PREVALENCE OF SALMONELLA ON PIG CARCASSES AT A

SLAUGHTERHOUSE IN HANOI, VIETNAM

NGUYEN PHU THAI

MASTER OF VETERINARY PUBLIC HEALTH

CHIANG MAI UNIVERSITY AND FREIE UNIVERSITÄT

SEPTEMBER 2007
PREVALENCE OF SALMONELLA ON PIG CARCASSES AT A
SLAUGHTERHOUSE IN HANOI, VIETNAM

NGUYEN PHU THAI

A THESIS SUBMITTED TO CHIANG MAI UNIVERSITY AND

FREIE UNIVERSITÄT BERLIN IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF VETERINARY PUBLIC HEALTH

CHIANG MAI UNIVERSITY AND FREIE UNIVERSITÄT

SEPTEMBER 2007
 iii

ACKNOWLEDGEMENTS

I would like to give sincere thanks to the post-graduate studies in International

Animal Health (FU-Berlin) and the Veterinary Public Health Center for Asia Pacific
(CMU), for organizing a wonderful program which not only gives one a chance to
improve knowledge on VPH, particularly in terms of practice, but also to better
understand the colorful cultures and people among the diverse countries in which I
attended the courses. I would like to thank the Royal Thai Government for providing
me financial support for the master’s degree program in veterinary public health.

I express my deep thanks to Prof. Dr. Karl H. Zessin, the program director, Dr.
Maximillian Baumann, the program coordinator, Assoc. Prof. Dr. Lertrak
Srikitjakarn, Dean of the veterinary faculty and program coordinator; and Dr.
Khwanchai Kreausukon, VPHCAP Director, for fulfilling their responsibility to run
the course successfully and smoothly.

Moreover, I would like to give my sincere thanks to my academic advisors,

Dr. Lüppo Ellerbroek, Federal Institute for Risk Assessment, Germany; Assoc. Prof.
Dr. Renu Pinthong (CMU); and Co-advisor, Dr. Anucha Sirimalaisuwan (CMU) for
providing support and advice for my qualified research work, and the writing of the
thesis.

I would like to thank Dr. Moses N. Kyule, Dr. Pawin Padungtod, and Ms.
Rose Schmitz, for their help in epidemiological idea, study design, data analysis and
fruitful suggestions and corrections; warm thanks to Prof. Reinhard Fries for
providing me with valuable knowledge on veterinary public health and research.
 iv

I would like to thank Dr. Bui Quang Anh, Director General of the Department
of Animal Health Vietnam for permission of study, the National Center for Veterinary
Hygiene and Inspection No.1 for accommodating me, providing a labour force,
equipment and facilities for me during my work in my home country of Vietnam.

Many thanks to local advisor, Assoc. Prof. Dr. Dau Ngoc Hao, Deputy
Director General of DAH, for giving me general and macroscopic ideas and advice on
research.

I send my sincere thanks to the laboratory staff of the FU-Berlin, Chiang Mai
University and the Veterinary University Vienna for their great effort to make me
capable of implementing my research practice well.

My deep love to my wife, my son, parents and relatives for giving me the
encouragement, moral support, and cooperation so that I could keep my mind on my
studies to achieve a fruitful result. I wish to dedicate my work to my beloved family
members.

Nguyen Phu Thai

 v

Thesis Title Prevalence of Salmonella on Pig Carcasses at a

Slaughterhouse in Hanoi, Vietnam

Author Nguyen Phu Thai

Degree Master of Veterinary Public Health

Thesis Advisory Committee Dr. Lüppo Ellerbroek Chairperson (FU-Berlin)

Assoc.Prof.Dr.Renu Pinthong Chairperson (CMU)
Dr.Anucha Sirimalaisuwan Member (CMU)

ABSTRACT

The main purpose of this study was to investigate prevalence of Salmonella in

pig at slaughterhouse, and to find out risk factors related with Salmonella
contamination in Hanoi Vietnam, during the time from November 2006 to May 2007.
There were total 356 samples of pig lymph nodes and carcass swabs taken at a
slaughterhouse according to ISO 17604; Salmonella identification following ISO
6579:2002 and Decision 2006/668/EC. Salmonella serotypes were determined using
Enteroclon Antisera for slide agglutination (SIFIN® Manufacturer, Germany).

Out of 356 samples, 149 Salmonella isolates were confirmed to be Salmonella

positive. Prevalence of Salmonella from swabs and lymph nodes were 48.9% (95%
CI: 41.36% - 56.45%) and 34.8% (95% CI: 27.96% - 42.37%) respectively.
Salmonella Derby was the most common occurrence (49.7%), following by
Salmonella Typhimurium (37.6%). Two of these serotypes represented 87.3% of all
Salmonella isolates.

There was a significant difference between the prevalence of Salmonella in

backyard and intensive farms.
 vi

ชื่อเรื่องวิทยานิพนธ ความชุกของเชื้อซัลโมเนลลาในซากสุกรจากโรงฆาสัตว
ในฮานอย เวียดนาม

ผูเขียน นาย เหงียน ภู ไท

ปริญญา สัตวแพทยสาธารณสุขศาสตรมหาบัณฑิต

คณะกรรมการที่ปรึกษาวิทยานิพนธ ดร. ลุปโป เอลเลอโบรก ประธานกรรมการ (FU-Berlin)

รศ.ดร.เรณู ปน ทอง ประธานกรรมการ (CMU)
ดร.อนุชา ศิริมาลัยสุวรรณ กรรมการ (CMU)

บทคัดยอ

การศึกษานี้มจี ดุ ประสงคเพื่อสํารวจความชุกของซาลโมเนลลาในสุกรจากโรงฆาสัตว และ

ตองการสํารวจปจจัยเสี่ยงทีท่ ําใหเกิดการปนเปอนซัลโมเนลลาในซากสุกรไดทําการตรวจซัลโม-
เนลลาจากตัวอยางตอมน้ําเหลือง (lymph nodes) และจากซากสุกรจํานวน 356 ตัวอยาง จากโรงฆา-
สัตว ณ เมืองฮานอย ประเทศเวียตนาม ระหวางเดือนพฤศจิกายน 2549 - พฤษภาคม 2550 ทําการ
ตรวจซัลโมเนลลาตามวิธี ISO 6579:2002 และ Decision 2006/668/EC ไดตรวจชนิดซีโรไทป
(serotypes) โดยใช Enteroclon Antisera สําหรับตรวจแบบวิธี agglutination slide พบซัลโมเนลลา
ทั้งหมด 151 ไอโซเลท จากซากสุกรและตอมน้ําเหลืองจํานวน 48.9% (95% CI: 41.36% - 56.45%)
และ 34.8% (95% CI: 27.96% - 42.37 %)ตามลําดับพบซัลโมเนลลาสายพันธุเดอบีและไทฟมิวเรียม
จํานวน 87.3 %ของซาลโมเนลลาที่พบทั้งหมด ความชุกของซาลโมเนลลาจากสุกรที่เลี้ยงตามบาน
มีความแตกตางจากสุกรที่เลีย้ งในฟารมอยางมีนัยสําคัญ
 vii

TABLE OF CONTENTS

Page
ACKNOWLEDGEMENTS iii
ABSTRACT IN ENGLISH v
ABSTRACT IN THAI vi
LIST OF TABLES xi
LIST OF ILLUSTRATION xii
ABBREVIATIONS AND SYMBOLS xiii
1. INTRODUCTION AND OBJECTIVES 1
2. LITERATURE REVIEW 5
2.1 Salmonella genus: 5
2.1.1 Salmonella microbiology
5
2.1.2 Serotyping of Salmonella
8
2.1.3 Distribution of Salmonella serovars
9
2.1.3.1 Distribution of Salmonella serovars in the world
9
2.1.3.2 Distribution of Salmonella serovars in Vietnam
11
2.2 Salmonellosis
12
2.2.1 Epidemiology
12
2.2.2 Salmonellosis in humans.
13
2.2.3 Public health and economic impacts
15
2.2.4 Salmonella control in pig farms
17
2.2.5 Studies of Salmonella in pork in Vietnam and overseas
18
3. MATERIALS AND METHODS
20
3.1 Time and place of research
20
3.2 Study design
20
3.3 Sample size determination
20
3.4 The slaughtering process 21
 viii

Page
3.5 Collection of samples 22
3.5.1 Lymph node samples 22
3.5.2 Carcass swabs 23
3.6 Laboratory procedures for isolation and identification of 24
Salmonella:
3.6.1 Samples preparation 26
3.6.2 Pre-enrichment 26
3.6.3 Selective enrichment 26
3.6.4 Plating and identification 26
3.6.5 Confirmation 27
3.6.5.1 Nutrient Agar 27
3.6.5.2 Biochemical confirmation 27
3.6.5.3 Serological confirmation 28
3.7 Data management statistical analysis 30
4. RESULTS 31
4.1. Salmonella isolation 31
4.1.1 Prevalence of Salmonella from all samples 31
4.1.2 Salmonella from lymph nodes by farm type 33
4.1.3 Salmonella from lymph nodes by province 33
4.1.4 Salmonella from lymph nodes at the time of pig 34
transportation from farm to slaughterhouse
4.2. Serotyping 35
4.2.1 Overall Salmonella sero-group distribution 35
4.2.2 Salmonella serogroups from lymph nodes 36
4.2.3 Salmonella serogroups from swab 37
4.2.4 Salmonella serotypes distribution 38
5. DISCUSSIONS AND CONCLUSION 39
5.1 Discussions 39
5.2 Conclusion 42
 ix

