- Email: [email protected]
Changes in antigen and glycoprotein patterns during the development of Oesophagostomum dentatum
International Journal for Parasitology 17 "0887# 0742Ð0759
Changes in antigen and glycoprotein patterns during the development of Oesophagostomum dentatum A[ Joachim\ B[ Ruttkowski\ A[ Daugschies Institute of Parasitology\ Hannover School of Veterinary Medicine\ Bunteweg 06\ D!29448 Hannover\ Germany Received 08 June 0887^ received in revised form 17 August 0887^ accepted 2 September 0887
Abstract During its development from free!living infectious third!stage larvae to the adult worms in the large intestines of pigs\ Oesophagostomum dentatum experiences several environmental changes[ Di}erences in protein patterns can re~ect such changes[ Somatic and ES antigens and glycoproteins of pre!parasitic\ histotropic and intestinal stages were compared by single!dimension SDSÐPAGE and stage!speci_c proteins were de_ned[ Furthermore\ fourth!stage larvae derived from di}erent sources*in!vitro cultivation and intestinal contents*were compared and also found to be di}erent[ It is hypothesised that O[ dentatum reacts to environmental stimuli by di}erential expression of speci_c proteins as a possible mode of adaptation to the host[ Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[ Keywords] Oesophagostomum dentatum^ Antigens^ Glycoproteins^ Pigs^ Stages
0[ Introduction The strongyle nematode Oesophagostomum dentatum is one of the most abundant intestinal parasites in pigs\ even under intensive rearing con! ditions ð0\ 1Ł[ It is generally regarded as a well! adapted parasite\ since infections are readily estab! lished at high rates in helminth!na(ve as well as in previously infected pigs without signs of clinical disease ð2\ 3Ł[ Furthermore\ the parasite responds to the presence of parasitic competitors ð4Ł[ In!vitro experiments have shown that O[ denta! tum produces bioactive lipids in stage!speci_c patterns which are important for the parasite|s development and can modulate in~ammatation in
mammalian tissues ð5Ð7Ł[ Besides lipids\ proteins are supposed to be major modulators of the host|s immune response and are certainly important for the biology and development of nematodes\ and it has been proposed that changing protein patterns during nematode development re~ect the parasite|s adaptation to changes in the environment\ a pre! requisite for its survival ð8\ 09Ł[ In the present work we investigated protein changes in the course of development of O[ dentatum from the preparasitic to the adult stages in order to de_ne stage!speci_c patterns[ 1[ Materials and methods 1[0[ Parasite material
Corresponding author[ Tel] ¦38!400!842 7607^ fax] ¦38! 400!8427683^ e!mail] ardaugÝparasit[tiho!hannover[de[
For the following experiments\ the monospeci_c O[ dentatum strain OD!Hann was used[ Third!stage
S9919!6408:87:, ! see front matter Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[ PII] S 9 9 1 9 ! 6 4 0 8 " 8 7 # 9 9 0 5 2 ! 4
0743
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
larvae\ L3 cultured in vitro for 07Ð10 days "L3c# as described ð00Ł\ L3 isolated from intestinal contents of pigs slaughtered 8Ð00 days after oral infection "L3p# and adults from patently infected pigs were used[ 1[1[ Isolation and puri_cation of parasites Third!stage larvae were isolated from faeces incu! bated for 6 days at 16>C and 69) r[h[ using the Baerman technique\ puri_ed\ and exsheathed as described previously ð5Ł[ Larvae grown in vitro were washed several times in 9[8) sodium chloride "26>C# to remove debris\ the larger L3 were sep! arated from the smaller L2 by sedimentation and washed twice in warm sodium!chloride solution[ Fourth!stage larvae and adults were isolated from the intestinal contents of pigs as described by Slot! ved et al[ ð01Ł[ and washed in warm sodium!chloride solution until the supernatant was clear[ The viability of the parasites was surveyed micro! scopically[ For the preparation of ES proteins "ESP# the parasites were immediately transferred to medium prewarmed to 26>C "see below#[ For pro! tein lysates the parasites were frozen in liquid nitro! gen and stored at −79>C[ 1[2[ Lysates of somatic proteins Somatic protein "SP# lysates were prepared by grinding the parasites to a _ne powder on dry ice and adding lysis bu}er[ For each preparation\ 019 999 freshly exsheathed L2\ 799 L3c\ 799 L3p or 399 adults of mixed sexes were lysed in 0!ml lysis bu}er[ For preparation of total lysate a bu}er con! taining 14 mM!sodium phosphate:19 mM!sodium chloride "pH 6[9#\ 9[4) CHAPS "Boehringer Mannheim#\ 9[0) SDS "Sigma#\ and 9[3 mM!Pefa! blocþ "Boehringer Mannheim# was used[ After incubation at 3>C for 07 h the lysates were cen! trifuged at 5999 g for 34 min at 3>C and the super! natant was carefully removed[ For fractionation of protein preparations the ground parasites were lysed in three steps] in step one\ bu}er I ð14 mM! sodium phosphate:19 mM!sodium chloride "pH 6[9# and 9[3 mM!PefablocþŁ was added and the lysate was incubated for 3 h at 3>C\ centrifuged and
subsequently washed in a large volume of bu}er^ in step two\ bu}er II "bu}er I including 9[4) CHAPS# was added and the samples were incubated for 07 h at 3>C\ centrifuged and washed as above^ and in a third step\ bu}er III "bu}er II including 9[0) SDS# was added\ the samples were heated at 83>C for 09 min and the supernatant was removed[ The protein contents in the preparations were measured using the Biorad Protein Assay "Biorad#[ 1[3[ ExcretoryÐsecretory proteins "ESP# One!hundred!and!twenty!thousand sheathed L2\ 019 999 freshly exsheathed L2\ 799 L3c\ 799 L3p or 399 adults of mixed sexes were incubated for 5 or 13 h in 49!ml cell culture ~asks containing 4 ml RPMI!medium "Life Technologies# with 499 U ml−0 of penicillin\ 499 U ml−0 of strep! tomycin\ 9[0 mg ml−0 of oxytetracyclin\ and 199 U ml−0 of nystatin "Sigma# at 27>C and 09) CO1[ For each stage 04!ml medium in portions of 4 ml:~ask