Khóa luận tốt nghiệp Công nghệ sinh học: Detection of Salmonella enterica subsp. Enterica and salmonella enterica serovar enteritidis in swiftlet houses by multilex PCR and investigating the presence of virulence genes sipB and fliC

enterica AND Salmonella enterica serovar Enteritidis INSWIFTLET HOUSES BY MULTILEX PCR AND INVESTIGATING THE PRESENCE OF VIRULENCE GENES sipB AND fliC... enterica AND Salmonella enterica

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

DETECTION OF Salmonella enterica subsp enterica AND Salmonella enterica serovar Enteritidis INSWIFTLET HOUSES

BY MULTILEX PCR AND INVESTIGATING THE PRESENCE

OF VIRULENCE GENES sipB AND fliC

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

GRADUATION THESIS

DETECTION OF Salmonella enterica subsp enterica AND Salmonella enterica serovar Enteritidis INSWIFTLET HOUSES

BY MULTILEX PCR AND INVESTIGATING THE PRESENCE

OF VIRULENCE GENES sipB AND fliC

Advisor Student

Dr DINH XUAN PHAT DO THI TRINH

Thu Duc City, March 2023

I would like to express my sincere thanks to the Board of Nong Lam Universityand the Board of Directors in charge of the Faculty of Biological Sciences for creatingthe most favorable conditions for me to complete my thesis

I would also like to thank all the teachers of the Faculty of Biological Scienceswho have transferred valuable knowledge during my years at the university

And especially, I would like to express my deep gratitude to Dinh Xuan Phat.PhD for wholeheartedly teaching, guiding, supporting, and giving me inspirationthroughout the process of the thesis

Many thanks to Assoc Prof Dr Tran Thi Le Minh and all members of

DH18SHA have always been with me throughout the learning process at theuniversity

In particular, thanks to Ms Nguyen Thi Mi Mi, Ms Nguyen Hoai Thuong, Ms.Cao Tran Quynh Nhu, and Mr Hoang Gia Lam for always helping, sharing, andsupporting me

And finally, I would like to thank dad, mom, and family for always loving,

trusting, and encouraging me And my friends have accompanied me on this longjourney

Sincere

CONEIRMATION COMMITMENT

My name is Do Thi Trinh, Student ID: 18126192, Class: DH18SHA, Faculty

of Biological Sciences, Nong Lam University Ho Chi Minh City, I hereby declare: this

is the graduation thesis conducted by myself, the data and information in the researchare completely honest and objective This study was a part of the research “Isolationand molecular identification of potentially pathogenic Escherichia coli, Salmonella,and Clostridium perfrigens on swiftlet houses environments in Southern Vietnam” Itake full responsibility to the council for these commitments

Thu Duc City, March 2023Student’s signature

Do Thi Trinh

30 swiftlet houses in Southern Vietnam by multiplex PCR assay, and investigation ofthe prevalence of virulence genes as sipB and fliC genes The assay used two specificprimer pairs designed to detect the STM gene of S enterica subsp enterica and the

Insertion Element sequence of S Enteritidis, with product sizes of 137 bp and 316 bp,

respectively The thermal cycle was optimized as follows: initial denaturation at 95°Cfor 5 min, followed by 35 cycles of amplification encompassing denaturation at 95°Cfor 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 40 sec, and ending by afinal extension step at 72°C for 7 min The mPCR was established with the limit of

detection for each gene at 1*10° ng/uL The results of this study indicated that with

ninety field samples, 6.67% (6/90) of samples were found to be positive for S entericasubps enterica and samples positive with S Enteritidis accounted for 3.33% (3/90).For the virulence gene, samples positive with the sipB and fliC genes were 66.67%(4/6) and 50% (3/6), respectively The results demonstrated that the mPCR procedurewas optimized and functioned well in routine veterinary diagnosis

Keywords: mPCR, Salmonella enterica subsp enterica, S Enteritidis, swiftlet house

TÓM TẮT

Phát hiện Salmonella enterica subsp enterica và Salmonella enterica serovar

Enteritidis trong nhà Yến bang kỹ thuật Multiplex PCR va khảo sát

sự hiện diện của các gene độc lực sipB và fliCSalmonella một trong những nguyên nhân gây tình trạng nhiễm khuẩn trên gia cầm và chim hoang dã với các bệnh lâm sàng cấp tính và mãn tính, trong những năm gần đây, liên tiếp xuất hiện các đợt bùng phát dịch bệnh ở người do thực phẩm có liên quan đến Salmonella Vì vay, nghiên cứu nay được thực hiện để khảo sát sự hiện điện

của Salmonella enterica subsp enterica va Salmonella serovar Enteritidis từ mẫu phân

và mẫu phết bề mặt tô yến được thu thập từ 30 nha Yến ở miền Nam Việt Nam bằng

kỹ thuật multiplex PCR, đồng thời khảo sát sự lưu hành các gen độc lực sipB và fliC ở Salmonella Trong nghiên cứu này sử dụng hai cặp mồi đặc hiệu được thiết kế dé pháthiện gen STM của S enterica subsp enterica và trình tự Insertion Element của S.Enteritidis, với kích thước sản phâm khuếch đại lần lượt là 137 bp và 316 bp Chu trình nhiệt được tối ưu hóa như sau: tiền biến tính ở 95°C trong 5 phút, tiếp theo là 35 chu kỳ khuếch đại bao gồm biến tính ở 95°C trong 30 giây, bắt cặp ở 56°C trong 30 giây và kéo dai ở 72°C trong 40 giây, và cuối cùng là hậu kéo đài ở 72°C trong 7 phút Quy trình mPCR được thiết lập với giới han phát hiện cho từng gen ở 1*105 ng/uL Kết quả của nghiên cứu này khảo sát trên 90 mẫu thực địa, có 6,67% (6/90) mẫu

dương tính với S enterica subsp enterica và mau dương tính với S Enteritidis chiếm

3,33% (3/90) Đối với các gen độc lực, tỷ lệ mẫu dương tính với gen sipB và fliC lần lượt là 66,67% (4/6) và 50% (3/6) Kết quả trên chứng minh rằng quy trình mPCR đã tối ưu và hoạt động tốt trong chân đoán thú y thường quy.

