Use of Host-Specific Bacteroidales 16S rRNA for Microbial Source Tracking of Environmental Water in Hanoi, Vietnam

VIETNAM NATIONAL UNIVERSITY VIETNAM-JAPAN UNIVERSITY---Hanoi - 2019 PHAM MINH NGOC USE OF HOST-SPECIFIC BACTEROIDALES 16S rRNA FOR MICROBIAL SOURCE TRACKING OF ENVIRONMENTAL WATER IN H

VIETNAM NATIONAL UNIVERSITY VIETNAM-JAPAN UNIVERSITY -

Hanoi - 2019

PHAM MINH NGOC

USE OF HOST-SPECIFIC BACTEROIDALES 16S rRNA FOR MICROBIAL SOURCE TRACKING OF

ENVIRONMENTAL WATER IN

HANOI, VIETNAM

MASTER THESIS

VIETNAM NATIONAL UNIVERSITY VIETNAM-JAPAN UNIVERSITY -

PHAM MINH NGOC

USE OF HOST-SPECIFIC BACTEROIDALES 16S rRNA FOR MICROBIAL SOURCE TRACKING OF

ENVIRONMENTAL WATER IN

HANOI, VIETNAM

SUPERVISORS PROF HIROYUKI KATAYAMA ASSOC.PROF IKURO KASUGA

PROGRAM: MASTER OF ENVIRONMENTAL

ENGINEERING CODE: PILOT

TABLE CONTENT

LIST OF TABLES ii

LIST OF FIGURES iii

ABBREVIATION v

ACKNOWLEDGMENTS vi

INTRODUCTION vii

CHAPTER 1: LITERATURE REVIEW 1

1.1 Fecal indicator bacteria (Escherichia coli) 1

1.2 Bacteroidales bacteria 2

1.3 Microbial source tracking method 6

CHAPTER 2: MATERIAL AND METHODOLOGY 16

2.1 Material and Equipment 16

2.2 Methodology 16

CHAPTER 3: RESULTS AND DISCUSSIONS 33

3.1 Case study in Kanazawa 33

3.2 Case study in Hanoi 39

CHAPTER IV CONCLUSION 52

REFERENCE 53

LIST OF TABLES

Table 1.1 :The primer and probe sequence of host- specific Bacteroidales marker 10

Table 2.1: The information of sampling point for Pig-fecal source 18

Table 2.2: The information of sampling point for chicken-fecal source 18

Table 2.3: The information of sampling point for duck- fecal source 19

Table 2.4: The information of water sampling point 19

Table 2.5: The Primer and Probe of host-specific Bateroidales markers 29

Table 3.1: The information of sampling places in Kahokugata Lake 34

Table 3.2: Possitive ratios and mean concentrations of host-specific Bacteroidales markers in water sample 37

Table 3.3: Possitive ratios and mean concentrations of animal-specific Bacteroidales markers in fecal-source sample and water sample 47

LIST OF FIGURES

Figure 1.1 Main orders of Bacteroidetes 3

Figure 1.2: The concept of Microbial Source Tracking 7

Figure 2.1 :The sampling point at the South of Hanoi 17

Figure 2.2: The process of culture E coli for fecal sample 23

Figure 2.3: The process of culture E coli for water sample 23

Figure 2.4: The steps of diluted standard solution 31

Figure 3.1: The sampling points at Kahokugata Lake 33

Figure 3.2: The concentration of E coli and total coliform of water sample 35

Figure 3.3: Relation between concentration of pig-specific assay and E coli bacteria in water sample 38

Figure 3.4: The concentration of E coli and Total coliform of Pig-fecal source 39

Figure 3.5: The concentration of E coli and Total coliform of Chicken-fecal source 40

Figure 3.6: The concentration of E coli and Total coliform of Duck-fecal source 41

