Vol. 59, No. 12

Protective Immunity to a Blood-Feeding Nematode (Haemonchuscontortus) Induced by Parasite Gut Antigens

DOUGLAS P. JASMER* AND TRAVIS C. McGUIREDepartment of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164-7040

Received 17 May 1991/Accepted 11 September 1991

To determine the ability of gut antigens to induce a protective immune response against blood-feedingnematodes, isolated gut antigens were used to immunize goats against Haemonchus contortus. Immunization-induced antibody responses recognized parasite gut antigens which were associated predominantly with themicrovillous membrane region of the parasite gut. Antibody from immune serum also recognized seven

predominant gut proteins on a Western blot (immunoblot). Several of these proteins appeared to be integralmembrane proteins on the basis of their solubility in the detergent Triton X-114, indicating that thepresentation protocol stimulated an antibody response to microvillous membrane antigens. Three different age

groups of goats ranging from <6 months to >1 year were immunized for challenge experiments. After infectionwith 104 larvae, an 87 to 95% reduction in fecal egg counts for all age groups of goats was achieved in theimmunized compared with the control group. The reduction of worms in immunized goats ranged from 65%(kids) to 89% (yearlings) compared with controls. These results indicate that gut antigens can induce significantprotection against blood-feeding nematodes. Antibody to H. contortus gut antigens also cross-reacted withmicrovilli of other blood-feeding nematodes including Ostertagia ostertagi and small strongyles of horses, whichindicates that epitopes associated with the gut are phylogenetically conserved.

Blood-feeding nematodes cause some of the most impor-tant parasitic diseases of humans and domestic animals (1, 9,22, 28). Control of these parasites is complicated by the costof anthelmintics, a high capacity of some blood-feedingnematodes to develop drug resistance (7, 23, 29), and thelack of effective vaccines.Immunity to blood-feeding nematodes can be induced in

both young and adult hosts (3, 12, 15, 18-20). However, theonly effective vaccine developed so far involves the use ofirradiated third-stage larvae to protect against Ancylostomacaninum (15-17). The use of irradiated larvae to vaccinateagainst other blood-feeding nematodes has achieved variablesuccess (10, 12, 25). Furthermore, the long-term viability oflarvae represents a major obstacle to this approach. Theidentification of antigens capable of inducing a protectiveimmune response is currently the most significant problem toovercome for immunologic control of these parasites.The parasite gut provides a potential source of protective

antigens from blood-feeding nematodes (18, 20, 21, 30). It isthought that the blood-feeding process could deliver an

immune response directed against gut surface antigens of theparasite. Excellent progress in immunizing against tick in-fections by using this approach has been made (20, 21, 30).In addition, immunization experiments using putative para-site gut antigens of the blood-feeding nematode Haemon-chus contortus induced significant protection against chal-lenge infections (18). Here we demonstrate directly thatparasite gut antigens induce a protective immune response toH. contortus challenge in goat kids. Our data indicate thatgut membrane proteins were the dominant antigens recog-nized by antibody from immune serum and demonstrate thatthe epitopes of some gut antigens are conserved in otherblood-feeding nematodes.

* Corresponding author.

MATERIALS AND METHODS

Parasites and gut antigen. The strain of H. contortus usedin this research was obtained from Ray Gamble (8) (U.S.Department of Agriculture, Beltsville, Md.). The strain ofOstertagia ostertagi was originally isolated from Louisiana(26) and was obtained from John Dame (University ofFlorida, Gainesville). Procedures for maintaining these par-asites in the laboratory by passage through host animalswere previously described (11). Because of their relativelylarge size, adult female worms were dissected to obtain gutsamples. Gut samples consisted of intestine excluding theesophagus and were stored in phosphate-buffered saline(PBS) (pH 7.4) at -20°C. Adult small strongyle nematodes(species undetermined) were obtained from the large intes-tine of a horse submitted to the Washington State AnimalDisease and Diagnostic Laboratory.Sample preparations for antigen analysis. Gut samples

from adult worms or whole, third-stage larvae were dis-rupted in a ground-glass homogenizer in the presence ofproteinase inhibitors (1 mM phenylmethylsulfonylfluoride-1mM L-tosyl-p-lysylchloromethylketone) and either NonidetP-40 or Triton X-114. Triton X-114 extraction of integralmembrane proteins followed described protocols (4). Gutsamples (1 mg of protein) were homogenized in 1% TritonX-114-150 mM NaCl-10 mM Tris-HCl, pH 7.4, on ice. Thelysate was centrifuged at 100,000 x g for 30 min at 4°C topellet detergent-insoluble material. After the supernatantwas layered onto a sucrose cushion, cloud formation was

