Study on epidemiological characteristics of fasiolosis in buffaloes and bovines in thai nguyen, bac kan, tuyen quang province and preventive and treatment methods (2010 - 2013)

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MINISTRY OF EDUCATION AND TRAINING THAI NGUYEN UNIVERSITY MINISTRY OF EDUCATION AND TRAINING THAI NGUYEN UNIVERSIY -------------------- -------------------- PHAM DIEU THUY PHAM DIEU THUY STUDY ON EPIDEMIOLOGICAL CHARACTERISTICS OF FASCIOLOSIS IN BUFFALOES AND BOVINES IN THAI NGUYEN, BAC KAN, TUYEN QUANG PROVINCE AND PREVENTIVE AND TREATMENT METHODS (2010 - 2013) STUDY ON EPIDEMIOLOGICAL CHARACTERISTICS OF FASCIOLOSIS IN BUFFALOES AND BOVINES IN THAI NGUYEN, BAC KAN, TUYEN QUANG PROVINCE AND PREVENTIVE AND TREATMENT METHODS (2010 - 2013) Speciality: Parasites and Veterinary Microbiology Code: 62.64.01.04 SUMMARY OF PH.D. DISSERTATION IN VETERINARY MEDICINE SUMMARY OF PH.D. DISSERTATION IN ADVISOR: Prof. Nguyen Thi Kim Lan, Ph.D. VETERINARY MEDICINE Thai Nguyen, 2014 Thai Nguyen, 2014 3 LIST OF WORKS RELATED TO THE DISSERTATION 1. Nguyen Thi Kim Lan, Pham Dieu Thuy, Pham Thi Trang (2012), “Infection rate and intensity of Fasciola gigantica in buffaloes and bovines in Thai Nguyen”, Animal husbandry magazine July, pages 19 - 23. 2. Pham Dieu Thuy, Nguyen Thi Kim Lan, Hoang Thi Ngan (2012), “Status of Fasciola infection in buffaloes and bovines in Bac Kan province”, Animal husbandry magazine, August, pages, 26 - 31 3. Pham Dieu Thuy, Nguyen Thi Kim Lan, Tran Nhat Thang, Tran Thi Phuong Thao (2014), “Study on egg viability and development time of Fasciola gigantica larvae in the external environment and in the intermediate host”, Journal of Agriculture and Rural Development, June, pages 122 - 126. 4. Pham Dieu Thuy, Nguyen Thi Kim Lan, Tran Nhat Thang, Tran Thi Phuong Thao (2014), “Study on the contamination of Fasciola gigantica eggs and larvae in the environment”, Veterinary sciences and Techniques, Vol. XXI, No6, pages 76 - 81. 1 2 INTRODUCTION - Laboratory of Veterinary and Animal Science faculty - College of Agriculture and Forestry, Thai Nguyen University - Institute of Ecology and Biological Resources. The Fascioliasis in buffaloes and bovines (Fassciolosis) caused by two liver fluke species namely Fasciola hepatica and Fasciola gigantica, is considered as a common parasitic disease which causes great losses in the economy of buffalo and bovine production in the world (Soulsby E. J. L., 1987). In recent years, the Fascioliasis in buffaloes and bovines has occurred widely and increased in the number due to the climate change and the migration of animals between countries and/or regions (Kasib Muhammad Khan et al., 2013). There are some studies on the epidemiological characteristics of fascioliasis in buffaloes and bovines and the effectiveness of the treatment. But, only few works has been paid which considers the issue in the mountainous area such as Thai Nguyen, Bac Kan and Tuyen Quang province. Therefore, an effective measures of prevention and the control of this diease in the mountainous area are obviously needed. CHAPTER 1 OVERVIEW CHAPTER 2 MATERIALS, CONTENTS AND METHODS OF STUDY 2.1. Objects of study - Buffaloes and bovines in three provinces: Thai Nguyen, Bac Kan and Tuyen Quang - Fasciolosis in buffaloes and bovines. 2.1.2. Study period: From 2010 - 2013 2.1.3. Places of study 2.1.3.1. Places of implementation: Thai Nguyen, Bac Kan and Tuyen Quang province. 2.1.3.2. Places of samples - Sperstructure Laboratory - National Institute of Hygiene and Epidemiology. 2.2. Materials of study 2.2.1. Materials and samples of study * Animals of study: buffaloes and bovines at diffenrent age, and freshwater snails * Samples of study: samples of fresh feces from Buffaloes and bovines; samples of soil from animal house (sediment) floors, aquatic grass samples, samples of snails without covers of the mouth, Fasciola samples collected from necropsied buffaloes and bovines, and bile samples, etc. 2.2.2. Instruments and chemicals: Light microscopy, counting chamber Mc Master, cattle bedding, jars for specimen, surgical instrument set, glass sinks, glass pots, plastic trays, enamel tray, anthelmintic drugs against liver fluke, etc. 2.3. Contents of study 2.3.1. Determining parasitic liver flukes in buffaloes and bovines in three Northern mountainous provinces 2.3.2. Epidemiological characteristics of fasciolosis caused by F. gigantica in buffaloes and bovines 2.3.2.1. Investigating current status of animal husbandry and the prevention of buffaloes and bovines from parasiolosis in three studied provinces 2.3.2.2. Situation of F. gigantica infection in buffaloes and bovines 2.3.2.3. Study on eggs and larvae of F. gigantica in the external environment and the intermediate host 2.3.3. Studying correlation between the number of F. gigantica eggs in 1 gram of feces with the number of parasitic flukes buffaloes and bovines 3 4 2.3.4. Studying prevention and control measures of fasioliasis caused by F. gigantica in buffaloes and bovines 2.4.. Methods of study * Determining the rate and intensity of infection F. gigantica of bufalo in 2 months, 4 months after the trial * Sugegesting the prevention and control procedures of fascioliasis caused by F. gigantica in buffaloes and bovines. 