1st year phd presentation_mohammad shamsur rahman
TRANSCRIPT
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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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