1st year phd presentation_mohammad shamsur rahman

8/10/2019 1st Year PhD Presentation_Mohammad Shamsur RAHMAN

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Mohammad Shamsur RAHMAN

XXV cycle

Matricola: 967455

Identification & typing of shellfish

associated foodborne pathogens withanalysis of human health risks

University of PadovaDoctoral School of Veterinary Sciences

Public Health & Comparative Pathology Curricula


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Biomolecular approach to

identify Vibriosin Shellfish

Vibrio Shellfish


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Vibr io?

Introduction

V. choleraewith polar flagella

December 03, 2010 (Association of VibrioBiologists-AViB)

Name of Genus (8) Total No. species

Aliivibrio 5

Catenococcus 1

Enterovibrio 4

Grimontia 1

Listonella 2

Photobacterium 21 (2 subspecies)

Salinivibrio 5 (3 subspecies)

Vibrio 84 (2 subspecies)

Total 123

1854 Filippo Pacini, Italian physician, FlorenceDiscovered first Vibriosp. V. ch olerae


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Vib rio ? ......

Yellow areas: countries with cholera cases

Black dots: countries with imported cholera cases

Human pathogens: V. cholerae, V. parahaemolyticus, V. Vulnificus

Coral pathogens: V. mediterranei, V. Coralliilyticus

Pathogens of Aquaculture animals- fish, crustaceans, Molluscs(V. alginolyticus, V. splendidus)

Nutrient cycling: V.harveyi

Cholera in Haiti likely to infect 400,000

more than 1,415 people dead


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Background

Shellfish (clams & mussels) farming very important in

Veneto, Italy

Consumer prefer raw live molluscs, semi-processed or

slightly cooked

Vibriospp. very common in edible shellfish

-some species cause serious foodborne illness

So correct & reliable identification is very important

regarding public health risks


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Objectives

to Compareresults of two different approaches

to develop a MLSA(Multi locus sequence analysis)approach for complete identification of Vibrios in

Molluscs


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Materials and

Methods


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Materials and Methods

Vibrio Isolation Period: 2007

Area: Venice lagoon, Italy

172 field strains of Vibrios

11 reference strains

12 Vibrio sequences NCBI


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Mixed culture of Vibriospecies

from a mussel sample

Biochemical: Alsinas scheme29 different test: max 10 for a species


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Materials and Methods

MLSA(Multi locus Sequence Analysis)

gyrB, pyrH, recA & atpA

Target gene Amplicon length (bp)

gyrB 570

pyrH 537

recA 510

atpA 489

Total 2106General overview of the process for maintenance, queries, and analysis of gene

sequences using the MLSA Database server (Bennasar et al. 2010)


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RESULTS


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Sl. No. Species namenumber as Alsina's

scheme1 V. algino lyt icu s 29

2 V. anguil larum 10

3 V. campbelli 3

4 V. cholerae 4

5 V. fis ch eri 3

6 V. flu vialis 177 V. harveyi 6

8 V. logei 4

9 V. marinu s 1

10 V. meditarr anei 15

11 V. mim icus 3

12 V. ner eis 4

13 V. parahaemo lyt icu s 33

14 V. pelag ius 6

15 V. sp lendid us 10

16 V. vu lnif icu s 21

17 Vibr io spp. 3Total 172

Biochemical:Alsinas schemeResults........


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MLSA: 4 genes (100 strains)

- 13 clusters and 6 single strains

Results........

Thompson et al. 2009


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3 genes MLSA(157 strains)

-16 clusters & 8 single strains

Results........

R l


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Species Alsinasscheme MLSA3 genes

(157 strains)

Kappasvalue of

agreement

1 V. alginolyticus 25 32 0.77

2 V. parahaemolyticus 33 23 0.74

3 L. anguillarum 10 15 0.09

4 V. harveyi 6 22 0.08

5 V. vulnificus 14 1 0.123

6 V. splendidus 9 6 0.092

7 V. fluvialis 17 1 0.1

Species Alsinas

scheme

MLSA

4 Genes

(100 strains)

Kappas value of

agreement

1 V. alginolyticus 25 22 0.862 V. parahaemolyticus 20 16 0.80

3 L. anguillarum 4 12 0.2

Results........

Kappa

value

Level of

Agreement


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Importance

Importance

False negative results of pathogenic strains

could be a potential public health risk

Accurate & reliable taxonomic identification

of environmental Vibriostrains

V. vulnificusAlsinas scheme

MLSA

+ -

+ 1 0

- 13 143

V. parahaemolyticus

Alsinas scheme

MLSA

+ -

+ 22 1

- 11 123

V. harveyi

Alsinas scheme

MLSA

+ -

+ 2 20

- 4 131


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Sequence other reference strainsto make complete identification

Include other housekeeping genes & 16S rRNA to add more

information in phylogenetic analysis

Work on presence of pathogenic genes especially in V.

parahaemolyt icus

New sampling targeted V. parahaemolyticus (before and after

depuration process) - Qualitative (ISO) and quantitative (MPN-PCR,real time PCR)

Future..


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1st year phd presentation_mohammad shamsur rahman

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