Khóa luận tốt nghiệp Công nghệ sinh học: Sequencing and analyzing genetic characteristics of the stn gene of Salmonella enterica subsp. enterica isolated from swiftlet house environments

This study was conducted to sequencing and analyze genetic characteristics of thesin gene of Salmonella enterica subsp.. enterica were examined for the presence of the s7 gene, after the

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY - HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

I would like to express my sincere gratitude towards the Board of Nong LamUniversity Ho Chi Minh City and the Board of Faculty of Biological Sciences forcreating the most favorable conditions for me to finish this study

And especially, I would like to express my deep gratitude to Dinh Xuan Phat PhDfor wholeheartedly teaching, guiding, supporting, and giving me inspiration throughoutthe process of the thesis

It would be impossible for the study to be completed if it hadn’t been the help ofall people who has accompanied with me during this hard times And for that, I want tosend thanks of Ms Nguyen Thi Mi Mi and other members of Gene TechnologyLaboratory BIO313 for helping me to reach my goals

Finally, I want to thank my family for always believing in me and encouraging

me to continue on the route I've chosen Thank you for motivating me to keep trying.Thank you to my buddies for always supporting me, studying with me, and helping meachieve my goals

Although I still have many unanswered thanks, I hope those who have supported

me greatly remain well and successful in their careers

Sincere

CONEIRMATION COMMITMENT

My name is Chau Ngoc Huynh Nhu, Student ID: 19126125, Class: DH19SHB,Faculty of Biological Sciences, Nong Lam University Ho Chi Minh City I guarantee

that this research was conducted by myself and that the results presented are utterly

Thu Due City, February 2024

Student’s signature

This study was conducted to sequencing and analyze genetic characteristics of thesin gene of Salmonella enterica subsp enterica isolated from swiftlet houses inSouthern Vietnam Ninety samples including 67 fecal samples and 23 nest surface swab

samples were collected from 30 swiftlet houses in Binh Duong, Dong Nai provinces and

Ho Chi Minh City The samples were isolated and identified Salmonella enterica subsp.enterica using PCR method Samples subsequently tested positive for Salmonellaenterica subsp enterica were examined for the presence of the s7 gene, after thenamplified full-length gene, sequenced and genetic analyzed 6/90 (6.67%) were positivewith Salmonella spp and 6/6 (100%) were positive with Salmonella enterica subsp.enterica by PCR For the detection of the virulence stn gene of 6 isolated Salmonellasamples, the results showed that this gene appeared with a detection rate of 100% Inthis study, we had successfully designed primer for amplification and sequencing of stngene with length of 750 bp Then, we also constructed phylogenetic tree of this gene

and analyzed of more 34 sequences from GeneBank with identity nucleotides of 98.4

-100% and amino acids of 94.8 - -100% It was noted that there were 18 positions withamino acid change So, the study had successfully amplified and sequenced the full-length stn gene and analyzed the genetic characteristics of the stn gene in Salmonellaenterica subsp enterica

Key words: Salmonella, sequencing, stn gene, swiftlet house

TÓM TẮT

Nghiên cứu này được thực hiện nhằm phân tích trình tự và đặc điểm di truyền của

gen stn trên vi khuẩn Salmonella enterica subsp enterica trong nhà yên ở miền Nam Việt Nam Chín mươi mẫu bao gồm 67 mẫu phân và 23 mẫu phết bề mặt tô được thu thập từ 30 nhà yến ở các tỉnh Bình Dương, Đồng Nai và Thành phố Hồ Chí Minh Sau

đó, các mau được phân lập và phát hiện Salmonella enterica subsp enterica bằng

phương pháp PCR Các mẫu sau đó được xét nghiệm dương tính với vi khuẩn Salmonella enterica subsp enterica đã được kiểm tra sự hiện diện của gen sin, sau đó

khuếch đại gen có chiều dài đầy đủ, giải trình tự và phân tích di truyền Kết quả phân

lập ghi nhận 29/90 (32,22%) mẫu nghi ngờ dương tính với Salmonella 6/90 (6,67%) mẫu được phát hiện dương tinh với Salmonella spp và 6/90 (6,67%) dương tính với

Salmonella enterica subsp enterica bằng PCR Kết quả phát hiện gen độc luc sin của 6 mau Salmonella đương tính trước đó cho thay gen này xuất hiện với ty lệ phát hiện là 100% Trong nghiên cứu này, chúng tôi đã thiết kế thành công primer dé khuếch đại và giải trình tự gen sin có chiều là 750 bp Sau đó, nghiên cứu này còn xây dựng cây phát sinh loài của gen này và phân tích trình tự tương đồng của 34 trình tự có độ tương đồng nucleotide 98,4 - 100% va độ tương đồng các axit amin 94,8 - 100% Ghi nhận có 18 vi trí có sự thay đôi về axit amin Như vậy, nghiên cứu này đã thành công trong việc khuếch đại và giải trình tự toàn bộ gen sén, và phân tích các đặc điểm di truyền của gen sin ở

Salmonella enterica subsp enterica

Từ khoá: Salmonella, giải trình tự, stn gen, nhà yến.