were prepared[ A medium control with! out worms was also set up[ Prior to incubation the worms were washed in 09 ml of the culture medium at 26>C for 4 min[ After incubation the supernatant was removed and Pefablocþ was added to a _nal concentration of 9[3 mM[ The samples were cooled on ice\ centrifuged at 1999 g to remove debris and _ltered through a 9[34 mm _lter "Renner#[ They were then centrifuged in a Centriplusþ!09 column over! night at 0799 rpm at 3>C until the remaining volume was approximately 9[4 ml[ The columns had been washed with 1 ml of 0) bacitracin "Roth# and twice with 4 ml sterile distilled water[ After centrifugation the retentate "9[4 ml# was collected and subjected to SDSÐPAGE as described below[ 1[4[ SDSÐPAGE and Western blotting SDSÐPAGE was carried out in a Protean Mini Cell "Biorad# on a 3) stacking:04) running gel using pre!mixed acrylamide!bis "C 2[2)^ Biorad# as described by the manufacturer[ One and a half micrograms of protein of each sample "in a volume of 04 ml of lysis bu}er# was mixed with 2 ml loading bu}er containing 9[90) bromphenol blue\ 09) glycerol\ 149 mM!Tris "pH 5[7#\ 4) b!mercapto! ethanol "all chemicals purchased from Roth# and
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
1) SDS "Sigma#\ loaded and run at 199 V for 64 min[ For size estimation MW markers "Biorad# were included in the run[ Subsequently\ gels were either silver!stained or blotted onto a Hybondþ!N¦ positively charged nylon membrane "Amersham# in a semi!dry blotting device "Semiphorþ\ Pharmacia# at 009 mA for 34 min and blocked with PBS "9[8 mM!sodium phosphate\ 1[6 mM!sodium chlor! ide\ pH 6[1# containing 0) BSA "Sigma#[ Sera were derived from two pigs experimentally patently monoinfected with a daily dose of 09 999 L2 of O[ dentatum strain Od!Hann on 2 consecutive days 7 weeks prior to bleeding or from two helminth! na(ve pigs and used as pools[ Western blotting was carried out with serum diluted 0]199 using an ABC kit "Boehringer Ingelheim# according to the manu! facturer|s instructions[ Biotin!labelled goat!anti pig IgG "Boehringer Ingelheim# was used as secondary antibody\ and after incubation with an avidinÐ biotinÐhorseradish!peroxidase complex the sub! strate TMB "Boehringer Ingelheim# was converted to a blue derivate[ Glycoprotein blotting was per! formed using a Glycoprotein Detection Kit "Biorad# according to the manufacturer|s instruc! tions[ In short\ after blocking the proteins were biotinylated by treatment with periodate and sub! sequent incubation in hydrazide[ Enzyme!labelled avidin bound to the biotinylated glycosylated pro! teins and converted the substrate[ Ovalbumin was included as a positive control[ 1[5[ Western blots using blocked sera In order to detect only stage!speci_c proteins serum "dilution 0]1999# was incubated with 4 vol[ of the homogenate contained in one blot strip for 29 min at room temperature "as determined by titration studies^ data not shown#[ A set of blot strips with homogenates from L2\ L3c\ L3p\ and adults was incubated as follows] I] II] III] IV] V]
positive serum "positive control#^ negative serum "negative control 0#^ no serum "negative control 1#^ postive serum blocked with L2!homogenate^ positive serum blocked with L3c!homo! genate^ VI] positive serum blocked with L3p!homo! genate^
0744
VII] positive serum blocked with adult homo! genate^ VIII] positive serum blocked with a combination of all homogenates except the one on the respective membrane strip[
2[ Results 2[0[ SDSÐPAGE and silver staining To demonstrate that the protein preparations were largely undegraded\ a fraction of the lysates and the ESP was stained with silver after electro! phoretic separation[ The lysates contained proteins in the whole size range of the gel[ Due to the high number of bands\ however\ di}erences between the stages could not be demonstrated[ On the ESP gels only a band of approximately 099 kDa could be seen for the L3 and adult stages "not shown#[ 2[1[ Western blot analysis of total lysates The results of the blots of total lysates are given in Fig[ 0[ For the L2 three di}erent bands "049\ 29 and 19 kDa# were detected which were all speci_c for the L2 stages as they could only be detected with positive serum and were blocked with L2 homogenate[ The largest antigen could also be blocked with L3c homogenate[ For the L3c speci_c proteins of approximately 89 and 19 kDa were detected which could be blocked with homologous homogenate[ L3p also possesses two proteins "039 and 07 kDa# which are both blocked by L3c[ The 039 kDa antigen is also blocked by L3p protein[ Adults displayed a 049 kDa band that could not be blocked with homogenate and a double band at 099 kDa which was blocked with adult as well as L3p protein[ 2[2[ Western blot of fractionated lysates Figure 1a shows the results of this experiment[ The L2!speci_c 29!kDa band was found in the SDS! fraction\ as well as the smaller antigens of this stage[ The speci_c proteins of the L3c were all found in fraction II[ The 039!kDa antigen of the L3p was found in fraction I and smaller antigens in fractions
0745
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
Fig[ 0[ Western blot analysis of total homogenates "serum dilution] 0]1999#[ L2] third!stage larvae^ L3c] fourth!stage larvae from in! vitro cultivation^ L3p] fourth!stage larvae derived from intestines^ Ad] adult worms^ m] MW marker[ Arrows mark the sizes "kDa# of the detected bands[ I] positive serum^ II] negative serum^ III] no serum^ IV] positive serum blocked with L2!homogenate^ V] positive serum blocked with L3c^ VI] positive serum blocked with L3p!homogenate^ VII] positive serum blocked with adult homogenate^ VIII] positive serum blocked with a combination of all homogenates except the one on the respective membrane strip[
