Post on 05-Apr-2018
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Presented by
PALLAVI NANDI
R.D.V.V University Jabalpur
STUDY ON MALARIA SPOROZOITEAND MOLECULAR IDENTIFICATION OF
MALARIA VECTORS.
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CONTENTS
INTRODUCTION
AIM AND OBJECTIVE
METHODOLOGY
RESULTS
DISCUSSION
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INTRODUCTIONMalaria , a disease that has caused untold misery
throughout the world since antiquity.
Caused byPlasmodium (vivax , falciparum , ovale,malariae )
Transmitted exclusively by femaleAnophelesmosquitoes.
Approximately 350 500 million malaria cases & onemillion deaths occur every year due to malaria.
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Malaria is prevalent in 108 countries of the tropical &subtropical world.
According to the WHO 2010 World Malaria Report ,2.23% of deaths occur worldwide.
Southeast Asia contributes 2.5 million cases to theglobal burden of malaria. Of this, India alonecontributes 76% of the total cases.
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Malaria in central India
In MP, there are some districts where theproblem of malaria is worsening year by yearparticularly the hardcore tribal districts .
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Life cycle of malaria
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Sporozoites Derived from Greek word ( sporo = seed &
zoon = animal)
Shape : spindle-shaped, elongated.
Size: 11 m in length and 1 m in diameter
The infectious stage of the Plasmodium lifecycle, the form in which malaria is passed fromthe mosquito vector to the mammalian host .
Circulate through the body and invade liver in
a short time.
Despite their short persistence in circulatingblood,induce a strong immune response
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Where we can detect
sporozoites ?Salivary glands of femaleAnopheles mosquitoes.
The species ofAnopheles involved in thetransmission of human malaria world-wide areincredibly diverse .
There are 58 species of Indian anophelines indifferent ecological settings in India
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oA. culcifacies
o A. fluviatiliso A. stephensi
o A. dirus
o
A. minimuso A. sundaicus
oA. annularis
oA. varuna
oA.jeyporiensis
oA. philippinensis
oA. Subpictus*
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Malaria in central India are transmitted by two efficient
vectors i.e.Anopheles culicifacies and A. fluviatilis .
An. culicifacies is the vector of rural and peri-urban
malaria in peninsular India.
whileAn. fluviatilis is the vector of local importance in
the forests and forest fringes
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Why it is important to detect
sporozoites? Detection of sporozoites in infectedAnopheles , an integral component of malariaepidemiology to find out the transmission route.
Detection ofPlasmodium sporozoite of human origin in mosquito salivary glands is
important to determine which species ofAnopheles is more prevalent in a particularregion.
As the correct identification of any vector implicated in malaria transmission is key tosuccessful control.
Failure to recognise sps ofAnopheles may result in failure to distinguish between avector & a non-vector.
Hence the assessment of the impact of control measures may be seriously misleading.
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How can we detect the infection in
mosquito salivary gland ?
Quick and accurate methods is thus required tocheck the transmission intensity in thatparticular area.
Monitoring of the infective Anopheles species upto now required microscopic examination and
dissection of individual mosquitoes for thedetection of sporozoites.
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Thoughthey are accurate , but this procedure did
not identifies specific Plasmodium species.
Requires appropriate technical skills
Fresh specimens at time of dissection
Thus, time consuming & labour intensive.
Alternative methods have used specific Mabs
raised against CS proteins.
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Circumsporozoite elisa
As malaria sporozoites posses a major surface Ag , theCS protein which uniformly surrounds their externalcoat , that is infective to the vertebrate host.
MAbs raised against this protein are used in the fieldto detect which species of Anopheles vectors areinvolved in malaria transmission.
The most attractive alternative to microscopy is CSELISA sandwich assay.
Considered to be the gold standard
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Cs - protein
CS protein is an important multifunctionalmolecule for the parasite, fulfilling different roles(depending on the developmental stage) that are
vital for the parasite's development.
Several functions of CSP :-
(1)In the invasion of a mosquito's salivary glands .
(2)a key role in gliding motility of sporozoite.(3) Region II of the CS protein serves as a ligand forbinding sporozoite to hepatocytes
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WHY CS ELISA ?
Specific for oneof the several
species ofPlasmodia
infecting man
Requires easilytransportablereagents thatavoid disposalproblems associatedwith radioisotopes .
Detectsporozoitesin pooledsamples
Fast ,efficient,sensitive &stable
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Do not differentiate between the actual surface of
the sporozoite & CS protein that may be depositedon the mosquito tissue.