TABLE OF CONTENTS

PageACKNOWLEDGEMENT S sts:eausesecansansanasennancumanann an eneninecranecemnnenimers 1

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LIST OF ABBREVIATIONS cán na nh 2020060103 61115818 c6 556814386425130613 8558348500488 Vill

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2.1 Introduction to Salmonella and Salmonella enterica subsp enferica - 3

2.1.1 Taxonomy of Salmonella enterica subsp €FÍ€FÏC@ -.- 5-55 5<<<+ssssseerseesee 3

2.1.2 Basic biological characteristics of SalmOnell A cceccecceeceeseeeceeteeeeeeseeteeeseeseeeseees 4

2.1.3 Basic genomic characteristics Of S2Ï7owelÏ4 - +-++++s++ss++sexsseeesseeses 3

2.1.4 Salmonella enterotoxin (StH) 0 nnửutd4 6

2.2 SalmonellOsis 1 ?

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2.3 Diagnosis, prevention, and treatment the dIsease - - + ++++x++scc+cezeeezxrs 10

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2.3 Laboratory diagnostic methods 2 S2 3222231321121 221 2125111 1exre 12

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2.3.1.2 Stages o9 12

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2.3.2.2 Step of Sanger se€qU€TCIHE - - + 2+ + 323122 221231 2 12 121 11 ket 15

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24:5: LHIESFHRNGIIII TGSGBTDĂ sau beisinDidinDGG1114110014834603804554893894358 285836 GHEH.78S43/0 mn 17

CHAPTER 3 MATERIALS AND METHODS 572 Sc+cs+zsersrrrrrrrrrerree 18

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3:2 Materials and methods ::0sccsossnesprsseesmenaezenaneasnnnnrnmmwenram meee 18

3.2.1 Positive controls, negative controls, and field samples - + -+<-+ 18

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3.2.3 Equipment and instrument T0 19

3.3 Research worktlow sssusceccussmmsm em meensneu meme mee eee NEE 19

3.3.1 DNA extraction using WizPrepTM Viral DNA/RNA Mini Kit V2 19

3.3.2 Designing primers used for amplifying full-length gene - 20

3.3.3 Detection of the presence of Salmonella enterica subsp enterica in the swiftlethouse CNVITONMENE N06 21

3.3.4 Amplifying and sequencing Sf QeMe - + - + 5+ *S+*S2<£Es ren rrrkt 22

3.3.5 Phylogenetic analysis 17177 23

CHAPTER 4 RESULTS AND DISCUSSION ‘seesecsescussrusrnueeneneueneareraemernes: 24

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4.1.1 Designing primers used for amplifying full-length sin gene - 24

4.1.2 Detection of the presence of Salmonella enterica subsp enterica in swiftlet

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4.1.3 Amplifying and sequencing full-length øene - 555 5+ ++<£+x£+ec+eczeres 25

4.1.4 Phylogenetic ni Tnhh 26

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CHAPTER 5 CONLUSION AND RECOMMENDATION 3Ö

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: Lipopolysaccharide

: Heat-labile enterotoxin: Polymerase Chain Reaction: Salmonella enterotoxin: subspecies

: Chain-Termination Sequencing Method: Xylose Lysine Deoxycholate

: The White-Kauffmann-Le Minor: Centers for Disease Control

: Bismuth sulfate agar

: Salmonella Shigella agar

: Transfer RNA

Primers used for detection of Salmonella enterica subsp enterica 21

Thermal cycles used for detection of Salmonella enterica subsp enterica

The components used for PCR amplifying full-length sim gene 22

Thermal cycles PCR amplifying full-length sim gene 22Primers used for amplifying full-length stn øene ++-+>5-+<+ 24

