Thesis (M.Sc.) -- University of Adelaide, Dept. of Microbiology and Immunology, 198

Desakorn, Varunee

Adelaide Research &amp; Scholarship

)6\ilFACTORS INVOLVED IN IMMUNITY TO NEMATOSPIROIDES DUBIUSINFECTIONS IN MICE \ibyVarunee DesakornB.Sc.Med.Tech. (Thailand)M.P.H. (Phillppines)A thesis submj-tted for the degree ofMaster of ScienceinThe Department of Microbiology and ImmunologyThe University of Adelaide,Adelaide,South AustraliaDecember, 19831l-ABSTRACTThe present j-nvestigation examined the interaction betweennþuse peritoneal rnacrophages with various develo¡mental forms oflrTen¡atospiroides dubius (Heli,,Enoscxrei{gis polv€vf,us) a natural nernatodeparasite of the nrcuse and the ability of se:rum frcm mice irnrnrne tothis infection to danage exsheathed L, in the absence of phagocyticcells.Initial studies exarnined the adherence of perÍtoneal exudatecel1s to the surface of the parasite and the factors involved inprmoting this adhesion. Peritoneal exrdate cel1s frcrn norrnalmice did not adhere to the cuticle of exsheathed L3 in the absenceof serum, but did so when the larvae had been sensitised with norne.lmouse serum. In this case the cells bound to the larvae viatheir C3 receptors and confirnred previous observations that theparasite activated ccrnplerrent via the alternative pathway. It wasalso fourd that serum frcr¡ rnice innune to this infection containedantibodies directed against the cuticle of all larval stages asneasured by the adhesion of e,ells to Iarvae that had beensensitised with heat inactivated serum. The ceI1s in thisinstance adhered to the larvae via their Fc receptors. llYre factorsinvolved in prøroting adhesion of peritoneal e>mdate celIs frcrnnornøI mice and those frcrn mice resistant to this i-nfection weresimilar. Ttre ¡:eritoneal exudate ce11s adhering to the 1,arva.e werefound to be predcrninantly nacrophages.The celI adhesion str:dies denpnstrated that all of thedeveloping post-infective a¡rd the adult fortn of the parasite werecapable of actir¡ating ccnplenent by the alternative pathway with thenarked exception of lan¡ae coltected 96 hows post-infection.' Tttista1indicated. a possible change in the antigenic structtrre of thecuticle.Þçerirrents were carried out to deterrnine the abilÍty ofvarious innn:noglobulin isotypes frqn nìouse irlrnme to reinfection tonediate cell binding to all develo¡xrental fornrs and adult worrns ofthe parasite. It was fourd that the ability of IgM to prønotethe binding of cel-Is to the larvaL stages increased dranaticallyfrcnr the pre-infective stages including sheathed and exsheathed L,up to 96 hor:rs post-infective larvae, but prønoted little bindingto adult wonns. The IgG, prmoted cell binding in a sjmilarpa,ttern to that of IgM, but binding of the cells to 96 hotlrspost-infective lrawa.e was considerably reduced. Both IgG2, andISZU entranced the binding of cells to pre-infective Latstae, htwere not active in prcnroting cel1 binding to post-infective 1,awae.Hor,vever at a physiological level only IgG, and IgM were active inprmroting ce11 adhesion.An in vitro assay to determine the effect of cell bindingupon the infectivity of exsheathed þ indicated that peritonealexudate ce1ls frcn¡ mice infected with 2 doses of L, were capable ofdamging the larvae (as meastrred .by a l-oss in infectivity)providing the larvae had been sensitised with antibody and ccrnplenentor ccnplenent alone, but had no effect if the lar'.tae had beensensitised vi-ith antibody alone even though antibody nediated ce1Iadherenoe. Peritoneal e>mdate cells frqn norrml mice wereunable to danage the larvae