II and III[ The antigens of the adults were found in fractions I and III[ Bands of 79 kDa for the L2 and 49 kDa for the adults were also present on the blot incubated with negative serum "as indicated on the blot with positive serum and on the glycoblot\ Fig[ 1b#[ 2[3[ Glycoblot of lysates In addition to the non!speci_c bands as described above\ glycoproteins could be found for the L3c and L3p in the lysates of bu}er I "a band of ×59 kDa and a double band of 11 and 13 kDa# and of bu}er III "44 kDa# and a double band of 16 and 29 kDa for the adults in bu}er III[ 2[4[ Western blot and glycoblot of culture super! natants Before and after incubation\ at least 89) of the parasites were still motile[ The Western blot of the concentrated culture supernatants is shown in Fig[ 2[ In the L2 supernatants no protein could be
detected\ even after 13 h of incubation[ In the super! natants of the other stages a common 099!kDa protein was detected which was glycosylated "not shown#[ In the supernatants of L3c incubated for 5 h smaller proteins were stained only weakly\ whereas after 13 h of incubation a larger number of proteins ranging from 27 to 099 kDa was present[ In the samples from the L3p the same proteins were detected after 5 h of incubation\ and additionally smaller proteins "×03 kDa# were found[ L3p and adults were incubated for only 5 h to avoid bacterial contamination[ A medium control without worms and blots with negative serum revealed no bands "not shown#[
3[ Discussion At the point of transition from the free!living to the early parasitic stage\ marked by exsheathment of the L2\ O[ dentatum experiences crucial physical and chemical changes in its environment[ During the course of infection\ mainly in the histotropic
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
0746
Fig[ 1[ Western blot of fractioned somatic proteins[ "a# Blot with positive serum "dilution 0]199#[ "b# Glycoblot[ L2] third!stage larvae^ L3c] fourth!stage larvae from in!vitro cultivation^ L3p] fourth!stage larvae derived from intestines^ Ad] adult worms[ I] fraction I "water!soluble#^ II] fraction II "CHAPS#^ III] fraction III "SDS:83>C#[ Arrows mark bands that were also stained in blots with negative serum[
0747
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
Fig[ 2[ blot of excretoryÐsecretory proteins[ L2s] sheathed third! stage larvae^ L2e] exsheathed third!stage larvae^ L3c] fourth! stage larvae from in!vitro cultivation^ L3p] fourth!stage larvae derived from intestines^ Ad] adult worms^ m] MW marker[
stage\ it then faces its greatest challenge\ the encounter with the host|s immune system[ The his! totropic larva lives in closest contact with the tissue of the host\ and limitation of the host defence is vital for the parasite in order to maintain its integrity[ Simultaneously the parasite develops from micro! scopic L2 into considerably larger\ morphologically distinct pre!adult stages and _nally into sexually di}erentiated adults[ Both the developmentally regulated and the host!adaptive changes can result in changing protein patterns[ Lipids have been shown to be of importance for the development of O[ dentatum and may also con! tribute to hostÐparasite interactions ð5Ð7Ł[ In other nematodes\ proteins\ especially those with antigenic properties\ are supposed to be mediators of immu! nomodulation ð8\ 09\ 02Ł[ In the present paper we investigated antigens and glycoproteins of the parasitic stages of O[ dentatum[ We de_ned di}erent stages by their size and mor! phology ð03Ł as L2\ L3 and adults[ Furthermore\ we distinguished between L3 grown in vitro and in vivo[ In vitro\ larvae were cultured for 07Ð10 days\ when the development to L3 had reached its maximum of 4Ð09) of the initial L2 population[
With a few exceptions where L3 moult and develop to pre!adult L4\ this is the _nal stage in culture\ whereas in!vivo post!histotropic L3 can already be recovered from the intestinal contents in high num! bers on days 8Ð00 p[i[ We postulated that the di}erent population dynamics could be re~ected by di}erent protein patterns[ Changes in protein patterns during development have been described for gastrointestinal strongyles such as Oesophagostomum radiatum ð04Ł\ Ostertagia species[ ð05\ 06Ł and Haemonchus contortus ð07\ 08Ł[ Such changes are believed to serve as a mechanism of immune evasion and often involve proteins located at the parasite|s surface from where they can be shed into the environment\ or proteins actively secreted by the parasite "as reviewed by ð8\ 19\ 10Ł#[ We chose two groups of proteins\ antigens and glycoproteins\ for our investigations[ Antigens were chosen because the recognition of a protein as fore! ign and the subsequent induction of an immune response are indicative of hostÐparasite interaction ð7Ł[ Glycoproteins can be involved in hostÐparasite interactions ð11Ð13Ł and other biological functions of nematodes ð14\ 15Ł[ The avidinÐbiotin system which we employed for blotting enhances the sen! sitivity of protein detection compared with silver staining and other labelling techniques ð16Ł[ The glycoblot assay is limited to the detection of non! terminal carbohydrate moieties\ but it is less speci_c than lectin binding assays and therefore more suit! able for screening procedures[ Western blot analysis of fractionated proteins facilitates the comparison of protein preparations by using di}erential solu! bility of proteins as a biochemical feature ð17Ł[ With the applied techniques we found appreci! able di}erences between the stages and also between the L3 from di}erent sources[ In particular\ the strongly labelled somatic 29!kDa protein of the L2\ the 89!kDa antigen of the L3c and the ES 099!kDa glycoprotein of the advanced parasitic stages are of interest and will be characterised further[ While antigens were detected speci_cally with positive serum when total lysates were used\ fractionation of lysates revealed additional\ non!speci_c proteins which reacted with negative serum and also in the glycoblot when no antibodies were employed and are probably biotinylated[ This phenomenon has been described for other nematodes ð18Ł^ its func!