Từ khóa: nhà Yến, mPCR, Salmonella enterica subsp enterica, S Enteritidis

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS se bán gan he 11 19865111183156165143113E4RSS313556395358814464S8SEGEG1414EE0L5058 8380 i

CONFIRMATION COMMITMENT, - - cà 1 TT HH He ulABSTRACT soscoronncncenisenntenneansnrnnsnnaensessaaciendtanasennsnesacnnegnenesenentiaasenenstannaaganennhenransensta 1H

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Chapter 2 LITERATURE RE VIBW sssssesxssssss saves sie instiesoavesusinactasnasnatavnstastauhobasectassisessesinan 3

D1, Salmon aS PAthO GOR PP SA socsxa sn annsnes cevrucen senegeaacuneuaen coueseeees eancenre ốc 3

Decl Tes Maat OI porter epee cee ee oer cn a gee 32.1.2 Classification and nomenclature cccccecceccceccesceecceseeseeeseeseeeseeseceseeseeeseeaeeneeens 32.1.3 Bacterial morphology 00 cccecceesceesccesceeeseeeseeescecseesscecneeesceeseeceseeeeeeeseeeeeeeseeeees 4

ZA A, CU|HIESL-GHRERGSGTISHOGE sccccsancaenscss sean reesei eerste asa sen se anato raaeetoneIE TS 5z5: MSCHONIS SETUETfE tuisasystrtsdbsssttsinlbssbbkSsb33ASEEAsggis8tsegtsbBsslpssBkSãxtisisdihisbxistosxBsialdExEslIstrite 7

2 ASG LLEATISTHHSSTOTE51755+ s5: 5203871 1539015: G080103190588338ẸSESS539iUSS8.S163Ng.23151/-8G0G03151L03850133g1902088016801073.88 82.2 Salmonellosis 1177 10, Noo nh Ỏ 102.2.2 Virulence genes sipB and fliC of the SalmonelÏa - -++s=++x++sxsexseeesess 112.2.3 Clinical signs and macroscopic Ï@SIOTIS 5-22 S+£2+£++£++E+eEeeEererrrrrrrrrs 12

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2.2.4 Treatment and prevention of the đ1s€asSe 522 +22 *+2E*2E£*2E£zeszxezrrsres 13

Did « VGCAUM CID scans sconssncasmaemabeesermssasence nara ne memennamnee meen 13

DDD), (PC VICHIL|OTÏtxetrrsotidtntcdiSict8,8irgNhing3G104ctstBSUDESMGLSSSGSEoSVStgBSEBEENNGAISAIGUSSGS0A20r4GI0M5000463NG0003ã0800:8 132.3 Laboratory diagnostic methods - - 52+ 22 *+ 2+3 2 2xx rrrrrrrrvee 142.3.1 Polymerase Chain Reaction - PCT - - 5 2+ St + St Ssseksrrrrrrrrrrrrerreree 14259L: Prine plesOl PCR: piusxscsi62011150208513054359868S858838SAHBSNSXSGIGEEGRHESERSSGSRIERGGISSSEHISGIRISHSIS0858 15

DB led: Stages Ol PCR se uceesesce socom cguema steam mee uemeanuenon crn recent ceeeeua toner career 1623.1.5 Applicationot PCR: f6€HTHđDbsesssseseeniigissisiigiioEiDuSg0DE103G03880133:13g311680 461006383056 172.3.2 Other diagnostic methods T0 172.4 Current TESEALCh csencroensnneensunensenssnsenconcancenesvsatensensnseeneenantenageatucenssunseneasenens 18Chapter 3 MATERIALS AND METHODS tesscssseovennaanrsasrannerrsstacsreneeranesemmneveass 19Sul Time and.placeot researe lt ce.ss.sgsencormsumenlesaurwepanineacinats alas uinsnsamusereniauansimecsutnete 19

3.2 Materials, equipment, and 1nS(TUIN€TIES 5-5 22+ 22+ ++£++E+xEeeeeerrrrrerrerrke 19

cv 19E089 -.-ccsoceerereoneersonssonsenens ốẽố.ốẽốốố 193:2:3-.42qU10fiER{f 8H StU CIS bien sessssessx1t240156860G05EES4BEUSSESS6S2HS0SEBSRSSRGISDEHI-SSI38833003 20BiB UGS0lfCG DAW OLNS ve sizioszkcits22306 3 di2ckgiodsgozlfEtruksgrliirinkSshuzetSonÊtộötigudlctgaBSobmtigl0nö:EogiiufrEEnioS8tfouhrgtsdfsie 20

3.3.1 DNA extraction from field samples and recovery of DNA target - 223.3.2 Primers used and thermal cycles for the researchh -+-+++ss++<>+sc<+s 23