Figure 3.7: The concentration of E coli and Total coliform in Day river 42

Figure 3.8: The Pig-2-Bac marker’s standard curve 43

Figure 3.9: The amplification plot of Pig-2-Bac 44

Figure 3.10: The Chicken/ Duck-Bac marker’s standard curve 45

Figure 3.11: The amplification plot of Chicken/Duck-Bac marker 46

Figure 3.12: Relation between concentrations of pig-specific Bacteroidales marker (Pig-2-Bac) and E coli bacteria throughout the fecal sample and water sample 49

LIST OF PICTURES

Picture 2.1: The Duck farm 20

Picture 2.2: The Chicken farm 21

Picture 2.3: The Pig farm 21

Picture 2.4: One of the Day river's sampling point (R3) 22

ABBREVIATION

16S rRNA: 16S ribosomal RNA

E coli: Escherichia coli

FIB: Fecal Indicator Bacteria

LD: Library-dependent

LID: Library-independent

MST: Microbial Source Tracking

PCR: Polymerase Chains Reaction

qPCR: Real-time Polymerase Chains Reaction

ACKNOWLEDGMENTS

I would like to express my gratitude to all those who gave me the possibility to complete during my thesis I want to thank the Master of Environmental Engineering Lab of Vietnam-Japan University, Vietnam National University, and Nagasaki in National Institute of Hygiene and Epidemiology, Hanoi Besides, I want to thank the Microbial lab of professor Honda at Kanazawa University for giving me permission to carry out experiments to commence this thesis in the first instance Moreover, I appreciate the JICA company that supported me a lot in the Master program

I am deeply indebted to my supervisor – Prof Katayama and Assoc Prof Kasuga for their enthusiastic instruction throughout my thesis time

I also would like to thank all members in MEE laboratory for their help, support, interest and valuable hints

Hanoi, May 2019

Student

Phạm Minh Ngọc

INTRODUCTION

Vietnam is a developing country towards a sustainable development Therefore, the problem of environmental pollution is always an urgent issue and is concerned Currently, partly due to industrial development, tons of untreated wastewater are discharged directly into water inlet sluice, rivers, and lakes Pollutants such as organic substances and metals untreated penetrate directly into the water source In addition, in urban areas, waste is thrown away in many places, causing sewage congestion around areas such as To Lich River, Nhue River and Day River which have the phenomenon of pollution and stinking generated by garbage

In addition, in rural areas where livestock and poultry breeding, the water source was usually contaminated by human feces and animal feces Because waste treatment conditions in those areas have not been developed and backward, besides,

it has small livestock Instead of collecting and treating according to the system, waste will normally be discharged directly into the surrounding environment such

as groundwater and surface water (rivers and lakes) lead to the fecal pollution in environment water These are causes of the incidence of water-related diseases such

as E coli infected diarrhea, dermatitis, or eye diseases They are increasing and

likely to spread disease Hence, the pollution of the water environment is affecting directly to the health of people

In Hanoi, one of the largest rivers flowing through many communes of Hanoi is the Day River This river starts from the Red River, and it goes through many communes which have many livestock and poultry farms (mainly pigs, chickens, ducks, and cows) that discharge livestock waste directly into that river area Therefore, the Day river has a lot of potential for being contaminated by the feces source However, the current fecal pollution in Day River is only determined by the

FIB method, especially the use of E coli culture in the lab Therefore, the managers

still do not know exactly the pollution sources of there, whether the main source of pollution in the Day river basin comes from the treatment of livestock waste

Currently in the world, in many developed countries, in addition to using the FIB method to assess water quality, the Microbial source tracking (MST) method has been developed to find sources of fecal contamination From there, managers can assess water quality and human health risks The MST method is now widely used with specific hosts of the 16S rRNA gene of Bacteroidales bacteria which were found unique in feces, rumen and other cavities of humans and animals, often in greater abundance than traditionally used coliform bacteria [1] In previous studies, manure contamination was found from the source of some animals, was based on the PCR endpoint method of the 16S rRNA gene of Bacteroides In addition, more advanced with the qPCR method determined an accurate concentration of pollutants according to the number of copies and combined with microbial source tracking method as a marker and identify sources of pollution [2]