2.4.1. Method of necropsy and examination are used to collect and identify F. gigantica species in buffaloes and bovines in Thai Nguyen, Bac Kan and Tuyen Quang province 2.4.2. Method of investigating veterinary hygiene and prevention of fascioliasis caused by F. gigantica in cattle and bufaloes 2.5. Methods of data processing The data acquired were analyzed and processed using the method of Biostatistics (according to Nguyen Van Thien, 2008) based on Excel 2003 and Minitab 14.0 software. 2.4.3. Methodologies for studying epedemic charateristics of Fascipla spp. in buffaloes and bovines: use of Benedek sedimentation method (1943) for examination of Fasciola eggs. CHAPTER 3 RESULTS AND DISCUSSION 2.4.4. Studying eggs and larvae of F. gigantica in the external environment and the intermediate host 2.4.5. Studying lifetime of F. gigantica eggs in the external environment (when not released into water) 3.1. Results of the species determination of parasitic Fasciola spp. in buffaloes and bovines in three provinces (Thai Nguyen, Bac Kan and Tuyen Quang) 2.4.6. Study of time of miracidium released from eggs and survival in water 2.4.7. Study on the duration of F. gigantica larval in Lymnae viridis snails - intermediate hosts 2.4.8. Method of determining correlation between the number of F. gigantica eggs in 1 gram of feces and the number of parasitic liver flukes in buffaloes and bovines. 2.4.9. Method of determining the effect of medicines against F. gigantica 2.4.10. Trials on preventive measures of fascioliasis caused by F. gigantica in buffaloes and bovines. - Place of implementation: Dong Hy district - Thai Nguyen Nguyen province. - Trial object: buffalo at 2 - 4 years old. Content: * Arranging the test Table 3.1. Results of the necropsy examination of buffaloes and the collection of liver flukes Place (province) Thai Nguyen Bac Kan Tuyen Quang Total Number of necropsied buffaloes (bufalo) Number of infected buffaloes (bufalo) Percentage (%) Infection intensity (liver fluke/bufalo) 150 150 150 450 89 78 97 264 59.33 52.00 64.67 58.67 5 - 89 3 - 72 6 - 78 3 - 89 Table 3.2. Results of the necropsy examination of bovines and the collection of liver flukes Place (province) Thai Nguyen Bac Kan Tuyen Quang Total Number of necropsied bovines (bovine) Number of infected bovines (bovine) Percentage (%) Infection intensity (liver fluke/(bovine) 20 20 20 60 8 6 8 22 40.00 30.00 40.00 36.67 7 - 52 1 - 45 4 - 64 1 - 64 5 6 The results in table 3.1 and 3.2 show that at post - mortem examination of 450 buffaloes and 60 bovines in three provinces, the infection rate in buffaloes and bovines was 58.67% and 36.67%, respectively. compared with the 2 samples mentioned above. So the intermediate forms were also F. gigantica species. Table 3.3. Indentification of parasitic liver fluke species in buffaloes and bovines Place (province) Thai Nguyen Bac Kan Tuyen Quang Total Number of liver flukes for determinat ion (fluke) 262 356 538 1156 3.2. Epidemiological characteristics of fascioliasis caused by Fasciola in buffaloes and bovines 3.2.2. Situation of F. gigantica infection in three Northern mountainous provinces Results of species determination Fasciola Intermidiate Fasciola hepatica forms between gigantica species species the 2 species Number of flukes % Number of flukes % Number of flukes % 258 347 517 1122 98.47 97.47 96.10 97.06 0 0 0 0 0 0 0 0 4 9 21 34 1.19 2.29 4.06 2.94 Table 3.3 shows that in the total of 1156 liver fluckes classified, 97.06% of them was Fasciola gigantica, this was from 96.10% - 98.47% between the provinces, and there was no fluke belonging to Fasciola hepatica species. However, there were 34 flukes (2.94%) in the intermediate forms between the two species (F. gigantica and F. hepatica) (these flukes had "shoulder" but not obvious). So we had redefined these samples by biological molecular methods. The result of DNA sequencing of three reprenstative samples showed that 99% of the samples were homologous with the gen bank of F. gigantica. Thus, the flukes in the intermediate forms were also F. gigantica species. Comparing nucleotide sequence and amino acid sequence, it is obvious that 2 samples of F. gigantica having the same sequences. The other one had 5 nucleotides and 3 amino acids different 3.2.2.1. Infection rate and intensity of F. gigantica in buffaloes and bovines in Thai