Page
REFERENCES 44
APPENDIXES 56
Appendix A 56
Appendix B 58
Appendix C 60
Declaration 64
CURRICULUM VITAE 65
 x

LIST OF TABLES

Table Page

1. Salmonella serotypes in human and animals; clinical and other 6

consequences of infection

2. Number of Salmonella serovars 7

3. Antigen of some Salmonella serotypes 9

4. Distribution of Salmonella Serotypes in Vietnam 11

5. Total Number of Salmonella and Shigella confirmed in Thailand in 15

2005

6. Estimated annual costs due to Salmonella, April 2003 16

7. Prevalence of Salmonella in swab and lymph nodes 31
7a. Prevalence of Salmonella in swab and lymph nodes in cross table 32

8. Prevalence of Salmonella from lymph nodes by farm type 33

9. Prevalence of Salmonella from lymph nodes in different provinces 33

10. Prevalence of Salmonella from lymph nodes by the duration time of pig 34
transport

11. Salmonella serogroups from all samples 35

12. Salmonella somatic antigen from lymph nodes 36

13. Salmonella somatic antigen from swab 37

14. Distribution of Salmonella serovars in pigs at a slaughterhouse in 38

Hanoi, Vietnam
 xi

LIST OF ILLUSTRATIONS

Figure Page

1. Distribution of Global Salmonella serotypes from human and non- 10

human sources during 2000 - 2004

2. Slaughtering line 22
3. Swab sampling sites on the carcass 24

4. Salmonella isolation and identification (ISO 6579:2002) 25

5. Standard Operation Prescription on Salmonellae Serotyping 29

 xii

ABBREVIATIONS AND SYMBOL

- Negative
+ Positive
BfR Federal Institute for Risk Assessment
BPLS Brilliant green Phenol Red Lactose Sucrose
BPW Buffered Peptone Water
CDC Center for Disease Control and Prevention
DAH Department of Animal Health Vietnam
DIN Deutsches Institut für Normung
DNA Deoxyribonucleic Acid
EC European Commission
ERS Economic Research Service (USDA)
EU European Union
GAIN Global Agriculture Information Network
FDA Food and Drug Administration
GDP Gross Domestic Product
H Flagella
HHS United States Department of Health and Human Service
ISO International Standardization Organization
LIA Lysine Irion Agar
LPS Lipopolysaccharide
MARD Ministry of Agriculture and Rural Development
MKTT Muller Kauffmann Tetrathionate broth
ml Milliliter
MOFA Ministry of Foreign Affairs Vietnam
MOH Ministry of Health
 xiii

O Somatic
OR Odds ratio
The Australian Government Department of Health and Ageing
Ozfoodnet Publisher
P P-value
RVS Rappaport Vasiliadis
S. Salmonella
SPSS Statistical package for social sciences
spp. Species
Subsp. Subspecies
TSI Triple Sugar Iron Agar
UK United Kingdom
US United States
USA United States of America
USDA United States Department of Agriculture
Vi Capsular antigen or envelop antigen
WHO World Health Organization
XLT4 Xylose Lysine Turgitol 4 Agar
χ2-Test Chi-square Test
1. INTRODUCTION AND OBJECTIVES

Vietnam is an S-shaped country with an area of 330,000 square kilometers,

located in Southeast Asia, between latitude 102º08' - 109º28' east and longitude 8º02'
- 23º23' north. It has a 4550 km border to China in the north, Laos to the northwest,
Cambodia to the southwest, and a 3444 km coastline of the Eastern Sea (South China
Sea) and Bac Bo Gulf in the east, and the Thai Gulf in the south (MOFA, 2007).
Population was estimated to be 84 million in 2006 (GSO, 2005).

Pork dominates in meat consumption in Vietnam, contributing 81% to the total

livestock production consumed in 2005, and 80% of that in 2004 (GAIN, 2006; GSO,
2006). According to the Ministry of Agriculture and Rural Development Vietnam,
there were 27.4 million pigs in 2005, increasing 4.9% in comparison to that in 2004
(MARD, 2006), therefore, pork may be the main vector of transmission of Salmonella
to humans. Increasing demand for monitoring Salmonella infection throughout the
pork production chain is necessary.

Hanoi is the capital city of Vietnam with a population of more than 3 million
habitants (GSO, 2005). The population is increasing because of industrialization and
urbanization. As a result, Hanoi is continuously facing a high demand for food
supply.

Daily meat consumption in Hanoi is 280 - 300 tons, which includes 180 - 200
tons of pork (contributing to around 70% of all meat), 62 tons of poultry meat, and 40
tons of beef (Vnexpress, 2005).
 2

The equipment and facilities in animal slaughterhouses in Hanoi are old,

inadequate, and in a desolate condition because Vietnam is in the first stage of
transition from a centrally planned economy to a market-oriented system. There are 6
complex slaughterhouses, which consist of 8 - 12 lines for each slaughterhouse. The
Hanoi People’s Committee had a plan to build new slaughterhouses to satisfy the
demand for fresh food. However, there are many arguments about the plan
(Vnexpress, 2005).

In Vietnam, food safety is one of the major problems and more research is
needed to concentrate on food safety (Kim, 2002). However, there have been few
researches on Salmonella on pigs in Vietnam so far, particularly in the northern part
of the country. High contamination and unhygienic conditions have been observed in
tank water at slaughterhouses in Hanoi with more than 62% of the water tank samples
being positive for Salmonella spp. (Cédric, 2006). Water contamination at abattoirs
might be the result of slaughtering practices and responsible for the high rate of pig
carcass contamination (Cédric, 2006).

In a global context these days, food safety and Salmonella are becoming an
increasing concern for the global pork market today. Salmonellosis caused by
Salmonella serovars has become an important public health problem throughout the
world (Srifuengfung, 2005; van der Klooster and Roelofs, 1997; Workman, 1999).
Salmonellosis is a major cause of bacterial enteric illness in both humans and animals.
Even in developed countries where they have potential manpower and finances, the
infections of Salmonella have still been recognized as a major hazard to humans
(Bouvet, 2003).

It is estimated that 1.4 million cases of salmonellosis occur among humans in

the United States annually (Mead, 1999; ERS – USDA, 2003). The zoonoses, which
occur most frequently in the industrialized world today, are foodborne infections
caused by Salmonella and Campylobacter (Jørgensen, 2002).
 3

There are few researches on salmonellosis in humans in Vietnam. However,

typhoid caused by Salmonella Typhi continues to be a major cause of morbidity and
mortality. The majority of cases (90%) occur in the southern part of the country, in
the Mekong Delta region (Lin, 2000). For non-typhoid Salmonella infections in
humans, S. Typhimurium is the most common cause in South Vietnam (Vo, 2006).

According to the Ministry of Health Vietnam, salmonellosis is the biggest

problem in Vietnam followed by tuberculosis, leptospirosis, and rabies (Wegener,
1999) and 3.000 - 4.000 cases are caused by foodborne infections with at least 100 -
200 fatalities annually (MOH, 2005).

Objectives of this study

In response to the food safety term ‘from farm to fork’, this study has been
conceived with the aim of gaining more knowledge about Salmonella occurrence,
serotypes, and the risk factors related to Salmonella contamination of pigs.

The fundamental information obtained in this study would provide a scientific

database for Salmonella prevalence, serotype distributions and cross contamination in
the abattoir. The data of this research will give information about the hygiene status
in slaughterhouses in Hanoi City and neighbouring provinces, as well as about
Salmonella infection in pig farms.

Furthermore, the baseline information would be used in formulating

hypotheses as well as in designing long-term studies aimed at establishing monitoring
trends and setting up strategic measures against Salmonella.

In this study, three main purposes are:

• To determine the occurrences of Salmonella spp. of pig carcasses at a

slaughterhouse in Hanoi, Vietnam.
 4

• To determine serotypes of isolated Salmonella.

• To find out the risk factors related to Salmonella contamination in pig

carcasses.
 2. LITERATURE REVIEW

2.1 Salmonella genus:

2.1.1 Salmonella microbiology:

Salmonella bacteria which belong to the genus Salmonella, the family

enterobacteriaceae, are gram-negative (Yan, 2003). They are small facultative
anaerobic, straight rods, 0.7 - 1.5 x 2 - 5µm in size (Holt, 2002).

Most genera of Salmonella are usually motile by peritrichous flagella.

However, S. Gallinarum or S. Pullorum are always non-motile. (Krieg and Holt,
1984). Salmonellae produce hydrogen sulfide, but some S. Choleraesuis and most
strains of S. Paratyphi A are exceptions (ISO 6579:2002).

The bacteria grow optimally at 37 °C. Salmonella are oxidase negative,

catalase positive, indole and voges-proskauer negative, but methyl red and simmons
citrate positive (Holt, 2002).

Salmonellae live in the intestinal tracts of a wide range of warm and cold-
blooded animals. For example, S. Typhimurium can adapt to a wide host range.
Other species are specifically adapted to a particular host (WHO, 2005). For instance
Salmonella Typhi to primates, Salmonella Dublin to cattle, Salmonella Choleraesuis
to pigs.
 6

Table 1: Salmonella serotypes in human and animals; clinical and other

consequences of infection (Quinnand, 2003)

Salmonella serotype Host Consequences of infection

Salmonella Typhimurium Many animal species Enterocolitis and septicemia

Humans Food poisoning
Salmonella Dublin Cattle Many disease conditions
Enterocolitis and septicemia
Salmonella Choleraesuis Pigs Enterocolitis and septicemia
Salmonella Pullorum Chicken Pullorum disease
(bacillary white diarrhoea)
Salmonella Gallinarum Adult birds Fowl typhoid
Salmonella Arizonae Turkeys Arizona or paracolon infection
Salmonella Enteritidis Poultry Often sub-clinical in poultry
Many other species Clinical disease in mammals
Humans Food poisoning
Salmonella Brandenburg Sheep Abortion

Salmonella genus consists of two species Salmonella enterica and Salmonella

bongori. Salmonella enterica is divided into six subspecies which are S. enterica
subsp. Enterica, S. enterica subsp. Salamae, S. enterica subsp. arizonae, S. enterica
subsp. diarizonae, S. enterica subsp. houtenae, and S. enterica subsp. indica (Yan,
2003; Popoff, 2001).

