in vivo culture of p. berghei (praktikum)
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praktikumTRANSCRIPT
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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