Nguyen, Bac Kan and Tuyen Quang province Table 3.6. Infection rate and intensity of F. gigantica in buffaloes in the places studied Place (province, district) * Thai Nguyen Dong Hy Vo Nhai Dai Tu Number of buffaloes examined (buffalo) Number of infected buffaloes (buffalo) Percentage (%) n % n % n % 1800 600 600 600 851 335 238 278 47.28b 55.83c 39.67a 46.33b 447 162 151 134 52.52 48.36 63.45 48.20 262 111 58 93 30.79 33.13 2437 33.45 142 62 29 51 16.69 18.51 12.18 18.35 40.72a 53.33b 31.67a 37.17a 461 186 141 134 62.89 58.13 74.21 60.09 191 102 30 59 26.06 31.87 15.79 26.46 81 32 19 30 11.05 10.00 10.00 13.45 51.89c 56.00b 45.83a 568 228 166 60.81 67,86 60.36 289 89 82 30.94 26.49 29.82 77 19 27 8.25 5.65 9.82 3.83b 174 53.87 118 36.53 31 9.60 46.63 1.476 58.62 ≤ 200 Infection intensity (eggs/g feces) > 200 - 500 > 500 χ2= 31.779; P = 0.000 1800 733 600 320 600 190 600 223 χ2= 63.055; P= 0.000 * Tuyen Quang 1800 934 Yen Son 600 336 Ham Yen 600 275 * Bac Kan Cho Moi Bach Thong Ngan Son Tuyen Quang city 600 323 2 Total χ = 13.784; P = 0.001 5400 2518 742 29.47 300 χ2= 45.551; P = 0.000 * Notes: In vertical line, the numbers with superscriptions are different remarkably in stastistics. 11.91 7 8 Table 3.6 shows that in three provinces the infection rate of F. gigantica in buffaloes was 46.63%, ranging from 40.72% to 51.89%. The difference between the three provinces was significant (P < 0.001). Particularly, in Tuyen Quang, the infection rate in buffaloes was highest (51.89%), followed by Thai Nguyen (47.28%), the lowest was in Bac Kan proince (40.72%). (38.57%), and the lowest rate was in Bac Kan province (29.84%). However, the differences in prevalence of bovines Fasciola in Thai Nguyen and Tuyen Quang were not significant (P > 0.05). In different districts the prevalence was apparent (P < 0.001), but in Tuyen Quang province, the differences in prevalence between districts were not clear (P > 0.05). In term of infection intensity, there was lighter and more moderate intensity in bovines compared with buffaloes, but the severe infection intensity was less. In general, the infection intensity in buffaloes was mild. However, buffaloes infected intensity was accounted for 58.62%; the average intensity made up 29.47%; while the severe intensity accounted for 11.91%. 3.2.2.3. Infection rate and intensity of F. gigantica in buffaloes and bovines acording to seasons Table 3.7. Infection rate and intensity of F. gigantica in bovines in the places investigated Number of Number of infected Percentage Places (province, bovines district) examined bovines (%) (bovine) (bovine) * Thai Nguyen Dong Hy Vo Nhai Dai Tu 630 243 ≤ n 200 % Infection intensity eggs/g feces) > 200 - 500 n % n > 500 % 38.57b 144 59.26 75 30.86 24 9.88 b Table 3.10. Infection rate and intensity of F. gigantica in buffaloes according to seasons Number of Number Places buffaloes of infected Percentage Season (province) examined buffaloes (%) (buffalo (buffalo) 210 110 210 63 210 70 χ2= 25.846; P = 0.000 * Bac Kan 630 188 ChoMoi 210 80 Bach Thong 210 69 Ngan Son 210 39 χ2= 20.485; P= 0.000 Tuyen Quang 630 225 Yen Son 210 67 Ham Yen 210 75 52.38 30.00a 33.33a 47 45 52 42.73 71.43 74.29 44 15 16 40.00 23.81 22.86 19 3 2 17.27 4.76 2.85 Sping Summer Thai Nguyen Autumn Winter 29.84a 38.10b 32.86b 18.57a 123 48 52 23 65.43 60.00 75.36 58.97 51 27 12 12 27.13 33.75 17.39 30.77 14 5 5 4 7.44 6.25 7.25 10.26 Sping Summer Bac Kan Autumn Winter 35.71b 31.90 35.71 136 39 46 60.45 58.21 61.33 77 25 23 34.22 37.31 30.67 12 3 6 5.33 4.48 8.00 Tuyen Quang city 39.52 51 61.45 29 34.94 3 3.61 Sping Summer Autumn Winter 34,71 403 61.43 203 30.95 50 7.62 210 83 Tuyen Quang 2 Total χ = 2.655; P = 0.265 1.890 656 2 χ = 11.015; P = 0.004 * Notes:I vertical line, the numbers with superscription are different remarkably in statistics. Table 3.7 shows that the infection rate of F. gigantica in bovines was 34.71% (much lower than buffaloes), the highest rate found in Thai Nguyen Total Sping Summer Autumn Winter 450 173 486 291 414 218 450 169 χ2= 66.906; P= 0.000 378 115 468 245 450 202 504 171 χ2= 55..697; P = 0.000 503 254 453 271 412 220 432 189 χ2= 23.653; P = 0.000 1.331 542 1.407 807 1.276 640 1.386 529 χ2= 129.022; P = 0.000 Infection intensity (eggs/g feces) > 500 ≤ 200 > 200 - 500 n % n % n % 38.44a 59.88c 52.66b 37.56a 78 165 117 87 45.09 56.70 53.67 51.48 63 80 69 50 36.42 27.49 31.65 29.59 30.42a 52.35c 44.89b 33.93a 80 146 119 116 69.57 59.59 58.91 67.84 31 66 54 40 26.96 4 3.47 26.94 33 13.47 26.73 29 14.36 23.39 15 8.77 50.50b 59.82c 53.40bc 43.75a 136 187 138 107 53.54 69.00 62.73 56.61 93 63 67 66 36.61 23.25 30.45 34.92 25 21 15 16 9.85 7.75 6.82 8.47 40.72a 57.36c 50.16b 38.17a 294 498 374 310 54.24 61.71 58.44 58.60 187 209 190 156 34.5 25.9 29.69 29.49 61 100 74 65 11.26 12.39 11.87 11.91 32 46 32 32 18.49 15.81 14.68 18.93 * Notes: in vertical line, the numbers that carry different letters are different in stastistic signinficance. 