The results of Sa/monella isolation from swiftlet house samples 25

Percent similarity nucleotide and amino ac1d - +5 55s ++ss++s£+ss<+s 27

LIST OF FIGURES

PageFigure 2.1 Scanning electron microscopy of a Š.Typhimur1um - - 55s 5-5+

Figure 2.2 Circular map of the S Typhimurium P-stx-12 chromosome

Figure 2.3 Gross pathology of an American cowbird that died of salmonellos1s

Figure 2.4 Salmonella infection in wild passerine species .:c:cceseeceeseeeseeseeeeeeeees

Figure 2.5 Gross lesions of Sa/monella infection in wild birds - 555 <>>5

Figure 2.7 The Sanger sequencing method in 7 steps . - +- 5555 +++ss++s+zeszsss

Figure 4.1 Electrophoresis results of investigation of st øene ¿+ +cscc++xs++

Figure 4.2 Electrophoresis results of amplifying of st øene - -++-x<<x<sx<s+

Figure 4.3 Phylogenetic trees based on the nucleotide sequence of stn øenes

Figure 4.5 Distribution of amino acid variability along stn sequenec

-10

CHAPTER 1 BACKGROUND

1.1 Introduction

In Vietnam, bird's nest farming for commercial purposes has appeared since 2004

in some Southern provinces However, in the past 10 years, this profession hasdeveloped rapidly with many different types and scales According to the Ministry ofAgriculture and Rural Development, there are 42 out of 63 provinces nationwidecurrently involved in swiftlet farming with over 22,000 bird nest houses The estimatedproduction of bird nest products was estimated to be 150 tons per year, equivalent toabout 600 million USD The Mekong Delta had the highest number of swiftlet houses

at 10,572, accounting for 44.67% of the region's total swiftlet houses The South-CentralCoast has 5.965 houses, accounting for 25.21% (Vietnam Agriculture Newspaper:https://vietnamagriculture.nongnghiep.vn/)

The presence of bacteria in the bird's nest environment may produce low quality

of edible bird’s nests, in addition, these bacteria can also cause many foodborne diseasesposing risks to the public health (RAZAK, 2016; Worku ef a/, 2022)) Salmonellosis,

infection caused by a wide range of Salmonella enterica strains, is a prevalent

food-borne disease Salmonella infections are classified as typhoidal salmonellosis (TS,enteric fever) or non-typhoidal Salmonella (NTS) infections (Ngogo ef ai, 2020;Akinyemi et al., 2021) The major pathogenic serovars of Salmonella enterica thatinfected humans through the consumption of certain food products are SalmonellaEnteritidis and Salmonella Typhimurium (Thung ef a/., 2018)

The stn gene was found in Salmonella spp., regardless of their serotype (Dinjus

et ai, 1997; Makino et al., 1999) Salmonella enterotoxin (sin) is a virulence factor thatcontributes to diarrhea in its host (Chopra ef a/., 1994) Biological activities of stn arecritical for Salmonella virulence, particularly during the enteric phase of the disease(Chopra ef al., 1999) Therefore, the thesis “Sequencing and analyzing geneticcharacteristics of the stn gene of Salmonella enterica subsp enterica isolated fromswiftlet house environments” was conducted

1.2 Objectives

Successfully sequenced and analyzed genetic characteristics of the stn gene of

isolated Salmonella enterica subsp enterica from swiftlet house environments

1.3 Contents

Content 1: Amplifying and sequencing full-length gene

Content 2: Phylogenetic analysis

CHAPTER 2 LITERATURE REVIEW

2.1 Introduction to Salmonella and Salmonella enterica subsp enterica

Salmonella is named after American bacteriologist D E Salmon, who firstisolated the bacteria from the intestines of pigs in 1884 (Su and Chiu, 2007) It is aGram-negative, motile, hydrogen sulfide-producing, acid-labile and facultative

intracellular organism that commonly causes gastroenteritis worldwide and contributes

to cross-infection between humans and animals (AJmera and Shabbir, 2023)

2.1.1 Taxonomy of Salmonella enterica subsp enterica

The genus Salmonella belongs to the Enterobacteriaceae family, orderEnterobacteriales, class Gamma-Proteobacteria, phylum Proteobacteria, the KingdomMonera or Eubacteria (Tindall ef a/., 2005; Adeolu ef al., 2016) According to theCenters for Disease Control (CDC), the genus Sa/monella contained two species,Salmonella enterica and Salmonella bongori Salmonella enterica is divided into sixsubspecies: enterica (1), salamae (Il), arizonae (Hla), diarizonae (IIIb), houtenae (IV),and indica (VD, whereas S bongori has no subspecies The genus Salmonella contained

approximately 2600 serovars based on antigenic polymorphisms of their somatic O

antigens (lipopolysaccharide), H antigens (flagellar proteins), and Vi antigens (capsularpolysaccharides) (Brenner ef a/., 2000; Tindall ef al., 2005) The majority of the serovarsare Salmonella enterica subsp enterica (1), with the most common serogroups being A,