Microbial source tracking is a useful and important method and/or tool for environmental authorities to seek the source of possible outbreaks of animal diseases In Vietnam, a present outbreak of African pig cholera and past lessons of H5N1 chicken fever are firm evidence of the necessity of this research Therefore, this research topic on "Use of Host-Specific Bacteroidales 16S rRNA for Microbial Source Tracking (MST) of Environmental Water in Hanoi, Vietnam" which was to assess and detect the source of fecal pollution on Day River, which originates from pigs, chickens or ducks by application the method of biomarkers Using the specific host of the 16S rRNA Bacteroidales gene as a marker detect pollution sources by real-time PCR (qPCR) In addition, the study also assesses and compares the specificity and sensitivity of markers in Hanoi environment The study highlights the scientific significance and practicality of the topic

The structure of this study including four main parts and it will be shown as below:

CHAPTER 1: LITERATURE REVIEW

1.1 Fecal indicator bacteria

1.2 Bacteroidales bacteria

1.3 Microbial source tracking

CHAPTER 2: MATERIAL AND METHODOLOGY

2.1 Material and Equipment

CHAPTER 3: RESULTS AND DISCUSSIONS

3.1 Case study in Kanazawa

3.2 Case study in Hanoi

CHAPTER4: CONCLUSION

REFERENCE

CHAPTER 1 LITERATURE REVIEW

1.1 Fecal indicator bacteria (Escherichia coli)

In the environmental quality assessment criteria, Fecal Indicator Bacteria (FIB) method is a procedure for detecting the presence of pathogenic bacteria In other words, this method is supposed as a proxy in environmental samples such as water environment and soil environment Correspondingly, a similar behavior to pathogens is considered as a proxy As a matter of fact, the ideal bacteria indicate that the presenting microbe in the intestinal tract of people and animals as pathogens which exist in contaminated samples (as existing pathogens in contaminated samples) Besides that, they also have the survival model which is familiar to a pathogen outside the host as well as cannot reproduce and grow in an easy environment In addition, the indicator bacteria are selected when they are easy to detect, have a low bad impact on the researcher and it is important that the costs are appropriate (relatively cheap)[3]–[5]

The criteria for selecting FIB which mentioned above is to establish a determination

for the temperate system On the other hand, some chosen organisms such as E coli

or coliform have been applied to tropical systems without taking into account the potential properties of the tropics (eg humidity, temperature, and so forth) Also, all these factors affect the survival of the selected bacteria Moreover, some practical evidence has been shown that the bacteria indicated for countries in the temperate zone is suitable for the tropical system [6][7] For example, according to Carillo and

Jiminez's study [8][9], it has been shown that E coli may exist in tropical freshwater for some time, in addition, E coli is also found in epigenetic bromeliads

from tropical rain forests [10] Likewise, from the perspective of Winfield and

Groisman in 2003 [11], FIB and E coli, particularly can exist and develop in

tropical environments, where high temperatures and organic matter concentrations are high

E coli is known as Escherichia coli, a gram-negative, anaerobic bacteria that lives

mainly in the large intestine of humans and animals When E coli is released into

the environment, they survive and grow in wet fecal of man and animal feces for several days under anaerobic condition before self-degradation, so it is possible to

create free-living populations lead to the environmental pollution Most E coli

strains do not harm hosts although they are parasites, sometimes they help the host fight some pathogens and create symbiotic relationships with the host Nevertheless, there are still a few types that can cause food poisoning and some intestinal diseases

for humans and animals such as diarrhea [12], [13]