3.3.3 Optimization of the mPCR prOfOCOIÏL 22222223 *22 E22 E+zE+zverreerrrrrerrrrreree 243.3.3.1 Optimization of the annealing temperature for s-PCR reaction 243.3.3.2 Optimization of the annealing temperature for mPCR reaction 25

3.3.3.3 Optimization of the primer concentration for mPCR reaction 25

3.3.3.4 Evaluation of primer specificity in mPCR reaction 5+ +5s++s<++s 26

3.3.3.5 Determination of the detection limit for m-PCR reactIon - - 5: 21

3.3.4 Detection of S enterica subsp enterica and S Enteritidis in field samples 273.3.5 Determination of the presence virulence genes sipB and /Ï¡C - 27Chapter 4, RESULTS AND DISCUSSION vsessccesssnsceonsesansneanancenemanceateanersnonveccnmneemnase 28

an 284.1.1 Optimization of the annealing temperature for mPCR reaction - 284.1.2 Optimization of the primer concentration for mPCR reaction - 30

4.1.3 Examination of primer specificity in mPCR reaction 5 5-5555 <+<=+<s52 31

4.1.4 Determination of the detection limit for mPCR react1ON - 5-5 =<=ss 324.1.5 Result of detection of S enterica subsp enterica and S Enteritidis 33

4.1.6 Investigating the presence of virulence genes sipB and /ÏC -+ 35

Đo on 354.2.1 Result of the detection limit for mPCR TreactIOA: - 5 55555 <+s<+s£+e+eczerss 35

4.2.2 Results of detection of S enterica subsp enterica and S Enteritidis 36

4.2.3 Results of investigating the presence of virulence genes sipB and /liC 36Chapter 5 CONCLUSION AND RECOMMENDATIONS -~ 38

5.2 Recommendations 8 Ả.Ả 38REFERENCE 6 cung edy code BI onc 54C HESXGSỒNHRSHISSE.4SDIENGHHSENEHESREGEAHSISNGNSGESRSSIGBBSEUESHS4C3SEEN 39APPENDIX, s:ssezss:sz2szsse:Sos21680: 15g18 050501030 8016052350028 nSE0kh33SoSL|gi-3001S89n0083390000345880puag 43

: Central Nervous System: Diethyl pyrocarbonate: deoxynucleotide triphosphates: Deoxyribonucleic acid

: Enzyme-linked immunosorbent assay: lipopolysaccharide

: Multiplex Polymerase Chain Reaction: Salmonella enterica subsp enterica serovar Enteritidis: Salmonella pathogenicity island

: Random Amplified Polymorphic DNA: Xylose Lysine Deoxycholate

: Tris - Acetate - EDTA: Triple Sugar Iron: type III secretion systems

LIST OF FIGURES

PageFigure 2.1 Diagram representing the Salmonella gefus - - 5555 + ++s++ss++ss+ssss2 4Figure 2.2 Scanning of electron micrographs of Sa/monella Enteritidis cells 4Figure 2.3 Colony morphologies of Salmonella Enter1tId1s -++-+>+-<5-+2 5

Figure 2.4 Isolation of the Salmonella from clinical specImens - - + 6

Figure 2.5 Colonial morphology and biochemical features of Salmonella - 7Figure 2.6 Circular representation of the S Enteritidis PT4 chromosome 8Figure 2.7 Egg contamination by Sa/monella Enter1tId1s -«++-<++<c+<<<<s+ 9Figure 2.8 Gross lesions of Sa/monella infection in pouÏtry -+-<+5s<<s<++ 13Figure 2.9 The components of Polymerase Chain ReactIon - - 5555 5>+<<<ss 14Figare:2.10; Prineiple Gf PCR cscccssensnensescnsnessansnp vay tHE33SSSS4ESGEEBESSES/3H48S3S4BSGHSBS.SAĐ134018846380058538 16

Figure 3.1 Scheme described optimized processing mPCR reaction - 21

Figure 3.2 Scheme described the detection of sipB and fliC genes - 21Figure 4.1 Optimization of annealing temperature in s-PCR -=5-<5+ 29

Figure 4.2 Optimization of annealing temperature in mPCT - 5-5555 =+ss52 30

Figure 4.3 Products of s-PCRs and mPCRS :csccescesseeseeeseeseeseeeeesneeseesseeseeeseeees 30Figure 4.4 Result of specificity primers f€Sf - - - 55-252 <S2*22£+sccsrrrrrerrrrrke 32Figure 4.5 The detection limit of the mPCTR 52 5-+ 25+ *>+£+s++eczxerrerrxeexs 33Figure 4.6 Electrophoresis results of PCR products of field samples 34Figure 4.7 Electrophoresis results of PCR products for each virulence gene .35

Chapter 1 BACKGROUND

1.1 Introduction

In recent years, statistics have shown us that swiftlet farming and swiftletproducts have become an extremely developed industry in Vietnam and somecountries around the world According to the Vietnam News, in 2022, Vietnam hasmore than 22,000 swiftlet houses in 42 out of 63 provinces and cities with an annualyield of about 120 tons worth 500 million USD (The Vietnam News: https://vietnamnews.vn/) Thus, understanding infectious pathogens is important to protect this new

and prosperous profession Currently, Salmonellosis an infection caused by the

bacterium Salmonella remains one of the global problems in a wide range of hosts,causes huge losses in poultry and/or wild birds, and foodborne outbreaks of humanillness