even in the presence of antibody andcanplenent.tr\rther in vltro studies shqrred that fresh turtreated serurnfrcrn mice infected u¡ith 4 doses of h, IMS(4) was aþle to darmgeexsheathed L. as nea"sured by a reductj-on in thefu infectivity.Jl_v.Tfiis effect was not apparent when fresh untreated senût frqn micegiven 2 irnrn:nisÍng doses, IMS(2) ruas used. The larvicLda"Iactivity was found only i-n long-term N. dubius i-nfected mice sinceserun frcrn mice infected with Mesocestoides corti an u¡relatedhelmlnth parasite did not reduce the infectivity of the 1.axvøe.Ttre larvicidal activity of III{S(4) appeared to be dependent onspecific antibodies of the IgM class reacting with the cuticle of theLar:¡ae and binding cornplenent.Studies in .viJ¡o shorved that fresh IMS(4) given intravenouslyto r¡aive mice protected thern against a subsequent intravenouschallenge with exsheathed L' but not against a challenge withsheathed þ. It was al-so found that serum frqn innnrne mice frcrnwtrich the greater proportion of the innunoglobulins had been renx¡vedwas unable to transfer innnrnity passively to naj-ve mice. Therelative importance of these studies to other investigations dealingwith nenatode parasites is discussed.IIIIIi1i1IIIjV.STATEMENTTt¡1s thesis contains no naterial previously suhnittedby ne for a degree in any university, and to the best of rrylarovûedge and belief it contains no rmterial previouslypublished or written by another trÞrson, e>rcept wtrere referenceis nøde in the text,Varunee DesakornDecenrber, 1983v1ACKNOWLEDGEMENTSI am deeply indebted to Dn. C.R. Jenkin for his tzupervisionand encouragerent throughout the course of this investi-gation.I am npst grateful to Dr. P.L. Ey for his understanding, advicecriticisn and enthusia.sn, and to the other nenrbers of theDepartnent of Microbiology and Innnmology for thei-r cqnnentsof this work.Tha¡ks are due to lvlrs. A. Ilallett for the culture ofparasite larvae, to lvlr. N. Shead for hÍs help with the preparationof graphs'and photographs, and to lv1rs. J. Fallon for the typing ofthis thesis. I should also aclalcnvledge the receipt of anAustralian Governr¡ent Award for SEAII'IED centre staff develo¡rnentprogÏa¡llne.Fina11y, I would like to thank my parents and friends for theirkindness and rnoral support.vt_t-BSAc.à.ÐTAWTAB,ISAIgIMSrMs(2)rr/Is(4)r3N.D.NMSPBStris\BSABBREVIATIONS USED IN THIS TTÍESISBovine serum albuninapproxina.telyethyl enediaminetet r a.aeætateethyleneglycol-bis (ß+minoethyl) N,N | -tetraacetateEnzyne-linked iÍnnrno-sorbent a,ssayInnnrnoglobuliniÍrnrme mouse serumsefllm frclrr, 2 tines N* d9þius infected micesentrn f.rqn 4 tines N. dubius infected micethird-stage (infective) N. dpb_ius larvaenot donenozmal nþuse serurnPhosphate-buf f ered sálineTlis(hydro><ynethyl)aminønethane Iø++, I,lg* - supplenented veronal-buffered salineTABLE OF CONTENTSTitleAbstractStatenentAclcrorledgenentsAbbreviations used in this thesisTable of ContentsChapter 1 : IntroductionvLrL.pageiiivvir''triiviii1.1.7.2.1.3.7.4.1.5.General IntroductionVaccination of hosts against nernatode, infectj.onMechanisrs of innn:nity to Nippostrongylus brasiliensisMechanisrns of i¡nrunity to ffic]:inella .,spiralis12474Nernatospiroides dubiu.s a r¡atural .nenatodeof the mouse1.5.1. Life Cyclet.5.2, Pathology of the infection1.5.3. Induction of inmunity in mice to infection withN. dubiusL.5.4. Changes in irnrnmoglobul-in levels during infection1.5.5. Cfia^nges in ce11 levels during infection1.5.6. The role of phagocytic ce1ls and antibody inÍnnnrnity tq L-Ot bius infectionCltzpter 2 : lvfaterials and Methods2.!. fncubation