E coli and total coliform were selected as one of the FIBs to assess the

environmental quality of water with standards that depend on the country, for example, follow the U.S Environmental Protection Agency (EPA) in 2009, the

level of E coli for drinking water is zero As a matter of fact, in Vietnam, the level

of E coli for drinking water is 0 CFU/100ml base one the National Technical

Regulation 01:2009/Ministry of Health Particularly, one of the main methods used

to detect FIB (E coli) is to use a laboratory culture, which is a simple method

Indeed, this method involves culturing a known volume of sample into the culture medium, then carrying out the incubation method within 18-24 hours at 37oC To put in another way, for the method of using FIB, it does not identify the source of contamination, thus some newer methods can track the source of infection through the use of biomarkers As a consequence, the use of specific gene markers of specific hosts such as specific signs for human or pig manure, etc has been strongly developed to identify sources of fecal contamination [2], [14] Besides, one of the specific genes currently being used in this way is Bacteroidales bacteria's 16S rRNA genes

1.2 Bacteroidales bacteria

According to Michael J Coyne and Laurie E.Comstock [15] in 2007, in the human body, the gastrointestinal tract has a lot of populations of organisms residence In the gastrointestinal system, the microbial density of the colon microflora is higher

than in other locations To put it differently, they include hundreds of other species

of microorganisms that coexist and have a mutually reciprocal relationship that benefits the host In this case, an oral cavity is also a place where a large number of organisms are accumulated over time, although they also have a mutual relationship, they do not benefit their hosts and cause frequent injury sick Not to mention, in both of these ecosystems, members of Bacteroidetes phylum which contain more than 700 special species play an important role More specific, the phylum contains many diverse organisms, including human pathogens, mammalian symbiosis, etc Markedly, the phylum Bacteroidetes divide 4 main orders: Sphingobacteriales, Cytophagales, Flavobacteriales, Bacteroidales (Fig 1.1) [16]

Figure 1.1 Main orders of Bacteroidetes [16]

In the Bacteroidales order, the members are gram-negative organisms, living mainly

in the human colon microflora where they provide beneficial properties to the host Identically, the fermentation of sugar, protein, saccharolytic derivatives which are the energy source for Bacteroides, are common compounds in the human gut In fact, through measurements for Bacteroides species, it was found that its number was 1010-1011 cells per gram of human feces In addition, previous studies have shown that the host's diet is related to intestinal microbiota composition For

example, Prevotella species are dominant in people who eat a lot of food contain a

lot of carbohydrates, while Bacteroides are dominant in those who consume a lot of protein and animal fat [16], [17]

Normally, the organisms of the intestinal microflora (such as Bacteroides bacteria)

often build a sustainable relationship with the host, in addition to competion with other members to get nutrition as well as a living environment For instance,

Bacteroides thetaiotaomicron which is one of the most salient species of Bacteroides in the lumen of the large intestine can transform the sugar (eg

polysaccharide) to the fermented product which benefits for human [16] Besides, they are subjected to attacks from various sources such as antimicrobial products from hosts, phages or harmful products of other bacteria Therefore, microorganisms change their surface as such as some pathogens, which is a survival advantage in this environment For example, some species have changed surfaces

like B fragilis which is considered as an opportunistic pathogen, they cause

gastrointestinal infection, or peritoneal cavity, and have the potential to cause appendicitis In other words, Bacteroides of Bacteroidales order are capable of resistance to many different antibiotics, resulting in potential pathogenicity [18] Currently, the complete or partial genome sequences of most of Bacteroidales are available To demonstrate, they have been stored, analyzed and classified in the gene library (NCBI) according to phylum Bacteroidetes and some have shown the gene publicly As an illustration, the sequence of the 16S rRNA gene of Bacteroidales was used and developed with the qPCR technique, which is one of the

common bacterial targets in the MST method at this time Specifically, Bacteroidales orders are the most widely distributed units to determine the source of fecal contamination of cattle and poultry In particular, Bacteroides is considered a major genus of bacteria in human feces [15]

