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 DI

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

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:

1 Learning Resource Center, Can Tho University

2 National Library of Vietnam

LIST OF PUBLISHED SCIENTIFIC ARTICLES

1 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

2 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, 11:73-

77

3 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

4 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

5 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 1 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 do 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 3 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, 5 (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)

3.3.1.2 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 grazing ducks in the Mekong Delta

free The crossfree sectional investigation was done to clarify the prevalence of the botulism disease on ducks in 4 provinces

- The number of samples were showed in Table 3.1

Table 3.1 The distribution of collected samples in 4 provinces

Step 1 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 1

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

(All steps were done and incubated in the anaerobic condition at 37 o C/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

Contents

in intestine

and

liver

Culture

on the blood agar and SFP

Typical colonies

Gram stain (bacilli,

Gr + , spores)

Biochemical characteristics

by usingAPI 20A

Clostridium botulinum

and keeping strains

in CMM/

thioglycolate

CMM media Soil,

water,

crabs,

snails

Subculture (identical colonies)

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 3 days to adopt the environment before using in the experiment

- Experimental design

Table 3.3 The experimental design

Group No of examined

mice Dose (ml/mouse)

- The filtrated supernatants were check the aseptic by culturing

on blood agar and SFP medium Those samples were incubated at

in Group II and Control were healthy without abnormal symptoms; it

indicated that botulin toxin was in that serum sample

d Determination of botulin toxin type

The sera having botulin toxin were chosen to determine the toxin types The sera were filtrated and divided into 3 equal parts to test with 3 kinds of antitoxins including C, D and E type with concentration of 1:1 Each suspension of serum and antitoxin was injected to 2 mice The standard antitoxin was diluted with the biophysical saline at the concentration of 1/100 before testing The experimental distribution was described in Table 3.4 Determination

of botulin toxin types was summarized in Fig 3.6

Table 3.4 The experimental design to determine the botulin toxin types

No of

samples

Type C (mice)

Type D (mice)

Type E (mice)

Control (mice)

Dose/mouse (ml)

Type C: serum + antitoxin type C; Type D: serum + antitoxin type D; Type E: serum + antitoxin type E; Putting those tests at the room temperature in 30-60min (CDC, 1998); Control (without antitoxin): injected the biophysical saline 0,9%

Fig 3.5: Determination of botulin type in the duck serum (CDC, 1998)

Serum + Antitoxin type C Serum + Antitoxin type D Serum + Antitoxin type E

Botulin toxin was destroyed by the heat

Died or abnormal mice Healthy mice

Autopsy, recording abnormal symptoms, and checking the aseptic of bacteia

suspension

Positive (having toxin in the serum)

Positive sera in Step 1 was filtrated via 0.45µm filter and divided into 3 parts to

examine the antitoxins

IP injected: 2 mice (1ml/mouse)

IP injected: 2 mice (1ml/mouse) (1ml/con)

IP injected: 2 mice (1ml/mouse)

Healthy mice Healthy mice

thường Healthy mice

Specimens has the toxin of

Duck serum (botulism infected)

Filtration (0.45µm filter), divided into 2 parts

9(qua lưới lọc Specimens suspension Group I: IP injected:

0.5 ml/mouse (2 mice/sample)

Group II: IP injected:

0.5 ml/mouse (2 mice/sample)

With heat treatment at 100 o C/10min Without heat treatment

Step

2

Step

1

Examined results

After 7 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

of samples were showed in Table 3.5

Table 3.5 The environment samples collected in this study

Place No of soil

samples

No of water samples

No of crab samples

No of snail samples

Total

An Giang 159 159 63 106 645 Can Thơ 141 141 42 94 563 Hau Giang 144 144 50 96 582 Kien Giang 156 156 61 104 646

- 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