IN VIVO CULTURE OF

PLASMODIUM BERGHEI
Mahardika AW
Dept of Parasitology
Fac.of Medicine Universitas Gadjah Mada

10/27/14

DR.Dika-FK-UGM

In Vivo
Culture of
Plasmodium

Human/
Primate

Rodent

P. falciparum

P. Berghei
P. yoelii
P. Vinkei

P. knowlesi

P. chabaudi
10/27/14

DR.Dika-FK-UGM

In Vivo Study
Drug sensitivity assay
Drug Action
Mechanism
Drug:Toxicity
Pharmacocinetic
Immune Response

ED50
Hemozoin Inhibition
Enzyme inhibition
Acute/ Sub acute/ Chronic Tox.
Side effect
Half life
Concentration in the plasma
Humoral Immunity
Cellular Immunity

Molecular Biology

DNA isolation-- PCR

Separation & Mapping chromosom

Clinical manifestation

Symptoms & signs

Pathology

Pathology
in the organs
10/27/14 DR.Dika-FK-UGM

Culture In vivo in Swiss Mice

Equipments :
Spuit

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

10/27/14

DR.Dika-FK-UGM

Materials & Reagents

Giemsa

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

10/27/14

DR.Dika-FK-UGM

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)

10/27/14

DR.Dika-FK-UGM

Counting erythrocyte
Dilute

10 l of blood in 990 l RPMI (102

dilution) : (A)
Dilute 10 l of (A) in 990 l RPMI (104
dilution) : (B)
Count erythrocyte (B) in
haemocytometer chamber

10/27/14

DR.Dika-FK-UGM

Counting erythrocytes
B

A
990 l RPMI
10 l mouse blood

102 dilution

990 l RPMI
10 l (A)

104 dilution

10 l (B)
On Hemocytometer
chamber

Counting Erythrocytes using microscope

(40x objective lense)
10/27/14

DR.Dika-FK-UGM

Haemocytometer
chamber

Number of Erythrocytes / mL:

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

10/27/14

DR.Dika-FK-UGM

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 l i.p. per mouse
Parasites appear after 2-3 days.
Mice succumbing to inf. Usually die on days 9-12

10/27/14

DR.Dika-FK-UGM

10

Preparation of Swiss Mice

10/27/14

DR.Dika-FK-UGM

11

Mouse Malaria Model

P.chabaudi

P. berghei

C57Bl/6
Resistant mice

Swiss mice
Suceptibel

Balb/C
semisuceptibel

Balb/C
Suceptibel

A/J
100% Suceptibel
10/27/14

DR.Dika-FK-UGM

12

Handle the mouse

Intraperitoneal injection
10/27/14

Desinfection with 70% ethanol

Infected mice
DR.Dika-FK-UGM

13

In vivo Test ( Peters,1987 )

4-day Suppresive Test

P.berghei 1x107

i.p
Swiss mice ( 25 gram )

Tratment: D-0 to 3

Group-1
10 mice
A mg/kgBB

Group- 2
10 mice
B mg/kgBB

Group-3
10 mice
C mg/kgBB

Group- 4
10 mice
D mg/kgBB

Group-5
10 mice
E mg/kgBB
Group- 6
10 mice
0,9% saline

Day-1 t0 4
-Thin blood smear
10/27/14 DR.Dika-FK-UGM
-ED50

14

P. berghei

10/27/14

DR.Dika-FK-UGM

15

Parasitemia : ( In Vivo Culture)

Number of pRBC
x 100%
Number of RBC ( 1000)

Growth Inhibition (%)

Prst. in control Prst.with drug
Prst. In control

10/27/14

x 100 %

DR.Dika-FK-UGM

16

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

Dose (mg/
Kg)

Parasitemia
(%)

Growth
Inhibition (%)

0 (K-)

34,6

6,25

32,1

7,23

12,5

26,6

23,12

25

17,4

49,71

50

8,8

74,6

100

3,6

89,9

10/27/14

DR.Dika-FK-UGM

17

Calculation of ED50
1. Parasitemia
40
35

34.6
32.1

% Parasitemia

30
26.6

25
20

17.4
15
10

8.8

3.6

0
0

10

20

30

40

50

60

70

80

90

100

Dose (mg/Kg)

10/27/14

DR.Dika-FK-UGM

18

Calculation of ED50
Dose Effect Relationship
Probit Analysis

% inhibition of parasite Growth

120
100
80
60
40
20
0
0

10

20

30

40

50

60

70

80

90

100

Dose (mg/Kg)

10/27/14

DR.Dika-FK-UGM

19

In vivo Antiplasmodial Activity of

Naturally Medicinal Plants
(Munoz et al., 1999)

ED50

Activity

>500 mg/kg

Not Potential

500 mg/kg

Moderate

250 mg/kg

Good

100 mg/kg

Very good
10/27/14

DR.Dika-FK-UGM

20

Pathology in organ
Collect

internal organs: brain, spleen, liver,

kidney, etc
Put in preservatives : formalin 5%
Speciment processes
Identification of pathologic response

10/27/14

DR.Dika-FK-UGM

21

Malaria in the liver

Cerebral malaria

Pf in capilary alveolar wall

Cytoadherence of Pf in capilary
10/27/14

DR.Dika-FK-UGM

22

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
10/27/14

DR.Dika-FK-UGM

23

10/27/14

DR.Dika-FK-UGM

24