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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 m in length and 1 m 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 ?

o o o o The species of Anopheles involved in the transmission of o human malaria world-wide are o incredibly diverse . o o There are 58 species of Indian o anophelines in different o ecological settings in India o Salivary glands of female Anopheles mosquitoes. A. culcifacies A. fluviatilis A. stephensi A. dirus A. minimus A. sundaicus A. annularis A. varuna A.jeyporiensis A. philippinensis 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. MAbs 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
(1) Adsorption of capture MAb to the wells of a microtiter plate. (6) Test samples are then added to the wells (7) Well contents aspirated & washed twice with PBST:20

(2) Incubated overnight

(5)

(8) Peroxidase conjugated MAb added & incubated for 1 hr at RT

Blocking buffer was aspirated from the well

(3) MAb solution was aspirated from the well

(4) Each well was then completely filled with blocking buffer

(9) Well contents were aspirated & washed 3-4 times with PBST -20

(10)

Substrate TMB solution was added to each well

(11) (12)

Incubated for 15 min in dark, colour change observed

Stop solution 2M H2SO4 added

Isolation of DNA from Anopheles mosquito


Individual mosquito placed in 1.5 ml tube with 25l G.B Grinded with atleast 10 turns of pestle Pestle washed with additional 25l G.B

Incubated on ice for 30 min to precipitate SDS

7 l of 8M potassium acetate & mix by tapping.

Incubated at 65 C for 30 mins

Centrifuged at 12000 rpm for 15 min at 30C

Supernatant was transferred to a fresh 1.5 ml tube.

100 l of ethanol added & incubated at - 20C

Centrifuged at 12000 rpm for 15 min at 30 C

supernatant removed

Air dried

Ethanol removed carefully without disturbing the DNA pellet.

100 l of 100% ethanol added & incubated for 10 -15 min at RT

Resuspended in atleast 150 l TE buffer. (stored at 20C).

100 l of 70 % ethanol added & incubated for 10 15 min at RT

centrifuged at 14000 rpm for 5 min at 30C

Amplification of D3 domain of 28S rDNA


REAGENTS REQUIRED CONCENTRATION
1X

Taq buffer

Mgcl2 DNTPs

1mM 160 M

Forward Primer D3 A Reverse Primer D3 B Taq polymerase DNA Template

200 nM

200 nM 1 U /reaction

50-200 ng

Amplification conditions for Primary PCR


CONDITION 1)Initial denaturation TEMPERATURE (IN C) 95 C DURATION 10 minutes

2)Denaturation 3)Annealing 4)Extension 5)Final extension

95 C 55 C 72 C 4 C

30 seconds 30 seconds 1 minutes infinity 35 cycles

Number of cycles = 35 cycles 1.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 )

DNA band transferred in gel extraction unit

Centrifuged at 5000g for 10 minutes

The slurry collected in the extraction column and eluted DNA in collection tube

The eluted DNA transferred in micro tube and stored at 4C

Sequencing PCR
Ingredients 1. Reaction reagent 1X 0.5 14X (required volume in l) 7

2. Primer 3. Buffer 4. Water 5. DNA

2 1.75 5.75 10

28 24.5 192.5 2

Total volume of the mixture =20l

Cycling Condition for Sequencing PCR


Condition 1. Initial Denaturation Temperature (in 0 C) 96 Duration 2 secs

2. Denaturation 3. Annealing 4. Extension 5. Final Extension Number of cycles =25

96 50 60 4

10secs 5secs 4mins 10 minutes 25

20l of sample was added

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

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

Centrifuge at 12000 rpm for 20min+decant supernatant carefully

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

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

RESULTS
Isolation of DNA :-

228 A. fluviatilis

307 A.culcifacies

535 mosquito

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.

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