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
tion is not clear[ The L2 of O[ dentatum have pre! viously been described as sources of non!speci_c or cross!reactive antigens ð29Ł[ Whether the di}erences between the L3 from di}erent sources are an artefactual e}ect of cul! tivation or of the method of recovery is yet to be determined[ It is interesting to note that in prep! arations of ESP the L3p had a much more complex protein pattern than the L3c\ and that the frac! tionated preparations of L3p contained water!sol! uble proteins ³29 kDa which may correspond to the smaller ESP of this stage[ It is not clear whether the stage di}erences in protein expression are due to changes in the parasite|s development or induced by the host[ The di}erences between the L3 stages indicate host in~uence as it has been described for Ascaris ESP ð20Ł^ however\ some proteins such as the glycosylated ESP present in the advanced stages may be due to environmental changes[ Early and late L2 and L3 will be investigated further to deter! mine onset and termination of the expression of stage!speci_c proteins[ To gain some insight into the function of these proteins\ their location in situ will be determined[ The de_nition and charac! terisation of speci_c proteins and their isolation permit detailed studies on worm biology and hostÐ parasite interactions in bioassays\ such as lym! phoproliferation assays or larval migration studies[
Acknowledgements The authors thank Professor Dr M[ Rommel and Professor Dr K[T[ Friedho} for critical reading of the manuscript[ Parts of this study were funded by the Deutsche Forschungsgemeinschaft "grant No[ Jo 156:2!0#[ Dr A[ Joachim is recipient of a Karl Enigk scholarship[
References ð0Ł Barutzki D\ Randelzhofer A\ Gothe R[ Artenspektrum und Befallshau_gkeit von Helminthen bei Mutterschweinen und ihren Ferkeln in Suddeutschland[ Tierarztl Umschau 0881^36]068Ð089[ ð1Ł Roepstor} A\ Nansen P[ Epidemiology and control of hel! minth infections in pigs under intensive and non!intensive production systems[ Vet Parasitol 0883^43]58Ð74[
0748
ð2Ł Christensen CM\ Barnes EH\ Nansen P\ Gro ndahl!Nielsen C[ Growth and fecundity of Oesophagostomum dentatum in high!level infections and after transplantation into na(ve pigs[ Parasitol Res 0885^71]253Ð257[ ð3Ł Christensen CM\ Barnes EH\ Nansen P[ Experimental Oesophagostomum infections in the pig] worm populations at regular intervals during trickle infections with three doses of larvae[ Parasitology 0886^006]434Ð441[ ð4Ł Christensen CM\ Nansen P\ Barnes EH[ The e}ect of con! current or sequential Oesophagostomum dentatum and O[ quadrispinulatum infections on the worm burdens of the two species in pigs[ Parasitology 0886^003]162Ð167[ ð5Ł Daugschies A[ Oesophagostomum dentatum] population dynamics and synthesis of prostanoids by histotropic stages cultured in vitro[ Exp Parasitol 0884^70]463Ð472[ ð6Ł Daugschies A[ Investigations into the production and func! tion of leukotrienes during histotropic development of Oesophagostomum dentatum[ Parasitol Res 0885^71]305Ð 311[ ð7Ł Daugschies A\ Ruttkowski B[ Modulation of migration of Oesophagostomum dentatum larvae by inhibitors and products of eicosanoid metabolism[ Int J Parasitol 0887^17]244Ð251[ ð8Ł Philipp M\ Rumjaneck FD[ Antigenic and dynamic proper! ties of helminth surface structures[ Mol Biochem Parasitol 0873^09]134Ð157[ ð09Ł Maizels RM\ Selkirk ME[ Immunobiology of nematode antigens[ In] Englund PT\ Sher A\ editors[ The biology of parasitism[ New York] Alan Liss\ 0877^174Ð297[ ð00Ł Daugschies A\ Watzel C[ In vitro development of his! totropic larvae of Oesophagostomum dentatum under vari! ous conditions of cultivation[ Parasitol Res\ in press[ ð01Ł Slotved HC\ Barnes EH\ Bjo rn H\ et al[ Recovery of Oeso! phagostomum dentatum from pigs by isolation of parasites migrating from large intestinal contents embedded in agar! gel[ Vet Parasitol 0885^52]126Ð134[ ð02Ł Philipp M\ Parkhouse RME\ Ogilvie BM[ Changing pro! teins on the surface of a parasitic nematode[ Nature 0879^176]427Ð439[ ð03Ł Kotlan A[ Studies on the life!history and pathological sig! ni_cance of Oesophagostomum spp[ of the domestic pig[ Acta Vet "Budapest# 0836^0]547Ð553[ ð04Ł East IJ\ Berrie DA[ Antigenic di}erences among the life cycle stages of Oesophagostomum radiatum[ Res Vet Sci 0875^30]018Ð029[ ð05Ł Canals A\ Gasbarre LC[ Ostertagia ostertagi] isolation and partial characterization of somatic and metabolic antigens[ Int J Parasitol 0889^19]0936Ð0943[ ð06Ł Keith KA\ Duncan MC\ Murray M\ Bairden K\ Tait A[ Stage!speci_c cuticular proteins of Ostertagia circumcincta and Ostertagia ostertagi[ Int J Parasitol 0889^19]0926Ð0934[ ð07Ł Schallig HD\ Baczkowska A\ Wedrychowicz H[ Bio! chemical characterization of surface antigens of infective larvae and adults of Haemonchus contortus[ Acta Parasitol 0884^39]106Ð113[ ð08Ł Raleigh JM\ Brandon MR\ Meeusen E[ Stage!speci_c expression of surface molecules by the larval stages of Haemonchus contortus[ Parasite Immunol 0885^07]014Ð021[
0759
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
ð19Ł Behnke JM\ Barnard CJ\ Wakelin D[ Understanding chronic nematode infections] evolutionary considerations\ current hypotheses and the way forward[ Int J Parasitol 0881^11]750Ð896[ ð10Ł Blaxter ML\ Page AP\ Rudin W\ Maizels RM[ Nematode surface coats] actively evading immunity[ Parasitol Today 0881^7]132Ð136[ ð11Ł Jarvis LM\ Pritchard DI[ An evaluation of the role of carbo! hydrate epitopes in immunity to Trichinella spiralis[ Parasite Immunol 0881^03]378Ð490[ ð12Ł Fetterer RH\ Rhoads ML[ Biochemistry of the nematode cuticle] relevance to parasitic nematodes of livestock[ Vet Parasitol 0882^35]092Ð000[ ð13Ł Schallig HDFH\ van Leeuwen MAW[ Carbohydrate epi! topes on Haemonchus antigens[ Parasitol Res 0885^71]27Ð 31[ ð14Ł Jeyaprakas A\ Jansson HB\ Marban!Mendoza N\ Zuck! erman BM[ Caenorhabditis elegans] lectin!mediated modi! _cation of chemotaxis[ Exp Parasitol 0874^48]89Ð86[ ð15Ł Kerwin JL\ Zurakowski KA\ Washino RK[ Carbohydrate!