Salmonella contains 2501 serotypes which have been identified up to 2004

(WHO, 2005; Popoff, 2001). S. enterica subsp. enterica, which is highly pathogenic
to warm-blooded animals, has 1478 serotypes. The S. enterica subsp. salamae has
been found in 498 serotypes; S. enterica ssp. arizonae (94 serotypes found), S.
enterica subsp. diarizonae (327 serotypes found), S. enterica subsp. houtenae (69
serotypes found), S. enterica subsp. indica (11 serotypes found).
 7

Table 2: Number of Salmonella serovars

Salmonella species subspecies Number of serovars

S. enterica enterica 1,478

salamae 498

arizonae 94

diarizonae 327

houtenae 71

indica 12

S. bongori 21

Total 2,501

(Source: WHO collaborating Center for Reference and Research on Salmonella,

Michel Y. Popoff. 8th Edition, pp.11)

The genus Salmonella of the family enterobacteriaceae can be roughly

classified into three categories or groups as well, according to their adaptation to
animal hosts (WHO, 2005; Hafez, 2000):

• Group 1: The highly host-adapted and invasive serovars include species of

restricted and invasive Salmonella such as S. Pullorum, S. Gallinarum, S.
Typhi, and S. Paratyphi in humans.

• Group 2: The non-host-adapted and invasive serovars consist of

approximately 10 - 20 serovars that are able to cause an invasive infection in
animals and humans. Currently, the most important serovars are S.
Typhimurium and S. Enteritidis.
 8

• Group 3: The non-host adapted and non-invasive serovars include most

serovars of the genus Salmonella. They may be pathogenic for animal and
humans.

2.1.2 Serotyping of Salmonella

The Kauffmann–White serotyping scheme for the designation of Salmonella

serotypes is used by most laboratories for the characterization of Salmonella isolates
(Silva, 2006). A serotype is firstly determined on the basis of the O group and then
the flagellar (H) factor antigens, which completely define the serotype identity of the
Salmonella strain in the cell wall of Salmonella organisms (Silva, 2006; Agasan,
2002).

The genus Salmonella has three kinds of major antigens which are somatic (O)
or cell wall antigens, surface (envelope) antigens, and flagellar (H) antigens (Jay,
1992).

Somatic (O) or Cell Wall Antigens

Somatic antigens, which are specific polysaccharides comprised of
lipopolysaccharide (LPS), an important component of gram-negative bacteria, are
heat stable and alcohol resistant (Todar, 2005). Somatic antigen is a polymer of O
sub-units and each O sub-unit consists of four to six sugars. Variation of the sugar
components of the O sub-unit leads to variation in O antigen (CDC, 2006).

Flagellar (H) Antigens

Most Salmonella have flagellar antigens which are unstable proteins under
heat and can switch between two types, known as phase 1 and phase 2. The H
antigens of phase 1 are designated with small letters, and those of phase 2 are
designated by Arabic numerals (Jay, 1992). A few Salmonella do not have flagella
antigens when they express non-motile. Most H antigens are diphasic strains which
are two different types of H antigens, but some H antigens are monophasic (express
 9

one phase of H antigen only, i.e. Salmonella Enteritidis, Salmonella Risen have only
phase 1)(Sifin, 2004).

Surface (envelope or capsula) Antigens

Envelope antigens in Salmonella may mask O antigens, and the bacteria will
not be agglutinated with O antisera. One specific surface antigen is well known: the
Vi antigen. The Vi antigen occurs only in Salmonella serovars: Typhi, Paratyphi C,
and rarely in S. Dublin (Todar, 2005; Selander, 1992; Morris, 2003; Sifin, 2004). In
case, Vi antigens mask O antigens, we use heat (boiling at 100 °C for 60 minutes) to
destroy surface antigens, then we test O antigen. (Sifin, 2004; Popoff, 2001).

Table 3: Antigen of some Salmonella serotypes

Somatic Flagella (H) antigens

Serotype Serogroup
antigens(O) Phase 1 Phase
S. Paratyphi A A 1, 2, 12 a (1,5)
S. Typhimurium B 1, 4, (5), 12 i 1,2
S. Agona B 4,12 f, g, s -
S. Derby B 1, 4, (5), 12 f, g (1,2)
S. Typhi D 9, 12, (Vi) c 1,2
S. Enteriditis D 1, 9, 12 g, m (1,7)
(Jay, 1992)

2.1.3 Distribution of Salmonella Serovars

2.1.3.1 Distribution of Salmonella serovars in the world

Among 1,100,000 Salmonella isolates from human and non-human (animal,

food, feed and environment) sources, Salmonella Enteritidis and Salmonella
Typhimurium were identified as the most common serotypes globally during 2000 -
2005 (WHO, 2006). (Figure 1)
 10

Non-human sources

Typhimurium
23%

Others
41%
Heidelberg
9%

Enteritidis
8%

Infantis
Agona 5%
3% Anatum
Derby
Brandenburg 4%
4%
3%

Human source
Others
15%
Virchow
2%
Heidelberg
2%
Newport
2%

Typhimurium Enteritidis
18% 61%

Figure 1: Distribution of Global Salmonella serotypes from human and non-human

sources during 2000-2004 (WHO, 2006)
 11

2.1.3.2 Distribution of Salmonella serovars in Vietnam

There has not been comprehensive national surveillance on Salmonella in

Vietnam so far, most researches were conducted in the southern part of the country.
However, Vo reported that the proportion of Salmonella Typhimurium, Anatum,
Weltevreden is 15.8%, 15.5%, and 11.4% respectively among 297 Salmonella isolates
from human, cattle, pigs, and poultry (Vo, 2006).

Table 4: Distribution of Salmonella Serotypes in Vietnam (Vo, 2006)

Serovars Human Cattle Pigs Poultry Total (%)

Typhimurium 21 3 23 47 (15.8)
Anatum 1 15 29 1 46 (15.5)
Weltevreden 4 11 17 2 34 (11.4)
Emek 2 26 28 (9.4)
Rissen 5 13 1 19 (6.4)
Derby 3 13 1 17 (5.7)
Blockley 1 14 15 (5.1)
S. (I), 4, 5, 12: b, - 2 3 4 1 10 (3.4)
Lexington 10 10 (3.4)
Hadar 2 6 8 (2.7)
Newport 1 1 6 0 8 (2.7)
London 1 2 4 7 (2.4)
Enteritidis 7 7 (2.4)
Albany 1 3 4 (1.3)
Panama 1 2 3 (1.0)
Rubislaw 3 3 (1.0)
Kedougou 2 2 (0.7)
Schwarzengrund 2 2 (0.7)
Tallahassee 2 2 (0.7)
Others 11 8 2 4 25 (8.3)
Total 56 63 111 67 297 (100%)
 12

2.2 Salmonellosis

2.2.1 Epidemiology

Salmonella are tenacious bacteria and can infect a wide range of hosts
including man, insects, reptiles and birds, and can persist in the environment (Fries,
2006). They are usually transmitted to humans through the digestive system, for
instance eating foods contaminated with animal feces (CDC, 2007).

On the other hand, the aerosol transmission of Salmonella has been observed
(Davies, 1999). The demonstration of S. Typhimurium in the intestinal tract and
lymph nodes of pigs with high dose aerosol exposure is remarkable. Moreover, the
most evidence for the likely importance of aerosol transmission comes from
observation in poultry (Davies, 1999)

Contaminated foods which are of animal origin, such as beef, poultry, milk, or
eggs, could not be recognized because they often look and smell normal. Many raw
foods of animal origin are frequently contaminated, but Salmonella is killed by heat
when cooked well. However, improper hygiene of infected food handlers may
become the source of contamination (CDC and HHS, 2007).

Salmonella are shed in the feces of warm and cold-blooded animals, and these
feces are also the source of Salmonella transmission to humans. Reptiles are
particularly likely to harbour Salmonella (CDC and HHS, 2007).

Diarrhea, fever, and abdominal cramps 12 to 72 hours after infection are the
most common symptoms when people are infected by Salmonella, and the illness
usually lasts 4 to 7 days. In some cases, symptoms may be so severe that the patients
must be hospitalised or some of them even die. The elderly, infants, and those with
impaired immune systems are more likely to have a severe infection (CDC, 2007;
WHO, 2005).
 13

It was reported that Salmonella was likely involved in the bird flu outbreak
(van Pelt, 2003). By the end of 2003, there was a remarkable increase of Salmonella
detection in Holland. 7500 extra cases of gastroenteritis that were caused by S.
Enteritidis were confirmed in the whole of the Netherlands, rising 50% in comparison
to 2002 (van Pelt, 2003).

2.2.2 Salmonellosis in Humans

The clinical symptoms of human salmonellosis depend on Salmonella

serotypes, from serious fever infection (i.e. typhoid fever) up to non-typhoid
gastroenteritis with possible progression (Lund, 2000). However, most of the
Salmonella infectious forms in humans are due to non-typhoid salmonellosis, which
are commonly associated with the consumption of foods from animal origins and
contaminated water (Vaeteewootacharn, 2005).