B, Cl, C2, D, and E (Tindall et al., 2005)

Serotyping has long been used to classify Salmonella The White-Kauffmann-LeMinor (WKL) scheme, which is based on serological characterization of the O and Hantigens, is the international standard for the naming of Salmonella serotypes (Le Minor,1970) The WKL method employed the agglutination reaction with antisera againstantigenic variants O and H (Le Minor ef a/., 1982) There were 46 O antigens and 114

H antigens among the known Salmonella, and over 2,600 "serovars" of the species hadbeen identified and described among the conceivable combinations (Diep ef a/., 2019).Although it has been the main method for serological characterization of Salmonella for

decades, this method is rapidly being replaced by the widely used genomic methods forserotyping species In the early 2000s, molecular detection-based strategies using

3

genetic sequences as surrogate antigen markers were developed, including several based methods (Kim ef a/., 2006; B Liu ef al., 2011; Salazar et al., 2015) as well as aliquid suspension DNA hybridization method based on X-map (e.g., Luminex)technology (McQuiston ef al., 2011; Moore ef a/., 2019).

PCR-Salmonella is a rod-shaped and Gram-negative bacterium It has a cell wall, cellmembrane, cytoplasm, ribosomes, plasmids, and nucleoid region The diameter of

Salmonella ranges from approximately 0.7 uM to 1.5 uM, and its length ranges from 2

to 5 uM, and flagella, which allows for motility (Whitman ef a/., 2015) Salmonella

typically produces hydrogen sulfide, breaking down D-glucose into hydrogen andcarbon dioxide, and reducing nitrate to nitrite (Pui ef al., 2011) With the exception of

Salmonella serovar Typhimurium, which does not create gas, practically all Salmonella

serovars are aerogeni (Popoff and LeMinor, 2005) According to 16S rDNA sequencing

analysis, Sa/monella belongs to the Gammaproteobacteria class (Cosby ef al., 2015)

Salmonella grows readily on blood and MacConkey agar (Yoshikawa ef al.,

1980) Salmonella isolation might also be accomplished using bismuth sulfate agar(BSA), Salmonella Shigella agar (SSA), and xyline lysine deoxycholate (XLD) agar.They ferment glucose and mannose but not lactose and sucrose on these agars (Ewing,

1986; Addis and Sisay, 2015) Salmonella serotypes grow best at temperatures between

35°C and 40°C Depending on the growth medium and serotype involved, they can grow

at temperatures ranging from 2°C to 54°C (Cosby ef al., 2015) Salmonella did not

develop at extremely low temperatures (Sörqvist, 2003) However, due to its hardiness,cold does not necessarily have a negative impact on the organism (Obafemi and Davies,1986)

2.1.3 Basic genomic characteristics of Salmonella

cps

MH tRNA rRNA

Wccskev-Figure 2.2 Circular map of the S Typhimurium P-stx-12 chromosome The

first and second circles, from inside to outside, represent GC skew and G+C

content, respectively The third circle depicts the reverse strand CDS, tRNA, and

rRNA; the fourth circle depicts the forward strand CDS, tRNA, and rRNA

CGView was used to create this image (Ong et al., 2013a; Stothard and Wishart,

2005)

The complete genome of S enterica serovar Typhimurium P-stx-12 had a single

circular chromosome with a GC content of 52.1% and a circular plasmid with a GC

value of 46.4% The chromosome has 4,885 predicted genes, including 4,691 coding genes, 22 rRNA genes, and 76 tRNA genes An amount of 75.34% of the genes

protein-on the chromosome were allocated specific COGs, and 25% of these genes were alsoawarded enzyme classification numbers that were engaged in 268 metabolic pathways(Ong ef al., 2013b)

The genome of Salmonella is relatively small, ranging in size from 4.7 to 5.3million base pairs (McClelland e/ al., 2001; Naushad et al., 2020; Lee ef al., 2022) Ithas a single circular chromosome that encoded proteins that involve in a variety ofcellular activities including metabolism, regulation, and pathogenicity (Den Bakker er

ai, 2011; X Liu et al., 2015; Sherry ef ai, 2023) Several studies had examinedSalmonella's genomic characteristics and discovered genes critical for its survival,pathogenicity, and antibiotic resistance (Morgan ef al., 2004; Valdez ef al., 2009; Ilyas

et al., 2017; Gao et al., 2022) These included genes that encoded toxins and othervirulent factors that allowed bacteria to infiltrate and infect host tissues, as well as genes

that regulated food intake and metabolism The existence of prophages, which are

bacteriophages that have integrated into the bacterial genome, is one of Sa/monella'smost well-known genomic traits (Ong ef a/., 2013b; Mottawea ef al., 2018)