initiated by heating at 37°C for 5 min. Phase separation was

accomplished by centrifugation at 2,000 x g for 5 min.Aqueous, sucrose, and detergent phases were collectedseparately, and the aqueous phase was extracted two addi-tional times with Triton X-114 as described above. Individ-ual Triton X-114 fractions were adjusted to 1% Triton X-114in a buffer identical to that used for lysis, and the phaseseparation procedure was repeated. The resulting TritonX-114 samples were stored at -20°C. The aqueous phasewas concentrated with a Centriprep-10 (Amicon) filter prior

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to storage. Protein concentrations were determined by thebicinchoninic acid assay (Pierce).Western immunoblots. Gut lysates were boiled in sample

buffer (2% [wt/vol] SDS [sodium dodecyl sulfate], 2.5%2-mercaptoethanol [vol/vol], 25 mM Tris-HCl [pH 6.8], 15%glycerol [vol/vol], a few crystals of bromophenol blue), andantigens were separated on a 7.5 to 17% gradient SDS-polyacrylamide gel. Antigens were electroblotted onto 0.45-,um-pore-size nitrocellulose filters (30 V for 16 h) in a buffercontaining 25 mM Tris-HCl (pH 8.5), 190 mM glycine, and20% methanol (voUvol). Filters were blocked in buffercontaining 10 mM Tris-HCl (pH 7.5), 170 mM NaCl, 0.1 mMPMSF, and 1% horse hemoglobin (wt/vol); rinsed in thesame buffer; and reacted with antisera from immunized andcontrol goats at various dilutions. Antisera were diluted inblocking buffer containing 0.1% SDS (wt/vol), 0.1% TritonX-100 (vol/vol), and 1 mM EDTA. Filters were rinsed inbuffer four times for 3 min and reacted with 125I-labeledprotein G. After excess protein G was rinsed away, filterswere dried and autoradiographed. Molecular weights ofproteins were determined from a minimum of three differentexperiments.

In situ antigen localization. Adult and third-stage larvae ofH. contortus, adult 0. ostertagi, and adult small horsestrongyles were fresh-frozen in embedding medium (O.C.T.;Miles) for cryosectioning. Sections were cut 5 ,um thick,applied to poly-L-lysine-coated microscope slides, fixed inmethanol-acetone-water (1:6:3), and used in direct fluores-cent-antibody-binding assays. Antibodies from immunizedand control goat sera were isolated as described previously(24) by ammonium sulfate precipitation and then by DEAEanion-exchange chromatography. Isolated immunoglobulinG (IgG) was conjugated with fluorescein isothiocyanate(FITC) (6), and unreacted FITC conjugate was eliminated bydialysis. FITC-labeled antibody was adjusted to 0.2 mg/ml inPBS containing a 1:10 dilution of normal goat serum andreacted with cryosections for 30 min at room temperature.After three 15-min washes in PBS, cryosections were exam-ined with a fluorescence microscope.Immunization and challenge experiments. For immuniza-

tion, 25 mg of whole gut was homogenized in 1 ml of PBSwith a ground-glass tissue homogenizer. The homogenatewas then mixed with an equal volume of Freund's completeadjuvant and sonicated at 50 W until emulsified. The antigenpreparation was divided among six goats for primary immu-nization by intramuscular injection. Ovalbumin (1 mg pergoat) was used as the control antigen and was prepared inadjuvant as described above. Immunized and control goatsreceived booster immunizations with their respective anti-gens, using Freund's incomplete adjuvant. Saanen kid goatsused in the first and third immunization trials were part of abreeding herd maintained at Washington State University inconfinement. Experimental kid goats were all maintained inisolation facilities from birth. Kid goats were shown to befecal egg count negative by sugar flotation (2) at the begin-ning of the experiment. Immunized and control kids receiveda primary immunization shortly after weaning at 2 monthsold. They were boosted seven times at 2-week intervals, andkids received their final boost when about 5 months old.Three weeks after the final immunization, each goat was

