackish ecological region 9.1%. The results of statistically analyzing also pointed out that this difference was statistical discrepancy (p
4.1.2 The results of autopsy to determine the prevalence of Fasciola infection in cattle in Mekong Delta Table 4.7 Prevalence of Fasciola infection in cattle in the Mekong Delta provinces (by autopsy) Prevalence Intensity Provines Examined cattle Infection Cattle (%) (X±SE) AG 279 75 26.88a 12.88±0.79 ĐDT 257 70 27.24a 12.37±0.88 VL 263 68 25.86a 11.91±0.83 BT 270 48 17.78b 7.02±0.48 TV 257 45 17.51b 6.71±0.56 ST 246 40 16.26b 6.40±0.61 Total 1,572 346 22.01 9.93±0.34 a,b in the same column showed the statistically significant difference at P< 0.05 Table 4.7 showed that cattle in 6 provinces had the Fasciola infection rate of 22.01% with infection intensity: 0.34 ± 9.93. Cattle in An Giang, Dong Thap and Vinh Long province were high infectious rate group. Specifically, the cattle in An Giang had the highest infection rate of 26.88% with the intensity of infection (IF) 12.88±0.79, following closely by Dong Thap with 27.24%, IF 12.37±0.88, and the lowest one in this group with 25.86% and IF 11,91±0,83. Cattle in Ben Tre, Tra Vinh and Soc Trang province belonged to low Fasciola infection group. In this group, the infectious rate of cattle in Ben Tre, Tra Vinh and Soc Trang were 17,78% with IF 7.02±0.48; 17.51% with IF 6.71±0.56; 16.26% with IF 6.40±0.61; respectively. 4.2 Results of liver fluke identification 4.2.1 Liver fluke identification by morphological method Totally 2253 liver flukes were collected from 6 provinces in Mekong Delta, then were measure the length and analyzed the morphological characteristics. The results found that the size of those liver flukes had the length: 33.21±0.48mm, width: 9.24±0.09mm, oral sucker 1.056 ± 0, 008mm, ventral sucker 1.468 ± 0, 011mm and lengh/width rates/3.49 ± 0.04 (the size fluctuating from 28.64 ± 0, 16 mm to 38.43 ± 0, 61mm for length and from 6.44 ± 0, 29mm to 10.77 ± 0, 14 mm for width, length/width ratio ranged from 2.83 ± 0.043 to 4.22 ± 0.105). Liver flukes are leaf-shaped, broader anterior than posteriorly, intestine branching and has distributed the vitelline glands throughout the body. Ventral sucker is usually bigger than oral sucker. The testes and ovaries are located just behind the ventral sucker and the feeling in the middle of the body expels the leaves. According to Phan The Viet et al. (1977) and Nguyen Thi Le 14
(2000), 2,259 Fasciola obtained in 6 provinces of the Mekong Delta are species of Fasciola gigantica. 4.2.2 The results of liver fluke identification by molecular biology 4.2.2.1 Analyzing PCR-RFLP results in liver fluke identification on cattle in the Mekong Delta 360b p170b p60bp Figure 4.2: The PCR products was digested by using restriction enzyme RsaI From left to right, M: Marker 100bp, Wells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10: samples: 11: negative control, 12: positive control- Fasciola gigantica. Totally 180 liver fluke samples collecting from 6 provinces were analyzed using PCR to multiply the ITS1 gene fragment by specific primer ITS1-F and-R ITS1 (Itagaki et al., 2005) designed to Fasciola spp. detection with the size of PCR products around 680 bp (Ichikawa and Itagaki , 2010). Through the results of electrophoresis PCR-RFLP products, 180/180 samples had the cutting pattern of Fasciola gigantica which had 3 different bands with size 360, 170, 60 bp. The results in this study strongly confirmed that liver flukes in Mekong Delta was Fasciola gigantica 4.2.2.2 Analysing the ITS1 gene sequencing of Fasciola gigantica The PCR products from 12 liver flukes in 6 provinces were purified and sequenced and BLAST with the nucleotide sequence of ITS-1 in Genbank. The sequencing results showed that 12/12 samples were Fasciola gigantica. This result pointed out that PCR-RFLP is the effective methods to distinguish the differences between F.gigantica and F.hepatica. The ITS-1 sequence of F.gigantica (this study) 660 bp had high level of similarity, just different from 1-2 nucletotide in the gene fragment was assigned area of. Among them, 8/12 samples had sequence perfectly match with samples of Fasciola gigantica in Thailand (F-T), Vietnam (F-VN)up to 99.8% to 100% (Ichikawa & Itagaki, 2010). This demonstrates that this gene has a very high similarity conservation among collected F.gigantica, which does not depend on the location of sampling. Fasciola gigantica expels patterns in the Mekong Delta provinces have similar proportions 15