2.1.4 Salmonella enterotoxin (stn)

Salmonella produces both exotoxins and endotoxins The endotoxin, known aslipid A, is a component of the outer membrane lipopolysaccharide (LPS) in Salmonella

It triggers a variety of biological reactions, both in vivo and in vitro (Hitchcock ef al.,

1986) Exotoxins are classified into two types: cytotoxins and enterotoxins However,

there is limited knowledge about the mode of action of Salmonella exotoxins (Ashkenazi

et al., 1988)

The most extensively researched exotoxin of Salmonella was the heat-labileSalmonella enterotoxin (sin), which has a molecular mass of approximately 29 kDa and

is encoded by the sin gene (Chary ef al., 1993; Prager et al., 1995) This stn gene from

Salmonella Typhimurium has a rare start codon TTG, is located at approximately 89

minutes on the chromosome, and has a size of 749 bp The s/z gene was present in all

84 strains tested, representing 14 Salmonella enterica serotypes, including SalmonellaEnteritidis and Salmonella Typhimurium, as detected by PCR and Southern blot (Prager

et al., 1995) At the amino acid level, homology was found between stn and the CT-Aand CT-B subunits of cholera toxin, LT-1 (heat-labile toxin of Escherichia coli),Pseudomonas aeruginosa exotoxin A, and diphtheria toxin Diphtheria Corynebacterium(Chopra et ai., 1994b) Recombinant stn has biological features comparable to CT, such

as elongation of Chinese hamster ovary cells and induction of a fluid secretory response

in ligated rabbit ileal loops (Chary ef al., 1993; Rahman and Sharma, 1995) Antibodies

6

produced against CT have the ability to neutralize the biological activity of stn (Rahman

and Sharma, 1995) St exerts its effects by increasing cAMP and increasing the

synthesis and release of prostaglandins The exact pathway by which s/z leads to fluidand electrolyte secretion remains to be determined (Prasad e/ a/., 1990; Darwin andMiller, 1999)

2.2 Salmonellosis

Salmonellosis is the general term for infections caused by Sa/monella, which are

classified into two types: typhoidal and non-typhoidal Typhoid fever is caused mostly

by Salmonella Typhimurium and is characterized by symptoms such as weakness,abdominal pain, fever, and loss of appetite (Matheson ef al., 2010; Eng et al., 2015;Barnett, 2016) Non-typhoidal salmonellosis, on the other hand, is caused by a range ofSalmonella serotypes and often produces food poisoning symptoms such as diarrhea,stomach cramps, and fever (Majowicz ef al., 2010; Feasey ef al., 2012) Differentsymptoms will appear in different animals and birds

2.2.1 Clinical signs

Clinical signs of salmonellosis in birds might include ruffled or fluffed feathers,

shivering, fast or heavy breathing, weakness, diarrhea, plaques on the oral mucous

membranes and crop, lethargy, and death The most common symptom of enteritis isdiarrhea, due to the form of avian droppings in general, enteritis in birds 1s more difficult

to be idented than that in mammals

Diarrhea can cause dirty feathers surrounding the vent, or it can cause the vent toget plastered with fluid feces or urates Salmonella Typhimurium can develop in other

organs, such as joints (Kang ef a/., 2015) Exposure to Salmonella spp can cause acute

or chronic sickness, but the disease can also remain an asymptomatic carrier stage in thebird's digestive system Emaciation, hemorrhagic or necrotizing enteritis orenterocolitis, hepatic necrosis and inflammation, and arthritis are all found during

autopsies of the dead individuals (Rabinowitz and Conti, 2010; Kang ef a/., 2015)

2.2.2 Gross lesions

Acutely dying birds have minimal visible lesions Nonspecific lesions such asenlarged, congested, mottled livers and spleens with or without tiny hemorrhagic ornecrotic foci may be detected Tan to white foci or nodules occur in the liver, spleen,pectoral muscles, subcutaneous tissue, brain, and other places in more chronic illness

|

circumstances Infection transmitted by egg transmission manifests as a creamy orcaseous yolk sac that is not absorbed into the celomic cavity upon hatching (Thomas efal., 2008).