challenged orally with 10,000 third-stage larvae of H. con-tortus. After week 3, when infections became patent, fecalegg counts were determined twice weekly for 3 weeks.Packed cell volumes were also obtained for kids during thisperiod. At the end of the observation period, abomasa wereobtained and processed for worm counts. The entire con-

tents of each abomasum were saved, and then abomasa weresoaked individually in tap water overnight to release fourth-stage larvae from the mucosa. For each goat, all wormscontained in 10% of the abomasal content and mucosaldigest were counted and the total worm counts were calcu-lated. Fecundity of adult female worms was assessed bycounting the eggs contained in the uteri of 10 females fromeach goat, except for some immunized goats which hadinsufficient worms to obtain 10 females.Mean parasite egg and worm counts were compared

between immunized and control groups by using Student's ttest (27). Means were considered significantly different at P< 0.05. When variances of means tested were significantlydifferent (P < 0.05 by using thef test), data were normalizedby log1o transformation and retested. In all cases, resultsfrom t tests with transformed data were in agreement withthose using nontransformed data.

Yearling Pygmy goats were used in the second immuniza-tion trial. These goats were obtained as kids from local goatherds. All were fecal egg count negative, with the exceptionof one which had nine nematode eggs per gram of feces (thespecies was undetermined). The one goat with low eggcounts was treated with fenbendazole, which eliminated theeggs in feces. This goat was assigned to the control group 6weeks after treatment to allow time for drug elimination.Pygmy goats were separated into immunized and controlgroups containing six goats each, and this immunization trialwas conducted as described for the first immunization trial.

In a third trial, confinement-raised Saanen goats wereinitially immunized at 6 months of age and received a total offour immunizations for both the immunized and controlgroups. In this case, fecal egg counts, but not worm counts,were obtained for goats after challenge.

RESULTS

To assess the immune responses of immunized goats togut antigens, postchallenge sera from Saanen kids (Table 1,experiment 1) were used in Western blots (Fig. 1). Theprofiles of proteins were similar, although not identical, foreach of the goats in the immunized group. Dominant anti-gens recognized by most immune sera included 33-, 37-, 40-,43-, 50- to 53-, 56-, and 98-kDa proteins. Although broadbands may contain more than one protein, resolving thesewill require further analysis. Antibody from kid 6 exhibitedthe strongest reaction and recognized two additional gutantigens of 103 and 173 kDa. While antibody from controlgoats showed little reactivity to H. contortus gut proteins,weak reactivity on longer exposures of Western blots wasdetected.To determine whether antibodies in sera of immunized

goats were induced against integral membrane proteins,whole gut samples were extracted with Triton X-114. TritonX-114-soluble and aqueous-soluble fractions were analyzedwith sera from immunized goat 6 and control goat 13 (Table1, experiment 1) on Western blots (Fig. 2). Proteins of 40, 43,56, and 98 kDa were enriched in the Triton X-114 phasecomnpared with the aqueous phase. Other proteins of 33, 36,and 50 kDa (the 33- and 36-kDa proteins are more apparentin longer exposures) appeared to be distributed equally inboth phases. Partial partitioning of these gut proteins intoTriton X-114 likely reflects specific characteristics such asheavy glycosylation or conformation (5, 14).To localize parasite gut antigens recognized by immunized

goats, antibody from immunized and control kids (6 and 13,respectively) were used in in situ antibody-binding assays.

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TABLE 1. Reduction in fecal egg counts, worm counts, and worm fecundity in kids vaccinated with gut antignes from H. contortus

Expt 1 Expt 2Parasiteparameter Mean (SD) count' Mean (SD) counta

Control Immunized Reduction Control Immunized Reduction pb

EPGc 3,373 (2,714) 170 (157) 95 0.05 1,549 (1,412) 75 (85) 95 0.05Adult worms 2,777 (1,074) 973 (918) 65 0.05 398 (334) 43 (29) 89 0.05Fourth-stage larvae 5.0 (2.6) 1.7 (1.3) 66 0.4 10 (4.0) 5.0 (2.6) 50 0.4Uterine eggsd 257 (91) 183 (63) 29 0.10 376 (124) 188 (167) 52 0.001

a n = 6 goats.b Probability values for means were compared by Student's t test, as described in Materials and Methods.c EPG (eggs per gram) values shown only for 3 weeks postpatency.d Eggs from 10 worms for each kid were counted when available. For experiment 1, n = 60 and 55 for control and immunized groups, respectively; for

expenment 2, n = 51 (control) and 39 (immunized).

