Proceeding s of Th e 5th Annu al I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2 01 5
I n conj unct ion w it h The 8th I n t ern at ion al Con feren ce of Ch em ical Eng ineering o n Science and Applicat ions ( Ch ESA) 2 0 15 Sept em ber 9 - 1 1, 2 01 5, Banda Aceh, I ndon esia
M or ph ology a n d Pa r a sit a e m ia D e v e lopm e n t of
Pla sm od iu m
b e r g h e i
in Ba lb / c M ice (
M u s m u scu lu s
)
1*
Rosnizar , and
1Kart ini Eriani
Depart m ent of Biology, Facult y of Mat hem at ic and Science, Syiah Kuala Universit y,
Darussalam , Banda Aceh 23111, I ndonesia;
* Corresponding Aut hor
: rosnizarj am il@gm ail.com
Abst r a ct
Malaria is one of t he m ost sev ere public healt h problem s w orldw ide. I t is a leading cause of deat h and disease in m any dev eloping count ries, w here y oung children and pregnant w om en are t he groups m ost affect ed. Malaria disease caused by Plasm odium parasit e hav e sy m pt om s t hat t y pically include fev er, fat igue, v om it ing and headaches. I n sev ere cases, it can cause y ellow sk in, seizures, com a or deat h. The present st udy is aim ed t o m onit or ing parasit em ia lev el and percent age of parasit e m orphology as parasit aem ia progresses. This research u sed Plasm odium berghei NK st rain obt ained from Nat ional Univ ersit y of Malay sia w hich originally from MR4, USA. Design used in t his research w as com plet ely random ized d esign, w it h 2 t reat m ent s w hich w ere m ice w it h out infect ion and m ice w it h infect ion of P. berghei parasit e. Met hod used in t his resear ch w as st aining m et hod of t hin sm ear of blood using Giem sa st ained and observ ed by m icroscopic. Param et er of observ at ion w ere m orphology and dev elopm ent of P. berghei at different lev el of parasit aem ia ( 10% , 2 0% , 30% and 40% ) . The result sh ow ed from m icroscopic ex am inat ion of blood slides prepared from t he st udy anim als indicat ed t h at t he ring form w as dom inant st age obt ained at all different st ages of infect ion follow ed by t r ophozoit e st age. Meanw hile schizont st age w as t he low est st age obt ained at all different st ages observ ed.
Ke y w ords: Malaria, Plasm odium berghei, parasit aem ia, t rophozoit e
I n t r od u ct ion
Malaria is a deadly disease that can lead to deat h ( Philips, 2001; WHO, 2004) . Children and pregnant wom en are t he m ost vulnerable group of vict im s ( CDC 2004). The disease is caused by infect ion wit h prot ozoan parasit es of t he genus Plasm odium (Carvalho et al., 2007). The World Health Organizat ion ( WHO) has report ed about 4 billion people from 90 different count ries are at risk of cont ract ing t he disease and as m any as 500 m illion cases of m alaria are reported each year ( Carvalho et al., 2007; Esm ark et al. 2006), and t he deat h of at least 1 up t o 2.5 m illion people each year (Curt idor et al., 2006; Esm ark et al., 2006; WHO, 2009) .
