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 u cosa l M a st Ce lls Con t r ibu t ion in I n t e st in a l D e fe n se of
Ch ick e n s (
Ga llu s d om e st icu s
) I n fe ct e d N a t u r a lly b y
Asca r id ia g a lli
1
Um m u Balqis,
2Masda Adm i,
3Darm awi, and
4*Muham m ad Ham bal
1 Laborat ory of Pat hology, Facult y of Veterinary Medicine, Syiah Kuala Universit y,
Darussalam , Banda Aceh 23111, I ndonesia; 2Alm uslim Universit y, Bireuen, Aceh, I ndonesia;
3Laboratory of Microbiology, Facult y of Veterinary Medicine, Syiah Kuala Universit y, Banda
Aceh 23111, I ndonesia;
4Laboratory of Parasit ology, Facult y of Veterinary Medicine, Syiah Kuala Universit y, Banda
Aceh 23111, I ndonesia;
* Corresponding Aut hor: d_darm awi@yahoo.com
Abst r a ct
This st udy was aim ed at finding out t he inv est igat ion of m ucosal m ast cells in int est ines of chick en t hat were nat urally infect ed by Ascaridia galli. Am ount of t en int est ine of freshly slaught ered chick ens (Gallus dom est icu s) found from local abat oir in Banda Aceh were div ided int o t wo groups cont aining fiv e int est ines of each. Mucosal m ast cells cou nt were done of which hist ologic slides were m ade in st ained serial hist ological sect ions wit h Alcian blue ( pH 0,3) and Safranin- O ( pH 0,1) of t he int est ines. The result showed t hat t he m ucosal m ast cells in creased significant ly ( P < 0.0 5) in int est ines of chick ens infect ed nat urally by surv iv al A. galli adult worm s. I t was con cluded t hat t he int est inal defense of chick ens against parasit e infect ion is associat ed wit h t he m u cosal m ast cells cont ribut ion by cr eat ing an env ironm ent host ile t o t he est ablishm ent and surv iv al of int est inal nem at odes, A. galli.
Ke yw o rds: m ast cell, Ascaridia galli, chick en, int est ine
I n t r od u ct ion
Ascaridia galli is an im portant intest inal nem atode of poult ry in m any parts of t he word and am ong the m ost pat hogenic of parasites t o localize in intest ine of laying hens. Chronic infect ion develops after cont inuous ingest ion of infect ive st ages larvae and usually result in anem ia, edem a, wieght - loss, and diarrhea, which in severe cases, can result in deat h.
Mast cell responses have been suggest ed in protect ion against A. galli infect ions. I nfect ions wit h 1000 dosis A. galli em bryonated egg in laying hens have shown t hat m ast cells accum ulate in t he sm all int est ine following infect ion ( Darm awi et al., 2013). Previously, De- yuan et al. ( 2003) described t hat m ast cells in t he t hym ic m edulla and jej unal m ucosa of chickens infected experim ent ally wit h A. galli, suggest ing t hat m ast cells responses m ay be involved in cont rolling t hese infect ion.
According t o our previously invest igat ion known that the int est ines infected by A. galli showed hist opathological changes of villi t he cover of desquam at ion, hyperplasia and fusion. Dem age level of t he sm all intest ine chicken was largely det erm ined by t he num ber of infect ing by A. galli. On t he ot her hand, t he m ore num ber of A galli infect ed t o chickens, t he higher level of dam age would be occured in t he sm all intest ine of chicken (Balqis et al., 2013) . Ham bal et al. ( 2013) explained t hat duodenum , j ej unum and ileum infected by A. galli showed hyperem ia, and inflam m at ory cell infilt rat ion in part of A. galli infect ion. I n addit ion, Darm awi et al. ( 2013) observed t hat m ucosal m ast cells involved in int est inal defense m echanism . The chickens infected orally wit h 1,000 em bryonat ed eggs of A. galli were able t o increase significant ly m ast cells response on days 14 post infect ion. The A. galli infect ion could t rigger the involm ent of m ucosal m ast cells response in j ejunal defense of laying hens against parasit ic diseases caused by A. galli. I n t his st udy, we asseyed t he m ucosal m ast cells m ade a cont ribut ion to int est inal defense of chickens against parasite infect ion caused by A. galli.