IgG isolated from serum was labeled with FITC and reactedwith cryosections of adult H. contortus worms (Fig. 3). Thegut of H. contortus is a syncytium composed of an externalcellular layer and a lumenal microvillous layer (Fig. 3D).Antibody-binding activity of the immunized goat was local-ized predominantly in the microvillous layer of the gut, andvery little reactivity was detected in other parts of the gut.Labeled control antibody did not bind detectably to gutantigens.

Next, the ability of immunized goats to resist challengeinfections was analyzed. In the first immunization trial,2-month-old Saanen kid goats were tested. The final boosterimmunization was given when the kids were 5 months old,and they were challenged at about 6 months of age. Theresults of challenge infections are shown in Fig. 4 and Table1. Mean fecal egg counts were significantly (P < 0.05)reduced in kids immunized with H. contortus gut antigensfrom the time of patency throughout the sampling period(Fig. 4). The percentages of decrease in the immunizedgroup ranged from 86 to 95% when compared with thecontrol group.Mean worm numbers were reduced by 65% in the immu-

nized group. The reduction in adult worms in immunizedkids was not due to a delay in the development of fourth-stage larvae in the mucosa, since there was no increase in thenumber of these larvae for the immunized compared with thecontrol group. Although surviving female worms in theimmunized group had a mean of 29% fewer eggs occurring intheir uteri than female worms from the control group, thedifference was not statistically significant (P > 0.05) in thisexperiment. No significant decrease in packed cell volumesin the control or immunized groups in this or the subsequentexperiments was observed.The immunization and challenge experiment was repeated

with yearling Pygmy goats, and the results are summarizedin Table 1. In this experiment, mean fecal egg counts, wormcounts, and uterine egg counts were significantly reducedby 95% (P < 0.05), 89% (P < 0.05), and 52% (P < 0.001),respectively, in the immunized compared with the controlgroup. In this case, the significant reduction in uterine eggcounts for the immunized group may indicate reduced fecun-dity of female worms in these goats. While the numbers ofworms in the abomasa and eggs in the feces were lower forcontrol Pygmy goats compared with control Saanen kids

3 4 5 10 1 12

200-

92.5.-

69

46- -

!.Y30 U-

6 7 9 13 16 18

200>

69i--

30 3

FIG. 1. Antigens in H. contortus gut homogenate recognized onWestern blots by antibody from protected kids. Gut antigens wereused at 20 ,ug of protein per lane, and a 1:1,000 dilution of sera fromimmunized (lanes 3 to 7 and 9) and control (lanes 10 to 13, 16, and18) kids was reacted with individual filters. Molecular weightstandards are shown.

1 2 3 4 5 6

200-

92.5

69>

46'-

30-

FIG. 2. Identification of integral membrane protein antigens inH. contortus gut homogenate. Ten micrograms of protein of total guthomogenate (lanes 1 and 4) and Triton X-114-soluble (lanes 2 and 5)and aqueous-soluble (lanes 3 and 6) fractions were Western blottedwith serum (1:1,000 dilution) from kid 6 (immunized, lanes 1 to 3) orkid 13 (control, lanes 4 to 6). Molecular weight standards are shownat the left.

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1 23

200 .

4 5 6

FIG. 3. Reactivity of antibody from an immune goat to microvilliof the H. contortus gut. Cryosections of adult H. contortus werereacted with FITC-labeled IgG from kid 6 immunized with H.contortus gut homogenate (A and C) or kid 13 immunized withovalbumin (B). The gut lumen is more clearly seen in panel C. Asection of adult H. contortus showing the gut, which is composed ofa peripheral syncytial layer and a microvillous layer (m), is alsoshown (D). Bar, 200 ,um.

(Table 1), the differences in these parameters for the immu-nized groups were similar in both experiments.A third experiment involving Saanen kid goats assessed

only fecal egg outputs in the immunized and control groups.In this case, the immunized group had a mean of 107 ± 97eggs per gram and the control group had a mean of 831 ± 298

10000

. IMMUNIZED=CONTROL

10000

0- - 100

10

21 23 28 33 35

DAYSFIG. 4. Fecal egg counts in immunized and control goats in

experiment 1. Mean (log1o) eggs per gram of feces (EPG) are shownfor the indicated days postinfection. Standard deviations are indi-cated above the bars. Reductions in mean eggs per granm of theimmunized compared with the control group ranged from 89 to 95%.Differences were statistically significant at the P < 0.05 level for alltimes tested.

