Nghiên cứu bệnh nhiễm độc tố botulin của vi khuẩn clostridium botulinum trên vịt tại một số tỉnh đồng bằng sông cửu long tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY DOCTOR THESIS SUMMARY Major: PATHOLOGY AND TREATMENT OF ANIMALS Code: 62 64 01 02 NGUYEN THU TAM STUDY ON BOTULIN POISONING DISEASE OF Clostridium botulinum ON DUCKS IN THE MEKONG DELTA, VIETNAM Can Tho- 2020 THIS STUDY WAS ACCOMPLISHED AT CANTHO UNIVERSITY SCIENCE INSTRUCTOR: ASSOC PROF NGUYEN ĐUC HIEN The thesis was defended with the doctoral examination committee at the school level Place:…………………………………., Can Tho University At… … day …… month … year ……… Opponent 1: Opponent 2: Opponent 3: The thesis could be found at the library: Learning Resource Center, Can Tho University National Library of Vietnam LIST OF PUBLISHED SCIENTIFIC ARTICLES Nguyen Thu Tam, Dang Ngoc Le 2014 Survey on the leisons in mice after injected the intestinal suspension originated from the ducks infected botulin toxin Can Tho University – Journal of Science, Agriculture Edition 2014: 107-110 Nguyen Thu Tam, Ly Thi Lien Khai, Nguyen Duc Hien 2016 The isolation and identification of Clostridium spp in the field soil in Phu Tan and Chau Phu district, An Giang province Science and Technology Journal of Agriculture and Rural Development, 11:7377 Nguyen Thu Tam, Tran Thi Phan, Nguyen Duc Hien 2016 The isolation and identification of Clostridium botulinum from limberneck ducks in Tan Phu and Tri Ton district, An Giang province Science and Technology Journal of Agriculture and Rural Development, 11:147-150 Nguyen Thu Tam, Nguyen Duc Hien, Ho Thi Viet Thu 2016 Diagnosis of “Cum can” disease on ducks via the experiment in mice Can Tho University – Journal of Science, Agriculture Edition 2016: 125-130 Nguyen Thu Tam, Nguyen Duc Hien, Ho Thi Viet Thu The isolation and identification of Clostridium botulinum on snails (Pila conica) and crabs (Somannia theplusa) in Can Tho city, An Giang and Kien Giang province Can Tho University – Journal of Science, Agriculture Edition 2016: 131-134 Chapter INTRODUCTION 1.1 The imperative of this study The Mekong Delta area has interlacing river systems, tropical climate, a wide area of rice cultivation, aquatic animals, and so on Therefore, it is convenient for raising free-grazing ducks The number of free-grazing ducks is about 31.5 million, got 70% of a total of ducks in this region as well as 40% of a total of ducks in Vietnam The free-grazing method has some benefits such as taking natural feed or spilled rice after harvesting to significantly reduce costs in the livestock However, it has potential risks due to uncontrol the grazing environment, and diseases can outbreak severely Recently, “Limberneck” disease or “Cum Can” (in Vietnamese) has been one of the common diseases that occurred in the free-grazing ducks in the Mekong Delta This disease is in the waterfowl caused by botulin toxin of Clostridium botulinum; thus, it is also called the botulism disease Clostridium botulinum is completely anaerobic and producing spores with the oval shapes This pathogen usually exists in the soil, especially in the sedimentary mud areas, mollusks corpses, and in the anaimal intestine It can produce severe botulinum neurotoxin to destroy all the central nervous system (Todar, 2009) Ducks infected with this toxin show some symptoms such as paralysis of the neck, eyelids, wings, legs, and a high rate of mortality; it causes a significant loss for the farmers (Rocke and Friend, 1998) In human and veterinary medicine, the botulism disease has been studied in humans, poultry, waterfowl However, the research of the botulinum disease as well as risk factors, bio-characteristics of C botulinum are limited in the Mekong Delta and also in Vietnam Research is necessary to for supplying information about C botulinum and the botulism disease in the free-grazing ducks in the Mekong Delta Therefore, the study “Study on botulin poisoning disease of Clostridium botulinum on ducks in the Mekong Delta” was carried out 1.2 The aim of this study - Determination of the frequency of the botulism disease on the free-grazing ducks in the Mekong Delta - Determination of the prevalence of Clostridium botulinum and the type of botulin toxin on the free-grazing ducks - Determination of the prevalence of C botulinum in the grazing environment - Determination of the pathogenicity of isolated C botulinum strains originated in the Mekong Delta 1.3 The new scientific