Investigations of Salmonella shown that they are found in all species ofmammals, pigs, poultry, wild birds, reptiles, amphibians, and humans all over theworld (Porwollik et al., 2004; Hamer et al., 2012; Krawiec et al., 2015) and in theenvironment in sites such as moist soil, water, fecal particles, and animal feeds, etc.(Abulreesh, 2012) In 2013, Obukhovska conducted research to identify naturalreservoirs of Sa/monella Enteritidis in wild birds in National Park 'Askania Nova' and

peninsula 'Arabat arrow' (the Azov Sea coast) and confirmed circulation of Salmonella

in this group of wild birds

Currently, many swiftlet houses were built within or near poultry farms which

poses a potential risk of the transmission of pathogenic viruses Thus, understanding

the presence of Salmonella in swiftlet houses is important for a good disease controlstrategy in the future Some studies have shown the possibility to detect S entericasubsp enterica and S Enteritidis by polymerase chain reaction via the STM gene thatencoded putative inner membrane protein (Kim et al., 2006) and the Insertion Elementsequence (Wang and Yeh, 2002; Mirmomeni et al., 2008), respectively Moreover,several studies have evaluated the presence of virulence genes such as the sipBencoding Salmonella invasion protein (Chen et al., 1996; Skyberg et al., 2006); thefliC gene encoding flagellin in Salmonella (Paiao et al., 2013) In this study, we

1

developed a multiplex PCR method and used it for the detection of Salmonellaenterica subsp enterica and Salmonella Enteritidis in samples collected in swiftlethouses and investigating the presence of virulence genes sipB and fliC genes.

1.2 Objectives

Successfully established mPCR protocol for detection of Salmonella enterica

subsp enterica and Salmonella enterica serovar Enteritidis and PCR to amplify sipB

and fliC genes of Salmonella

1.3 Contents

Content 1: Optimize the conditions of mPCR procedure for detection of S.enterica subsp enterica and S Enteritis

Content 2: Application of the optimal mPCR procedures to confirm S enterica

subsp enterica and S Enteritidis in field samples

Content 3: Examine the virulence genes sipB and /liC genes in samples positivefor Salmonella

Chapter 2 LITERATURE REVIEW

Salmonella spp are members of the family Enterobacteriaceae, a rod-shapedgram-negative bacterium, facultatively anaerobic rods (Holt et al., 1994) Salmonella

is currently classified into two species, Salmonella bongori and Salmonella enterica

As the Fig 2.1, S enterica had six subspecies: S enterica subsp enterica (I), S.enterica subsp salamae (II), S enterica subsp arizonae (IIa), S enterica subsp.diarizonae (IIIb), S enterica subsp houtenae (IV) and S enterica subsp indica (VDbased on biochemical reactions and contains over 2500 serovars (Grimont and Weill,2007) The number of serotypes continues to rise and these new serotypes are updates

of the Kauffmann-White scheme (Popoff et al., 2003), besides, can help identify the

source of disease outbreaks and track antibiotic resistance trends (Markey et al., 2013).Salmonella spp are classified into serovars based on the lipopolysaccharide (O),flagellar protein (H), and capsular (Vi) antigens (Brenner et al., 2000) Besides,agglutination reactions to antibodies or antisera specific to the somatic O antigens withinto six serogroups, designated A, B, Cl, C2, D, and E This grouping system allowed

genus identification of Salmonella and provided value for epidemiological studies

(Wattiau et al., 2011) In addition, there may be strains that differ in virulence And

formal names of serovars are often shortened, for example, Salmonella enterica subsp.enterica serovar Enteritidis can be called Salmonella serovar Enteritidis or Salmonella

Enteritidis

Salmonella enterica with almost over 2500 serovars, together with in

Salmonella bongori, are classified into subspecies based on somatic (O) antigen,flagellar (H) phase 1 antigens, flagellar (H) phase 2 antigens, and other antigensseparated by a colon (Giannella, 1996)

J Salmonella |

Salmonella bongori Salmonella enterica

M TT TTEETNRNE.

Subsp V

(20) Subsp.lI | Subsp.lI Subsp Illa Subsp lllb Subsp.IV Subsp VI

enterica | salamae arizonae diarizonae hautenae _ indica

(1454) (489) (94) (324) (70) (12)

{ 99% of human and animal infections |

Typhoidal Salmonellae Nontyphoidal Salmonellae (NTS)

Enteric fever Gastroenteritis/bacteremia

Human restricted/adapted Humans/animals

Figure 2.1 Diagram representing the Salmonella genus (Langridge et al., 2012;

Gyles et al., 2010)

2.1.3 Bacterial morphology

Enterobacteriaceae Salmonella is straight, nonspore-forming rods, measuring about

0.7 - 1.5 x 2.0 - 5.0 um (Gast and Porter Jr, 2020) Paratyphoid Sa/monella is usuallymotility by peritrichous flagella and motile, although in nature, there are occasionalmutations that are non-mobile And both Salmonella Pullorum and SalmonellaGallinarum are characteristically non-motile (Gast and Porter Jr, 2020) According toinvestigation of Salmonella has shown that they are found in all species of mammals,birds, reptiles, amphibians, and humans all over the world (Reeves et al., 1989).Among them, Salmonella serovar Typhimurium and Enteritidis account for mosthuman and domestic animal Salmonella infections (Porwollik et al., 2004) MostSalmonella serovars could cause disease in a broad range of hosts and some serovarshave a narrow host range (Spickler and Larson, 2013) In the environment in sites such

as moist soil, water, fecal particles, and animal feeds, Salmonellae could survive fornine months or more (Markey et al., 2013)