Otherwise, the 16S ribosome RNA gene sequence (16S rRNA gene) is the gene that encodes the RNA components of 30S subunit of ribosome Particularly, it is present

in all bacterial species and each species of bacteria has one or more copies of the 16S rRNA gene [19] According to a study by Weisburg in 1991[20], it was demonstrated that Gene 16S rRNA was used in studies of phylogenies because it is highly conserved among archaea and different bacterial species Besides, the sequence of 16S rRNA genes is also used to classify bacteria, identify and quantify them in complex biological mixtures, such as human intestinal samples, environmental samples (water samples) In addition, Cox.MJ's research in 2013 [21] indicates that this gene has a greatly conserved component of the DNA-based life-forms, hence, it is very suitable to be the target gene to DNA sequencing in samples containing many different organisms Based on conserved regions and highly variable regions as well as the detection specificity of 16S rRNA, universal PCR primers are designed to target those conservation areas to amplify molecules of other organisms of each other exists in the same sample environment Meanwhile,

in variable regions, sequencing helps to distinguish different specific microorganisms such as archaea, microorganisms, and bacteria [22]

In the past, for environmental samples, people often cultured and isolated to identify species, which took time and effort, money However, with the combination of PCR and 16S rRNA with the next generation sequencing method, it is possible to conduct research with multiple samples with lower cost and time [23] With this method, many new sequences have been found that do not belong to any culture species In addition, one of the first independent library methods was developed to detect the human source of fecal contamination based on endpoint detection of

Bacteroidales 16S rRNA gene by PCR method [24] This method is also one of the ways of MST tool methods MST method will be mentioned in the next section.

1.3 Microbial source tracking method

Microbial source tracking (MST) is one of the methods used to assess water quality and human health risks Markedly, this method is described as a tool to find and accurately identify sources of pollutants for the environment based on the linkage of some types of microorganisms to specific hosts such as DNA chains of the microorganism Likewise, MST appeared in the late 20th century with the aim of (1) identifying sources of fecal contamination in water environments that affect the health of people exposed to water, (2) improving the properties of FIB in the water bodies to the exactly fecal source [24]–[27]

On the other hand, MST's basic principle is to select microorganisms in the feces that are closely related to specific hosts and use their properties as markers for fecal contamination from the host To explain that, MST has two main methods: library-dependent (LD) and library-independent (LID) Especially, the first method LD needs to collect and type many FIB isolates for a number of specific properties such

as microbial resistance to antibiotics [24], or heredity, or use of carbon resources [28], [29] Coupled with, the second method LID is based on a specific trait of a specific species or type of bacteria, for example, using 16S rRNA genetically modified regions of Bacteroidales [23] With this intention, the concept of two methods of MST was explained clearly in below with Fig 1.2 [30]

1st: Library-dependent, culture-based: the water samples are collected from the unknown source as lakes, rivers, etc, the bacteria in these samples are grown and isolated in the laboratory The results are compared with the library which is built a base on the known source

2nd: Library-independent, culture-based: the water sample are collected, and the bacteria are grown or culture in the lab as the same 1st, however, the bacteria grown

are known its specific host or its fecal contamination source by the using DNA to identify its source

3rd: Library-independent, culture-independent: water samples from an unknown source are collected then using molecular techniques to identify DNA and, isolate directly

Figure 1.2: The concept of Microbial Source Tracking

This study focused on the library-independent, culture-independent method and using Bacteroidales bacteria with its specific host at the 16S rRNA point Many previous studies, many primers, and probes of 16S rRNA gene of Bacteroidales are related to hosts such as humans and animals that have been designed and tested with high specificity and sensitivity According to the Google Scholar, the following primers and probes have been designed and used common in highly reliable articles and used as cited evidence about 147 to 660 of different studies as shown below in Table 1.1