Necrotic plaques in the esophagus and crop lesions are the most commonlyassociated with salmonellosis in songbirds (Hurvell ef ai, 1974; Fichtel, 1978).Passerine species (cowbirds, goldfinches, and English sparrows) were found to have

multifocal necrotizing esophagitis and ingluvitis (Figure 2.3), S enterica serovar

Typhimurium was also isolated from necrotic lesions The epithelial surface was focallyulcerated, leaving a dense coating of necrotic cellular debris mixed with degenerate and

intact leukocytes and a plethora of gram-negative bacterial rods The underlying

submucosa was infiltrated by heterophils, with less lymphocytes and plasma cells Otherrecognized infections, such as avian poxvirus, which can cause esophageal lesions inpasserine animals, were not discovered (Hudson ef al/., 2000)

Figure 2.3 Gross pathology of an American cowbird that died

of salmonellosis The arrows delineate multiple necrotic plaques

on the esophageal mucosa from which S enterica serovarTyphimurium was isolated (Hudson et al., 2000)

A multifocal to diffuse fibrinnecrotic thickening of the crop mucosa (ingluvitis)(Figure 2.4 A, B, C), frequently with inflammation spreading to the underlyingconnective tissue and muscle Birds with these crop lesions exhibited fibrinnecrotichepatitis (Figure 2.4 D) and/or splenitis, and some had moderate to severe, locallywidespread small intestine hemorrhage (Hughes ef a/., 2008)

Figure 2.4 Salmonella infection in wild passerine species A: Greenfinch

crop: multifocal necrotic ingluvitis B: Greenfinch crop: diffuse necrotic

ingluvitis C: Greenfinch crop (Haematoxylin and Eosin stain): One central

and one peripheral nodule of necrotic crop mucosa and submucosa with

hemorrhage, surrounded by zones of infiltrating leucocytes Greenfinch liver

(Haematoxylin and Eosin stain): widespread hepatic necrosis surrounded by

a zone of macrophages and multinucleate large cells encircled by lymphocytes

diameter and frequently protruding into the crop lumen (Figure 2.5 D) Nodules were

found in the subcutis of the neck (4 - 5 mm in diameter) and the coelomic cavity (3 - 4

mm in diameter; Figure 2.5 E) (Giovannini ef al., 2013)

2.3 Diagnosis, prevention, and treatment the disease

2.3.1 Diagnosis

Salmonella spp may be isolated from blood, urine, bone marrow aspirates, stool

and other sterile sites from the hosts The basic Sa/monella infection diagnostic testing

is blood or stool cultures followed by conventional microbiological diagnosis and

serology (Acosta ef al., 2003) Blood cultures have a low sensitivity of 40 - 60% inenteric fever patients, however bone marrow aspirate cultures have a sensitivity of morethan 80%, making this type of culture the gold standard for enteric fever diagnosis(Baker ef al., 2010; Guerra-Caceres ef al., 1979; Parry et al., 2011; Vallenas ef al,1985) Only 30% to 35% of stool cultures are positive Because of the limited sensitivity,which is attributed in part to occasional bacterial shedding, the assessment of Salmonellaspp carriers needs numerous testing samples (Gilman ef a/., 1975; Vallenas ef al.,

10

1985) Urine culture sensitivity is likewise modest, ranging from 7% to 10% Positivecultures in the urine or feces may suggest an acute infection or chronic carriage (Gilman

et al., 1975)

Serologic tests have been utilized for diagnosis for almost a century The Widaltest, invented in 1896, uses a suspension of deceased S typhi as antigen to detect serumantibodies against flagellar and somatic antigens, which results in agglutination in

positive samples during the acute and convalescent phases of sickness The diagnosis

based only on the Widal test is usually mistaken due to the prevalence of false positiveand false negative findings There 1s also a large degree of variation across commercialformulations and a lack of standardization (Olopoenia and King, 2000; Wain andHosoglu, 2008) Rapid diagnostic testing employing PCR stools and blood specimens,

on the other hand, has been demonstrated to have good specificity and sensitivity (Hattaand Smits, 2007; Gomez-Duarte et a/., 2009; Gordon, 2011)

2.3.2 Prevention

Despite varied protection levels, vaccination is a helpful tool to aid with thechallenging work of managing and preventing Sa/monella infections in chicken flocks