Each year, there are 12 – 33 million cases of typhoid fever in the world
(Huang, 2005). In the USA, there are 1.4 million cases of non-typhoidal
salmonellosis annually, resulting in 168,000 visits to physicians, 15,000
hospitalisations and 580 deaths (WHO, 2005), and 500 cases of typhoid fever
(Huang, 2005)

In Japan, Salmonella Enfantis and Typhimurium have been major causes of

salmonellosis, although since 1989 Salmonella Enteritidis has predominated
(Shahada, 2006). Outbreaks of sporadic cases of salmonellosis have increased in
frequency and caused an important health problem (Shahada, 2006). According to
food poisoning statistics of the Infectious Disease Surveillance Center, of the 93 444
cases of bacterial foodborne illnesses during the period 1999 – 2002, salmonellosis
made up an average of 32% of these cases.

Salmonellosis has increased dramatically in the number of human cases in

most western countries. In 2001 Salmonella Enteritidis and Typhimurium caused
 14

64.2 and 21.5% of the Belgian human salmonellosis cases respectively (Botteldoorn,
2003).

In Germany it was estimated that 15–20% of all human cases of salmonellosis

were associated with the consumption of pork (Botteldoorn, 2003). Salmonella had
been the most popular cause of human cases since 1992 but in 2005, for the first time,
it becomes less than Campylobacter (BfR, 2006).

In the Netherlands salmonellosis is one of the major causes of bacterial

enteritis in humans, with an average incidence of 450 cases per 100,000 persons per
year at risk. An estimated 15% of the cases of salmonellosis in The Netherlands are
associated with consuming pork. (Berends, 1998).

In Australia, according to the annual report from the Department of Health and
Ageing Australia, there were 24,313 notifications of eight potentially foodborne
diseases, along with 118 outbreaks of foodborne disease in the country. Overall,
reports of both notifications and outbreaks were higher than years before.
Salmonellosis is one of the most common sporadic diseases with 7842 cases, and has
significantly increased (13.1%) in comparison with the previous year (Ozfoodnet,
2004).

It is reported that Salmonella Typhimurium was the most common pathogen

among foodborne outbreaks, and restaurants were the place to introduce the infection
(Ozfoodnet, 2005).

In Thailand, Salmonella are the main bacteria which cause gastrointestinal and
systemic infections. In 2005, out of 5,896 samples collected from food products and
clinical specimens, there were 5,270 cases of non-typhoidal Salmonella contributing
89.39%, and 35 cases of typhoidal Salmonella contributing 0.59% (Pathom, 2005).
(Table 5)
 15

Table 5: Total number of Salmonella and Shigella confirmed in 2005

Organisms Serovars Isolates Percentage

Non-typhoidal Salmonella 125 5,270 89.39%
Typhoidal Salmonella 2 35 0.59%
Shigella spp. 13 345 5.85%
Other bacterial 203 3.44%
No growth 43 0.73%
Total of samples 5,896 100.00%
(Source: The National Salmonella and Shigella Center, National Institute of Health,
Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand,
2005)

In Vietnam, the Ministry of Health recorded 3.000 - 4.000 cases caused by

foodborne infections with at least 100 - 200 fatalities annually. For example, there
were 145 outbreaks with 3,584 cases and 41 deaths in 2004. A proportion of 55.8%
of these cases were caused by several pathogens (MOH, 2005). Typhoid, caused by
Salmonella Typhi, continues to be a major cause of both morbidity and mortality.
The majority of cases (90%) occur in the southern part of the country, in the Mekong
Delta region (Lin, 2000). For non-typhoid Salmonella infections in humans, S.
Typhimurium is the most common cause in South Vietnam (Vo, 2006).

2.2.3 Public health and economic impacts

The infections caused by Salmonella serovars are implicated as important

Public health problems worldwide (van der Klooster and Roelofs, 1997; Workman,
1999).

Each year in the United States, microbial pathogens cause millions of cases of
foodborne disease and result in many hospitalizations and deaths (Lin, 2005). Cost
estimates per case of human salmonellosis range from approximately US$ 40 for
uncomplicated cases to US$ 4.6 million for cases ending with hospitalization and
 16

death (WHO, 2005). ERS - USDA estimated that the annual economic cost due to
Salmonella infections is $3 billion in USA (ERS – USDA, 2003).

Table 6: Estimated annual costs due to Salmonella, April 2003

Estimated annual costs

(Million USD)
Severity/cost component Number of Lost
Total
Cases Medical productivity;
premature Cost
death
No physician; recover fully 1,294,107 0 55.6 55.6
Visit physician; recover fully 101,903 26.3 14.6 40.9
Hospitalized; recover fully 15,906 202.5 6.2 208.6
Hospitalized; die 582 6.7 2,691.3 2,698.0
Total 1,412,498 235.5 2,767.6 3,003.1
(Source: ERS – USDA, Foodborne Illness Cost Calculator for Salmonella)

According to David Byrne, EU Commissioner for Health and Consumer

Protection, the costs of foodborne Salmonella alone are estimated to reach up to € 2.8
billion annually in EU countries altogether (European Commission, 2003).
In Denmark, the annual estimated cost of foodborne salmonellosis is US$ 15.5
million (2001) representing approximately 0.009% of the country GDP. A
Salmonella control program has been conducted for several years in the country, and
the annual cost of this control program is estimated around US$ 14.1 million (WHO,
2005).
In the Netherlands, annual social costs caused by human salmonellosis are
estimated between 32 and 90 million Euro (van Pelt and Valkenburgh, 2001).

However, very few countries report data on the economic cost of Salmonella;
data related to the cost of foodborne disease are generally not available from
developing countries (WHO, 2005).
 17

2.2.4 Salmonella control in pig farms

Nowadays, developing initiatives particularly to reduce Salmonella prevalence

on pig farms has been done in Northern Europe, most notably Sweden and Denmark,
and become a critical issue for U.S. farms (Bahnson, 2001). The potential to develop
and adopt reduction programs in the U.S. is becoming increasingly real (Bahnson,
2001).

Bio-security is considered to be the worldwide trend for Salmonella control in

the farm by compiling steps to find potential risk factors to prevent the introduction of
pathogenic agents to the herd (Davies, 1999). In order to control Salmonella in pig
farm, the initial step is to find out the risk factors which can be introduced in pork
(Davies, 1999 and Zheng, 2007). The important risk factors can be identified:

2.2.4.1 Feed: feeds of animal and plant origin are often contaminated with
Salmonella (Davies, 1999). It was reported that there was significant association
between fermented feed and Salmonella in the gastrointestinal tracts of pigs (Winsen,
2001).

2.2.4.2 Water supply: Salmonella were found in 63% of the water samples
collected from water bowls in the farm (Feder, 2001); while 62% of those were
detected in water tanks at a slaughterhouse (Cédric, 2006).

2.2.4.3 Expanded Herd: Salmonella can be introduced through infected pigs

mixing with the herd. It is found that the purchase of pigs which are Salmonella
positive increases the Salmonella prevalence at slaughter (Heijden, 2005).

2.2.4.4 Transportation: It was reported that transport stress e.g. noise, smells,
animal mixing, may cause pigs to shed Salmonella more frequently (Berends, 1997
and Mulder, 1995).
 18

2.2.4.5 Contacts: The contacts of domestic and wild animals and equipment
might cause the introduction of Salmonella. Rodents are carriers of Salmonella
(Leirs, 2004). Wild birds were also a higher risk for Salmonella contaminated feed on
the farm (Harris, 1997). Salmonella were detected in 11% of boots at
slaughterhouses and pig farms (Barber, 2002).

2.2.4.6 Floor and bedding materials: It was reported that the risk of
Salmonella infection increased in pigs from farms with solid floors (Cook and Miller,
2005).

2.2.5 Studies of Salmonella in pork in Vietnam and overseas

Pork is considered to be one of the main sources of Salmonella infection in

humans, (Wegener and Baggesen, 1996) therefore, more and more researches on
Salmonella in pig have been done. In the 1990’s, swine-specific Salmonella research
increased more than fivefold compared to ten years ago (Bahnson, 2001).

There was a national survey on Salmonella in Danish slaughter swine herds in

1995. It is reported that the proportion of serologically positive meat-juice samples
ranged from a mean of 2.9% in smaller herds (101 - 200 swine slaughtered per year)
to 6.1% in large herds (more than 5000 swine slaughtered per year) (Mousing, 1997).

In Germany, a research found 7% Salmonella in pork meat juice by ELISA

and 16.4% in lymph nodes by PCR method (Bernhard, 2007). The overall prevalence
of Salmonellae was estimated 6.2% in the slaughter pigs which are German in origin
(Käsbohrer, 2000).

In Belgium, Prevalence of Salmonella in pigs is 37% of the carcass samples as

a mean value; there are high variations from different slaughterhouses which are
between 0 and 70%. Distribution of S. Typhimurium predominated on pig carcasses
with 71% (Botteldoorn, 2003).
 19

It was reported that Salmonella on pig farms in Alberta, USA is 14.3%

(Andrijana, 2005). In Thailand, prevalence of Salmonella on pigs in farms,
slaughterhouses and pork in market are 6%, 28% and 29%, respectively (Padungtod,
2005)

In Vietnam, it is reported that the prevalence of Salmonella in pig farms and

pork in the market in the Mekong Delta River is 5.2% and 69.9%, respectively (Tran,
2004) and Tran, 2005). Overall Salmonella in pigs is 49.4% in the Mekong Delta,
Vietnam (Vo, 2006).