ð16Ł
ð17Ł
ð18Ł
ð29Ł
ð20Ł
mediated recognition of larval mosquito hosts by Rom! anomermis culicivorax[ J Nematol 0889^11]008Ð015[ Hsu SM\ Raine L\ Fanger H[ A comparative study of the peroxidaseÐantiperoxidase method and an avidinÐbiotin complex method for studying polypeptide hormones with radioimmunoassay antibodies[ Am J Clin Pathol 0870^64]623Ð627[ Northern C\ Grove DI[ Strongyloides stercoralis] antigenic analysis of infective larvae and adult worms[ Int J Parasitol 0889^19]270Ð276[ Romar(s F\ Iglesias R\ Garc(a LO\ Leiro J\ Santamarina MT\ Paniagua E\ Ubeir FM[ Free and bound biotin mol! ecules in helminths] a source of artifacts for avidin biotin! based immunoassays[ Parasitol Res 0885^71]506Ð511[ Larsen RH\ Christensen CM\ Lind P[ Serological assays for the identi_cation of Oesophagostomum dentatum in pigs[ Res Vet Sci 0886^52]4Ð09[ Kennedy MW\ Qureshi F[ Stage!speci_c secreted antigens of the parasitic larval stages of the nematode Ascaris[ Immunology 0875^47]404Ð411[
Changes in antigen and glycoprotein patterns during the development of Oesophagostomum dentatum A[ Joachim\ B[ Ruttkowski\ A[ Daugschies Institute of Parasitology\ Hannover School of Veterinary Medicine\ Bunteweg 06\ D!29448 Hannover\ Germany Received 08 June 0887^ received in revised form 17 August 0887^ accepted 2 September 0887
Abstract During its development from free!living infectious third!stage larvae to the adult worms in the large intestines of pigs\ Oesophagostomum dentatum experiences several environmental changes[ Di}erences in protein patterns can re~ect such changes[ Somatic and ES antigens and glycoproteins of pre!parasitic\ histotropic and intestinal stages were compared by single!dimension SDSÐPAGE and stage!speci_c proteins were de_ned[ Furthermore\ fourth!stage larvae derived from di}erent sources*in!vitro cultivation and intestinal contents*were compared and also found to be di}erent[ It is hypothesised that O[ dentatum reacts to environmental stimuli by di}erential expression of speci_c proteins as a possible mode of adaptation to the host[ Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[ Keywords] Oesophagostomum dentatum^ Antigens^ Glycoproteins^ Pigs^ Stages
0[ Introduction The strongyle nematode Oesophagostomum dentatum is one of the most abundant intestinal parasites in pigs\ even under intensive rearing con! ditions ð0\ 1Ł[ It is generally regarded as a well! adapted parasite\ since infections are readily estab! lished at high rates in helminth!na(ve as well as in previously infected pigs without signs of clinical disease ð2\ 3Ł[ Furthermore\ the parasite responds to the presence of parasitic competitors ð4Ł[ In!vitro experiments have shown that O[ denta! tum produces bioactive lipids in stage!speci_c patterns which are important for the parasite|s development and can modulate in~ammatation in
mammalian tissues ð5Ð7Ł[ Besides lipids\ proteins are supposed to be major modulators of the host|s immune response and are certainly important for the biology and development of nematodes\ and it has been proposed that changing protein patterns during nematode development re~ect the parasite|s adaptation to changes in the environment\ a pre! requisite for its survival ð8\ 09Ł[ In the present work we investigated protein changes in the course of development of O[ dentatum from the preparasitic to the adult stages in order to de_ne stage!speci_c patterns[ 1[ Materials and methods 1[0[ Parasite material
Corresponding author[ Tel] ¦38!400!842 7607^ fax] ¦38! 400!8427683^ e!mail] ardaugÝparasit[tiho!hannover[de[
For the following experiments\ the monospeci_c O[ dentatum strain OD!Hann was used[ Third!stage
S9919!6408:87:, ! see front matter Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[ PII] S 9 9 1 9 ! 6 4 0 8 " 8 7 # 9 9 0 5 2 ! 4
0743
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
larvae\ L3 cultured in vitro for 07Ð10 days "L3c# as described ð00Ł\ L3 isolated from intestinal contents of pigs slaughtered 8Ð00 days after oral infection "L3p# and adults from patently infected pigs were used[ 1[1[ Isolation and puri_cation of parasites Third!stage larvae were isolated from faeces incu! bated for 6 days at 16>C and 69) r[h[ using the Baerman technique\ puri_ed\ and exsheathed as described previously ð5Ł[ Larvae grown in vitro were washed several times in 9[8) sodium chloride "26>C# to remove debris\ the larger L3 were sep! arated from the smaller L2 by sedimentation and washed twice in warm sodium!chloride solution[ Fourth!stage larvae and adults were isolated from the intestinal contents of pigs as described by Slot! ved et al[ ð01Ł[ and washed in warm sodium!chloride solution until the supernatant was clear[ The viability of the parasites was surveyed micro! scopically[ For the preparation of ES proteins "ESP# the parasites were immediately transferred to medium prewarmed to 26>C "see below#[ For pro! tein lysates the parasites were frozen in liquid nitro! gen and stored at −79>C[ 1[2[ Lysates of somatic proteins Somatic protein "SP# lysates were prepared by grinding the parasites to a _ne powder on dry ice and adding lysis bu}er[ For each preparation\ 019 999 freshly exsheathed L2\ 799 L3c\ 799 L3p or 399 adults of mixed sexes were lysed in 0!ml lysis bu}er[ For preparation of total lysate a bu}er con! taining 14 mM!sodium phosphate:19 mM!sodium chloride "pH 6[9#\ 9[4) CHAPS "Boehringer Mannheim#\ 9[0) SDS "Sigma#\ and 9[3 mM!Pefa! blocþ "Boehringer Mannheim# was used[ After incubation at 3>C for 07 h the lysates were cen! trifuged at 5999 g for 34 min at 3>C and the super! natant was carefully removed[ For fractionation of protein preparations the ground parasites were lysed in three steps] in step one\ bu}er I ð14 mM! sodium phosphate:19 mM!sodium chloride "pH 6[9# and 9[3 mM!PefablocþŁ was added and the lysate was incubated for 3 h at 3>C\ centrifuged and
