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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.

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