distributions - Scientific pieces of evidence firstly about the prevalence of the botulism disease on the free-grazing ducks in the Mekong Delta - Determination of botulin type in the infected ducks in the Mekong Delta - Determination of botulin types of C botulinum in the infected ducks in the Mekong Delta - Application of the mouse bioassay to indicate the botulin infection due to Clostridium botulinum in ducks in Vietnam 1.4 The scientific meaning of this study It is the first study that systematic research about the botulism disease on the free-grazing ducks in the Mekong Delta, Vietnam From those results, a scientific process can be formed for diagnosing this disease as well as preventing and treatments of the botulism disease on the free-grazing ducks in the Mekong Delta and Vietnam Chapter RESEARCH CONTENTS AND METHODS 3.1 The research materials 3.1.1 The research period and places: This study was carried out from October 2013 to October 2018 3.1.2 Research places 3.1.2.1 Sample collected places Samples were collected in An Giang, Can Tho, Hau Giang, and Kien Giang province of the Mekong Delta, Vietnam 3.1.2.2 Samples analysis and the experiment on mice The isolation, identification and toxin examination of C botulinum were done at the Specialized Veterinary laboratory 3, Department of Veterinary Medicine, College of Agriculture, Can Tho University 3.1.2.3 The field-experiment The toxin test of C botulinum on laying ducks were done at Vemedim Corporation Company, Thoi Thanh ward, Thoi Lai District, Can Tho city 3.2 The research equipments Equipments and requirements - The questionnaire (Appendix 1) - The information was collected from the Statistical Yearbook and Sub-Department of Animal Health about the total of poultry/waterfowl population, climate, duck breedings, and diseases in the sample collection places - Microbiology stuffs Chemicals and media - Alcohol 96o, alcohol 70o, distiled water, cedar oil, Crystal violet, Lugol, Safranine, Bromocresole purple, Gelatin Phosphat Buffer… (Merck, Germany); sheep blood (Nam Khoa, Vietnam) - Examined antibiotics: amikacin, ampicillin, amoxicillin, ceftiofur, cephalexin, doxycycline, florfenicol, fosfomycin, marbofloxacin, norfloxacin (Oxoid, Bristish) - Mac Farland 0, (Biorad) - Media: NB (Nutrient broth, Merck, Germany), TSA (Tryptis Soy Agar, Merck, Germany), CMM (Cooked Meat Media, Oxoid, Bristish), EYA (Egg Yorlk Agar, Merck, Gemany), Thioglycollate (Merck, Germany), SFP Agar Base (Difco, USA), MHA (Mueller Hinton Agar, Merck, Germany) - Carbohydrate tests: Lactose, glucose, maltose, saccarose (Merck, Germany) - Biochemical test kit: API 20A (Biorad, USA) - TPGY broth: 5% Trypticase, 0.5% Pepton, 0.4% Glucose, 2% Yeast extract, 0.1% Sodium thioglycolate; CMM (Cooked Meat Medium, Oxoid, Bristish) - Antitoxins: type C, D, E (10UI/ml) (Statens Serum Institute, Denmark) 3.3 Research contents 3.3.1 Content 1: The botulism disease on the free-grazing ducks in the Mekong Delta 3.3.1.1 The research aim: Evaluation of the frequency of the botulism disease on the free-grazing ducks in the Mekong Delta 3.3.1.2 The research objects: The free-grazing ducks infected botulism disease in the Mekong Delta Those ducks included meat ducks and laying ducks The meat duck were raised around 4-12 weeks old while laying ducks were chosen after 12 weeks old 3.3.1.3 The research method a The livestock situation of ducks in the Mekong Delta The data were collected via the retrospective investigation from the Statistic Department of Sub-Department of Animal Health in those provinces including the total number of free-grazing ducks, advantages or disadvantages of natural condition for raising those ducks from 2012 to 2014 b The prevalence of the botulism disease on the freegrazing ducks in the Mekong Delta - The cross-sectional investigation was done to clarify the prevalence of the botulism disease on ducks in provinces - The number of samples were showed in Table 3.1 Table 3.1 The distribution of collected samples in provinces Places An Giang Can Tho Hau Giang Kien Giang Total No of meat ducks 20,000 19,000 18,000 22,000 79,000 No of laying ducks 25,800 28,700 25,350 31,200 108,505 Step Investigation Coporating with local veterinarians and owners to collect information about the number of duck flocks, health condition, nervous performances, activities If ducks showed symptoms of the botulism disease such as ruffled feathers, less eating, weak legs, paralysis of