9 10 The results in table 3.10 shows that regarding infection rate: overall, the infection rates of liver flukes in buffaloes in three provinces were different with seasons. The highest rate was in Summer (57.36%), followed by Autumn (50.16%); Spring (40.72%) and the lowest was in Winter (38.17%). However the infection rate of liver flukes in buffaloes between Winter and Spring was not markedly different (P > 0.05). comparing shows that the prevalence between bulls and cows was not markedly different (P > 0.05). Statistically there were also no significant differences of infection intensity between bulls and cows. This result was consistent with study findings on cattle by Pham Ngoc Vinh, Nguyen Trong Kim (1997). The study results on goats by Nguyen Thi Kim Lan et al. (1999). Khan M. K. et al. (2009). * Regarding the intensity of infection: Buffaloes infected mainly in mild and moderate intensity in all four seasons of the year. In severe intensity the infection rates of liver flukes in buffaloes and bovines in summer and were the lowest in Winter. 3.2.2.4. Infection rate and intensity of F. gigantica in buffaloes and bovines with different sex. Table 3.13. Infection rate and intensity of F. gigantica in buffaloes with sex of cattle Places (province) Sex Number of Number of bovine Percentage infected (%) examined cattle(cattle) (cattle) Infection intensity (eggs/g feces) ≤ 200 n % > 200 - 500 n n > 500 % n Bull 239 85 35.56 51 60.00 26 30.59 8 9.41 Cow 391 158 40.41 93 58.86 49 31.01 16 10.13 Bull 284 86 30.28 53 61.63 28 32.56 5 5.81 Cow 346 102 29.48 70 68.63 23 22.55 9 8.82 Bull 164 58 35.37 34 58.62 21 36.21 4 5.17 Cow 466 167 35.84 102 61.08 56 33.53 8 5.39 Bull 687 229 33.33 138 60.26 75 32.75 17 6.99 Cow 1203 427 35.49 265 62.06 128 29.98 33 7.96 Thai Nguyen Bac Kan Tuyen Quang Total χ2= 0.901; P = 0.342 Table 3.13 shows that the infection rates of F. gigantica in bulls and cows were 33.33% and 35.49% respectively. Statistical 3.2.3. Study on eggs and larvae of F. gigantica in the external environment and in intermediate hosts 3.2.3.2. Contamination of F. gigantica eggs on grazing land Table 3.16. Contamination of F. gigantica eggs in grazing land of buffaloes and bovines Top soil from grazing land Pool Place Number of Number of Number of Number of Percentage percentage (province, district) samples positive samples positive (%) (%) examined samples (+) examined samples (+) * Thai Nguyen 450 33 7.33 450 61 13.56 Dong Hỷ 150 11 7.33 150 20 13.33 Vo Nhai 150 10 6.67 150 16 10.67 Dai Tu 150 12 8.00 150 25 16.67 * Bac Kan 450 38 8.44 450 63 14.00 Cho Moi 150 14 9.06 150 23 15.33 Bach Thong 150 13 8.84 150 25 16.67 Ngan Son 150 11 7.56 150 15 10.00 * TuyenQuang 450 42 9.33 450 78 17.33 Yen Son 150 16 10.67 150 32 21.33 Ham Yen 150 14 9.33 150 24 16.00 Tuyen 150 12 8.00 150 22 14.67 Quang city Total 1.350 113 8.37 1.350 202 14.96 χ2= 1.178; P = 0.555 χ2= 3.016; P = 0.221 Table 3.16 shows that overall 8.37% of the samples of grazing land surface and 14.96% of water sampes from the hollows on grazing land in the places of the three provinces under study were contaminated with liver fluke germs. 11 12 3.2.3.3. The distribution of freshwater snails - intermediate hosts of F. gigantica 3.2.3.4. The prevalence of liver fluke larvae in freshwater snails intermediate hosts Table 3.17. Results of freshwater snail classification Number of Lymnaea viridis Lymnaea Places snails species swinhoei species (province, district) classified n (%) n (%) (snail) * Thai Nguyen 2160 768 35.56 621 28.75 Dong Hỷ 720 224 31.11 195 27.08 Vo Nhai 720 293 40.69 235 32.64 Dai Tu 720 251 34.86 191 26.53 * Bac Kan 2160 663 30.70 437 20.23 Cho Moi 720 221 30.69 174 24.17 Bach Thong 720 230 31.94 147 20.42 Ngan Son 720 212 29.45 116 16.11 *Tuyen Quang 2160 520 24.07 978 45.28 Yen Son 720 234 32.50 286 39.72 Ham Yen 720 120 16.67 365 50.69 Tuyen Quang city 720 166 23.05 327 45.42 Total 6480 1951 30.11 2036 31.42 Other species* n (%) Table 3.19. The prevalence of F. gigantica larvae in freshwater snails Place (province) Description Snail species 771 301 192 278 1.060 325 343 392 662 200 235 227 2493 35.69 41.81 26.67 38.61 49.07 45.14 47.64 54.44 30.65 27.78 32.64 31.53 38.47 Table 3.17 shows that in 6480 snails collected 30.11% belonged to the L. viridis species; 31.42% belonged to L. swinhoei species 38.47% were the other snail species (not the intermediate host of liver fluke). Thus more than 60% of the snails collected belonged to two species L. viridis and L. swinhoei. According to Dang Ngoc Thanh et al. (1980), Nguyen Trong Kim and Pham Ngoc Vinh (1997), intermediate hosts of Fasciola in Northern part of Viet Nam are two snails species: L. viridis and L. swinhoei. Results of snail classification in Thai Nguyen, Bac Kan and Tuyen Quang showed that two snail species accounted for a high proportion of snails collected. Common distribution with large numbers of two snail species creates favorable conditions for F. gigantica complete their life cycle. Number of snails examined (snail) Number of snails infected with F. gigantica larvae(snail) Percentage (%) n (snail) Sporocyst (%) n (snail) Redia % n (con) Cercaria (%) Thai Nguyen Bac Kan Tuyen Quang Lymnaea viridis Lymnaea swinhoei Lymnaea viridis Lymnaea swinhoei Lymnaea viridis Lymnaea swinhoei 768 621 663 437 520 978 108 40 173 41 208 318 14.06 55 50.93 63 58.33 66 61.11 6.44 13 32.50 19 47.50 17 42.50 26.09 95 54.91 97 56.07 104 60.12 9.38 18 43.90 25 60.98 22 53.66 40.00 76 36.54 96 46.15 103 49.52 32.52 124 38.99 131 41.19 141 44.34 Table 3:19 shows that on the prevalence of larvae in snails there were 14.06% of L. viridis and 6.44% of L. swinhoei in Thai Nguyen; 26.09% of L. viridis 9.38% L. swinhoei in Bac Kan; 40% of L. viridis and 32.52% of L. swinhoei in Tuyen Quang being infected with F. gigantica larvae. Thus, both species of snails were infected with liver fluke larvae ranging 20% 70%. In which L. viridis was infected with liver flukes with significantly higher rate compared with L. swinhoei (this shows in all three provinces studied). It is believed that L. viridis species was more susceptible to F. gigantica compared with L. swinhoei. - Regarding the prevalence of larval forms of F. gigantica in snail: L. viridis was infected with sporocyst larvae from 36.54% to 54.91%; Redia larvae from 46.15% to 58.33% and Cercaria larvae 49.52% to 61.11%. L. swinhoei infected with Sporocyst larvae from 32.50% to 43.90%; Redia larvae from 41.19% to 47.50% and Cercaria larvae from 42.50% to 53.66%. 13 14 3.2.3.8. Study on the time of Miracidium hatched released and surviving in water 3.2.3.9. Study of time for development of F. gigantica larvae in snail - intermediate hosts Table 3.26. Time to complete the larval stages of F. gigantica (from the time the eggs fall into the water) Table 3.24. Time of Miracidium hatched and released into water (from releasing eggs in water) Initial time of Time for Miracidium all Miracidium Number Temperature releaseed from released from eggs Season Experiment of and pH eggs samples water X ± mX total ( X ± mX ) total (day) (day) (day) (day) 15.40 ± 0.45 14.40 ± 0.36 I 10 22 - 23oC, Spring 14.60 ± 0.41 14 16.80 ± 0.25 16 II 10 6-7 15.20 ± 0.25 15.20 ± 0.62 III 10 ( Summer Autumn Winter I 10 II 10 III 10 I 10 II 10 III 10 I 10 II 10 III 10 o 26 - 27 C, 6-7 ) 8.80 ± 0.33 8.90 ± 0.31 Season Number of samples Spring 5 Summer 5 Atumn 5 Winter 5 9.70 ± 1.58 8 9.50 ± 0.23 24 - 25oC, 12.00 ± 0.58 6-7 13.70 ± 0.30 11 11.40 ± 0.21 20.00 ± 0.47 18 - 19oC, 19.30 ± 0.74 19 6-7 19.90 ± 0.42 8.30 ± 1.78 10 10.10 ± 2.18 14.10 ± 2.79 12.20 ± 2.81 15 15.40 ± 2.92 19.70 ± 0.63 21.10 ± 0.79 21 20.30 ± 1.57 Table 3.24 shows that in Spring Miracidium hached and released from egg earliest was 14 days, no later than 16 days; In Summer the earliest was 8 days and no later than 10 days; In Autumn, the earliest time was 11 days and no later than 15 days; In Winter the earliest was 19 days and no later than 21 days. Thus, the time required for development from the egg into the water to hatch into miracidium varied from 8 to 21 days. In winter, due to cold weather the development of eggs in water lasted longer. Whereas, in Summer the weather was hot, the development of eggs was markedly shorter. Stage of larvae Egg à Miracidium Miracidium à Sporocyst Sporocyst à Redia Redia à Cercaria Cercaria à Adolescaria * Egg à Adolescaria Egg à Miracidium Miracidium à Sporocyst Sporocyst à Redia Redia à Cercaria Cercaria à Adolescaria * Egg à Adolescaria Egg à Miracidium Miracidium à Sporocyst Sporocyst à Redia Redia à Cercaria Cercaria à Adolescaria * Egg à Adolescaria egg à Miracidium Miracidium à Sporocyst Sporocyst à Redia Redia à Cercaria Cercaria à Adolescaria * Egg à Adolescaria Time to complete (day) 14 - 16 3-4 6-8 18 - 20 2 - 4* 41 - 48 8 - 10 2-3 4 -7 15 - 17 2 – 4* 29 - 37 11 - 15 3–4 5–8 17 - 18 2 - 4* 36 - 45 19 - 21 4-5 8-9 20 - 21 3 - 5* 51 - 56 The results in table 3.26 shows that in winter, the time of developing egg and larval forms in the snail were the longest. The rule of the development time of larvae in intermediate hosts with seasons reveals that the temperature is an important factor and a major influence on the development of eggs and larvae of F. gigantica in the external environment and in intermediate hosts. 