Immunization of breeding stock using inactivated vaccines has shown excellent results

when used alongside with other biosecurity measures such as: proper cleaning anddisinfection, rodent control, sanitary barriers, culling of positive birds or flocks, andbacteriology surveillance as part of a program to reduce the incidence of Salmonellaenteritidis infections in commercial poultry flocks (McIlroy ef a/., 1989; Mead andBarrow, 1990; Gast ef al., 1992) Nonetheless, live vaccines elicit a more comprehensiveimmune response because they induce bacterial clearance earlier than inactivatedvaccines (Babu ef ạ., 2004)

2.3.3 Treatment of disease

Antimicrobial medications were administered to week - old hens infected withSalmonella infantis when they were one day old, either alone or in combination withperoral inoculation of bacterial culture The bacteria were derived from adult chickencecal contents Neomycin, neomycin plus polymyxin, neomycin plus oxytetracycline,and sulfadiazine plus trimethoprim were the antibacterial medicines that are often used.Although the efficiency varied amongst the parallel studies, the combination therapy ofoxytetracycline plus neomycin and bacterial culture appeared to be the most beneficial

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Sulfadiazine with trimethoprim, followed by bacterial culture therapy, reduced the

infection incidence The bacterial culture demonstrated a little anti-Salmonella impact

on its own Salmonella quickly resurfaced in the intestines after just antimicrobials wereadministered (Seuna et al., 1980)

2.3 Laboratory diagnostic methods

2.3.1 Polymerase chain reaction - PCR

In the 1980s, Kary Mullis developed PCR, for which he received the Nobel Prize

in 1993 (Evans, 2009; Mullis and Faloona, 1987) The advancement of DNAamplification tools has resulted in significant advantages in gene analysis, the diagnosis

of genetic diseases, and the detection of bacterial, viral, and fungal pathogens (Speers

et al., 2003; Atkins and Clark, 2004; Niesters, 2004) Additionally, PCR has proven to

be a valuable tool for cloning specific DNA fragments, enabling the examination of geneexpression and showing promise 1n the field of forensic medicine (Agne ef al., 2009).2.3.1.1 Principle of PCR

PCR allows for the synthesis of targeted DNA fragments through the use of aDNA polymerase enzyme, which plays a crucial role in the replication of geneticmaterial within cells (Agne ef a/., 2009) This enzyme synthesizes complementary DNAsequences by attaching a small fragment (a primer) to one of the DNA strands thatselects a specific site to begin synthesis Primers limit the sequences to be copied, withthe result that billions of copies of a specific DNA sequence are amplified (Mullis,1990)

2.3.1.2 Stages of PCR

12

-DNA template

<———T'rimer

-dNTP -DNA polymerase

Double stranded DNA

5) 3 Pron

—

5 3

Double stranded DNA

denature double-stranded DNA into single-stranded DNA,annealing with primers, and extension the DNA strand(Dey, 2018)

There are three main steps

extension The process is repeated using an automated thermal cycler until sufficient

in a PCR reaction: denaturation, annealing, and

amplification has occurred (Canene-Adams, 2013)

During the denaturation step,the reaction mixture is heated with an temperature

above 90°C to unwind the double helix structure of DNA by breaking apart the hydrogenbonds Then, to allow for primer annealing, the reaction mixture was cooled down to 45

- 65°C In this range of temperature, the forward and reverse primers hybridize to theopposite strands of DNA via complementary base pairing They must complement the

3” ends of the antiparallel strand of the template DNA When the mixture is heated to

72°C, the optimal for the DNA polymerase activity, the enzyme will bind to the

primer-template hybrid complex and creates a complementary DNA strand using the free

nucleotides in the reaction mixture (Jalali ef a/., 2017)

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After the extension step, the reaction is returned to the denaturation step and PCRcontinues Each cycle roughly doubles the amount of DNA because a new strand ofDNA serves as a template for replication in the next cycle This leads to an exponentialincrease in the quantity of DNA A total of 25 - 40 PCR cycles are carried out, depending

on the expected yield of the PCR product (Baynes, 2009.)