subsequently washed in a large volume of bu}er^ in step two\ bu}er II "bu}er I including 9[4) CHAPS# was added and the samples were incubated for 07 h at 3>C\ centrifuged and washed as above^ and in a third step\ bu}er III "bu}er II including 9[0) SDS# was added\ the samples were heated at 83>C for 09 min and the supernatant was removed[ The protein contents in the preparations were measured using the Biorad Protein Assay "Biorad#[ 1[3[ ExcretoryÐsecretory proteins "ESP# One!hundred!and!twenty!thousand sheathed L2\ 019 999 freshly exsheathed L2\ 799 L3c\ 799 L3p or 399 adults of mixed sexes were incubated for 5 or 13 h in 49!ml cell culture ~asks containing 4 ml RPMI!medium "Life Technologies# with 499 U ml−0 of penicillin\ 499 U ml−0 of strep! tomycin\ 9[0 mg ml−0 of oxytetracyclin\ and 199 U ml−0 of nystatin "Sigma# at 27>C and 09) CO1[ For each stage 04!ml medium in portions of 4 ml:~ask were prepared[ A medium control with! out worms was also set up[ Prior to incubation the worms were washed in 09 ml of the culture medium at 26>C for 4 min[ After incubation the supernatant was removed and Pefablocþ was added to a _nal concentration of 9[3 mM[ The samples were cooled on ice\ centrifuged at 1999 g to remove debris and _ltered through a 9[34 mm _lter "Renner#[ They were then centrifuged in a Centriplusþ!09 column over! night at 0799 rpm at 3>C until the remaining volume was approximately 9[4 ml[ The columns had been washed with 1 ml of 0) bacitracin "Roth# and twice with 4 ml sterile distilled water[ After centrifugation the retentate "9[4 ml# was collected and subjected to SDSÐPAGE as described below[ 1[4[ SDSÐPAGE and Western blotting SDSÐPAGE was carried out in a Protean Mini Cell "Biorad# on a 3) stacking:04) running gel using pre!mixed acrylamide!bis "C 2[2)^ Biorad# as described by the manufacturer[ One and a half micrograms of protein of each sample "in a volume of 04 ml of lysis bu}er# was mixed with 2 ml loading bu}er containing 9[90) bromphenol blue\ 09) glycerol\ 149 mM!Tris "pH 5[7#\ 4) b!mercapto! ethanol "all chemicals purchased from Roth# and
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
1) SDS "Sigma#\ loaded and run at 199 V for 64 min[ For size estimation MW markers "Biorad# were included in the run[ Subsequently\ gels were either silver!stained or blotted onto a Hybondþ!N¦ positively charged nylon membrane "Amersham# in a semi!dry blotting device "Semiphorþ\ Pharmacia# at 009 mA for 34 min and blocked with PBS "9[8 mM!sodium phosphate\ 1[6 mM!sodium chlor! ide\ pH 6[1# containing 0) BSA "Sigma#[ Sera were derived from two pigs experimentally patently monoinfected with a daily dose of 09 999 L2 of O[ dentatum strain Od!Hann on 2 consecutive days 7 weeks prior to bleeding or from two helminth! na(ve pigs and used as pools[ Western blotting was carried out with serum diluted 0]199 using an ABC kit "Boehringer Ingelheim# according to the manu! facturer|s instructions[ Biotin!labelled goat!anti pig IgG "Boehringer Ingelheim# was used as secondary antibody\ and after incubation with an avidinÐ biotinÐhorseradish!peroxidase complex the sub! strate TMB "Boehringer Ingelheim# was converted to a blue derivate[ Glycoprotein blotting was per! formed using a Glycoprotein Detection Kit "Biorad# according to the manufacturer|s instruc! tions[ In short\ after blocking the proteins were biotinylated by treatment with periodate and sub! sequent incubation in hydrazide[ Enzyme!labelled avidin bound to the biotinylated glycosylated pro! teins and converted the substrate[ Ovalbumin was included as a positive control[ 1[5[ Western blots using blocked sera In order to detect only stage!speci_c proteins serum "dilution 0]1999# was incubated with 4 vol[ of the homogenate contained in one blot strip for 29 min at room temperature "as determined by titration studies^ data not shown#[ A set of blot strips with homogenates from L2\ L3c\ L3p\ and adults was incubated as follows] I] II] III] IV] V]
positive serum "positive control#^ negative serum "negative control 0#^ no serum "negative control 1#^ postive serum blocked with L2!homogenate^ positive serum blocked with L3c!homo! genate^ VI] positive serum blocked with L3p!homo! genate^
0744
VII] positive serum blocked with adult homo! genate^ VIII] positive serum blocked with a combination of all homogenates except the one on the respective membrane strip[
2[ Results 2[0[ SDSÐPAGE and silver staining To demonstrate that the protein preparations were largely undegraded\ a fraction of the lysates and the ESP was stained with silver after electro! phoretic separation[ The lysates contained proteins in the whole size range of the gel[ Due to the high number of bands\ however\ di}erences between the stages could not be demonstrated[ On the ESP gels only a band of approximately 099 kDa could be seen for the L3 and adult stages "not shown#[ 2[1[ Western blot analysis of total lysates The results of the blots of total lysates are given in Fig[ 0[ For the L2 three di}erent bands "049\ 29 and 19 kDa# were detected which were all speci_c for the L2 stages as they could only be detected with positive serum and were blocked with L2 homogenate[ The largest antigen could also be blocked with L3c homogenate[ For the L3c speci_c proteins of approximately 89 and 19 kDa were detected which could be blocked with homologous homogenate[ L3p also possesses two proteins "039 and 07 kDa# which are both blocked by L3c[ The 039 kDa antigen is also blocked by L3p protein[ Adults displayed a 049 kDa band that could not be blocked with homogenate and a double band at 099 kDa which was blocked with adult as well as L3p protein[ 2[2[ Western blot of fractionated lysates Figure 1a shows the results of this experiment[ The L2!speci_c 29!kDa band was found in the SDS! fraction\ as well as the smaller antigens of this stage[ The speci_c proteins of the L3c were all found in fraction II[ The 039!kDa antigen of the L3p was found in fraction I and smaller antigens in fractions
0745
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
Fig[ 0[ Western blot analysis of total homogenates "serum dilution] 0]1999#[ L2] third!stage larvae^ L3c] fourth!stage larvae from in! vitro cultivation^ L3p] fourth!stage larvae derived from intestines^ Ad] adult worms^ m] MW marker[ Arrows mark the sizes "kDa# of the detected bands[ I] positive serum^ II] negative serum^ III] no serum^ IV] positive serum blocked with L2!homogenate^ V] positive serum blocked with L3c^ VI] positive serum blocked with L3p!homogenate^ VII] positive serum blocked with adult homogenate^ VIII] positive serum blocked with a combination of all homogenates except the one on the respective membrane strip[