15 16 3.3. Studies of correlation between number of parasitic F. gigantica in buffaloes and bovines with number of eggs in 1 g of feces. number of parasitic Fasciola / buffalo and number of eggs /g feces was correlated. The correlation coefficient of r = 0.96 indicates a very close correlation. * Correlation between number of eggs /g faeces and number of parasitic Fasciola / bovine was as follows: The linear regression equation y = a + bx (y: number of eggs/g faeces. x: number of parasitic Fasciola /bovine) Among them: a = 4.145 b = 8.094 → the linear regression equation: y = 4.145 + 8.094x The correlation coefficient: r = 0.969 * The correlation between the number of parasitic Fasciola /buffalo with number of eggs in 1 gram of feces was determined on Minitab 14.0 software. The results were as follows: Linear regression equation: y = a + bx (y: number of eggs / g of feces. x: number of parasitic Fasciola / buffalo) Among them: a = 0.194 b = 8.101 → linear regression equation: y = 0.194 + 8.101 x Correlation coefficient: r = 0.96 The correlation between the number of parasitic Fasciola / buffalo and number of eggs /g feces is illustrated in Figure 3.10 Correlation between number of F. gigantica eggs /g faeces and number of parasitic Fasciola /bovine is illustrated in figure 3.11. 8 00 5 00 7 00 6 00 4 00 3 00 4 00 y y 5 00 3 00 2 00 2 00 1 00 1 00 0 0 10 20 30 40 50 60 70 80 90 x 0 0 10 20 30 40 50 60 70 x Figure 3.10. The graph illustrating the equation y = a + bx of the relationship between the number of parasitic Fasciola /buffalo with number of eggs /g faeces. The graph 3.10 shows that number of corresponding points between the parasite count through buffaloes at postmortern examination with number of eggs in 1 gram of feces were mostly located around the curvature of the linear regression equation y = a + bx, away from lower left to upper right, there were no points lying far from this curvature. That means that the correlation between the Figure 3.11. The graph illustrating the equation y = a + bx of the relationship between number of parasitic Fasciola /bovine with number of eggs /g faeces. The graph 3.11 shows that number of corresponding points between the parasite count through bovines at necropsy with number of eggs in 1 gram of feces were mostly located around the curvature of the linear regression equation y = a + bx, from lower left to upper right, there was only one point lying a bit far from this curvature. 17 18 That means that the correlation between the number of parasitic fasciola /bovines and number of eggs /g of feces was direct correlated. The correlation coefficient r = 0.969 shows that this is closely corelated. 3.4. Study of prevention and treatment measures of fascioliasis caused by F. gigantica in buffaloes and bovines 3.4.1. Identifying drug for prevention and treatment of fascioliasis caused by F. gigantica that is highly effective and safe 3.4.1.2. Trial on efficacy and safety of some drugs against F. gigantica in buffaloes and bovines * Testing on a small scale Table 3.30. Efficacy of three drugs against F. gigantica in experimental bovine Table 3.30 shows that three drugs used for treatment of cattle infected with F. gigantica at dose above, efficacy achieved similar to the test on buffaloes (15/15 removed parasite eggs in feces after 10 days of dosing, 3/15 of cattle that were necropsied to examine liver flukes in bile ducts after 25-35 days using dewormers). At the same time, all 15 cows were no signs of any unusual after dosing. Thus, doses of three drugs used for cattle were safe. * Trial on a large scale We tested the efficacy of triclabendazole treatment at dose 15 mg /kg B.W albendazole and nitroxinil - 25 at dose 12mg /kg B.W in 450 buffaloes and 270 bovines in the studied places. The results are illustrated in table 3.31 and 3.32. Table 3.31. Efficacy of three dewormers angainst F. gigantica in cattle on a large scale Day after treatment Necropsy after treatment Prior to (eggs / g feces) Order treatment Drugs and dosage number Day (eggs / g Comparison of bovine at necropsy feces) 5 10 15 sluke/cattle after (fluke) treatment Albendazol (12mg/kg B.W) Triclabendazole (15 mg/kg kg B.W) Nitroxinil - 25 (12mg/kg kg B.W) 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 285 315 375 405 255 450 410 380 295 320 260 350 300 430 330 30 30 45 60 15 55 60 40 30 35 25 35 30 45 40 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 35 25 30 - Before dewworming Time 0 12 50 465.00 ± 56.00 3 45.60 ±10.30 47 94.00 Triclabendazole 15 50 418.30 ± 48.65 0 0 50 100 Nitroxinil - 25 12 50 445.28 ± 30.50 3 35.20 ±8.70 47 94.00 Albendazol 12 50 520.80 ± 53.10 5 60.80 ±14.40 45 90.00 II Triclabendazole 15 50 560.00 ± 46.50 0 0 50 100 Nitroxinil - 25 12 50 490.35 ± 28.50 4 50.00 ±13.40 46 92.00 Albendazol 12 50 420.30 ± 46.00 5 25.80 ±6.72 45 90.00 III Triclabendazole 15 50 380.50 ± 60.78 0 0 50 100 12 50 415.27 ± 23.60 5 40.50 ± 9.86 45 90.00 Nitroxinil - 25 0 Dewworming eficacy Number of Number of Infection intensty Number of Intensity Dose (mg/ buffaloes infected infected ( X ± mx ) ( X ± m x ) cleared from Efficacyy kg B.W) buffaloes buffaloes (%) eggs (buffaloes) (eggs/ feces g ) (buffaloes (eggs/ feces g ) (buffalo) Albendazol I 0 Type of drug 15 days after deworming Effectiveness of albendazole was quite high, reaching 90 - 94%. the infection intensity decreased from 420-465 eggs /g feces to 2560 eggs /g faeces. Nitroxinil - 25 also had althelmintic effectiveness reaching 90 - 94%, reducing infection intensity from 415 - 490 eggs 19 20 The results in table 3:31 shows that in all three treatments. triclabendazole has the highest althelmintic efficacy (100%). 