nedia and diluents2.t.L. Incubation nedia2,L.2. Di-luents2.2. MÍce2,3. lvlaintenance of the parasite Nerratospiroides dubit¡spa.rasite222223252931323535.tû3536(Heliprrpscrnoides polyg¡nrus Ir>(2,4.2.5.2.6.2.7,2.8.,oz,LO .2.t1.2,72.2.13 .Collection of exsheathed i.anra.e of N. dubiusCollection of post-infective larrraeMethod for innn¡rising rnice against infection withN. dubiusAssay of resistance to infecti-on $rith N. dubius37373939394040404L41424242434343444646Collection of serrumInactivation of complenent by nethylanriheCollection of ¡:eritoneal enrdate cel1sCeII counting and viabilitykeparation of siliconj-sed glass tubesT?eatrnent of mice with Salrmnella enteritidis 11 Ð(for the collection of activated peritoneal exudateceIls2.11. Measurenent of henplyLic car¡rlenent activity2.L5. Concentration of sertrn or ilrrilunoglobulin fractionsby ultrafiltration2.L6. Annroniun sulphate precipitation of innn:noglobulins2,L7. Pre¡nration of purified nþuse innnrnoglobulins2.L7.L. Sepha.dex Cr2OO chrqnatography2.17.2 kotein A-Sepharose chrcrnatography2,t8. kepa,ration of purified rþuse IgG and IgM .innunoglobulins2.19. Radial funrnmodiffusion analyses (Orchterlony test)2,2O. Erzyne linked i¡nrnrnosorbent assays (H,ISA)2.2L. Adsorption of irnrunoglobulin frcrn innn¡ne m)useserum by affinity chrcnatography2.22. Adsorption of ccmplenent ccnponent C3 frcrn nptrsesennn by affinity chrcrnatography2.?.3. Statistics4747xChapter 3 in the: adherence ofenrdate cel-ls to the l:âr'ra.L and adult ofN. dubius3.1. Intrrcduction3.2. MethodAssay for the adherence of peritonear exrdate ceIlsto different develogrental forrns of N. dubÍus3.3. Res[lts3.3.1. trb.ctors i-nvolved in the a.dherence of peritonealexudate cell_s frcrn norrm,I. rnice to exsheath.d h3.3.2. Ttre ability of sentrn frcrn norrnal mice or rniceresistant to re-infection to prønote theadherence of nornar peritoneal exudate celrs todifferent developnental forrns of N. dubiusC]oa,pter 4 The abilitv different isotjæes4848494951to prm¡ote the adhesion of nornal peritonealexudate cells to N. dubius4.I. Introduction4.2. Results4.2.L. Ttre efficacy of different innnrnoglobulinisotypes in prøoting the a.dherence ofnorrm.l peritoneal endate cells to exsheathed L34.2,2. Ttre ability of different innnrnoglobulinisotypes to prønote the a.dherence of norrm,Iperitoneal exudate cells to differentdevelo¡rrental forms of N. dubiùs4.2,3. Ccmparison between the ability of kiIIed, L,and living % to provide the productÍon.ofantibodies enha¡lcing adhesion of normalperitoneal ex¡date cel1s to exsheathed It4.2.4. Ttre i¡raþi1ity of killed la¡r¡ae to protectrnice against infection with N. dubiusilæv555658Chapter 5 : Tt¡e effect ,in vitro of peritoneal e>aldate ce11sfrcrn irnrune mice on the infectivity of þ ofN. dubius sensitised wÍth either specific antibodyxl-595.1.5.2.5.3.and/or cqnplernentIntroductionMethodIn vj-tro assay for the effect of peritoneal exudatecells on the infectivity of exsheathed L,Besultsthe effect of incubating peritoneal exudate cellSfrcrn resistant rnice for different periods of tirnewith exsheathed h or their infectivity aftersensitisation with IMS(2)The effect in yitlo of peritoneal exudate cellsfrcrn mice given 2 inr¡nrnising doses on theinfectivity of exsheathed L, sensitised withNMS or IMS(2) treated. or untreated at 5605.3.3. Car¡nrison between the effect in vitro ofperitoneal e>nrdate cells frcrn mice given 2irrmunising doses of % and cells frorn rnlcegiven 4 irnnrnising doses .f h on the infectivityof exsheathed h sensitised with either serumfrcrn mice given 2 irnrrunising doses of