II and III[ The antigens of the adults were found in fractions I and III[ Bands of 79 kDa for the L2 and 49 kDa for the adults were also present on the blot incubated with negative serum "as indicated on the blot with positive serum and on the glycoblot\ Fig[ 1b#[ 2[3[ Glycoblot of lysates In addition to the non!speci_c bands as described above\ glycoproteins could be found for the L3c and L3p in the lysates of bu}er I "a band of ×59 kDa and a double band of 11 and 13 kDa# and of bu}er III "44 kDa# and a double band of 16 and 29 kDa for the adults in bu}er III[ 2[4[ Western blot and glycoblot of culture super! natants Before and after incubation\ at least 89) of the parasites were still motile[ The Western blot of the concentrated culture supernatants is shown in Fig[ 2[ In the L2 supernatants no protein could be
detected\ even after 13 h of incubation[ In the super! natants of the other stages a common 099!kDa protein was detected which was glycosylated "not shown#[ In the supernatants of L3c incubated for 5 h smaller proteins were stained only weakly\ whereas after 13 h of incubation a larger number of proteins ranging from 27 to 099 kDa was present[ In the samples from the L3p the same proteins were detected after 5 h of incubation\ and additionally smaller proteins "×03 kDa# were found[ L3p and adults were incubated for only 5 h to avoid bacterial contamination[ A medium control without worms and blots with negative serum revealed no bands "not shown#[
3[ Discussion At the point of transition from the free!living to the early parasitic stage\ marked by exsheathment of the L2\ O[ dentatum experiences crucial physical and chemical changes in its environment[ During the course of infection\ mainly in the histotropic
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
0746
Fig[ 1[ Western blot of fractioned somatic proteins[ "a# Blot with positive serum "dilution 0]199#[ "b# Glycoblot[ L2] third!stage larvae^ L3c] fourth!stage larvae from in!vitro cultivation^ L3p] fourth!stage larvae derived from intestines^ Ad] adult worms[ I] fraction I "water!soluble#^ II] fraction II "CHAPS#^ III] fraction III "SDS:83>C#[ Arrows mark bands that were also stained in blots with negative serum[
0747
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
Fig[ 2[ blot of excretoryÐsecretory proteins[ L2s] sheathed third! stage larvae^ L2e] exsheathed third!stage larvae^ L3c] fourth! stage larvae from in!vitro cultivation^ L3p] fourth!stage larvae derived from intestines^ Ad] adult worms^ m] MW marker[
stage\ it then faces its greatest challenge\ the encounter with the host|s immune system[ The his! totropic larva lives in closest contact with the tissue of the host\ and limitation of the host defence is vital for the parasite in order to maintain its integrity[ Simultaneously the parasite develops from micro! scopic L2 into considerably larger\ morphologically distinct pre!adult stages and _nally into sexually di}erentiated adults[ Both the developmentally regulated and the host!adaptive changes can result in changing protein patterns[ Lipids have been shown to be of importance for the development of O[ dentatum and may also con! tribute to hostÐparasite interactions ð5Ð7Ł[ In other nematodes\ proteins\ especially those with antigenic properties\ are supposed to be mediators of immu! nomodulation ð8\ 09\ 02Ł[ In the present paper we investigated antigens and glycoproteins of the parasitic stages of O[ dentatum[ We de_ned di}erent stages by their size and mor! phology ð03Ł as L2\ L3 and adults[ Furthermore\ we distinguished between L3 grown in vitro and in vivo[ In vitro\ larvae were cultured for 07Ð10 days\ when the development to L3 had reached its maximum of 4Ð09) of the initial L2 population[
With a few exceptions where L3 moult and develop to pre!adult L4\ this is the _nal stage in culture\ whereas in!vivo post!histotropic L3 can already be recovered from the intestinal contents in high num! bers on days 8Ð00 p[i[ We postulated that the di}erent population dynamics could be re~ected by di}erent protein patterns[ Changes in protein patterns during development have been described for gastrointestinal strongyles such as Oesophagostomum radiatum ð04Ł\ Ostertagia species[ ð05\ 06Ł and Haemonchus contortus ð07\ 08Ł[ Such changes are believed to serve as a mechanism of immune evasion and often involve proteins located at the parasite|s surface from where they can be shed into the environment\ or proteins actively secreted by the parasite "as reviewed by ð8\ 19\ 10Ł#[ We chose two groups of proteins\ antigens and glycoproteins\ for our investigations[ Antigens were chosen because the recognition of a protein as fore! ign and the subsequent induction of an immune response are indicative of hostÐparasite interaction ð7Ł[ Glycoproteins can be involved in hostÐparasite interactions ð11Ð13Ł and other biological functions of nematodes ð14\ 15Ł[ The avidinÐbiotin system which we employed for blotting enhances the sen! sitivity of protein detection compared with silver staining and other labelling techniques ð16Ł[ The glycoblot assay is limited to the detection of non! terminal carbohydrate moieties\ but it is less speci_c than lectin binding assays and therefore more suit! able for screening procedures[ Western blot analysis of fractionated proteins facilitates the comparison of protein preparations by using di}erential solu! bility of proteins as a biochemical feature ð17Ł[ With the applied techniques we found appreci! able di}erences between the stages and also between the L3 from di}erent sources[ In particular\ the strongly labelled somatic 29!kDa protein of the L2\ the 89!kDa antigen of the L3c and the ES 099!kDa glycoprotein of the advanced parasitic stages are of interest and will be characterised further[ While antigens were detected speci_cally with positive serum when total lysates were used\ fractionation of lysates revealed additional\ non!speci_c proteins which reacted with negative serum and also in the glycoblot when no antibodies were employed and are probably biotinylated[ This phenomenon has been described for other nematodes ð18Ł^ its func!