15 days after dosing, examining feces, eggs were not detected. The althelmintic /g of feces to 35-50 eggs /g of feces 15 days after deworming. Observing buffaloes before and after dosing, we found that the activities of the whole buffaloes were normal, eating and ruminating normally, no buffaloes had any side effects after receiving the drug. So we evaluated: dose levels of three drugs used were 100% safe for cattle. Table 3.35. Infection rate and intensity of F. gigantica after 4 months of the experiment 3.4.2. Trial on therapeutic measures in treating fascioliasis caused by F. gigantica in buffaloes Infection rate and intensity in buffaloes of the experimental group and the control prior to the experiment are illustrated in table 3.33. Table 3.33. Infection rate and intensity of F. gigantica in buffaloes prior to the experiment Group Description Number of buffaloes examined (buffalo) Number of infected buffaloes ((buffalo) Percentage (%) n ≤ 200 % Infection n intensity >200 (number of eggs/ - 500 % gram of feces) n > 500 % Experime ntal group Control Statistical significance (P) 80 80 - 31 30 - 38.75 18 58.06 11 35.48 2 6.45 37.50 17 56.67 11 36.67 2 6.67 > 0.05 > 0.05 > 0.05 > 0.05 Table 3.33 shows that infection rate and intensity of F. gigantica in the experimental group and control group prior the experiment were equivalent 3.4.2.2. Infection rate and intensity of F. gigantica after 4 months of the experiment Group Description Number of buffaloes examined (buffalo) Experimental group Control Statistical significance (P) 80 80 - 14 36 - 17.50 13 92.86 1 7.14 0 0.00 45.00 24 66.67 10 27.78 2 5.56 < 0.001 < 0.001 < 0.001 < 0.001 Number of infected buffaloes (buffalo) Percentage (%) Infection intensity (number of eggs/ gram of feces) ≤ 200 >200 -500 > 500 n % n % n % Table 3.35 shows that the infection rate after 4 months of experiment, the prevalence of F. gigantica in buffaloes in the experimental group was 17.50%, while the prevalence in the control group was 45.00%. This difference was very clear (P <0.001). The intensity of infection: + Mild infection intensity: 92.86% of buffaloes were infected with liver fluke in experimental groups in light intensity, whereas in the control group, mild prevalence was 66.67%, this difference was very clear (P <0.001). + Average intensity of infection: In experimental group mean intensity of infection was 7.14%, while that in the control group was 27.78%. + Heavy infection intensity: There were no buffaloes in experimental group being infected in severe intensity, while the proportion of severe infections in the control group was 5.56%. The difference had statistical significance (P <0.001). 21 22 3.4.3. Recommendation of combining preventive and control procedures to prevent fascioliasis caused by F. gigantica in buffaloes. 5. Killing intermediate hosts of Fasciola, drainage of water, drying muddy, wet pastures and grazing land are effective measures to kill Lymnaea spp... Fresh snails can be killed by using lime poder, copper sulffate... or rearing waterfowls (ducks, muscovy ducks, geese) and black carps (mylopharyngodon piceus). However it depends on place to apply appropriate measures in order to kill intermediate hosts of Fasciola effectively. From the results of the study we recommend combining preventive and control procedures against fascioliasis in buffaloes and bovines follows: 1 Deworming F. gigantica for buffaloes and bovines: Deworm periodically liver fluke twice/ year (phase 1 in April- May; phase 2 in September - October) for the whole herd of buffaloes and bovine once examining feces shows that 20-30% of these animals are infected with liver fluke. Three drugs at dose levels having been tested (triclabendazole, at dose 15 mg /kg B.W, albendazol and nitroxinil - 25, at dose of 12 mg /kg) all were effective in removing liver fluke. Depending on the locale and on the specific conditions, one of those drugs to remove liver fluke from buffaloes can be selected. However, the use of triclabendazole is the best effective. 