M a t e r ia ls a nd M e t h ods
Am ount of t en intest ine of freshly slaughtered chickens (Gallus dom est icus) found from local abatoir in Banda Aceh were divided into t wo groups cont aining five int est ines of each. The first group det erm ined by int est ines were not find A. galli adult worm infect ion as cont rols. The second group det erm ined by intest ines cont ained am ount of A. galli adult worm s infect ed nat urally.
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
H ist olog ica l Proce du r e
I nt est ine’s segm ent was dissected, flushed wit h cold st erile saline solut ion, opened longit udinally, and placed, m ucosa side up, ont o sm all pieces of blot t ing paper. The segm ents were t hen fixed in 10% buffered norm al form alin. This process was perform ed for each laying hen using sterile inst rum ents for each dissect ion. Fixed sam ples were dehydrat ed in t he ascending concent rat ions of et hanol ( 50% , 60% , 70% , 80% , 96% ( 1) , 96% ( 2) and 100% ) . The sam ples were cleared in xylol and were em bedded in paraffin wax. Three of each hist ological sect ions ( 3- 5 µm of t hickness) were stained with Alcian blue ( pH 0,3) and Safranin- O ( pH 0,1) ( Sigm a) . Aft er washing, sect ions were counterst ained wit h eosin and m ounted as described by Darm awi et al. ( 2013) wit h certain m odificat ions.
M a st Ce ll St a in ing
The num ber of m ast cells per 10 villus crypt units ( VCUs) was count ed on each sect ion. Mast cell counts were perform ed under light m icroscopy using an eyepiece square grat icule (eyepiece × 10, object ive × 40), and data expressed as m ean num ber of m ucosal m ast cells ( MMCs) per VCU as described by previous aut hors (McDerm ot t et al., 2003; Noviana et al., 2004; Li et al., 2004;
Königová et al., 2008; Darm awi et al., 2013) wit h cert ain m odificat ions.
St a t ist ica l an a lysis
Dat a were analyzed by t he St udent t t est , where t t ests were used for com parisons of m ast cell num bers. P values of < 0.05 were t aken t o indicat e a significant difference.
Resu lts a nd D iscussion
I n t his st udy we found t hat t he num ber of m ucosal m ast cells in healt hy chickens is st able, but t heir num bers increase in A. galli infect ion ( Table 1).
Table 1. Mucosal m ast cell num ber/ 10 villus crypt unit ( m ean ± SD) in t he intest ines from uninfect ed, and infect ed nat urally by A. galli in chickens
Groups Mucosal m ast cell num ber in intest ines
Uninfected 321.32 ± 57.42*
I nfect ed 391.41 ± 89.73*
* Significant ly different from uninfected and infected chickens ( P < 0.05) .
Results shown are represent at ive of two independent experim ents. MMC/ 10VCU ± SD
I n t his st udy, m ucosal m ast cells significant ly increased in int est ines of chickens infected nat urally by A. galli in com parison wit h uninfect ed chickens as shown in Table 1. Our result s in t his study agree wit h and support t hose of Onah & Nawa, ( 2004), Li et al., 2004), ( Vukm an et al., 2013), Ball et al., ( 2013) , De- yuan et al. ( 2003), Suzuki et al. ( 2008), and Königová et al. ( 2008) who showed m ast cells are cont ribut ed in intest inal defense against worm infect ion. Our previous study explained that large num bers of m ast cells were observed in t he j ej unum of infected laying hens. Laying hens infected wit h em bryonated eggs of A. galli accum ulat ed m ast cells in the j ejunum ( Darm awi et al., 2013).
I n t he chickens, A. galli adult worm s est ablish in t he lum en of intest ine known “ lum en phase” . However, A. galli parasit e was not only able to survive in t he lum en but could also penet rate in t he barrier int est inal m ucosal defense wit h m igrat ion t o t he t issue “ hist ot rophic phase” . Previously invest igat or described t hat A. galli infect ive larvae invaded in t he epit helium and located in t he lam ina propia after hatching in lum en ( Luna- Olivares et al., 2012) . There are som e hist opat hological changes in int est ine of Gallus dom est icus caused by A. galli infect ion. I n confirm at ion of our previous st udy, we found t hat t he chicken (G. dom est icus) infect ed nat urally by A. galli caused t he infilt rat ion of inflam at ory cell and hem orrhagy in t he intest ine (Ham bal et al., 2013) . I n addit ion, the m ore sm all int est ine of t he chickens suffered hist opat hological changes nam ely desquam at ion, hiperplasia, fusion t hat occurs in t he j ejunum ( Balqis et al., 2013) .