Typical Salmonella colonies on agar media are about 2 - 4 mm in diameter,round with smooth edges, slightly raised, and glistening (Gast and Porter Jr, 2020)

2.1.4 Cultural characteristics

The steps for the isolation of Salmonella from clinical specimens (feces,tissues, swabs, etc.) are indicated in Fig 2.5, cultured in Buffered Peptone Water(BPW) is the pre-enrichment broth of choice in conjunction with Rappaport-Vassiliadis (RV) selective enrichment medium (Vassiliadis et al., 1987) Incubationfor 18 - 24 hours and the temperature for pre-enrichment should range between 35°Cand 37°C, which is the optimal growth temperature of Salmonella spp This stepassures the resuscitation of bacteria and their multiplication which will be later sub-cultured onto selective/ indicator media (MacConKey, XLD, BG medium) (Markey etal., 2013)

Faeces, tissues, specimens from abortion cases and heart blood

Direct plating on ppapor

GN broth (BBL) selectivelindicator media Sees

| Rappaport

Subculture

“ —— 24 and 48 hours

Incubate at 37°C for 48-72 hours

Pig isolates Most Salmonella speciesSLE yl ack one - Suspicious colonies | momen(S Typhisuis) XLD: red colony/black centre

Inoculate TSI agar and lysine decarboxylase broth

Most: R/Y/H2S + and lysine +

S Choleraesuis R/Y/H,S — and lysine +

S Typhisuis R/Y/H,S + and lysine —

Identify by biochemical tests

if considered necessary

Salmonella polyvalent antisera

Negative

Figure 2.4 Isolation of the Salmonella from clinical specimens BG:

brilliant green agar, BG (modified): brilliant green agar, modified (Oxoid),XLD: xylose lysine deoxycholate medium, TSI: triple sugar iron agar.(Markey et al., 2013)

Colonial morphology on selective/indicator media:

On XLD medium: after 24 to 48 hours of culture, small colonies appear on thesurface of the agar, the majority of Salmonella serotypes produce H2S and have red

colonies with a black centre (Fig 2.6A) The typical reaction for Salmonella in TSIagar is a red (alkaline) slant, yellow (acid), and superimposed (black) H2S production

(R/Y/H2S+), however, Salmonella serovar Choleraesuis does not produce H2S (Fig

2.6B) Thus, further biochemical tests or molecular methods should be carried out fordeeper identification.

Genome sizes of Salmonella varied among serovars with ranges of 4460 to

4857 Kbp and genome size of S Enteritidis PT4 1s 4,685,848 bp (Thomson et al.,2008) Salmonella Enteritidis frequently contain plasmids that range from 55 - 60 Kbp

A phage-typing scheme originally developed by Ward et al 1987 hepted todifferentiate 27 phage types (PT) within serovar Enteritidis Currently, predominantphage types isolated worldwide in Enteritidis serovar are PT4 and PT8, which wereinitially the most commonly found in the United States and the United Kingdom

Today, these two phage types and PT13a account for the majority 1n Enteritidisserovar The genomes of the different serovar Enteritidis phage types were found to behighly similar

a

Figure 2.6 Circular representation of the S Enteritidis PT4 chromosome From the

outside in, the outer circle 1 marks the position of regions of difference Circle 2 showedthe size in base pairs Circles 3 and 4 showed the position of CDS transcribed in aclockwise and anti-clockwise direction, respectively; circle 5 shows the position of S.Enteritidis PT4 pseudogenes Circles 6 and 8 showed the position of S Enteritidis PT4genes that had orthologs in S Typhimurium strain LT2 (all CDS colored green) and S.Gallinarum strain 287/91 (all CDS colored blue), respectively Circles 7 and 9 showed theposition of S Enteritidis PT4 genes that lacked orthologs in S Typhimurium strain LT2 (allCDS colored pink) and S Gallinarum strain 287/91 (all CDS colored gray), respectively.Circle 10 showed the position of S Enteritidis PT4 rRNA operons (red) Circle 11 showed

a plot of G + C content (in a 10-kb window) Circle 12 showed a plot of GC skew ([GC]/[G + C]; in a 10-Kb window).(Thomson et al., 2008)

2.1.6 Transmission

Salmonella spp has been the major cause of the food-borne salmonellosis

pandemic They are transmitted mainly through the fecal-oral route, the Salmonellafrom fecal contamination can colonize the gastrointestinal tract of chickens, humans,and animals consuming water and food contaminated with bacteria (Abulreesh, 2012).Birds and other animals could become infected by ingestion of contaminated feed,drinking water, or contact with an infected animal (Hinton, 1988; Kinde et al., 1996)

Besides, several studies suggested the ability to transmit viruses of fomites andmechanical vectors (e.g., insects such as flies) (Holt et al., 2007).