Most of deat hs occurred in south Africa of t he Sahara desert t hat is 80 percent of t he tot al deaths from m alaria worldwide (Pett ersson 2005). I n children, death is usually caused by cerebral m alaria, acute anem ia and respiratory disorders, m eanwhile deat hs in adults ot her t han respirat ory dist ress syndrom e, the sym pt om s are m ore prevalent such as kidney dam age and lung dam age ( Becker et al. 2004) . I n addit ion, t he disease can cause an enlarged spleen and liver ( Engwerda et al. 2004) . This condit ion is caused by a buildup of m alaria pigm ent granules. Plasm odium t hat at tack sm all m am m als such as m ice am ong which are P. berghei ( Cruz et al. 2000; Lau et al. 2001; Sherm an 1998) . Plasm odium has been used in studies t o replace the use of P. falciparum in m any aspect s such as drug resist ance, t he diversit y of ant igens, the m echanism s of ent ry int o t he erythrocyte and st udy of organelles plast ids. The result s of an assessm ent of the rodent parasite and t he host is very helpful in assessing t he relat ionship bet ween Plasm odium in hum an ( Leiden Universit y, 2005) .I nt raerit rosit st age is t he m ost violent stage of infect ion (Menard, 2000; Vaid, 2007). Merozoit es are released from t he infected eryt hrocyte will be in plasm a and bind eryt hrocyte and entering new ones. I n circulat ion, m erozoites quickly at taches and ent ers eryt hrocytes and replicat e t o produce new infect ive m erozoites ( Cowm an and Crabb, 2006) . This cycle cont inues unt il create clinical sym pt om s in pat ients ( Greenwood et al. 2005) . The t ransit ion st age in different cells and t issues in t he m osquito vector and t he m am m alian host shows a high abilit y of m alaria parasit e t o adapt t o its environm ent ( Mat uschewski 2006; Vaughan et al., 2008) . Merozoit es are released int o t he bloodst ream t o infect eryt hrocyt es eit her repeat the cycle int raerit rosit t hrough the est ablishm ent of schizont - m erozoit es, or undergo a gam et ogenesis process for t he form at ion of gam et ocyt es ( Bannister & Mitchell, 2003; Fuj iako and Aikawa, 1999) .
M a t e r ia ls a nd M e t h ods
The st udy began wit h t he isolat ion and preservat ion of in v ivo cult ure parasite P. berghei in m ice st ocks and m ake infect ion in m ice with a predet erm ined dose of 1 x 106 infected eryt hrocytes/ m ice. The research was conducted at t he Laborat ory of Microt echnique, FMI PA, Unsyiah. The m at erials used
Proceeding s of Th e 5th Annu al I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2 01 5
I n conj unct ion w it h The 8th I n t ern at ion al Con feren ce of Ch em ical Eng ineering o n Science and Applicat ions ( Ch ESA) 2 0 15 Sept em ber 9 - 1 1, 2 01 5, Banda Aceh, I ndon esia
137
in t his st udy are t he cryopreservat ion of parasit e Plasm odium berghei, m et hanol, Giem sa dye and physiologycal saline solut ion.
I n ocu la tion of P. b e rg h ei Par a sit e
White m ale m ice Balb / c for about 8 weeks old were dist ributed in t wo t reatm ent s, t he m ice wit hout infect ion with P. berghei- infect ed m ice as cont rols and P. berghei infect ed m ice. Furt herm ore, P. berghei- infect ed m ice were divided int o 4 groups of sam pling at 10% parasit em ia, 20% parasit em ia, 30% parasitaem ia and 40% parasitaem ia. Each sam pling involves 5 m ice.
P. berghei which cryopreserved in liquid of nit rogen first heat ed in t he bat h 37 ° C for 10 m inut es. Furt herm ore, t he solut ion cont aining t he parasite is m ixed wit h NaCl NaCl physiological saline solut ion in t he rat io 1: 1 and cent rifuged for 10 m inut es at speeds of 650 x g. Pellet s com posed of P. berghei-infected eryt hrocytes then m ixed again wit h physiological NaCl in t he rat io of 1: 1. The pellets are t hen inj ected in healt hy m ice as st ocks by int raperit oneal route. P. berghei inoculum s are t hen prepared from stock m ice which have parasitem ia about 30% . The num ber of erythrocytes calculated using Haem asitom eter and level of parasitaem ia observed t hroughout t hin blood sm ear of slide wit h Giem sa st aining ( Field & Shut e 1955). Furt herm ore, parasit ic blood was t aken from the t ail end of a sm all piece of m ice st ock and dilut ed with physiological saline solut ion at t he dose of 1 x 106 eryt hrocyt es infect ed wit h m alaria parasit es/ m ice and t hen inject ed int o m ice in each t reatm ent .
Gie m sa st a in in g
Thin blood sm ear of slide t reated and cont rol m ice were st ained by Giem sa. Before stained, slides were fixed wit h m et hanol and allowed t o dry for 1 m inut e in room t em perat ure. After t hat , slide colored with Giem sa dye which diluted in a volum e of 1: 9 with a phosphat e buffer solut ion (PBS), pH 6.8, and left for 20 t o 30 m inutes. The slide is t hen washed wit h tap water flows slowly and dried at room t em perat ure. The percentage of parasit aem ia observed furt her under t he light m icroscope wit h 100x m agnificat ion.