Various aut hors described that increased num bers of m ucosal m ast cells are oft en observed in affected t issues during helm int h infect ions (Onah & Nawa, 2004; Li et al., 2004; Vukm an et al., 2013; Ball et al., 2013; De- yuan et al., 2003; Suzuki et al., 2008; Königová et al., 2008) . This phenom enon supported by Darm awi et al. ( 2013) who observed that m ast ocyt osis occurred in j ejunal sect ions of laying hens challenged wit h em bryonated of A. galli. I nfect ion induces m ucosal m ast cells degranulat ion in t he int est inal t hat is considered t o be a host defense m echanism against t he parasit e. I n support of t his hypot hesis, various authors described t hat m ast cells involved in m ucosal defense m echanism . Various aut hors described t hat m ucosal m ast cells play an im portant role as effect or cell act ivated in response t o m ost helm int h infect ions, cont ribut e t o expulsion of a num ber of
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
gast rointest inal nem atode parasites. Migrat ion of m ast cells induced by t egum ental coat ant igen of Fasciola hepat ica (Vukm an et al., 2013) . Mast ocyt osis was act ivat ed in jej unal of m ice challenged wit h St rongyloides venezuelensis ( Onah & Nawa, 2004). Mucosal m ast cells are an im port ant effect or for Trichinella spiralis expulsion in rat s and m ice ( Suzuki et al., 2008) . The larger num bers of m ucosal m ast cells in abom asal m ucosa of lam bs infected wit h Haem onchus cont ort us (Shakya et al., 2009) , followed by H. cont ort us expulsion from abom asum s of sheep ( Ort olani et al., 2013).
Con clusion
The m ucosal m ast cells cont ribut e t o intest inal defense in chickens by creat ing an environm ent host ile t o t he est ablishm ent and survival of int est inal nem atodes, A. galli.
Ack n ow led g em e n ts
We wish t o t hank the chairm an and all theacing st aff of Pat hology Laborarory, and Research Laborat ory Facult y of Veterinary Medicine t hat frendly pleased us t o conduct this research.
Re fe r e n ce s
Balqis, U., M. Ham bal, Dar m awi, and C. S. Ut am i. 2013. Hist opat hological changes in int est ine of chick en (Gallus dom est icu s) infect ed nat urally by Ascaridia galli. Proceedings of The 3rd Annuual I nt er nat ional Conference
Sy iah Kuala Univ ersit y ( AI C Unsy iah) I n conj u nct ion wit h The 2nd I nt ernat ional Conference on
Mult idiscipplinary Research ( I CMR) , Pages: 343- 34 8, Oct ober 2- 4, 2013, Banda Aceh, I ndonesia.
Ball, D.H., H.K. Tay , K.S. Bell, M. L. Coat es, L. Al- Riy am i, J. R.W. Harnet t , & M.M. Harnet t . 2013. Mast cell subset s and t heir funct ional m odulat ion by t he Acant hocheilonem a v it eae product ES- 62. J. Parasit ol. Res. 1- 13 . ht t p: / / dx .doi.org/ 10.1 155/ 2 013/ 96 126 [ 15- 05- 201 3] .
Darm awi, U. Balqis, M. Ham bal, R. Tiuria, Frengk i, B.P. Priosoery ant o. 201 3. Mucosal Mast Cell Response in Jej unum of Ascaridia galli- infect ed Lay ing Hens. J. Med. Pet . 36( 2) : 113- 1 19. DOI : 10.5398/ m edpet .2013.3 6.2.11 3
De- y uan, O.U., G.A.O Deng- hui, W.A.N.G. Kai- gong, X.U. Le- ren. 2003. Mast cells in t he t hy m ic m edulla and j ej unal m ucosa of chick ens infect ed ex perim ent ally wit h Ascaridia galli. Chinese Journal of Vet erinary Science. Abst ract . ht t p: / / en .cnk i.com .cn/ Art icle_en/ CJFDTOTAL- ZSYX20 030101 7.ht m [ 1 5- 09- 2 013] . Ham bal, M., U. Balqis, Darm awi, and C. S. Ut am i. 2013 . I n filt rat ion of inflam m at ory cells in int est ines of chick ens
infect ed nat urally by Ascaridia galli. Pr oceedings of The 3rd Annuual I nt ernat ional Conferen ce Sy iah Kuala
Univ ersit y ( AI C Unsy iah) I n conj un ct ion wit h The 2nd I nt ernat ional Conference on Mult idiscipplinary
Research ( I CMR) , Pages: 362- 36 4, Oct ober 2- 4 , 2013, Band a Aceh, I ndonesia.
