STUDY ON MALARIA SPOROZOITE AND MOLECULAR IDENTIFICATION

OF MALARIA VECTORS.

STUDY ON MALARIA SPOROZOITE AND MOLECULAR IDENTIFICATION

OF MALARIA VECTORS.

CONTENTS INTRODUCTION

AIM AND OBJECTIVE

METHODOLOGY

RESULTS

DISCUSSION

INTRODUCTION Malaria , a disease that has caused untold misery throughout the world

since antiquity.

Caused by Plasmodium (vivax , falciparum , ovale, malariae )

Transmitted by female Anopheles mosquitoes.

Approximately 350 – 500 million malaria cases & one million deaths occur every year due to malaria.

Sporozoites

Derived from Greek word ( sporo = seed & zoon = animal)

Shape : spindle-shaped, elongated.

Size: 11 μm in length and 1 μm in diameter

The infectious stage of the Plasmodium life cycle, the form in which malaria is passed from the mosquito vector to the mammalian host .

Circulate through the body and invade liver in a short time.

Despite their short persistence in circulating blood,

induce a strong immune response

Where we can detect sporozoites ?

Salivary glands of female Anopheles mosquitoes.

The species of Anopheles involved in the transmission of human malaria world-wide are incredibly diverse .

There are 58 species of Indian anophelines in different ecological settings in India

o A. culcifacieso A. fluviatiliso A. stephensio A. diruso A. minimuso A. sundaicuso A. annulariso A. varunao A.jeyporiensiso A. philippinensiso A. Subpictus*

Aim and objective To identify infected Anopheles which are carriers of sporozoites which

would determine the sporozoite infection rate.

To identify which Anopheles vector species are prevalent in this area & are responsible in malaria transmission .

Methodology

To perform circumsporozoite(CS) protein ELISA

To isolate DNA from Anopheles mosquito

To identify the species of Anopheles by PCR

Analysis of PCR products by agarose gel electrophoresis.

Sequencing of PCR product

CIRCUMSPOROZOITE ELISA

As malaria sporozoites posses a major surface Ag , the CS protein which uniformly surrounds their external coat , that is infective to the vertebrate host.

MAb’s raised against this protein are used in the field to detect which species of Anopheles vectors are involved in malaria transmission.

The most attractive alternative to microscopy is CS ELISA sandwich assay.

Considered to be the ‘gold standard’

CS – ELISA

Isolation of DNA from Anopheles mosquito

Amplification of D3 domain of 28S rDNA

REAGENTS

REQUIRED CONCENTRATION

Taq buffer

1X

Mgcl2

1mM

DNTPs

160 µM

Forward Primer D3 A

200 nM

Reverse Primer D3 B

200 nM

Taq polymerase 1 U /reaction

DNA Template  

50-200 ng  

Amplification conditions for Primary PCR

CONDITION 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 ∞ infinityNumber 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

Sequencing PCR

Ingredients 1X 14X (required volume in µl)

1. Reaction reagent 0.5 7

2. Primer 2 283. Buffer 1.75 24.54. Water 5.75 192.55. DNA 10 2

Total volume of the mixture =20µl

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. Extension 60 4mins

5. Final Extension 4 10 minutes

Number of cycles =25

25

20µl of sample was added20µl of sample was added

Mix the 50μl absolute ethanol +incubated at room temp. Mix the 50μl absolute ethanol +incubated at room temp.

Centrifuge at 12000 rpm for 20min+decant supernatant carefullyCentrifuge at 12000 rpm for 20min+decant supernatant carefully

Hi-Di formide (12µl)was added. Tubes were snapchilled & proceeded for sequencingHi-Di formide (12µl)was added. Tubes were snapchilled & proceeded for sequencing

125mM EDTA(2µl.)was added to each tube +3M Sodium acetate(2μl) added125mM EDTA(2µl.)was added to each tube +3M Sodium acetate(2μl) added

Washing was done with 70% ethanol and spin at 12000 rpm for 10 min.Washing was done with 70% ethanol and spin at 12000 rpm for 10 min.

RESULTS• Isolation of DNA :-

Figure :- showing amplification of isolated DNA samples from both A.fluvitalis & A .culcifacies

Lane 1 -5 = shows isolated DNA samples from A. fluvitalis (375 bp product)

Lane 6 = 100 base pair ladder.

Lane 7 – 11 = shows isolated DNA samples from A. culcifacies (372 bp product)

Figure : nucleotide alignment of Anopheles fluviatilis species complex

Why it is important to detect sporozoites?

Detection of sporozoites in infected Anopheles , an integral component of malaria epidemiology to find out the transmission route.

Detection of Plasmodium sporozoite of human origin in mosquito salivary glands is important to determine which species of Anopheles is more prevalent in a particular region.

As the correct identification of any vector implicated in malaria transmission is key to successful control.

Failure to recognise sps of Anopheles may result in failure to distinguish between a vector & a non-vector.

Hence the assessment of the impact of control measures may be seriously misleading.