46 '

30

FIG. 5. Antigens of H. contortus third-stage larvae and adult0. ostertagi recognized by immune serum to adult H. contortus gutantigen. Homogenates containing 10 ,ug of protein of H. contortusgut (lane 1), whole third-stage larvae (lane 2), and 0. ostertagi gut(lane 3) were reacted with serum (1:1,000) from goat kid 6 (immu-nized) or 13 (control). Molecular weight standards are shown at theleft.

eggs per gram, representing a mean reduction of 87% (P <0.05) for the immunized group.Because of the significant protection observed, the expres-

sion of gut antigens in another life cycle stage (third-stagelarvae) of H. contortus was determined. The FITC-labeledantibody from goat 6 bound to internal organs of third-stagelarvae, while no binding of an FITC-labeled goat antibodyfrom a control goat was detected (results not shown).Although the small size of these larvae prevented identifica-tion of the specific organs involved, the gut is the dominantinternal organ occurring in this life cycle stage. In Westernblots, antibody from immune serum recognized antigens of33, 45, 50, and 98 kDa in homogenates of third-stage larvaewhich were also present in the adult H. contortus gut (Fig.5). An antigen of 173 kDa was detected in third-stage larvaeand not detected in the adult gut in this Western blot;however, a protein of this molecular mass was detected inthe adult gut in other experiments (Fig. 1, lane 6). Thisprotein could be expressed at higher levels in larvae com-pared with adults.To determine whether gut antigens of H. contortus might

be conserved in related hemophagous nematodes, antiseraagainst H. contortus gut homogenate were used on cryosec-tions and Western blots of gut from 0. ostertagi, which is theprimary stomach worm of cattle. In cryosections, FITC-conjugated IgG reacted predominantly with the microvillousregion of the 0. ostertagi gut (Fig. 6), which is identicalto the location for H. contortus. In Western blots, four0. ostertagi gut antigens of 50, 98, 105, and >200 kDa wereclearly detected with immune serum to H. contortus gutantigen (Fig. 5). Two of these 0. ostertagi gut antigens (50and 98 kDa) had apparent molecular masses similar to gutantigens of H. contortus. Adult small strongyle nematodes(species not determined) from a horse were also tested in thein situ assay, and fluorescent antibody to H. contortus gutcross-reacted with the microvillous gut region of thesenematodes (results not shown).

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FIG. 6. Cryosections of adult 0. ostertagi were reacted withFITC-labeled IgG from kid 6 immunized with H. contortus guthomogenate (A) or kid 13 immunized with ovalbumin (B). Bar,200 ,um.

DISCUSSION

The most important contribution of this research is directevidence that gut antigen of a blood-feeding nematode caninduce significant protection against challenge infections inyoung animals. Other antigen preparations have been shownto induce protective immunity against H. contortus. One ofthese was a preparation of high molecular mass (>30,000kDa), soluble somatic antigen extract (30,000 x g) andexcretory-secretory products from fourth-stage larvae (19).Another was derived by differental centrifugation fromwhole adult H. contortus, representing material which pel-leted between 10,000 and 50,000 x g and contained putativegut antigens (18). Both of these antigen preparations inducedsignificant protective immunity against lambs <6 monthsold. However, the tissue origin of protective antigens wasnot determined in either study. The reductions in meanworm counts (65%) and mean fecal egg counts (95%) shownhere support the earlier proposal that gut antigens areprospective targets for vaccination against H. contortus andother blood-feeding nematodes.

Significant reductions in fecal egg counts and worm num-bers were also achieved in immunized yearling goats, and asignificant reduction in fecal egg counts (the only parameterexamined) was observed in a third experiment. While previ-ous exposure to H. contortus cannot be ruled out for theyearling Pygmy goats, a comparison of immunized andcontrol goats clearly indicates that gut antigens induced theprotective immunity observed. Even though packed cellvolumes were not significantly decreased in control goats inany of the experiments, the level of adult worm reductions inimmunized goats suggests that gut antigens will be capable ofinducing protection against clinical disease.The variability in reductions of fecal egg counts and

worms among the immunized goats most likely reflectsdifferences in the immune responses of individual goats.Currently, this variability does not limit the potential of gutantigens in vaccine development for the following reasons. Asignificant decrease of fecal egg counts in young animals,even with relatively high individual variability, would trans-late into a significant decrease in subsequent pasture expo-sure to H. contortus for the herd. Also, the antigen sourceused in these trials is complex and not amenable for use on