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
tion is not clear[ The L2 of O[ dentatum have pre! viously been described as sources of non!speci_c or cross!reactive antigens ð29Ł[ Whether the di}erences between the L3 from di}erent sources are an artefactual e}ect of cul! tivation or of the method of recovery is yet to be determined[ It is interesting to note that in prep! arations of ESP the L3p had a much more complex protein pattern than the L3c\ and that the frac! tionated preparations of L3p contained water!sol! uble proteins ³29 kDa which may correspond to the smaller ESP of this stage[ It is not clear whether the stage di}erences in protein expression are due to changes in the parasite|s development or induced by the host[ The di}erences between the L3 stages indicate host in~uence as it has been described for Ascaris ESP ð20Ł^ however\ some proteins such as the glycosylated ESP present in the advanced stages may be due to environmental changes[ Early and late L2 and L3 will be investigated further to deter! mine onset and termination of the expression of stage!speci_c proteins[ To gain some insight into the function of these proteins\ their location in situ will be determined[ The de_nition and charac! terisation of speci_c proteins and their isolation permit detailed studies on worm biology and hostÐ parasite interactions in bioassays\ such as lym! phoproliferation assays or larval migration studies[
Acknowledgements The authors thank Professor Dr M[ Rommel and Professor Dr K[T[ Friedho} for critical reading of the manuscript[ Parts of this study were funded by the Deutsche Forschungsgemeinschaft "grant No[ Jo 156:2!0#[ Dr A[ Joachim is recipient of a Karl Enigk scholarship[
References ð0Ł Barutzki D\ Randelzhofer A\ Gothe R[ Artenspektrum und Befallshau_gkeit von Helminthen bei Mutterschweinen und ihren Ferkeln in Suddeutschland[ Tierarztl Umschau 0881^36]068Ð089[ ð1Ł Roepstor} A\ Nansen P[ Epidemiology and control of hel! minth infections in pigs under intensive and non!intensive production systems[ Vet Parasitol 0883^43]58Ð74[
0748
ð2Ł Christensen CM\ Barnes EH\ Nansen P\ Gro ndahl!Nielsen C[ Growth and fecundity of Oesophagostomum dentatum in high!level infections and after transplantation into na(ve pigs[ Parasitol Res 0885^71]253Ð257[ ð3Ł Christensen CM\ Barnes EH\ Nansen P[ Experimental Oesophagostomum infections in the pig] worm populations at regular intervals during trickle infections with three doses of larvae[ Parasitology 0886^006]434Ð441[ ð4Ł Christensen CM\ Nansen P\ Barnes EH[ The e}ect of con! current or sequential Oesophagostomum dentatum and O[ quadrispinulatum infections on the worm burdens of the two species in pigs[ Parasitology 0886^003]162Ð167[ ð5Ł Daugschies A[ Oesophagostomum dentatum] population dynamics and synthesis of prostanoids by histotropic stages cultured in vitro[ Exp Parasitol 0884^70]463Ð472[ ð6Ł Daugschies A[ Investigations into the production and func! tion of leukotrienes during histotropic development of Oesophagostomum dentatum[ Parasitol Res 0885^71]305Ð 311[ ð7Ł Daugschies A\ Ruttkowski B[ Modulation of migration of Oesophagostomum dentatum larvae by inhibitors and products of eicosanoid metabolism[ Int J Parasitol 0887^17]244Ð251[ ð8Ł Philipp M\ Rumjaneck FD[ Antigenic and dynamic proper! ties of helminth surface structures[ Mol Biochem Parasitol 0873^09]134Ð157[ ð09Ł Maizels RM\ Selkirk ME[ Immunobiology of nematode antigens[ In] Englund PT\ Sher A\ editors[ The biology of parasitism[ New York] Alan Liss\ 0877^174Ð297[ ð00Ł Daugschies A\ Watzel C[ In vitro development of his! totropic larvae of Oesophagostomum dentatum under vari! ous conditions of cultivation[ Parasitol Res\ in press[ ð01Ł Slotved HC\ Barnes EH\ Bjo rn H\ et al[ Recovery of Oeso! phagostomum dentatum from pigs by isolation of parasites migrating from large intestinal contents embedded in agar! gel[ Vet Parasitol 0885^52]126Ð134[ ð02Ł Philipp M\ Parkhouse RME\ Ogilvie BM[ Changing pro! teins on the surface of a parasitic nematode[ Nature 0879^176]427Ð439[ ð03Ł Kotlan A[ Studies on the life!history and pathological sig! ni_cance of Oesophagostomum spp[ of the domestic pig[ Acta Vet "Budapest# 0836^0]547Ð553[ ð04Ł East IJ\ Berrie DA[ Antigenic di}erences among the life cycle stages of Oesophagostomum radiatum[ Res Vet Sci 0875^30]018Ð029[ ð05Ł Canals A\ Gasbarre LC[ Ostertagia ostertagi] isolation and partial characterization of somatic and metabolic antigens[ Int J Parasitol 0889^19]0936Ð0943[ ð06Ł Keith KA\ Duncan MC\ Murray M\ Bairden K\ Tait A[ Stage!speci_c cuticular proteins of Ostertagia circumcincta and Ostertagia ostertagi[ Int J Parasitol 0889^19]0926Ð0934[ ð07Ł Schallig HD\ Baczkowska A\ Wedrychowicz H[ Bio! chemical characterization of surface antigens of infective larvae and adults of Haemonchus contortus[ Acta Parasitol 0884^39]106Ð113[ ð08Ł Raleigh JM\ Brandon MR\ Meeusen E[ Stage!speci_c expression of surface molecules by the larval stages of Haemonchus contortus[ Parasite Immunol 0885^07]014Ð021[
0759
A[ Joachim et al[:International Journal for Parasitology 17 "0887# 0742Ð0759
ð19Ł Behnke JM\ Barnard CJ\ Wakelin D[ Understanding chronic nematode infections] evolutionary considerations\ current hypotheses and the way forward[ Int J Parasitol 0881^11]750Ð896[ ð10Ł Blaxter ML\ Page AP\ Rudin W\ Maizels RM[ Nematode surface coats] actively evading immunity[ Parasitol Today 0881^7]132Ð136[ ð11Ł Jarvis LM\ Pritchard DI[ An evaluation of the role of carbo! hydrate epitopes in immunity to Trichinella spiralis[ Parasite Immunol 0881^03]378Ð490[ ð12Ł Fetterer RH\ Rhoads ML[ Biochemistry of the nematode cuticle] relevance to parasitic nematodes of livestock[ Vet Parasitol 0882^35]092Ð000[ ð13Ł Schallig HDFH\ van Leeuwen MAW[ Carbohydrate epi! topes on Haemonchus antigens[ Parasitol Res 0885^71]27Ð 31[ ð14Ł Jeyaprakas A\ Jansson HB\ Marban!Mendoza N\ Zuck! erman BM[ Caenorhabditis elegans] lectin!mediated modi! _cation of chemotaxis[ Exp Parasitol 0874^48]89Ð86[ ð15Ł Kerwin JL\ Zurakowski KA\ Washino RK[ Carbohydrate!
ð16Ł
ð17Ł
ð18Ł
ð29Ł
ð20Ł
mediated recognition of larval mosquito hosts by Rom! anomermis culicivorax[ J Nematol 0889^11]008Ð015[ Hsu SM\ Raine L\ Fanger H[ A comparative study of the peroxidaseÐantiperoxidase method and an avidinÐbiotin complex method for studying polypeptide hormones with radioimmunoassay antibodies[ Am J Clin Pathol 0870^64]623Ð627[ Northern C\ Grove DI[ Strongyloides stercoralis] antigenic analysis of infective larvae and adult worms[ Int J Parasitol 0889^19]270Ð276[ Romar(s F\ Iglesias R\ Garc(a LO\ Leiro J\ Santamarina MT\ Paniagua E\ Ubeir FM[ Free and bound biotin mol! ecules in helminths] a source of artifacts for avidin biotin! based immunoassays[ Parasitol Res 0885^71]506Ð511[ Larsen RH\ Christensen CM\ Lind P[ Serological assays for the identi_cation of Oesophagostomum dentatum in pigs[ Res Vet Sci 0886^52]4Ð09[ Kennedy MW\ Qureshi F[ Stage!speci_c secreted antigens of the parasitic larval stages of the nematode Ascaris[ Immunology 0875^47]404Ð411[