with the samples of Thailand, SLGL China, Japan, South Korea, Australia and Vietnam range from 99.0% to 98.9%. Therefore, large liver fluke circulation in cattle in the Mekong Delta region was F.gigantica. Table 4.8 Comparison the ITS1 nucleotide sequence of Fasciola sp. in the study and other strains of Fasciola worldwide Position of Nucleotide variation No 13 48 149 173 265 359 437 457 1 F-A A T G T T C T C 2 F-C A T G T T C T C 3 F-J A Y G Y W Y W Y 4 F-K A Y G Y W Y W Y 5 BT2 G C G T T T A T 6 TV2 G C G T T T A T 7 AG1 C C G T T T A T 8 DT1 A C A T T T A T 9 ST2 A C G T T T A T 10 ST1 A C G T T T A T 11 VL2 A C G T T T A T 12 BT1 A C G T T T A T 13 AG2 A C G T T T A T 14 F-T A C G T T T A T 15 F-VN A C G T T T A T 16 DT2 A C G T T T A T 17 VL1 A C G T T T A T 18 TV1 A C G T T T A T Annotation: AG1 AG2 liver flukes, collected in An Giang Province; BT1, BT2 samples in Ben Tre province; ĐT1, ĐT2: samples in Dong Thap province; ST1, ST2: samples collected in the province; TV1 TV2: samples in Tra Vinh province; VL1 VL2 samples in Vinh Long province; F-T: Fasciola gigantica in Thailand; F- VN: Fasciola gigantica in Vietnam; F-A: Fasciola hepatica in Australia; F-C: Fasciola hepatica; F-J: hybrid Fasciola in Japan; F-K:. hybrid Fasciola in South Korea 4.3 Life cycle of Fasciola 4.3.1 The distribution of freshwater snail species-intermediate host of F. gigantica Totally 15,848 snails were collected in the Mekong Delta provinces. Through the identification of classification based on the classification 16
system of the freshwater snails by Dang Ngoc Thanh et al. (1980) and John (1982), the results showed that collected snails in the Mekong Delta provinces were Lymnaea swinhoei (15.61%), followed by species of Lymnaea viridis (9.71%). Table 4.9 The development of Fasciola sp. egg in water environment Experiment STKS TGTBĐN (day) TGTN (50%) (day) 1 600 15.18±0.16 19.05±0.129 2 600 10.57±0.14 15.88±0.10 3 600 - - Notes: Experiment 1: Fasciola sp. Eggs in petri disk with water levels of 0, 5 cm, no illumination, pH from 6-8, the temperature from 26-290C. Experiment 2: Fasciola sp. egg in petri disk above 0, 5 cm, lighting 4 hours/day, pH from 6-8, the temperature from 26-290C. Experiment 3: Fasciola sp. egg in petri without water. STKS: survey number of eggs; TGTBĐN: the time the eggs begin to hatch, TGTN (50%): the time the eggs hatch 50% rate. Table 4.9 showed that Fasciola eggs cultivated in petri disk in experiment 1, the length of time for egg hatching was 15.18 ± 0.16 days. Fasciola sp. egg hatching time reached 50% rate is 19.05 ± 0.129 days. In experiment 2, the eggs started to hatch at 10.57 ± 0.14 days, the 50% of eggs hatching was after 15.88 ± 0.10 days. In experiment 3, there is no egg hatching. So, in the same condition as when illuminated, the time the eggs start to hatch the egg hatching time and reaches 50% earlier than when those were not illuminated. Thus, the water and light were identify as crucial element which has a great influence to the development of Fasciola sp. egg and miracidium shell escape. The light is an important factor in facilitating the miracidium escaping from shell eggs. Through the observation, we found that the development of Fasciola sp. eggs  miracidium were not happened at the same time. A B C D Figure 4.3: The development stages of Fasciola sp. egg form miracidium A. Eggs Fasciola sp.; B. the miracidium in eggs; C. the Miracidium in eggs are about escaping the shell; D. Miracidium 17