a large scale. As specific protective antigens are identifiedand produced, presentation protocols, which may reduce thevariability observed, can be optimized.An analysis of the antibody response of immune goats

indicates that a vigorous immune response was generatedagainst gut membrane antigens. Antibodies from an immuneserum selectively reacted with the microvillous region of thegut, and these antibodies recognized several gut antigens,some of which were integral membrane proteins defined bysolubility in Triton X-114. It is unlikely that these antigensoriginate from the esophagus, since there was no reactivityto this region of the worm in in situ antibody-binding assays.While only one protected goat serum was tested in someanalyses, the similar gut antigen profiles recognized byantibodies from all of the immunized goats from experiment1 indicate that it is representative of the group. We have alsoconducted preliminary experiments in which FITC-labeledantibody from immune serum reacted to the surface ofmicrovilli in freshly dissected pieces of H. contortus gut(results not shown). These results are encouraging, since gutmembrane surface antigens would be accessible to an im-mune response, and protective immunity against Boophilusticks has been induced with gut surface antigens of thisblood-feeding parasite (20, 21, 30).Although antibody was used to identify gut antigens

recognized by a protective host immune response, it was notassumed that the immunity was antibody mediated. There isevidence for mucosal immunity and both cell-mediated andhumoral immunity to gastrointestinal nematodes (13), withthe significance of each depending upon parasite and hostspecies. Protective immunity induced by gut antigens maybe different from that in natural immunity, especially if theparasite damage is restricted to the gut and caused byimmune mechanisms delivered during blood feeding. Forinstance, physical or biochemical constraints imposed byfeeding and digestive processes of the parasite may eliminatesome cellular and/or antibody-dependent immune mecha-nisms, including mucosal immunity. Alternatively, the loca-tion of antigens recognized by the immune response may notbe limited to the gut. In our in situ studies, some antibody togut antigen reacted weakly with the inner body wall of H.contortus. On the basis of our method of antigen prepara-tion, this result suggests that cross-reactive gut epitopesoccur in other tissues of the parasite, and preliminary resultswith monoclonal antibodies to gut surface epitopes supportthis possibility. In some cases, monoclonal antibodies to gutsurface epitopes bind specifically to a variety of tissues,including some recognition of cuticular antigens (unpub-lished data). Consequently, immune mechanisms induced bygut antigens may be directed at regions other than the gutand could involve mechanisms similar to those induced innatural infections.

Results indicating that gut antigens expressed in adult H.contortus were present in third-stage larvae may be impor-tant. Experiments described here were not designed to testwhether preadult parasite stages were affected by immunityto adult gut antigens. However, our results suggest this ispossible, since both third- and fourth-stage larvae are tissuedwelling. The presentation of protective gut antigens ex-pressed in these tissue stages could stimulate anamnesticimmune responses in vaccinated animals when they areexposed to natural challenge. Therefore, selecting specificantigens expressed in the adult gut and in larvae may be animportant strategy in vaccine development.

Species cross-reactivity of gut antigens is also of specialinterest. Blood-feeding nematodes in the order Strongylida

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belong to three superfamilies, including the Ancylostoma-toidea, Strongyloidea, and Trichostrongyloidea. Nematodesfrom the last two superfamilies were shown in this study toshare cross-reactive microvillous gut antigens. Currently, no

vaccines are available for nematodes in any of these super-

families, and control procedures have limitations similar tothose for H. contortus. Since gut membrane antigen epitopesmay be phylogenetically conserved, protective gut antigensfrom one blood-feeding nematode could have direct applica-tion either to vaccination against other such parasites or inidentifying gut surface antigens from these parasites.

ACKNOWLEDGMENTS

We express our appreciation to Richard Dixon, Jan Carlson,Alberta Brassfield, James Wood, Eldon Libstaff, Stewart Bohnet,Sufang Yao, and Melissa Swan for their excellent technical assis-tance. We also thank Guy Palmer for critically reading the manu-

script.This research was supported by grants from the Medical and

Veterinary Biotechnology Center of the Washington TechnologyCenters, USAID-Small Ruminant Cooperative Research SupportProgram DAN 1328-G-SS-4093-00, and the College of AgricultureResearch Center (project no. 0114).

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