in vivo culture of p. berghei (praktikum)

IN VIVO CULTURE OF PLASMODIUM BERGHEI

Mahardika AW Dept of Parasitology

Fac.of Medicine Universitas Gadjah Mada

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In Vivo Culture of

Plasmodium

Human/ Primate

P. falciparum P. knowlesi

Rodent

P. Berghei P. yoelii P. Vinkei

P. chabaudi

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•  ED50 Drug sensitivity assay

•  Hemozoin Inhibition •  Enzyme inhibition

Drug Action Mechanism

•  Acute/ Sub acute/ Chronic Tox. •  Side effect Drug: Toxicity

•  Half life •  Concentration in the plasma Pharmacocinetic

•  Humoral Immunity •  Cellular Immunity Immune Response

•  DNA isolation-- PCR •  Separation & Mapping chromosom Molecular Biology

•  Symptoms & signs Clinical manifestation

•  Pathology in the organs Pathology

In Vivo Study

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Culture In vivo in Swiss Mice Equipments :

�  Spuit injection � Ependorf tube � Hemocytometer � Micro pippet � Blue & Yellow tip � Object glass � Microscope

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Materials & Reagents

� Giemsa stain � Methanol abs. � Heparinized tube � RPMI � Normal saline �  P. berghei-Infected mice � Normal mice

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Thin Blood smear & Counting parasitemia :

� Thin blood film from tail �  Fix with abs. methanol : 1’ �  Stain with 5% Giemsa : 30’ � Count percent infected cells ( 100x oil

immersion)

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Counting erythrocyte

� Dilute 10 µl of blood in 990 µl RPMI (102

dilution) : (A) � Dilute 10 µl of (A) in 990 µl RPMI (104

dilution) : (B) � Count erythrocyte (B) in

haemocytometer chamber

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Counting erythrocytes

990 µl RPMI

10 µl mouse blood

990 µl RPMI 10 µl (A)

A B

10 µl (B)

On Hemocytometer chamber

Counting Erythrocytes using microscope

(40x objective lense)

102 dilution 104 dilution

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Haemocytometer chamber

Number of Erythrocytes / mL:

Σ in 25 Squares x dilution (102/104) x 104

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Passage of infection into new mice

�  Bleed the donor mouse �  Count parasitemia and the number of erythrocyte/

ml �  Dilute the blood in sterile RPMI to 107 inf.eryth./ml �  Inject 200 µl i.p. per mouse �  Parasites appear after 2-3 days. �  Mice succumbing to inf. Usually die on days 9-12

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Preparation of Swiss Mice

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P.chabaudi

C57Bl/6 Resistant mice

Balb/C semisuceptibel

A/J 100% Suceptibel

P. berghei

Swiss mice Suceptibel

Balb/C Suceptibel

Mouse Malaria Model

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Handle the mouse Desinfection with 70% ethanol

Intraperitoneal injection Infected mice

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In vivo Test ( Peters,1987 )

4-day Suppresive Test

P.berghei 1x107

Swiss mice (± 25 gram )

Tratment: D-0 to 3

Group- 4 10 mice D mg/kgBB

Group-3 10 mice C mg/kgBB Group- 2

10 mice B mg/kgBB

Group-5 10 mice E mg/kgBB

Group- 6 10 mice 0,9% saline

Day-1 t0 4 -Thin blood smear

-ED50

i.p

Group-1 10 mice A mg/kgBB

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P. berghei

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Parasitemia : ( In Vivo Culture) Number of pRBC x 100% Number of RBC (± 1000)

Growth Inhibition (%) Prst. in control – Prst.with drug x 100 % Prst. In control

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Dose (mg/Kg)

0 (K-)

6,25

12,5

25

50

100

Parasitemia (%)

34,6

32,1

26,6

17,4

8,8

3,6

Growth Inhibition (%)

0

7,23

23,12

49,71

74,6

89,9

Counting : 1. Parasitemia (%) 2. Growth Inhibition (%)

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Calculation of ED50

34.6 32.1

26.6

17.4

8.8

3.6

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 90 100

% P

aras

item

ia

Dose (mg/Kg)

1. Parasitemia

Calculation of ED50

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Dose – Effect Relationship Probit Analysis

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70 80 90 100

% in

hibi

tion

of p

aras

ite

Gro

wth

Dose (mg/Kg)

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In vivo Antiplasmodial Activity of Naturally Medicinal Plants

(Munoz et al., 1999)

ED50 >500 mg/kg

≤ 500 mg/kg

≤ 250 mg/kg

≤ 100 mg/kg

Activity Not Potential

Moderate

Good

Very good

Pathology in organ

� Collect internal organs: brain, spleen, liver, kidney, etc

� Put in preservatives : formalin 5% �  Speciment processes �  Identification of pathologic response

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Pf in capilary alveolar wall Cytoadherence of Pf in capilary

Cerebral malaria Malaria in the liver

Criteria of Malaria in the internal organs

� Quantitative/ semiquantitative: ◦ Number of cell with malarial pigment ◦ Number of inflamatory cells ◦  Plus system : +++

� Qualitative ◦ Normal ◦  Low ◦ Moderate ◦  Severe

� Descriptive

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