D e t e rm in a t ion of Pa r asit em ia Lev e l
A drop of infected blood of m ice were obt ained asept ically from t he tail end of m ice and sm eared on st erile glass objects. Thin blood sm ear of slide then st ained by Giem sa and observed under a light m icroscope with a m agnificat ion of 100 t im es. Parasit aem ia level calculat ed by t he form ula of Hauda et .al ( 1993) as follows:
Resu lts a nd D iscussion
Monit oring of t he developm ent of parasitaem ia in t he infected m ice was done after inoculat ion of parasite P. berghei. Treat ed m ice that have reached percent age of parasit aem ia at 10% , 20% , 30% and 40% was m onitored by m aking t hin blood sm ear of slide as m ent ioned above. The m orphology of t he parasit e at every st age parasitaem ia was also m onit ored in each group of anim als. Figures and t ables below showed t he m orphological form s of ring and t rophozoite stages of P. berghei.
10% Parasitemia 20% Parasitemia
30% Parasitemia 40% Parasitemia
Figu r e 1 . Morph ology of int raery t hrocy t e P. berghei parasit e at 10% , 20% , 30% and 40% parasit aem ia w hich observ ed using light m icroscope at 100 x m agnificat ion.
Proceeding s of Th e 5th Annu al I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2 01 5
I n conj unct ion w it h The 8th I n t ern at ion al Con feren ce of Ch em ical Eng ineering o n Science and Applicat ions ( Ch ESA) 2 0 15 Sept em ber 9 - 1 1, 2 01 5, Banda Aceh, I ndon esia
Ta b le 1 . Percentage of P. berghei m orphology in infect ed eryt hrocyt e (% )
Pa r asit a e m ia ( % )
M orp h olog y of P. b e rgh e i in in fe ct ed er y th r ocy t e ( % )
Ring for m ( n= 5 ) Tr op h oz oit e ( n = 5 ) Sch yz on ( n= 5 )
1 0 72.15 + 2.11 24.26 + 2.10 0.34 + 0.17
2 0 55.72 + 2.34 40.37 + 2.15 0.50 + 0.22
3 0 54.76 + 2.45 40.23 + 3.05 1.05 + 0.45
4 0 53.18 + 3.22 44.56 + 3.45 0.29 + 0.12
Bot h of t hese st ages were always present highest at every level of parasit aem ia, while t he schizont st age was t he lowest st age present s and only can be detect ed less t han 1% of all group of parasit ic m orphology. The lowest level of int raerit rosit schizont stage was t hought t o occured because t he parasite- infect ed erythrocytes have been lost at t his st age in t he blood circulat ion syst em and t hen gathered in organ capillaries such as t he lungs, liver and spleen t o undergo t he process of int raerit rosit schyzogoni ( Jansen & Waters, 2002). The release of m erozoit es in schizont stage causing t he at tendance of early st age of infect ion which is a ring and t rophozoit e form s in erythrocyt es. Most of t he parasit e infected eryt hrocyt es cont aining ring form t hat is 53- 73% and 23- 44% of t rophozoit e form . The presence of m ore ring form s at t he 10% level of parasitaem ia com pared wit h 20% t o 40% parasit aem ia was also t hought t o be an early- phase release of m erozoit es in eryt hrocyt es infect ed by P. berghei parasites. This process result ed in t he presence of m ore rings and young t rophozoites stage were observed in t hin sm ear of blood on each level of parasit aem ia.
Con clusions
The developm ent of parasit aem ia in t he infect ed m ice observed from m icroscopic exam inat ion of blood slides prepared from the st udy anim als indicated that t he ring form was dom inant stage obt ained at all different st ages of infect ion followed by t rophozoite st age. Meanwhile schizont stage was the lowest st age obtained at all different st ages observed.
Ack n ow led g em e n ts
we thank t he Minist ry of Educat ion and Culture for financial assistance under t he Fundam ent al Research Grant of Universitas Syiah Kuala, Nom bor: 035/ SP2H/ PL/ Dit.Lit abm as/ I I / 2015 date 5 Pebruari 2015.
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