With its wide host range, paratyphoid Salmonella has created reservoirs ofdisease in poultry and wild birds Serovar Enteritidis adapted well to the hen house

environment, the bird, and the egg Most commonly, hens are infected with S.Enteritidis by vertical transmission Vertical transmission occurs in birds through

transovarian infection of eggs Transovarian transmission is the direct contamination

of the yolk, albumen, and eggshell membranes (Okamura et al., 2001) According tothe Center for Disease Control and Prevention [CDC], USA, 2013, from 1985 to 2003

in 75% of S Enteritidis outbreak cases, eggs were confirmed as the primary ingredient

or food vehicle of contamination and a major outbreak occurred in 1994, S Enteritidis

contaminated liquid eggs caused the cross-contamination of ice-cream prepared at thesame facility (Hennessy et al., 1996)

Penetration of Salmonella through egg shell and membranes

2 "<<

Salmonella in faeces or vagina

Eggs post lay

Internal contamination of the eggs prlatartati atathrough infection of reproductive organs Viteline membrane

Motility towards and

the vitelline membrane

in yolk

Figure 2.7 Egg contamination by Salmonella Enteritidis (a) Salmonella is orally taken

up by the hen and enters the intestinal tract; (b) Egg contamination by Salmonellapenetration through the eggshell and shell membranes after outer shell contamination (c)Direct contamination of the yolk, yolk membranes, albumen, shell membranes, and eggshelloriginating from infection of the ovary, infundibulum, magnum, isthmus, and shell gland,respectively; (d) Salmonella bacteria deposited in the albumen and on the vitelline membrane.(Gruenheid and Finlay, 2003)

In humans, the consumption of contaminated animal foods (eggs, poultry, pork,beef, etc.), and contaminated water increased the risk of infection and outbreaksassociated with this food have been increasingly reported in recent years (Spickler andLarson, 2013).

2.2 Salmonellosis

2.2.1 Pathogenesis

Salmonellosis is an infection caused by Gram-negative bacteria from the genusSalmonella that usually affects the intestines in poultry with high mortality Almost allstrains of the Salmonella are pathogenic as the invasion of non-phagocytic human hostcells (Hansen et al., 2002) The Salmonella infection process was associated withSalmonella pathogenicity islands (SPIs), gene clusters located at the largechromosomal DNA region and encoding for the structures involved in the invasionprocess (Grassl and Finlay, 2008) SPIs encoded for type III secretion systems (TTSS)and two key SPIs were SPI-1 and SPI-2 which contained the type III secretion systems(TTSS) systems TTSS-1 and TTSS-2, respectively SPI-I TTSS is encoded by 30

genes whose products mediate the delivery of effector proteins into the host cell whileSPI-2 promoted invasion in a number of Salmonella serotypes including Salmonella

Enteritidis (Ochman and Groisman, 1996) The transferred proteins are referred to aseffector proteins and some effector proteins have been characterized of bacterialspecies (Galan, 2007) and TTSS - 1 and its effectors also play a role in the interactionbetween Salmonella and phagocytic cells (Barrow et al., 2010)

When Salmonella entered the digestive tract of host cells, they tend topenetrate the epithelial cells lining the intestinal wall, SPIs - multi-channel proteinsthat allowed Salmonella to carry its effectors across the intestinal epithelial cellmembrane into the cytoplasm And then, its effectors activated the signal transductionpathway, resulting in the outward extension of the epithelial cell membrane to engulfthe bacteria (Takaya et al., 2003)

In 2008, Bakowski et al stated that the viability of Salmonella strains in hostcells is important for the pathogenesis of the disease because, in the absence ofviability, Salmonella is nonvirulent In fact, when foreign bacteria entered the hostcell, the bacterrum was encased in a membrane compartment called a vacuole, at thesame time, the host cell would activate an immune response, resulting in the fusion of

1

the lysosomes and the secretion of enzymes to degrade the intracellular bacteria,however, in this case, TTSS of Salmonella to inject other effectors into the vacuole,causing the alteration of the compartment structure, blocks the fusion of thelysosomes, help them survive and replicate in the host (Gyles et al., 2010).

2.2.2 Virulence genes sipB and fliC of the Salmonella

Endotoxins play roles in the pathogenicity of Salmonella, associated with thelipid A portion of Salmonella cell wall lipopolysaccharide (LPS) LPS contributed tothe inhibition of the bacterial cell wall to attack and digestion by host phagocytes whenSalmonella entered host cells Furthermore, several proteinaceous toxins had also beenreported in Salmonella Salmonella Enteritidis pathogenicity has been related to anumber of virulence factors that adherence to intestinal epithelial cells and invasionmechanisms are important first steps in this process Flagella and some types offimbria may play roles in S Enteritidis invasion and dissemination to internal organs

and Flagella-deficient mutants were reduced the ability to invade the organs

containing adaptor protein inducing interferon and Tram - Trif-related adaptor

molecule) (Cook et al., 2007) Promoting host-cell apoptosis and necrosis via 1-mediated activation ofIL-1B and IL-18 (Dreher et al., 2002)

caspase-Salmonella had several peritrichous flagella and the length of the filament was

10 to 15 um long The /liC gene encoded the flagellar filament protein, which was the

main structural subunit and the primary globular protein forming the filament of theflagellum in the majority of Salmonella (Haiko and Westerlund, 2013) It was a potenttrigger of innate immune responses and could be used as an effective vaccine adjuvant

to induce cellular immune responses (Honko and Mizel, 2005; Taherkhani et al.,2014)

2.2.3 Clinical signs and macroscopic lesions

2.2.3.1 Clinical signs

In poultry, Salmonella infections of poultry typically cause clinical signs only

in very young chicks, Salmonella contamination within eggs may lead to embryomortality or rapid death among newly hatched birds Clinical signs of severeSalmonella infection in chicks include progressive lassitude, drooping wings, ruffledfeathers, shivering and huddling, lethargy, anorexia, emaciation, profuse waterydiarrhea, and CNS signs (Spickler and Larson, 2013) Some Salmonella Enteritidisstrains have caused anorexia, diarrhea, and reduced egg production in laying hens