Pork is one of the sources of foodborne salmonellosis in humans (van der

Gaag, 2004a and Berends, 1998a). The risk of transmission of Salmonella from pork
to humans can be quantified (Berends, 1997). Hald and Wegener used Danish
estimation principles to quantify sources of human salmonellosis and assessed the role
of pork in the transmission of Salmonella to humans in Denmark, the Netherlands,
Germany, Sweden and England and Wales. Although Salmonella types can occur in
almost all food-producing animals, there are often rather strong associations between
certain types and a particular animal reservoir (Hald, 1999; Hald, 2003a).

In Denmark, the Netherlands, and Germany, it was estimated that 10 to 15%,

14 to 19% , and 18 to 23%, respectively, of human infections could be attributed to
pork and pork products; while pork was presumably not an important association in
England and Wales, and was negligible in Sweden (Hald, 1999; Hald, 2003a).
3. MATERIALS AND METHODS

3.1 Time and place of research:

This study was implemented from November 2006 to May 2007. Ten
carcasses (from fattening pigs), which are equal to 20 samples (10 swabs and 10
samples of lymph nodes), were taken in a week from a slaughterhouse in Hanoi.

During the whole sampling period, 356 total samples from 178 swab samples
and 178 samples of lymph nodes were collected from one slaughterhouse in Hanoi,
Vietnam. Data was recorded in questionnaire (Appendix B) to find out risk factors
related to Salmonella in fattening pigs.

Isolation and biochemical confirmation were done in the laboratory of the

Department of Animal Health (DAH) in Hanoi, Vietnam. Salmonella serotyping was
carried out in the Faculty of Veterinary Medicine, Chiang Mai University and
Institute of Meat technology and Hygiene, Free University Berlin.

3.2 Study design

This study was a cross-sectional study for microbiological examination of

samples from carcasses.
 21

3.3 Sample size determination

4 PQ
Using the formula:
L2
Where:
P is assumed prevalence, based on previous studies or researches; P=80%
Q = 100 - P =100 - 80=20%
L is error rate; L=6%.

Assuming that the prevalence of Salmonella is 80 % in pre-slaughter pigs in

Hanoi (Cédric, 2006); at a 95% level of confidence and tolerable error rate of 6%, the
calculated sample size for this study was 178 pigs. Sample size is also equal to 178
when using a software package Win Episcope, version 2.0, 1998.

3.4 The slaughtering process

Description of a slaughterhouse in Hanoi, Vietnam

In this study, a “slaughterhouse” consists of 12 slaughter compartments
(slaughter slabs), which are concentrated in the same area of about 1000m2. Each
slaughter compartment has a pen for keeping pigs before slaughter; a boiling water
pool; a human consumption water tank for washing after de-hairing and evisceration
(Water for human consumption is used for carcass washing); one well from which
water is used for cleaning transportation vehicles and the floor. Water is used in the
slaughterhouse without chlorine or other decontamination chemicals. There are about
70 to 100 pigs to be slaughtered in each slaughterhouse per shift. There are 2 shifts of
slaughter; one shift is in the night from 1.00 to 6.00 and the other during the day from
11.00 to 14.00.

Samples are taken from only one slaughter compartment out of 12 slabs during
the day.
 22

Description of slaughtering line (Figure 2)

- Pigs are kept in pens, stunned by hit on the head, and then bled.
- Boiling water is taken in the bucket, and then poured on the carcass.
- De-hairing by knife and hand.
- Evisceration on the floor without hanging.
- Last washing, then transport of meat/carcasses to the market without chilling
The procedure of the slaughterline is hand work.

Pens Stunning Bleeding De-hairing

Market Washing2 Evisceration Washing1

Figure 2: Slaughtering line

3.5 Collection of samples

Ten carcasses were selected at one collection time (one visit to

slaughterhouse) by systematic sampling. If 100 pigs per day were slaughtered, one
sample was taken from 1 pig out of every 10 pigs. The first slaughtered pig was
chosen, and then followed by the tenth and the twentieth pig and so on.

3.5.1 Lymph node samples

The procedure is based on the commission decision of the European

Communities (Decision 2006/668/EC). At least 25 grams of ileocaecal/intestinal
lymph nodes without fat or connective tissues were collected from the same carcass or
at least five individual ileocaecal lymph nodes of each selected pig. Without a knife,
 23

but with gloved fingers, the lymph nodes were ‘harvested’ bluntly. Each sample was
collected in a plastic bag with a sample identification code, recorded date, and kept
refrigerated at 4°C in an icebox container.

3.5.2 Carcass swabs

Collection: Sample swabbing of the carcasses was performed after

evisceration. A surface of about 100 cm2 per site was swabbed using one single
abrasive sponge for all the following sites as indicated and numbered below in
accordance with Annex A of standard ISO 17604 (Figure 3):

• Hind limb, medial (1),

• Abdomen, lateral (belly, 2),
• Mid-dorsal region (mid-back, 3),
• Jowl (4).

(The method is mentioned in Decision 2006/668/EC, dated 29 September 2006).

When the head was removed before swab taking, the lowest part of the neck
was swabbed instead of the jowl in this case.

Cotton swabs were moistened in normal sterile saline solution prior to

sampling. Two sites of the carcass were swabbed with one side of the swab, which
was then turned over for the remaining four sites of the carcass (each side of a swab
cotton swabbed two sites of pig carcass). The cotton swab was wiped over each
sampling site for a total of approximately 10 times in the vertical and 10 times in the
horizontal direction. Each of four sites is at least 100 cm2. (Decision 2006/668/EC).

All samples were then kept in a separate icebox at 4°C and transported to the
laboratory within 24 hours after collection.
 24

1 3

a) Medial b) Lateral

Figure 3: Swab sampling sites on the carcass

(Source: ISO 17604:2003)

Overall number of sample

Overall 356 samples were taken for Salmonella investigation (178 samples of
lymph nodes for detecting the infection, and 178 samples of carcass swabs for
detecting contamination). Swabs were taken after evisceration.

3.6 Laboratory procedures for Isolation and Identification of Salmonella:

In this study, we followed the protocol of ISO 6579:2002 (Microbiology of

food and animal feeding stuffs - horizontal method for the detection of Salmonella
spp.) to detect Salmonella spp. (Figure 4.)
 25

Salmonella isolation and identification (ISO 6579:2002)

25g sample of lymph nodes Swab samples

225ml Buffer Peptone Water 100ml Buffer Peptone Water

Incubation for 18h±2h at 37oC

0.1 ml culture and 10ml Rappaport 1ml culture and 10 ml

Vassilialis Broth. Tetrathionate Broth.
Incubation for 24h±3h at 42oC Incubation for 24h±3h at 37oC

XLT4 medium and

BPLS agar (Brilliant Green Agar)
Incubation for 24h±3h at 37oC

Each plate is tested for a characteristic colony,

If negative, test the other four marked colonies

Nutrient agar.
Incubation for 24h±3h at 37oC

Biochemical confirmation Serological confirmation

Interpretation of results

Figure 4: Salmonella isolation and identification (ISO 6579:2002)

 26

3.6.1 Samples preparation

Lymph nodes were surface decontaminated before analysis by dipping the

lymph nodes into absolute alcohol and letting it evaporate. Decontaminated lymph
nodes were weighed and placed in a sterile stomacher bag with Buffered Peptone
Water (BPW; Merck KGaA, Germany) in a dilution of 1:10 (25 g of the sample was
transferred into a stomacher bag filled with 225 ml Buffered Peptone Water) and
homogenized by a stomacher machine.

3.6.2 Pre-enrichment

• The homogenized sample was incubated at 37°C for 18h±2h.

• Regarding swabs samples, each sample was put in 100 ml of BPW. The
sample was incubated at 37°C for 18h±2h (ISO 6579: 2002).

(Media preparation is shown in appendix A)

3.6.3 Selective enrichment

The pre-enrichment broth was mixed. Then:

• 0.1 ml was transferred to 10 ml pre-warmed Rappaport Vasiliadis (RV; Merck
KGaA, Germany) enrichment broth, incubation at 42°C for 24h±3h.
• One ml of the broth was also transferred to 10 ml of Muller Kauffmann
Tetrathionate broth /MKTTn (MKTT; Oxoid, UK) broth for the secondary
selective enrichment. It was incubated at 37°C for 24h±3h.

3.6.4 Plating and Identification

After incubation for 24 h, a loop of material from the RVS broth and MKTT
was transferred and streaked separately onto the surface of XLT4 agar (Xilose lysine
tergitol 4 agar) and BPLS agar (Brilliant green Phenol Red Lactose Sucrose agar).
 27

The plates were incubated in an inverted position at 370C for 24 h. After incubation,
the plate was checked for growth of typical Salmonella colonies.

Typical colonies of Salmonella grown on XLT4 agar show a black centre and
a lightly transparent zone of reddish colour due to the colour change of the indicator
(Salmonella H2S negative variants (e.g. S. Paratyphi A). On XLT4 agar, typical
colonies of S. Paratyphi A are pink with a darker centre. Lactose-positive Salmonella
grown on XLT4 agar are yellow with or without blackening).

Typical colonies of Salmonella grown on BPLS agar have a reddish color and
translucent colony.

3.6.5 Confirmation

3.6.5.1 Nutrition Agar:

Suspected colonies were streaked on the surface of pre-dried nutrient agar

plates and incubated at 37±1 °C for 24 ± 3 hrs, in a manner that allowed the isolated
colonies to develop. Up to five colonies per plate were purely cultured and used for
biochemical and serological confirmation.