Königov á, A., G. Hrck ov a, S. Velebný , J. Corba, & M. Várady . 2008. Ex perim ent al infect ion of Haem onch us cont ort u s st rains resist ant and suscept ible t o benzim idazoles and t he effect on m ast cells dist ribut ion in t he st om ach of Mongolian gerbils (Meriones unguiculat us) . Parasit ol. Res. 102: 587–5 95. doi 10.1007/ s004 36- 007 - 0792- 4 [ 28- 05- 2013] .
Li, E., P. Zhou, Z. Pet rin, & S.M. Singer. 2004. Mast cell- dependent cont rol of Giardia lam blia infect ions in m ice. I AI . 72( 1 1) : 6642– 6649. doi: 10 .1128/ I AI . 72.11. 6642– 664 9.2004 [ 22- 05- 20 13] .
Luna- Oliv ares, L.A., T. Ferdushy , N. C. Ky v sgaard, P. Nej su m , S.M. Tham sborg, A. Roepst orff, & T.M. I burg. 201 2. Localizat ion of Ascar idia galli larv ae in t he j ej unu m of chick ens 3 day s post infect ion. Vet . Parasit ol. 185( 2–4) : 186–19 3.
McDer m ot t , J. R., R.E. Bart ram , P.A. Knight , H.R.P. Miller, D.R. Garrod, & R. K. Grencis. 2003. Mast cells disrupt epit helial barrier funct ion during ent eric nem at ode infect ion. PNAS. ( 100) 13: 7761–7766 . www.pnas. org/ cgi/ doi/ 10.107 3/ pnas. [ 15- 05- 20 13] .
Nov iana, D., K. Mam ba, S. Mak im ura, & Y. Horii. 2004. Dist ribut ion, hist ochem ical and enzy m e hist ochem ical charact erizat ion of m ast cells in dogs. J. Mol. Hist ol. 35: 123 –132.
Onah, D.N., & Y. Nawa. 2004. Mucosal m ast cell- deriv ed chondroit in sulphat e lev els in and worm ex pulsion from FcRγ- k nock out m ice following oral challenge wit h St rongy loides v enezuelensis. J. Vet . Sci. 5( 3) : 221– 226. Ort olani, E. L., M.L. Leal, A.H. Minerv ino, A.R. Aires, R.L. Coop, F. Jack son, & N.F. Sut t le. 2013. Effect s of
parasit ism on cellular im m une response in sheep ex perim ent ally infect ed wit h Haem onchus cont ort u s. Vet . Parasit ol. Abst ract . doi: 10. 1016/ j .v et par.2013 .02.01 4. [ 15- 05- 2013] .
Shak y a, K.P., J.E. Miller, & D.W. Hor ohov . 2009 . A Th2 t y pe of im m une response is associat ed wit h in creased resist an ce t o Haem on chus cont ort us in nat urally infect ed gulf coast nat iv e lam b s. Vet . Parasit ol. 16 3( 1- 2) : 57- 66. doi: 10.1 016/ j .v et par.2009. 03.052 . [ 15- 05- 2013] .
Suzuk i, T., T. Sasak i, H. Tak agi, K. Sat o, & K. Ueda. 2008 . The effect ors respon sible for gast r oint est inal nem at ode parasit es, Trichinella spiralis, ex pulsion in rat s. Parasit ol. Res. 103: 128 9–129 5. doi 10.100 7/ s0 0436- 0 08-1130- 1 [ 28- 05- 20 13] .
Vuk m an, K.V., P. N. Adam s, D. Dowling, M. Met z, M. Maurer, & S.M. O’Neill. 2013. The effect s of Fasciola hepat ica
t egum ent al ant igens on m ast cell funct ion. I nt . J. Parasit ol. 43( 7 ) : 531–53 9. ht t p: / / dx .doi.org/ 10.1 016/ j .ij para. 2013. 01.011 [ 1 5- 05- 2 01 3] .