L3, II\4S(2);or serum frqn rnice given 4 innn:nising doses ofL3, rMS(4)The effect in vitro of peri-toneal exrdate celIsfrqr¡ mice given living SaLnonella enteritidis 11 Ð(on the infectivity of exsheathed þ5.4. ConclusionChapter 6 : The effect of'serum on the infectivitV of þfollorÍng incubation in vitro5.3.15 .3.2.5.3.4.596061636á676.1. Introdrrctilon 686.2, MethodIn vitrosystem on the infeetivity of exsheathed þ6.3. Resultsassay for the effect of a cell-free serurnxl_I .7368696.3.1.6,3.2.6.3.3.6.3.4.6.3.5,6.3.6.6.3.7.6.3.8.6.3.9.6.3.10.6.3.11.ïhe optimal incubatj-on time required for sennnfrqn mice infected with 4 j¡rrunÍsing doses toOarnage LBLength of tirne required for llvtS(4) to impairthe infectivity of hThe lan¡ùcidal activity of IIr{S(4) on varyingnumbers of exsheathed LBThe effect of the further addition of IIr{S(4)during the course of j-ncubation on theinfectivity of exsheathed L,TitratÍon of the larvicÍdal activity of seruntaken at r¡arious tines during the course ofinfection frqn mic,e infected v/'ith varSzingnr.¡rnber of doses of 200 N. dubius L,Cønparison between the effects of sentrn frqn72mice infected with 4 doses of N. dubius þ andserum frqn mice infected with Mesocestoides cortion the infectivity of exsheathed þ Z4The effect of storing IIvIS(4) on ice for.varyingperiods of tfure on its larvicidal activity T6The effect of storing IIvtS(4) at -2Oo on itsability to danage þ i.n vitro ZTThe effect of heat treaürent at differenttenperatures on the la,:n¡icidal activity of I[{S(4) 7ZThe effect of dialysis on the larvicidal activityof Ilr4S(4) 7Athe requirenent for ccnrplenænt in rrediating danageto exsheathed þ in vitro by senrn frqn rniceinfected ïirith 4 inrnurÍsing doses of N. dubir:s L, Tg69707L6.3.L2. The ability of II\4S(4) a^tter rern¡rral of theimunoglobulins to damage exsheathed Lo6.4. ConclusionsChapter 7 : kotection afforded to norral mlce to N. dubiusinfection follqirine the passive transfer ofserumfrmr mice resistant to re-infection7.1. Introduction7.2. Results7.2.1.8081xiii.87r¿Passive transfer of innnnity to naive micegiven IÀ{S(4) intraperitoneallyPassive transfer of j¡rrn¡nit¡z to naive mic,eby the intravenous injection of IMS(4)Passive transfer of innunity to naive rnice bythe ,intravenous injection of II\4S(4) follovedby a challenge infection of either exsheathed Ijor sheathed I*.JThe ability of various dilutions of IMS.(4) totransfer irnrrunity passively to naÍve miceThe effect of freezirrg and thawing on.theability of I[4S(4) to transfer protection tonaive rniceCan¡:arison between the in vivo.and in- vitrolarvicidal effect of IIvIS(4) after chrcrmtographyon either an anti-npuse C3 or an a¡rti-rnouseF(abr )2 colunntr\¡rther evidence that ccnplenent is involvedin the lanricidal activity of llvlS(4)The effect of an innunoglobutÍn preparationor an IgG and IgM fracti-on pre¡nred frcrn II\4S(4)on the infectivity of exsheathed L, in thepresence and absence of NIt'lSThe effect of purified IgM prepared frcm, I[4S(4)on the i-nfectivity of exsheathed Lrin,thepresence of NIIIS4æu857.2.2.7.2.3,7.2,4.7 .2.6.7.2.6.7.2,7.7.2,8.7.2.9,85868890gL7 .3. OonclusionsChapter 8 : DiscussionBibliographyx].v.%97111

Factors involved in immunity to Nematospiroides dubius infections in mice

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Factors involved in immunity to Nematospiroides dubius infections in mice

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Factors involved in immunity to Nematospiroides dubius infections in mice

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Adelaide Research & Scholarship