2.3.2 Sanger sequencing

In 1977, Sanger sequencing was developed based on selective “chain

termination” reactions occurring via in vitro DNA replication by DNA polymerase This

is accomplished by the introduction of dideoxynucleotides (ddNTPs) in the replication

reaction Four types of ddNTPs are used, each corresponding to a specific nucleotide

(ddATP, ddGTP, ddCTP, and ddTTP) These analogs of deoxyribonucleotides (dNTPs)

lack the 3'-hydroxyl group, making them unable to bond with the 5' - phosphate of the

following dNTP Previously, the products of these reactions were submitted topolyacrylamide gel electrophoresis to infer the DNA sequence based on the positions ofthe bands Currently, each dNTP is typically linked to a specific fluorescent markerthrough capillary electrophoresis in order to identify the base that terminates the chain

(Van Dik et ạ., 2014; Reuter ef al., 2015)

2.3.2.1 Principle of Sanger sequencing

According to Atkinson ef al (1969) the inhibitory effect of dideoxythymidine triphosphate (ddTTP) on DNA polymerase I depends on itsincorporation into the extending oligonucleotide chain in place of thymidylic acid (dT).Because ddT lacks a 3' - hydroxyl group, the chain cannot be extended further, resulting

2',3'-in specific term2',3'-ination at positions where dT is 2',3'-incorporated When a primer andtemplate are combined with DNA polymerase, the enzyme incubates a mixture ofddTTP and dTTP, as well as the other three deoxyribonucleoside triphosphates (one of

which is tagged with *’p) This results in a mixture of DNA fragments with identical 5'ends and ddT residues at the 3' ends When this mixture is fractionated through

electrophoresis with denaturing acrylamide gels, the resulting band pattern reveals thedistribution of dTs in the newly synthesized DNA By utilizing similar terminators for

the remaining nucleotides in separate incubations and conducting parallel gel runs, a

band pattern is produced and allows for the reading of the sequence of the DNA

14

2.3.2.2 Step of Sanger sequencing

eo make multiple copies attach a add to four

of a segment primer polymerase solution

denature dsDNA

grow complementary denaturate the electrophorese the

chains until termination dye grown chains four solutions

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Figure 2.7 The Sanger sequencing method in 7 steps (Gauthier, 2007)

Sanger proposed a technique called the Chain-Termination SequencingMethod (CTSM), which consists of seven steps: (1) denature the dsDNA fragment that

needs to be sequenced using heat, (1) amplify one of the ssDNA fragments byperforming Polymerase Chain Reaction, (11) attach a primer that corresponds to one end

of the sequence, (iv) divide the fragments into four solutions containing DNApolymerase (an enzyme that helps a complementary DNA strand develop) and the fourdeoxynucleotides (dATP, dTTP, dCTP, and dGTP) A low concentration of one of the

four termination dideoxynucleotides (ddATP, ddTTP, ddCTP, and ddGTP) is also

present in each solution, (v) the polymerase grows a complementary chain until adideoxynucleotide is added to the growing chain One of the elements 1n the duplicated

chain, either the primer or the nucleotides (the normal or the termination ones), must be

tagged with a radioactive or fluorescent marker for detection at a later stage, (vi) theresulting chains are then denatured, resulting in a series of chains that all start at thesame point but end at different positions along the DNA sequence, ending in ddXTP,(vii) finally, the four DNA samples are separated by gel electrophoresis using fourdifferent columns filled with a polyacrylamide gel An external electric field is applied

15

to force the single-stranded DNA chains to migrate through the gel The speed ofmigration within the gel 1s determined by the length of the DNA chains, allowing thesequence to be determined by ranking the electrophoretic bands from fastest to slowest.

This can be done by comparing the band positions to a known DNA ladder Gelelectrophoresis 1s a widely used technique in molecular biology for separating andanalyzing DNA fragments (Sanger ef al., 1977; Gauthier, 2007)

examined, 51 were positive for Salmonella PCR tests were employed to identifySalmonella enterotoxin (stn) genes, with these genes found in 95% of the isolates

According to research “Toxicity and antimicrobial susceptibility of salmonella

spp strains isolated from diarrhoeic dairy calves in Duc Trong district, Lam Dong

Province” by Nguyen Xuan Hoa ef a/ in 2020 The research focused on examining 38

strains of Salmonella bacteria obtained from 74 diarrheic calf samples Through PCRanalysis, it was determined that 12 of these bacterial strains contained a gene encoding

the toxin Additionally, 8 strains tested positive for the gene associated with Salmonella

enterotoxin

In 2021, a study was conducted by Le The Bien and Truong Xuan Sy to evaluatethe presence of virulence genes in Salmonella isolates obtained from fresh meat

samples These samples were collected from both slaughterhouses and markets located

in Binh Thuan Province Out of 107 isolates encompassing 6 serotypes (8S.Typhimurium, S Weltevreden, S Anatum, S Braenderup, S Newport, S.Schwavzengrund), a total of 15 were examined using polymerase chain reaction (PCR)

to identify the presence of Sa/monella invasion gene A (invA) and Salmonella virulence

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