4.3.3 Time of miracidium in water environment Tracking time of miracidium in conditions 26-290C showed that the miracidium lived from 10-12days in aquatic environment. In first 10 hours after hatching miracidium, no death miracidium were recorded. The death after 10 hours, 10-11 hours, after 11-12 hours hatching were 8.22%, 41.56% and 52.89%; respectively. The research results of longevity of miracidium in water was shorter than other research results of foreign studies, but still longer than some documents of of Pham Van Khue and Phan Luc (1996); Pham Dieu Thuy et al. (2014). 4.3.4 The development of Fasciola gigantica larvae in the intermediate host Lymnaea swinhoei and Lymnaea viridis. Through experimental design, the larvae of Fasciola gigantica were infected on Lymnaea swinhoei and Lymnaea viridis. The results showed that the Lymnaea swinhoei and Lymnaea viridis larvae were infected by sporocyst after 10 days, redia found in snails from 14 to 38 days after infecting miracidium, the cercaria found in snails from 26-42 days after infecting miracidium. At the same time, observing the miracidium culture water, we detected the presence of cercaria-swimming in water using very strong activity tail, after 1-2 hours then the cercaria tail loss to form adolescaria. So the length of time for development from miracidium invasion to Lymnaea swinhoei and Lymnaea viridis to cercaria was from 26 to 42 days. The results showed that both Lymnaea swinhoei and Lymnaea viridis snails were infected by Fasciola sp. larvae as conducting experimental infection. Thus, the intermediate host of Fasciola gigantica in Mekong Delta region is Lymnaea swinhoei and Lymnae viridis. A B C Figure 4.4 The larval stages of Fasciola developed in Lymnaea swinhoei and Lymnaea viridis A. Sporocyst; B. The redia; C. Cercaria 4.3.5 Results of infection cause larvae Fasciola for cattle Totally 6 cattle were chose and infected by cercaria to observe the presence of Fasciola sp. eggs in cattle feces. In the experiment 1 (100 18
cercaria/ 1 cattle), Fasciola sp. eggs were detected from 114 to 115 days after infection with the intensity of infection (IF) at 1+ and 2+. In the experiment 2 (150 cercaria/1 cattle), the eggs were also detected at the same time with experiment 1, and the IF were 2+. In the experiment 3 (200 cercaria/1 cattle), the eggs were somehow detected earlier from 111 to 114 days with IF 3+. Therefore, the higher number of cercaria invaded to host, the higher intensity infection. Cercaria continued developing in cattle (definite host) and developed to mature live flukes and laid eggs in 111-115 days after infection. Table 4.10 Operating results Fasciola infection in experimental cattle Number of Number of liver fluke Prevalence of liver Experiment Cattle cercaria collect fluke collect (%) infection ĐC 0 0 0 1 1 100 29 29.0 2 100 30 30.0 Total 200 59 29.50a 1 150 47 31.33 2 2 150 46 30.67 Total 300 93 31.0a 1 200 63 31.5 3 2 200 65 32.5 Total 400 128 32.0a In the experiment 1, the number of adult liver flukes/ number of cercaria infected were 29.50% with the intensity infection 29-30 liver flukes. In the experiment 2, the proportion between adult liver flukes and cercaria infected were 31% with higher intensity 46-47 liver flukes. Similarity, in the experiment 3, this proportion rised upto 32% and the intensity 63-65 liver flukes. However, the statistically analyzing showed that there was no signicant difference among 3 experiment. The results of intensity of infection in 3 groups were also tested by fecal examination which also provided the same pattern. 4.3.6 Species of Fasciola determination results of infecting cattle 4.3.6.1 Results determine the species of liver fluke by using traditional methods Through the analysis of morphological characteristics and the structures of the 280 liver fluke infection-causing cattle obtained experimentally. All of liver flukes were Fasciola gigantica (basing on the features described in 19