3.6.5.2 Biochemical Confirmation

The pure colonies after incubation on nutrient agar were picked up and
inoculated into Triple Sugar Iron (TSI; Merck KGaA, Germany) slant, Lysine Iron
agar (Merck, Germany) and Urea (Urea; Merck KGaA, Germany) slant. All
inoculated biochemical media were incubated at 37°C for 18 - 24 hrs,
 28

The biochemical confirmation followed the isolation. In the case of

Salmonella, the reaction would present the following appearances.
1. Glucose: Positive (+)
2. Gas: Positive (+)
3. Lactose: Negative (-)
4. Sucrose: Negative (-)
5. H2S: Positive (+)
6. Urease: Negative (-)
7. LDC: Positive (+)
8. VPR: Negative (-)
9. Indole: Negative (-)

3.6.5.3 Serological confirmation

All strains which were isolated and suspected as Salmonella spp. were brought
to confirm at Chiang Mai University and FU-Berlin for serovar identification,
following the Kaufmann-White scheme and guidance from Sifin (Appendix C). The
serological confirmation of Salmonella antigens is conducted by slide agglutination
testing with the product Sifin, manufacturer Germany (Figure 5). The following
antisera were used to identify S. Serovars from Salmonella isolates:

a. Salmonella polyvalent I (A-E) and II (F-67)

b. Salmonella somatic Group A, B, C, D, E
c. Subgroups:
O4, O5, O27 (in group B);
O7, O8, O20, O61,62,7, O20 (in group C);
O9, O46, O27, Vi (in group D);
O10, O15, O34, O19 (in group E)
d. Salmonella flagella (H) Antisera kit
 29

The Suspected Salmonella Strains

Pure culture

Stocking culture

1. Elimination of NaCl
Auto-agglutination

+ -

2. Agglutination Biochemistry Anti-Sera I (A to E)

(Polyvalent
Antisera I, II)

+ - Anti-Sera II (F to 67)

3. Determination of Somatic antigens + -

3.1. Group characteristics for “A”, “B”, “C”, “D”, “E”
3.2. Characteristics of the subgroups in each group

Salmonella (-)

4. Determination of Flagella antigens

Biochemical
Phase 1 Phase 2 confirmation

5. Diagnosis of the serovar based on both

somatic and flagella antigens

Figure 5: Standard Operation Prescription on Salmonellae Serotyping

(Fries, 2006)
 30

3.7 Data management and statistical analysis

All the works and data in the slaughterhouse, laboratory and questionnaires
were managed using Microsoft Excel. The carcass samples and lymph node samples
from the same pigs were transferred into cross tables to compare, and the value was
expressed as percentages and relationship (correlation). Serovar groups of
Salmonella, were (i.e. Group A, B, C, D, E) also filled into cross table.

In order to find risk factors related to pig infection, data from lymph nodes
(including isolation and serotyping) were recorded in the frequency table.

McNemar test was used to compare prevalence of Salmonella between lymph

nodes and swabs from the same pigs. Yates corrected Chi-square test was used to
compare the prevalence of Salmonella in other tests in this study. Many statisticians
suggested using Fisher’s exact test which is always correct for both small and large
sample size. However, when sample size is large enough, Yates corrected Chi-square
test yields similar result to Fisher’s exact test.

Software: Epicalc version 2.0; SPSS version 11.5, were used to compute data.
Endnote version 9.0 was used to insert references.
4. RESULTS

4.1 Salmonella isolation

4.1.1 Prevalence of Salmonella from all samples

In a total of 178 carcass swabs taken, 87 of those were confirmed to be
Salmonella positive, showing 48.9% prevalence in swabs (95% CI: 41.36% -
56.45%), and 62 out of 178 lymph node samples were detected as Salmonella
positive, yielding a prevalence of 34.8% ; 95% CI: 27.96% - 42.37% (table 7)

The prevalence of Salmonella from carcass swabs and from lymph nodes was
significant difference in terms of statistics (p=0.007).

Table 7: Prevalence of Salmonella in swab and lymph nodes

No. of
No. of
Sample type Salmonella 95% CI * P-value
samples
positive (%)
Carcass
P= 0.007
Swab 178 87 (48.9%) 41.36% - 56.45%
(McNemar test)

Lymph nodes 178 62 (34.8%) 27.96% - 42.37%

*CI = Confidence Intervals
 32

In order to determine statistically significant difference between prevalence of

Salmonella in swab and lymph nodes, data was transfered to a 2x2 contingency table
to obtain a P-value (table 7a)

Table 7a shows that 34 samples were positive in both swab and lymph node,
63 samples were negative in both swab and lymph nodes. 28 samples were negative in
swab and positive in lymph node.

Table 7a: Prevalence of Salmonella in swab and lymph nodes in cross table
Swab
Total
+ -

+ 34 28 62
Lymph Node
- 53 63 116

Total 87 91 178
+ Salmonella Positive
- Salmonella Negative
 33

4.1.2 Salmonella from lymph node samples by farm type

Table 8 shows that the prevalence of Salmonella from lymph nodes in
intensive and backyard farms was 27.8% (CI: 19.45% - 38.02%) and 43.2% (CI:
32.40% - 54.67%) respectively. There was a statistically significant difference
between the prevalence of Salmonella in backyard and intensive farms.

Table 8: Prevalence of Salmonella from lymph nodes by farm type

Farm Odds
No. of No. of 95% CI *
type Salmonella P-value ratio
samples
positive (%) (95% CI)

Intensive 97 27 (27.8%) 19.45% - 38.02% P= 0.047 1.973

(Yates’
(1.056-
corrected 3.685)
Backyard 81 35 (43.2%) 32.40% - 54.67%
χ2-Test)

Total 178 62 (34.8%) 27.96% - 42.37%

*CI = Confidence Intervals

4.1.3 Salmonella from lymph node samples by province

Table 9 shows that the prevalence of Salmonella from Ha Nam and Bac Ninh
province was highest in comparison with other provinces (about 40%), while no
Salmonella was found out of 3 samples from Hanoi.

Table 9: Prevalence of Salmonella from lymph nodes in different provinces

No. of No. of Salmonella
Province %positive 95% CI *
samples positive

Bac Ninh 66 27 40.9 29.18 - 53.70

Ha Tay 76 22 29.0 19.40 - 40.65

Ha Nam 33 13 39.4 23.42 - 57.76

Ha Noi 3 0 --- ---

*CI = Confidence Intervals
 34

4.1.4 Salmonella from lymph node samples by duration time of pig

transportation from farm to slaughterhouse

We divided duration time of transportation from pig from farm to

slaughterhouse into 2 categories: category 1 (or “short transportation”) is less than or
equal to 1 hour and category 2 (or “long transportation”) is more than 1 hour (up to
2.5 hours) (Table 10).

Table 10 shows that prevalence of Salmonella was 26.9% (CI: 17.79% -

38.35%) and 41% (31.40% - 51.3%) in category 1 and category 2 respectively. There
was no significant difference between the prevalence of Salmonella according to
transportation time (p=0.07)

Table 10: Prevalence of Salmonella from lymph nodes by the duration time
of pig transport
Odds
Transport No. of No. of 95% CI *
Salmonella P-value ratio
time samples
positive (%) (95% CI)

1.886
P= 0.07
≤ 1 hour 78 21 (26.9%) 17.79% - 38.35%
(Yates’ (0.995-
3.576)
>1 up to corrected
100 41 (41%) 31.40% - 51.3%
2.5 hours χ2-Test)
Total 178 62 (34.8%) 27.96% - 42.37%
 4.2. Salmonella Serotyping

4.2.1 Overall Salmonella sero-group distribution

Table 11 shows that: For FARMTYPE, Salmonella group B represented 90.9 % and 90.4 % for intensive farm and backyard farm
respectively. Each other group contributed from 3.0 % to 6 %. For PROVINCE, Salmonella group B also dominated with 88.1 %, 90.7
%, and 94.3 % in BacNinh, HaTay, and HaNam respectively. Overall 149 isolates, group B represented 90.6%. Group A and group F-
67 were not found in this study.

Table 11: Salmonella serogroups from all samples

35
No. of No. of Salmonella No of Salmonella positive in each Group
Factors Level
samples positive B(%) C(%) D(%) E(%)

FARMTYPE Intensive 194 66 60 (90.9) 2 (3.0) 2 (3.0) 2 (4.5)

Backyard 162 83 75 (90.4) 3 (3.6) 5 (6.0) 0
Total 356 149 135 (90.6) 5 (3.4) 7 (4.7) 2 (1.3)
BacNinh 132 58 52 (91.2) 5 (8.8) 1 (1.8) 0
HaTay 152 54 49 (90.7) 0 3 (5.6) 2 (3.7)
PROVINCE
HaNam 66 35 33 (94.3) 0 2 (5.7) 0
Hanoi 6 2 1 (50.0) 0 1 (50) 0
Total 356 149 135 (90.6) 5 (3.4) 7 (4.7) 2 (1.3)
 4.2.2 Salmonella serogroups from lymph node samples

Serogroup from lymph nodes which is showed in table 12 that group B still dominates among serogroups, representing 87% but
there were no detection of group A and group F – 67.