Hence the CS Ag can be detected in thorax ofAnopheles without sporozoites in their salivaryglands.
This technique is also time consuming , requireslaboratory facilities to be performed.
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Aim and objective
To identify infected Anopheles which are carriersof sporozoites which would determine thesporozoite infection rate.
To identify whichAnopheles vector species areprevalent in this area & are responsible in
malaria transmission .
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Methodology
To perform circumsporozoite(CS) protein ELISA
To isolate DNA fromAnopheles mosquito
To identify the species ofAnopheles by PCR
Analysis of PCR products by agarose gel electrophoresis.
Sequencing of PCR product
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CS ELISA
(1)
Adsorption of captureMAb to the wells of a
microtiter plate.
(2)
Incubated overnight
(3)MAb solution wasaspirated from the
well
(4)Each well was then
completely filled withblocking buffer
(5)
Blocking buffer wasaspirated from the
well
(6)
Test samples are thenadded to the wells
(7)
Well contentsaspirated & washedtwice with PBST:20
(8)Peroxidase conjugated
MAb added &incubated for 1 hr at
RT
(9)Well contents were
aspirated & washed 3-4 times with PBST -20
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(10)
Substrate TMB solution was added toeach well
(11)
Incubated for 15 min in dark, colour
change observed
(12)
Stop solution 2M H2SO4 added
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Individualmosquito placed in1.5 ml tube with 25l
G.B
Grinded withatleast 10 turns of
pestle
Pestle washed withadditional
25l G.B
Incubated at
65 C for 30 mins
7 l of 8M potassiumacetate & mix by
tapping.
Incubated on ice for30 min to
precipitate SDS
Centrifuged at12000 rpm for 15
min at 30C
Supernatant wastransferred to a
fresh 1.5 ml tube.
100 l of ethanoladded & incubated at
- 20C
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Centrifuged at
12000 rpm for15 min at
30 C
Ethanol removedcarefully without
disturbing theDNA pellet.
100 l of 70 %ethanol added &incubated for
1015 min at RT
centrifuged at14000 rpm for 5
min at 30C
100 l of 100%ethanol added &
incubated for
10 -15 min at RT
supernatantremoved
Air dried
Resuspended inatleast 150 l TEbuffer. (stored at -
20C).
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Amplification of D3 domain of 28S
rDNAREAGENTS REQUIREDCONCENTRATION
Taq buffer 1X
Mgcl2 1mMDNTPs 160 M
Forward
Primer D3 A
200 nM
Reverse
Primer D3 B
200 nM
Taq polymerase 1 U /reactionDNA Template
50-200 ng
A lifi i di i f
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Amplification conditions for
Primary PCRCONDITION TEMPERATURE
(IN C)
DURATION
1)Initial denaturation 95 C 10 minutes
2)Denaturation 95 C 30 seconds
3)Annealing 55 C 30 seconds
4)Extension 72 C 1 minutes
5)Final extension 4 C infinity
Number of cycles = 35 cycles1.Forward Primer D3A ( 5 GAC CCG TCT TGA AAC ACG GA 3 )2.Reverse Primer D3B (5 TCG GAA GGA ACC AGC TAC TA 3 )
35 cycles
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DNA bandtransferred ingel extraction
unit
Centrifugedat 5000g for10 minutes
The slurrycollected in
the extractioncolumn and
eluted DNA incollection
tube
The elutedDNA
transferred inmicro tube
and stored at4C
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Sequencing PCR
Ingredients 1X 14X
(required volume in l)
1. Reaction reagent 0.5 72. Primer 2 283. Buffer 1.75 24.54. Water 5.75 192.55. DNA 10 2
Total volume of the mixture =20l
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Cycling Condition for Sequencing PCR
Condition Temperature (in 0 C) Duration
1. Initial Denaturation 96 2 secs
2. Denaturation 96 10secs
3. Annealing 50 5secs
4. Extension60 4mins
5. Final Extension 4 10 minutes
Number of cycles =25
25
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product
20l of sample was added
Mix the 50l absolute ethanol +incubated at room temp.
Centrifuge at 12000 rpm for 20min+decant supernatant carefully
Hi-Di formide (12l)was added. Tubes were snapchilled & proceeded for
sequencing
125mM EDTA(2l.)was added to each tube +3M Sodium acetate(2l)added
Washing was done with 70% ethanol and spin at 12000 rpm for 10 min.