neck/eyelids/wings/legs, researcher used the questionnaires to collect more data and bought the duck samples Step 2: Collecting samples Speciments were collected on alive or just died ducks (1) To alive ducks: blood was withdrawed 5-10ml from the neck veins; ducks were examined the gross lesions and also collected the intestinal content and liver (2) To just died ducks: ducks were dissected to check the gross lesions and collected samples as in alive ducks In each duck flocks, it collected 1-5 ducks 3.3.1.4 Observed factors + The ratio of the botulism disease on the free-grazing ducks in the Mekong Delta + The frequency of clinical symptoms of the botulism disease on ducks + The frequency of lesions of the botulism disease on ducks 3.3.2 Content 2: The isolation of C botulinum and determination of botulin toxin on the free-grazing ducks infected botulism disease 3.3.2.1 The research aim: Determination of the prevalence of C botulinum and botulin toxin types on the free-grazing ducks 3.3.2.2 The research objects: The sera and speciments of infected ducks in Content 3.3.2.3 The research method a The isolation of C botulinum from speciments of infected ducks The isolation of C botulinum from speciments of infected ducks was carried out following Lindstrom and Korkeala (2006) and having modifications This method was showed in Fig 3.1 Contents in intestine and liver Culture on the blood agar and SFP Soil, water, crabs, snails CMM media Typical colonies Gram stain (bacilli, Gr+, spores) Biochemical characteristics by usingAPI 20A Clostridium botulinum and keeping strains in CMM/ thioglycolate Subculture (identical colonies) (All steps were done and incubated in the anaerobic condition at 37oC/24-48h) Fig 3.1 The method of isolation and identification of Clostridium botulinum (Lindstrom and Korkeala, 2006) (having modification) Fig 3.2 C botulinum colonies on blood agar Fig 3.3 C botulinum colonies on SFP Fig 3.4 The spores of C botulinum under the microscope (X 100) b The biochemical characteristics of C botulinum: those characteristics were examined by using API 20A kit (following the manufacture) Table 3.2 The biochemical characteristics of C botulinum using API20A kit c Determination of botulin toxin of C botulinum - Sera: sera were melt and filtrated via 0.45µm filter to collect the clear supernatant - Experimental animals: SPF mice that bought in Pasteur Institute, Ho Chi Minh were raised in days to adopt the environment before using in the experiment - Experimental design Table 3.3 The experimental design Group Group I Group II Control No of examined mice 400 400 20 Dose (ml/mouse) 0,5 0,5 0,5 - The filtrated supernatants were check the aseptic by culturing on blood agar and SFP medium Those samples were incubated at 37oC, 24h - The serum used in this study must be aseptic Examined results Mice in Group I showed symptoms such as paralysis of legs/eyelids, ruffled feathers, abdominal breathing or died while mice Examined results After days observed, mice were healthy It indicated that sera had the corresponding of toxin types; toxin in serum was neutralized by the added antitoxin Mice in the control group: healthy without abnormal symptoms 3.3.2.4 Observed factors - The prevalence of C botulinum in specimens - The rate of sera having toxin - The prevalence of toxin types - The frequency of clinical symptoms on mice infected with botulin toxin - The frequency of gross lesions on mice infected with botulin toxin 3.3.3 Content 3: Determination of risk factors causing the botulism disease on the free-grazing ducks in the Mekong Delta 3.3.3.1 The research aim: Evaluation of the prevalence of C botulinum and the pathogenicity of isolated strains from the grazing environment 3.3.3.2 The research objects: the environment samples (soil, water, crabs, snails) 3.3.3.4 The research method a Sample collection At the same time of collecting botulism infected ducks, the environment samples were collected in the same fields The number of samples were showed in Table 3.5 Table 3.5 The environment samples collected in this study Place An Giang Can Thơ Hau Giang Kien Giang Total No of soil samples 159 141 144 156 600 No of water samples 159 141 144 156 600 No of crab samples 63 42 50 61 216 No of snail samples 106 94 96 104 400 Total 645 563 582 646 2,436 - Soil and water were collected in one field Wet fields: taking soil at the depth of 5-10 cm Wet fields with mud: taking the mud on the surface of the field Each