Table 12: Salmonella somatic antigen from lymph nodes

No. of No. of Salmonella No of Salmonella positive within each Group

Factors Level
samples positive B (%) C (%) D (%) E (%)

FARMTYPE Intensive 97 27 23 (85.2) 2 (7.4) 1 (3.7) 1 (3.7)

Backyard 81 35 31 (88.6) 3 (8.6) 1 (2.9) 0

1 (1.6)

36
Total 178 62 54 (87.0) 5 (8.0) 2 (3.2)

BacNinh 66 27 22 (81.5) 5 (18.5) 0 0

HaTay 76 22 19 (86.4) 0 2 (9.1) 1 (4.5)

PROVINCE
HaNam 33 13 13 (100) 0 0 0

Hanoi 3 0 0 0 0 0

Total 178 62 54 (87.0) 5 (8.0) 2 (3.2) 1 (1.6)

 4.2.3 Salmonella serogroups from swab samples

Table 13 shows Serogroup from swabs and group B also was the most common, representing 91% in total serogroups in swab
samples. Group A, C, and F-67 were not found in swab samples.

Table 13: Salmonella somatic antigen from swab

No. of No. of Salmonella No of Salmonella positive within each group

Factors Level
samples positive B (%) D (%) E (%)
Intensive 97 39 37 (94.9) 1 (2.6) 1 (2.6)
FARMTYPE

37
Backyard 81 48 44 (91.7) 4 (8.3) 0

Total 178 87 81 (93.1) 5 (5.7) 1 (1.1)

BacNinh 66 31 30 (96.8) 1 (3.2) 0

HaTay 76 32 30 (93.7) 1 (3.1) 1 (3.1)

PROVINCE
HaNam 33 22 20 (90.9) 2 (9.1) 0

Hanoi 3 2 1 (50) 1 (50) 0

Total 178 87 81 (93.1) 5 (5.7) 1 (1.1)

 38

4.2.4 Salmonella serotypes distribution

Table 13 shows that among 149 Salmonella isolates confirmed from a total of
356 samples, Salmonella Derby was most commonly identified in this study (49.7 %),
followed by Salmonella Typhimurium (37.6 %). Two of these serovars represented
87.3 % of Salmonella isolates (130 out of 149).

Table 13: Distribution of Salmonella serovars in pigs at a slaughterhouse in

Hanoi, Vietnam

No. of Serotypes Total Distribution

Salmonella
Group No. of of serotypes
Serovars Swab Lymph node serotypes (%)
Derby B 43 31 74 49.7
Typhimurium B 33 23 56 37.6
Rissen C 0 5 5 3.4
Agona B 2 0 2 1.3
Lexington E 1 1 2 1.3
Stanley B 2 0 2 1.3
*(Salmonella 4,5: b: -) B 1 0 1 0.7
**(Salmonella 9: v) D 5 2 7 4.7

Total 87 62 149

*For Salmonella group B, one isolate was not completely typed, O4, O5, and b (one H
phase) were confirmed. Therefore the names of serotypes were written as formula
(Salmonella 4,5: b: -)

**For Salmonella group D, seven isolates (4.7 %) were not completely identified, O:9
and v (one H phase) were confirmed. Therefore, they were written as formula
(Salmonella 9: v. )
 5. DISCUSSIONS AND CONCLUSION

5.1 Discussions

5.1.1 Salmonella in pigs in Vietnam

Prevalence of Salmonella in pig feces in Vietnam were found in a wide range,

from 5.2% in the Mekong Delta (Tran, 2004), to 50% in a slaughterhouse in Hanoi
(Cédric, 2006). This study found that a nearly 35% prevalence of Salmonella was
detected in lymph nodes, which showed the infection of pigs as Salmonella carriers of
farm origin. Prevalence of Salmonella from swab samples (48.9%) at slaughterhouses
in this study was lower than in previous research (95.7%) (Cédric, 2006). It might be
due to the fact that the hygiene level of slaughterhouses was improved or actual
prevalence of Salmonella reduced.

Carcass swab is used to assess Salmonella carcass contamination or cross-

contamination under slaughtering hygiene. When we receive the prevalence of
Salmonella from swab of pigs or the surfaces of carcasses after evisceration we can
evaluate cross-contamination by feces, infected tissues, and the slaughterhouse
environment (Oosterom, 1991).

Chantong (2005) found that prevalence of Salmonella from lymph nodes,

swab before spraying and swab after chilling was 64.1%, 33.1% and 13.3%
respectively (Chantong, 2005). In the present study, Salmonella from swab samples
was higher than that from lymph nodes. It shows high contamination during slaughter
processing.
 40

In this study, there was significant correlation between prevalence of

Salmonella from swabs and lymph nodes (McNemar p=0.007). It should be
explained that the contamination of carcasses might be from evisceration.
It was consistent with the report from Berends who found that inadequate
evisceration was an important source of carcass contamination (Berends, 1997).
However, Padungtod, who conducted the study in small - scale slaughter house in
Thailand, found no significant difference between pig carcass swabs and lymph nodes
(Padungtod, 2006).

5.1.2 Salmonella from lymph node samples by farm type

Salmonella from lymph nodes which are taken immediately at slaughter,

almost perfectly indicates the infection rates of pig herds at farm level. We found that
there was a statistically significant difference between the prevalence of Salmonella in
backyard farm (43.2%) and intensive farms (27.8%) (P = 0.047). Risk of Salmonella
in backyard farm was nearly 2 times higher than intensive farm (OR = 1.973 and 95%
CI not include 1).
It complied with a previous study in South Vietnam (Tran, 2004). It might be
the improvement on hygiene, feed and water supply in commercial farms.

5.1.3 Salmonella from lymph node samples at the time of pig transportation
from farm to slaughterhouse

Transport time did not influence the prevalence of Salmonella. We found no

significant difference between a long duration of transport and short transportation (P
= 0.07). It agreed with a research from the Ministry of Agriculture, Food and Rural
Affaires, Canada (Wayne Du, 2003). However, it was found that the increase of
Salmonella in pigs was related to transportation (Berends, 1997; Hurd, 2002).
Maybe duration time of transport from farms to slaughter house between two
categories was not much different, so it did no affect the occurrence of Salmonella. In
this study “long transportation” was more than 1 hour and maximum 2.5 hours while
“short transportation” was less than 1 hour.
 41

5.1.4 Overall Salmonella serotypes distribution

There are no data concerning about Salmonella serotypes through the pork
production chain in northern Vietnam so far. However, there was available literature
on Salmonella serovars in chicken meat in Hanoi, Vietnam. The result can also
compare with similar research on pork chain in Mekong Delta, southern Vietnam.

Out of 25 isolates from pigs in Mekong Delta, top three serotypes were S.
Javiana (36%), S. Derby (16%), S. Weltevreden (12%). (Tran, 2004). Overall 297
isolates from human, cattle, pigs, and poultry in south Vietnam, top three Salmonella
serotypes were S. Typhimurium (15.8%), S. Anatum (15.5%), S. Weltevreden (11.4%)
(Vo, 2006).

Most common Salmonella serovars in chicken meat in Hanoi, Vietnam were S.

Agona (31.01 %), S. London (18.6 %), S. Emek (17.83 %), and S. Typhimurium (7.75
%) (Huong, 2005).

In present study, most common serotypes were S. Derby (49.7%), S.

Typhimurium (37.6%). Two of these serovars represented 87.3 % of Salmonella
isolates.

Although S. Enteritidis has been one of the most common serovars worldwide
(WHO, 2006), it was not found in this study. The result is consistent with other
studies in Vietnam such as Bao (2005) and Tran (2004).
 42

5.2 Conclusion

Prevalence of Salmonella in lymph nodes and swab was 34.8% and 48.9%
respectively. Overall 149 isolates, group B was most common among serogroups,
representing 90.6%.

Salmonella Derby (49.7%) and Typhimurium (37.6%) were the most common
serovars in present study, representing 87.3% among 151 isolates.

There was significant correlation between prevalence of Salmonella from

lymph nodes and carcass swabs. Transportation did not influence to the increase of
Salmonella.

The prevalence of Salmonella in backyard was significantly higher than in the

intensive farms.

It is broadly acknowledged that Salmonella is the major cause of foodborne

diseases; the incidence of salmonellosis in human infections, which can be attributed
to pork and pork products, has increased in recent years, epidemiological data of
Salmonella in pigs and pork products should be available to inform public health
authorities about the level and magnitude of the problem for improving the current
situation. The following recommendations for hygiene improvement in the pig farms
and slaughterhouse standard are given in order to reduce the level of Salmonella in
pork.

Firstly, we should emphasize on strategies of Salmonella control at farm level

to reduce prevalence of Salmonella from farm origin. It is useful to study more about
surveillance on Salmonella at farm level.

Secondly, it is necessary to improve sanitary practice and system standards

strictly in the slaughterhouse such as HACCP, find out the adapted CCPs where the
 43

reverse pathogens can be reduced to acceptable level, carry out regular training for
people handling in slaughterhouse. A control of risk factor which may introduce
Salmonella such as rodents, flies is helpful. Using chlorinate water for carcass
washing should be applied at slaughterhouse.

Lastly, public education is strongly recommended to show people the

awareness of Salmonella. It is useful to suggest consumers and food business handlers
to follow safe practices on food handling such as appropriate storage and cooking of
pork. Since high prevalence of Salmonella on pork was detected, eating raw or under-
cooked pork is not recommended.
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 APPENDIXES

Appendix A: MEDIA PREPARATION

Following the manufacturer’s guidelines:
Buffered peptone water (BPW; Merck KGaA, Germany)
Preparation: Suspend 25.5 g in 1 liter of demineralized water; if desired dispense into
smaller vessels; autoclave (15 min. at 121 °C).
pH: 7.0 ± 0.2 at 25 °C

Rappaport Visiliadis broth (RV; Merck KGaA, Germany)

Preparation: Suspend 42.5 g in 1 liter of demineralized water; dispense into tubes or
flasks; autoclave (15 min. at 121 °C).
pH: 5.2 ± 0.2 at 25 °C

Muller Kauffmann Tetrathionate broth Base (Oxoid, UK)

Preparation: Suspend 82 g in 1 liter of demineralized water, heat briefly to boiling.
Do not autoclave! Cool below 45 °C, just prior to use add 19 ml/l iodine potassium
iodine solution and 9.5 ml/l of a 0.1% brilliant green solution.
Dispense any eventual precipitate.