All Bac is a primer which was designed to detect all Bacteroidales bacteria in the environment as a universal detection In like manner, it is shown in Layton’s research 2006 with 100% of sensitivity and in another search like Mieskin’s research, it also showed off its ability to detect in all samples For the human-associated markers, they are used detecting the human fecal contamination including human-specific HF183 which was appeared in Bernhard’s study in 2000

at USA with 100% of sensitivity, and it was tested in many countries like Australia [31], Frace [32], Ireland [32], etc, with high ability of detection were 100%, 94%, 100%, respectively In addition, TaqMan qPCR system for human-assays used BacHum marker [33] and HuBac marker [34] in the USA whose sensitivities were 98% and 33% , respectively Besides, in the Haramoto’s research at Japan, 2018, BacHum assay was shown its sensitivity with 100% too, in addition, it also showed its accuracy was 86%, that meaning it had a little cross-reaction with another contamination source

For the Pig host-specific, In the study of Mieszkin in Frace (2009), the Pig-2-Bac and Pig-1-Bac were used, and they were detected the Bacteroidales in the feces, slurry, a lagoon with 100% of sample positive with the TaqMan indicated probe Besides, in the study of P.Somnark, 2018 [35] and Haramoto, 2018 [36], the marker Pig-2-Bac is used too and its sensitivity is 100% for all sample feces Besides, they didn’t have more cross-reaction with another fecal source, with accuracy more than 75% With PF163 primer, it is designed by Dick,2005 [37] for the host-specific pig with the SYBR indicated probe, this primer is used with sensitive about 100% for all feces sample Furthermore, PF163 assay was done in the Sonmark’s research in Thailand, and its specificity is 77 % with 83% of accuracy

For the host-specific of the chicken and the duck, in the study of Kobayashi in Japan, 2012[38], the Chicken/Duck-Bac marker was designed for detecting all chicken and duck, however, this marker detected the positive samples for the duck and the chicken with 96%, 61% of sensitivity, respectively It was not highly specific for both chicken and duck In that study, two specific primers for duck-host

and chicken-host also were designed too for comparison with the universal primer for both Duck-Bac primer was used to detect for duck fecal source and showed 80% of sensitivity Besides, Chicken-Bac was shown 70% of sensitivity, nevertheless, it was found out having cross-reaction with the pig fecal pollution On the other hand, two primers Chicken/Duck-Bac and Duck-Bac were used with TaqMan probe system, and Chicken-Bac was used with SYBR probe system

For the ruminant-host specific, BacR and BacCow were utilized in the previous studies of Reischer in 2006 [39] and Bernhard’s study [37], respectively They were shown their highly specific with 100%, and 0% of cross-reaction with other feces sources for BacR In addition, for BacR primer, it was recommended testing for South Asian countries Moreover, these primers were used again in the B.Malla’s research [40], they detected all fecal samples with 100% specific and low cross-reaction with chicken and duck fecal samples, and non-cross-reaction with the human, dog, and pig-source In the study of Haramoto in Japan,2018, BacR and BacCow were shown 93% and 100% of sensitivity, respectively with high accuracy were 94%, and 66 %, respectively

Table 1.1 :The primer and probe sequence of host- specific Bacteroidales marker

amplicon (bp)

Reverse

primer

Pig-2-Bac 163Rm

Reverse

primer

Pig-1-Bac 163Rm

Forward

primer

CHAPTER 2 MATERIAL AND METHODOLOGY

2.1 Material and Equipment

All chemicals were used from Laboratory for Environmental Engineering of Master Environmental Engineering Program at Vietnam-Japan University- Vietnam National University with some equipment such as filtration system and the incubator Real-time polymerase chain reaction (qPCR) machine was used at Institute of Tropical Medicine, Nagasaki University in National Institute of Hygiene and Epidemiology, Hanoi

2.2 Methodology

2.2.1 Study location

The areas collected sample include four communes of Hanoi (My Duc Commune, Ta Thanh Oai Commune, Thanh Oai Commune, and Chuong My Commune) These places focus on livestock cattle and poultry Besides, almost communes stay around Day river whose water is used for irrigation and breeding At Ta Thanh Oai Commune, there are three farms which are the pig farm and duck farms collected In Thanh Oai Commune, sampling was carried out at six farms including two duck farms, three chicken farms, and one pig farm Besides, in Chuong My Commune with a lot of pig farms and chicken farm with a large scale, so that, three pig farms and one chicken farm were collected there Finally, in My Duc Commune, a pig farm with huge scale was chosen in this study In Day river, five places were marked to collect sample from upstream to downstream The detail information of each sampling point and the map were shown below