sample was 25-30g Water samples: taking 50-100ml and keeping in the sterile tubes - Crabs, snails were collected in one field Those crabs or snails were small or just died, and kept in the sterile tubes All samples were kept in the cool conditions (Franciosa et al., 1996) 10 b The isolation of C botulinum from the environment samples The method was same as the decription in 3.3.2.3 3.3.3.5 Observed factors - The prevalence of C botulinum in the soil and water - The prevalence of C botulinum in crabs and snails 3.3.4 Content 4: The pathogenicity of C botulinum isolated from infected ducks 3.3.4.1 The research aim: Determination of pathogenicity of isolated C botulinum strains 3.3.4.2 The research objects: isolated C botulinum strains in Content and Content 3.3.4.3 The research method a Antimicrobial susceptibility of C botulinum The disk-diffusion method of Kirby-Bauer was used in this study (Bauer et al., 1966) The sensibility of C botulinum to antibiotics were determined following the standards of Clinical and Laboratory Standards Institute (CLSI, 2019) - Method: the suspension of C botulinum was adjusted with MacFarland 0.5 to equal 108CFU/ml and spread on MHA medium After that, antibotics dics were put on the media how to be 2.5-3.5cm each other and 2cm to the edge of the petri disk Those samples were incubated at 37oC, 24h with CO2 After 24h, the zone diameter of antibiotics were measured to determine the susceptibility Table 3.6: The standard of zone diameter of antibiotics (CLSI, 2019) Zone diameter (mm) Content Antibiotics (μg) Sensitive Intermediate Amikacin 30 ≥ 17 15-16 Ampicillin 10 ≥ 17 14-16 Amoxicillin 25 ≥ 18 14-17 ceftiofur 30 ≥21 18-20 Cephalexin 30 ≥ 18 15-17 Doxycycline 30 ≥13 10-12 Florfenicol 30 ≥19 15-18 Fosformycin 200 ≥16 13-15 Marbofloxacin ≥18 16-17 Norfloxacin 10 ≥17 13-16 11 Resistant ≤ 14 ≤ 13 ≤ 13 ≤17 ≤ 14 ≤9 ≤14 ≤12 ≤15 ≤12 b The experiment of botulin toxin on ducks - Suspension of botulin toxin The process followed the method of Cook et al (1998) and was showed in Fig 3.6 Selected C botulinum colonies on TSA (from strains caused mice dead) Subculture TPGY broth Anaerobic incubation at 37oC/24h CMM (Cooked Meat Medium) Abaerobic incubation at 35oC/5days Get 10 ml from CMM Centrifuge 12.000 rpm, 15min/4oC Collecting and filtrating supernatant Examined samples with botulin Fig 3.6The preparation method of botulin toxin Determination of LD50 on ducks The toxin suspension was prepared following the above method A total of 90 ducks were used to determine the LD50 via intravenous and oral injection Group I: C botulinum was oral injection at the dose of 3ml, 5ml, 7ml, 10ml Each dose was done with ducks and one toxin suspension Group II: C botulinum was intravenous injection at the dose of ml, 2ml, ml, 4ml, 5ml, 6ml to the wing veins tiêm trực 12 tiếp vào tĩnh mạch cánh vịt Each dose was done with ducks and one toxin suspension The experimental design was showed in Table 3.7 Table 3.7 The experimental design to determine LD50 of botulin toxin Experiment Injection route Intravenous Dose (ml) No of examined samples 3 3 3 No of examined ducks 9 9 9 Oral 9 10 LD50 was determined for each examined sample and got the average value The formula of LD50 calculation was follwed Reed and Muench (1938) LD50 = - x (b-a) + a A: Death ratio (%) next over 50% B: Death ratio (%) next under 50% a: Diluted concentration of A b: Diluted concentration of B Experimental ducks: 140 days old, small size, bright feathers, clear eyes, agility The cages and raising environment were prepared well for living and testing Before testing, ducks were kept in experimental cages days The injection of botulin toxin to ducks The toxin suspension was prepared following Fig 3.5., and injected following Table 3.6 After IV or PO with the toxin suspension, ducks were observed the clinical symptoms Those ducks was observed about diet, laying rate, symptoms and been necrospy at post-injection days to examine lesions Observed symptoms After injection, ducks were observed the clinical symptoms The information was resorded times/day (6h, 12h, 18h after infection, and untill ducks were death) The toxin suspension was prepared following Fig 3.5., and injected following Table 3.6 After IV or PO with the toxin suspension, ducks were observed the clinical symptoms Those ducks was observed about diet, laying rate, symptoms and been necrospy at post-injection days to examine lesions The clinical symptoms were observed