2 Treatment of cattle manure to kill eggs of F. gigantica: daily collect manure from stable and put in pitches of manure composting, or gather them in one place as a pile and cover with thick mud contained 5 -10 cm 3 Cleaning cattle stables every 2 months, using disinfectants to spray in order to kill F. gigantica eggs in the floor of cattle houses and their surrounding areas. 4. Improving pastures, grazing land for buffaloes and bovines: Removing stagnant water on pastures, collecting manure from grazing land for composting are needed in order to limit the spread and development of eggs and larvae of trematodes in the environment. 6. Strengthening the care and feeding animals: For buffaloes and bovines at different ages (especially calving cows), it is necessary to ensure adequate rations in terms of quantity and quality. Cattle over 8 years old must be culled so that they will not shed F. gigantica eggs into the environment. CONCLUSIONS AND RECOMMENDATIONS 1. Conclusion 1- 450 buffaloes and 60 cattle in 3 provinces were necropsied, the infection rate in buffaloes was 58.67% and in cattle was 36.67%. F. gigantica is the only species of trematode parasitising liver and bile ducts of buffaloes and bovines the places invstigated, common prevalence is 100% 2 - Epidemiological characteristics: + Status of animal husbandry and prevention of parasitosis in buffaloes and bovines in 3 provinces is not good, especially preventive measures of parasitosis in buffaloes and cattle. + The prevalence of F. gigantica in buffaloes and bovinein three provinces is 46.63%; in cattle is 34.71%. The rate and intensity of infection increases with aging. Buffaloes and bovines are infected with F. gigantica in larger number and more severe in summer. The rate and intensity of infection in the other seasons are lower and lighter. 23 24 Variation in the rate and intensity of infection of cows and bulls are markedly irregular. 3- Correlation between the number of eggs /g of feces with number of parasitic liver flukes / animal is correlated quite closely, in accordance with the linear regression equation y = a + bx. + The areas around stables and barns of buffaloes and bovines are contaminated with F. gigantica eggs at the rate of 20 - 43%. 8.37% of the soil samples from the surface of grazing land and 14.96% of water samples from the hollows on grazing land were contaminated with F. gigantica eggs. + L. viridis and L. swinhoei - intermediate hosts of F. gigantica are commonly distributed in all investigated places. The infection rate of liver fluke larvae in snails varies 20 - 70%. Infection rate of L. viridis is 61%. + Samples of aquatic grasses in Thai Nguyen. Bac Kan and Tuyen Quang are contaminated with Adolescaria at the rate of 14 - 23%. + In Spring, survival of F. gigantica eggs lasts 12 - 48 days (in dry feces), 43 - 125 days (in wet feces). Survival of eggs is shorter than that in Summer, longer in Autumn and the longest in winter (19 - 80 days in dry feces, 75 - 160 days in wet feces). The survival time of eggs is short in dry soil, longer in wet soil. In Summer, the survival time of eggs in the ground is shorter than in other seasons. + In Spring, Miracidium hatches and is releases from the egg earliest in 14 days, no later than 16 days; in Summer time this is 8 and 10 days; in Autumn 11 and 15 days; in Winter 19 and 21 days. After hatching from the egg, Miracidium in the water is not more than 10 hours (in Spring), 11 hours (in Summer), 14 hours (in autumn) and 9 hours (in Winter). The time from releasing F. gigantica eggs in water until forming Adolescaria is the longest in winter (51 - 56 days), followed by Spring (41 - 48 days). Autumn (36 - 45 days) and the shortest is in Summer (29 - 37 days). 4- At the recommeded doses, efficacy of han - dertin B and fasciolid against F. gigantica is not high (78 - 86%). Efficacy of triclabendazole against F. gigantica at dose of 15 mg /kg B.W is 100%; Efficacy of albendazole and nitroxinil - 25 at the same dose (12mg /kg B.W) reaches 90 - 96%. All of 3 drugs are safe for cattle and buffaloes. 5- Prevention and control of F. gigantica in buffaloes and cattle should be performed by using five synchronized key measures (deworming liver flukes with triclabendazole, composting manure. killing snails, intermediate hosts, sanitation of feed, water, stables and grazing land), culling the animals older than 8 years old. 2. Recommendation In the Northern mountainous provinces, cattle are raised under grazing methods, utilizing the existence of natural conditions. Therefore, it is needed to implement the combined prevention and control of Fascioliasis gigantica for buffaloes and bovines as the six contents of the above conclusion. This is believed to help reducing the economic losses caused by the parasiolois disease, improving livestock productivity, and promoting the industry of animal husbandry. 25
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