Figure 2.1 :The sampling point at the South of Hanoi The sampling point

Table 2.1: The information of sampling point for Pig-fecal source

1 ng T m Xanh farm including 1200 pigs 70000m2

in My Duc Commune Producing breeding pigs and pig breeds imported from Thailand

2 The small farm in Thanh Oai Commune with 5 mother pigs and

more than 50 pigs In there, There, the farm is closed, the mother pig gives birth to piglets and the piglets continue to be kept at the farm until grew up to sell to the market

3 The Pig farm of Mr Toi in Chuong My Commune This farm has

600 pigs and the pig breeds imported CP company Vietnam

4 The Pig farm of Mr Tien in Chuong My Commune This farm

has 600 pigs and the pig breeds imported CP company Vietnam

5 The big farm in Thanh Oai Commune included 70 mother pigs

and 600 pigs for sell The pig breeds imported GreenFeed company of Vietnam

6 The Pig farm of Mr Tien in Ta Thanh Oai Commue This farm

has 200 pigs and 50 mother pigs from CP company

Table 2.2: The information of sampling point for chicken-fecal source

1 The chicken farm in Thanh Oai Commune has 500

chickens/140m2 The chicken breeds imported Thuy Phuong farm

in Son Tay Commune

2 The farm of Mr Lam in Thanh Oai Commune has 3000

chickens/300m2

3 The chicken farm of Mr Tien in Chuong My has 7000 chickens

/750m2

4 The chicken farm of Mr Toan has 7000 chickens in Thanh Oai

Commune Table 2.3: The information of sampling point for duck- fecal source

1 The duck farm in Thanh Oai Commune has 3500 ducks/20000m2

2 The farm of Mr Toan in Thanh Oai Commune has 1000 ducks

3 The duck farm in Ta Thanh Oai Commune has 3000 ducks

4 The duck farm has 2000 ducks in Ta Thanh Oai Commune

Table 2.4: The information of water sampling point

collected on 4 farms from adult chickens and young chickens, and chicken is bred in the hen-coop For the duck fecal source, almost duck farm in Vietnam is breeding outside with the pool However, the duck farm target was focused on the farm which bred inside the house to ensure the quality of the fecal source to avoid the possibility of cross-contamination at the source For example, if it is outside, it will be contamination from other pollution sources such as chicken fecal source, pig fecal source, or bird source too Duck fecal samples (n=9) were collected on 4 farms from the adult and young duck in the duck-spics All samples were collected by cotton and kept into 2ml phosphate buffer saline (PBS) solution in 15ml tube After collected sample, the sample kept in the icebox during the collecting time All samples were stored in the fridge until the qPCR was performed

Picture 2.1: The Duck farm

Picture 2.2: The Chicken farm

Picture 2.3: The Pig farm

b River water sample

River water samples were collected from Day River in Hanoi, Vietnam as shown in Figure 2.1 A total sample was 6 samples from upstream to downstream in April 2019 All samples were collected 100ml into the duran bottle (100ml) which was autoclave before using kept in the store at -4oC before DNA extraction was performed Besides,

at each sampling point, they were collected 15ml into 15ml tube to cultured E coli in

the laboratory during 24h after collected sample

Picture 2.4: One of the Day river's sampling point (R3)

2.2.3 E coli culture

Figure 2.2: The process of culture E coli for fecal sample

Figure 2.3: The process of culture E coli for water sample

a Agar preparation

- Prepare autoclaved bottle and glass petri dish for agar container in advance

- Autoclave the distilled water before using for dissolved agar

- Suspended chromocult coliform agar (CC agar) in demineralized (26.5g/l) by heating in a boiling water bath or in a current stream Stir the contents to assist