including movement, activities, feathers, squealing, chased reaction, breathing, feces, swimming 13 Observed lesions Ducks that were nearly death or just died, were dissected to determined the lesions in the organ systems such as the nervous system(brain), respiratory system (trachea, lung), circulation system (heart), digestive system (esophagus, proventriculus, gizzard, small intestine, large intestine, anal), skin, musscle, connective tissues, lympho and the liver, spleen, kidneys Those picture of lesions were captured to make the data 3.3.4.3 Observed factors - The mortality rate of ducks after days of injection with the typical symptoms of botulin poisoning - The frequency of typical symptoms of the botulism disease caused by C botulinum - The frequency of lesions in the internal organs 3.6 Data analysis Data were collected and basically analyzed by using Microsoft Excel Data were also statistically analyzed by using Chi-quare Yates and Fixer’s test, Minitab 16.0 software with the confidence level 95% 14 Chapter RESULTS AND DISCUSSION 4.1 The prevalence of the botulism disease on the free-grazing ducks in the Mekong Delta 4.1.1 The botulism disease on the free-grazing ducks in the Mekong Delta The prevalence of the botulism disease was showed in Table 4.1 Table 4.1 The prevalence of the botulism disease on the free-grazing ducks Botulism infected ducks No of Province No of examined ducks flocks No of ducks Ratio (%) An Giang 53 45,800 558 1.22 Can Tho 47 44,700 497 1.11 Hau Giang 48 43,350 547 1.26 Kien Giang 52 53,200 633 1.19 Total 200 187,050 2,235 1.19 The results of Table 4.1 showed that ducks were infected with botulism disease at a little higher rate in Hau Giang province (1.26%) However, the infected rate was not significantly different among those provinces (P>0.05) It indicated that the free-grazing ducks in the Mekong Delta could be infected botulin toxin of C botulinum at the same risk This disease causes the paralysis of neck, eyelids, wings, and legs; it called “Cum can” disease in those provinces 4.1.2 The prevalence of the botulism disease on the free-grazing ducks in the Mekong Delta by breedings Table 4.2 The rate of the botulism disease on the free-grazing ducks by breedings Breeds No of examined ducks No of infected ducks Meat duck 99,676 905 Laying duck 87,374 1,330 Total 187,050 2,235 Ratio (%) 0.91a 1.52b 1.19 Ducks were raised on the field from to 12 weeks old, after that, those ducks were selected for raising to collect eggs in many years Therefore, laying ducks started at 12 weeks old In Table 4.2, laying ducks were infected with botulism disease (1.52%) higher than meat ducks were (0.91%) (P0.05) Moreover, the prevalence of C botulinum in those specimens was not remarkably different among those provinces (P>0.05) (Table 4.5) 4.1.2 Determination of toxin in the sera of botulism infected ducks via the experiment on mice 4.1.2.1 The results of toxic test on mice Table 4.6 The percentage of mice infected botulin toxin from the sera of botulism infected ducks Group I (n = 200) Group II (n = 200) Control group (n = 20) Conditions No of Ratio No of Ratio No of Ratio mice (%) mice (%) mice (%) Death 126 63.00 0 0 Abnormal (*) 74 37.00 0 0 Normal 0 200 100 20 100 Infected disease 200 100 0 0 (*): mice with symptoms such as moody, skip eating, diarrhea, abdominal breath The results exhibited that mice in Group I (without heat treatment) were died (63%) and abnormal (37%); mice in Group II (with heat treatment) were healthy It indicated that there was botulin toxin in sera of botulism infected ducks as well as the decription of CDC (1998) Therefore, the free-grazing ducks that were neck paralysis was dueto botulin toxin of C botulinum 17 4.1.2.2 The determination of botulin toxin type via the neutral reaction on mice Table 4.7 The results of determination of botulin toxin type (n=126) Type C Type D Type E Type C+D Type C+E Control No No No No No No Results Ratio Ratio Ratio Ratio Ratio Ratio of of of of of of (%) (%) (%) (%) (%) (%) samples samples samples samples samples samples (1) (2) (3) 49 26 51 38.89 20.64 40.48a 72 22 32 57.14 17.46 25.40b 65 25 36 51.59 19.84 28.57b 3.97 (1) No neutralization (died mice), (2) Partial neutralization (mice with death), (3) healthy mice 0 0 12 100 1.59 clinical symptoms, no The results of Table 4.7 showed that the botulin toxin type C in ducks’ sera was present at the highest rate (40.48%) followed by type E (28.57%), type D (25,40%) (P