(Shivaprasad et al., 1990) Although adult birds are more resistant to infection with

Salmonella and often asymptomatic in infected birds, they can transmit the diseasethrough eggs to their young (Gast and Porter Jr, 2020)

Morbidity and mortality could be high during the first 2 - 3 weeks of life withbody weight loss or growth retardation In general, asymptomatic Salmonella

infections are common, in many cases, infections become symptomatic only when theanimal is stressed by external factors (including vaccinations, transfer of birds, and

rearing at a high density) (Suzuki, 1994)

2.2.3.2 Macroscopic lesions

Severe Salmonella infection in young chicks could involve septicemia with

high mortality and yolk sac infection is one of the typical signs in newly hatched

poultry, the navel skin is often red, and the yolk sac is typically filled with blood,

besides, enteritis with necrotic lesions in the mucosa, and cecal cores may occur as the

Salmonella infection becomes more severe Lesions in young birds might include theliver being enlarged with foci of necrosis, kidneys and splenic might be enlarged andcongested, and accumulation of fibrin on the surfaces of the heart and liver (Gast andPorter Jr, 2020)

Figure 2.8 Gross lesions of Salmonella infection in poultry (4) Hemorrhagic

yolk sac in a 5 day old broiler chick due to Paratyphoid Salmonella infection; (B)Enlarged liver with foci of necrosis in a bird; (C) Mucosal necrosis andhemorrhage in small intestine (Gast and Porter Jr, 2020)

2.2.4 Treatment and prevention of the disease

2.2.4.1 Treatment

For many years, the prevention and treatment of Salmonella infections inpoultry with antibiotics have been controversial, uncomplicated Salmonella infectionsare not recommended to be treated with antibiotics Undeniably their usefulness for

commercial purposes, however, antibiotics could affect the intestinal flora and increasethe emergence of antibiotic-resistant strains (Cosby et al., 2015)

In fact, many strains of Sa/monella are resistant to one or more antibiotics, soantibiotic treatment should be based on susceptibility testing Antibiotics wereemployed effectively for S Enteritidis control in poultry including Ampicillin,Enrofloxacin, Fluoroquinolone, and Gentamycin (Mcllroy et al., 1989; Seo et al.,2000) Besides, correction of electrolyte imbalances and fluid replacement are used in

cases of enteritis

2.2.4.2 Prevention

Farms have used biosecurity systems that effectively prevent Salmonella, therisk of introducing the bacterium into a flock can be decreased, quarantine newlytransferred animals, flock management, good hygiene, and minimizing stressfulevents And when a Salmonella outbreak occurs in a herd, it should be promptlyidentified, treated, or culled if necessary to decrease the spread in the herd Control

and prevent fecal contamination of feed and water, and clean contaminated equipment

and materials

Livestock vaccines can reduce the level of shedding of Salmonella, in somestudies showed that killed vaccines, vaccines composed of outer-membrane, fimbrial,

or flagellar proteins decreased incidences of S.Enteritidis infection in poultry

(Feberwee et al., 2000; Khan et al., 2003)

2.3 Laboratory diagnostic methods

2.3.1 Polymerase Chain Reaction - PCR

In the 1980s, Kary Mullis invented this technique, which was based on thethermal cycle and DNA replication reaction that takes place in a tube, PolymeraseChain Reaction (PCR) technique is a widely used method in testing, diagnostics, andespecially molecular biology by making multiple copies of certain target DNA via the

main enzyme DNA polymerase PCR could allow the isolation of DNA fragments

from genomic DNA by selective amplification of a specific region of DNA At the end

of the PCR reaction, the specific sequence will be accumulated in billions of copies(amplicons) It is applied widely in molecular biology and techniques, such as DNAcloning, DNA sequencing, microarrays, etc

St Sense strand 8!

TT CTT

ATG TAA

TRƠN ; “TT ® ®

5° primer (forward primer

1.9 (reverse primer) Nucleotides (dNTPs)

synthesizes new strands of DNA to the target sequence The most used of theseenzymes is Zag polymerase (from Thermus aquaticus), and Pfu polymerase (fromPyrococcus furiosus) is used because of its higher fidelity when copying DNA Primer

pair - oligonucleotide sequences that are complementary to the target sequence and the

polymerase begins synthesizing new DNA two primers, which is used to determine theDNA fragment to be amplified dNTPs mean deoxynucleotide triphosphates - singleunits of the bases A, T, G, and C, which 1s the building block of the DNA molecular.And Buffer solution that provides a suitable chemical environment for the activity ofDNA polymerase (Shaheen et al., 2020)

Since PCR was invented by Mr Mullis, since then, PCR has been improvedand developed into many new formats to enhance the utility of this method There aresome common types of PCR: Real-Time PCR (quantitative PCR or qPCR), Reverse-Transcriptase (RT-PCR), Multiplex PCR, Nested PCR, Touchdown PCR, Fast PCR,Hot Start PCR, etc As a result, PCR is a significant technology that is mostly used forexperiment laboratories of molecular biology, biomedicine, criminal science, and

DNA testing (Rahman et al., 2013)

2.3.1.1 Principles of PCR

Polymerase Chain Reaction (PCR) technique is a method for obtaining a largeamount of a specific DNA sequence from a DNA sample This amplification is based

on the replication of a double-stranded DNA template Primers pair contain a

complementary sequence with the 3’ end of the sense strands and anti-sense strands of

target DNA They have been divided into three main stages: a denaturation phase, ahybridization phase with primers (annealing), an elongation phase (extension), and thewhole phases have a cycle The product of the previous phase after one cycle willserve as a template for the next steps, thus exponential amplification is happened(Kadri, 2019)