Brilliant Green Phenol Red Lactose Sucrose Agar (BPLS; Merck KGaA, Ger.)
Preparation: Suspend 51 g in 1 liter of demineralized water by heating in a boiling
water bath or in a current of stream; autoclave (15 min. at 121 °C); pour to plates.
pH: 6.9 ± 0.2 at 25 °C
 57

Xylose-lysine-tergitol 4 (XLT4; Merck KGaA, Germany)

Preparation: Suspend 59 g in 1 liter of demineralized water, add 4.6 ml XLT4 Agar
Supplement solution and heat the medium in a boiling water-batch (not on a heating
plate!). Cool to approx. 50 °C and pour plates.
Do not overheat, do not autoclave!
pH: 7.4 ± 0.2 at 25 °C.

Nutrient Agar (NA; Merck KGaA, Germany)

Preparation: Suspend 20 g in 1 liter of demineralized water by heating in a boiling
water bath or in a current of stream; autoclave (15 min. at 121 °C). Pour to plates.
pH: 7.0 ± 0.2 at 25 °C.

Triple Sugar Iron Agar Slants (TSI; Merck KGaA, Germany)

Preparation: Suspend 65 g in 1 liter of demineralized water by heating in a boiling
water bath or in a current of stream; dispense into tubes; autoclave (15 min. at 121°C).
Allow solidifying to give agar slants.
pH: 7.4 ± 0.2 at 25 °C.

Urea Agar (Urea; Merck KGaA, Germany)

Suspend 21 g in 1 liter by heating in a boiling water bath or in a current of steam;
Auto clave for 15 min. at 121 °C. Prior to use, liquefy the medium, cool to 45-55 °C,
mix in 50 ml/l of a filter-sterilized 40% urea solution (Merck, Cat. No. 1.08487);
prepare slant agar tubes.
 58

Appendix B: QUESTIONNAIRE FORM

Questionnaire number ……………………

Isolation and Identification of Salmonella from pig carcasses at a slaughterhouse

in Hanoi – Vietnam

1 General information:

Item Response Remarks

1.1 Name of abattoir

1.2 Date of collection

……. / ……….. / 200

1.3 Province where pigs come □ Hanoi City

from (farm trace back) □ ………………….……. province
□ Intensive
1.4 Type of farm
□ Backyard

1.5 Destination of transport ……………………… km

□ Less than 12 hours

1.6 Time of pig rest
□ 12-24 hours
□ Yes
1.7 Transport means C&D
□ No
1.8 Type of samples taken □ lymph nodes
□ carcase swab
1.9 Date of shipment to the ……………….……
laboratory.
 59

2. Slaughterhouse questionnaire:

Item Response Remarks

2.1 Water
□ Tap
2.1.1 Source
□ Well
□ Yes
2.1.2 Disinfection
□ No
□ Yes
2.2 Stunning
□ No
□ On the line
2.3 Evisceration
□ On the table

2.4 Floor cleaned before □ Yes

slaughtering □ No

2.5 Floor cleaned after □ Yes

slaughtering □ No
□ Glove □ Mask
2.6 Human hygiene
□ Apron □ None
 60

Appendix C: SEROTYPING OF SOMATIC ANTIGENS (SIFIN, 2004)

1. Elimination of auto-agglutination Salmonella strains:

Place 1 drop of saline solution onto a clean glass slide. Disperse in the drop a loop of
pure colony. Rock the slide gently for 30-60 second. If bacteria have clumped into a
distinct unit, the strain is considered auto-agglutinated, and shall not be used for the
next tests.

2. Starting with the observation Polyvalent serum I (A-E):

- If positive, test group A, B, C, D, E.
- If negative, test by Polyvalent serum II (group F to 67).
- If antisera II negative, then conclude that the isolate is not Salmonella

Test Reagent Agglutinaion

Salmonella Anti-Sera Polyvalent I (A-E) + -
Salmonella Anti-Sera Polyvalent II (F-67) n.a.* +
Result Group A-E Group F-67
Frequency high low
* Not applicable: do not run this test (test is not required)

3. Determination of main groups (A, B, C, D, E)

For testing, use the instructions from the manufacturer of the antisera.
• Sequence of testing.
• Test the main groups until getting a positive reaction.
• Stop testing if one group reacts positive; do not test for the other ones.
 61

Test Reagent Agglutinaion

Anti-Sera group D + - - - -
Anti-Sera group B n.a. + - - -
Anti-Sera group C n.a. n.a. + - -
Anti-Sera group E n.a. n.a. n.a. + -
Anti-Sera group A n.a. n.a. n.a. n.a. +
Result Group D Group B Group C Group E Group A
Frequency high high low very low very low
n.a. = Not applicable: do not run this test (test is not required)

4. Determination of O antigens and Vi antigen using mono- antisera “within” the

main groups

4.1 Within Group A (O 2)

Possible antigen combination: 1,2,12 and 2,12

4.2. Within Group B (O 4)

Test Reagent Agglutinaion
Anti-Sera subgroup O 4 + + +
Anti-Sera subgroup O 5 + - -
Anti-Sera subgroup O 27 n.a.* + -
Result O 4,5,12 O 4,12,27 O4,12
Frequency high low
* Not applicable: do not run this test; O 5 and O27 do not occur together
 62

4.3 Within Group C

Test Reagent Agglutinaion
Anti-Sera subgroup O 7 + - - -
Anti-Sera subgroup O 8 n.a.* + + +
Anti-Sera subgroup O 61 n.a.* + - -
Anti-Sera subgroup O 20 n.a.* n.a.** + -
Result O 6,7 O 6,8 O 8,20 O8
Frequency high high low very low
* Not applicable: do not run this test (test is not required)
** Not applicable: do not run this test; O 6 and O20 do not occur together

4.4 Within group D: D1 (O9), D2 (O9,46), D3 (O9,46,27)

Test Reagent Agglutinaion
Anti-Sera + + - + +
subgroup O 9
Anti-Sera - + + - -
subgroup Vi
Anti-Sera n.a.* n.a.* n.a.* + -/(+)
subgroup O 46
Anti-Sera - n.a.* n.a.* n.a.* +
subgroup O 27
Result O 9,12 S.Typhi S. Typhi** or O9,46 O 9,12,46,27
S.Paratyphi C
Frequency very high low low very low insignificant
* Not applicable: do not run this test (test is not required)
** Vi antigen may inhibit O agglutination; suspend the pure culture in isotonic saline
(0.9% NaCl) and heat at 100 °C for 60 minutes. Vi antigen will be destroyed. Then
Test O9. If O9 positive, then isolate is S. Typhi. If O9 negative isolate is tested by
antisera O7. If O 7 positive, isolate can only be S. Paratyphi C.
 63

4.5 Within group E: E1 (O 3,10); E4 (O 1,3,19)

Test Reagent Agglutinaion
Anti-Sera + - - - + -
subgroup O 9
Anti-Sera n.a. + + - n.a. +
subgroup Vi
Anti-Sera n.a. - + n.a. n.a. -
subgroup O 46
Anti-Sera - - n.a. + + +
subgroup O 27
Result O 3,10 O 3,15 O 3,15,34 O1,3,19 O1,3,10,19 O1,3,15,19
Frequency high low low low extremely rare
n.a. = Not applicable: do not run this test (test is not required)
64
 65

CURICULUM VITAE

Full name: NGUYEN PHU THAI

Date of birth: 12 February, 1972

Sex: Male

Place of birth: Tan Yen district, Ha Bac (now known as Bac Giang)
province, Vietnam

Marital status Married

Nationality Vietnamese

Occupation Veterinarian (DVM), Specialist of DAH

Employer Department of Animal Health

Ministry of Agriculture and Rural Development

Postal address Home: To 25B, cum 3, TuLien, TayHo, HaNoi, Vietnam

Office: Cuc Thu Y (DAH), PhuongMai, DongDa, HaNoi,

Vietnam

Phone number 84-4-8687150 (Office); 84-4-7193870 (Home);

0983-720212 (mobile)

Fax number 84-4-8691311, 8685961 (Office)

Email ngphth2000@yahoo.com.au

EDUCATIONAL AND TRANING BACKGROUND

1978-1985 Primary School

1985-1989 Secondary School

1989-1994 Faculty of Veterinary Science and Husbandry,

Hanoi Agricultural University

2000 Training course on main infectious diseases in Vietnam held

by SVSV (Strengthening Veterinary Service in Vietnam) in
Ho Chi Minh city.
 66

2002 International Training Course on Meat Inspection and

Quarantine in Malaysia, held by FAO.

July-December, Economic and Trade Policy Focus at Victoria University of

2004 Wellington, New Zealand

2005-2007 Master Degree of Veterinary Public Health, Freie Universität

Berlin and Chiang Mai University

OCCUPATIONAL EXPERINCE

1994-2001 Worked for Hanoi Regional Veterinary Center (HRVC) as

Official Meat Inspector and as examiner at laboratory of
HRVC

2001-present Department of Animal Health Vietnam (DAH)

RESEARCH AND LABORATORY WORK

April-October, Aflatoxin on Chicken

1994

November 2006 – Salmonella Isolation at Laboratory of National Center for

April 2007 Veterinary Hygiene and Inspection No.1

April-June 2007 Salmonella serotyping in Chiang Mai University