@ Denaturation 2) Annealing © Elongation

Figure 2.10 Principle of PCR Amplification is achieved by a series of three

steps: (1) Denaturation, in which double-stranded DNA templates are heated toseparate the strands; (2) Annealing, in which short DNA molecules called primersbind to flanking regions of the target DNA; and (3) Extension, in which DNA

polymerase extends the 3' end of each primer along the template strands (The

WNTPROTEINS: http://wntproteins.com/)

Step 1: Denaturation of double-stranded DNA

The temperature at the stage ranges from 94 - 96°C using a thermal cycler, thetwo single strands of DNA are separated by raising the temperature to high, and thematrix DNA, which serves as a template during the replication, is denatured Becausethe hydrogen bonds can break down easily at a temperature higher than 80°C and

single strands of DNA are created

Step 2: Annealing phases (hybridization)

It is also called the primer hybridization temperature The primer is a shortnucleic acid sequence bound to the individual strands of DNA, DNA polymeraseenzyme bound and started synthesizing the nucleotides (A, T, C, G) from the addedmixture solution will pair with the individual separated strands of DNA, and thetemperature at this stage is between 40 and 70°C

Step 3: Elongation phases (extension)

In the third phase, a new duplicate double-stranded DNA molecule had beenformed from each of the single strands of the original sample molecule and the

reaction takes place at 72°C and Jaq polymerases will be extended DNA targets It

takes 20 - 40 cycles using the thermal cycler which automatically repeats the heating

and cooling cycles of the process to synthesized and generated an amount of DNA

(about 0.1 yg) For the synthesis of the whole new DNA strands, it should have an

elongation phase at 72°C again Thus, a single segment of DNA from one sample can

be amplified to form millions of copies after 35 cycles (Kadri, 2019)

2.3.1.3 Application of PCR technique

Polymerase Chain Reaction (PCR) is widely used for varied applicationsacross the field of molecular biology and PCR technology is extensively used innumerous applications The most common medical application of PCR is for genetictesting where a DNA sample is analyzed for the presence of genetic disease mutations,detection of genetic disease, diagnosis of retroviral infection, and cancers Besides,

this technique is also used in genetic research, gene therapy, evolutionary studies,

tissue typing, research applications, and sequencing (Bioinformatics, Human genomeprojects, and Genome cloning) (Solanki, 2012)

2.3.2 Other diagnostic methods

Detection of Salmonella spp from clinical specimens by isolation method:

pre-enrichment Salmonella in Buffered Peptone Water (BPW), conjunction with

Rappaport-Vassiliadis (RV) selective enrichment medium, incubation for 18 - 24hours and the temperature for pre-enrichment should range between 35°C and 37°C,which 1s the optimal growth temperature of Salmonella spp and 1Ÿ Proteus species is aproblem, the enrichment broths can be incubated at 43°C to make it more selective.And then subcultured onto selective/indicator media (MacConKey, XLD, BGmedium) through colony morphology Identification of Salmonella by biochemicalproperties tests (According to EN ISO-6579: 2002 - Microbiology— General Guidance

on Methods for the Detection of Salmonella International Organization forStandardization, Geneva, Switzerland) (Markey et al., 2013)

Table 2.1 Specific biochemical tests of Salmonella

=— Urease Oxidase Indole Catalase Mobility Citrate

1

Molecular diagnosis methods: Real-time PCR (qPCR) method was used fordetecting Salmonella bacteria and detection in the sample Involves an initialenrichment step followed by a PCR-based method; RAPD-PCR technique; nucleotide

sequencing techniques (Markey et al., 2013) One of the most commonly used

techniques is pulsed-field gel electrophoresis (PFGE), this method separates DNAunder conditions of alternating polarity, etc

Serological methods of diagnosis: Specific antibodies have been utilized in avariety of enzyme immunoassay (EIA); Immunomagnetic separation (IMS);Agglutination tests In 1991, Kim et al developed an ELISA (Enzyme-linkedimmunosorbent assay) process for the detection of S Enteritidis antibodies in chickens

was found to be sensitive and specific

2.4 Current research

Several reports were shown that Salmonella enterica subsp enterica and

Salmonella Enteritidis were found in wild birds when examined on the feces of wild

birds or the surface swab samples (Hamer et al., 2012; Obukhovska, 2013; Krawiec et

al., 2015; Santos et al., 2020) In addition to the traditional methods for the detection

of Salmonella were based on cultures using selective media and then performedbiochemical and/or serological tests, in some studies, the PCR assay has been used toscreen rapidly for large samples The PCR assay was developed for the detection ofSalmonella enterica subsp enterica via the STM gene that encoded putative inner

membrane protein (Kim et al., 2006) and for detection of Salmonella Enteritidis via

the Insertion Element (IE) gene sequence (Wang and Yeh, 2002) Moreover, a PCR

assay was established to detect the presence of Sa/monella Enteritidis via virulence

genes such as the sef4 (Mirmomeni et al., 2008) and the sđ/7 gene (De Freitas et al.,2010)

Some studies developed PCR methods for the detection of the presence ofvirulence genes Salmonella invasion protein B (sipB) played an important role in theSalmonella invasion of nonphagocytic cells (Skyberg et al., 2006; Obe et al., 2020).And the /liC gene was encoded for flagellin in Salmonella (Paiao et al., 2013)