INFECTION AND IMMUNITY, Aug. 1989, p. 2434-24400019-9567/89/082434-07$02.00/0Copyright ©) 1989, American Society for Microbiology

Immunization of Chickens with Live Escherichia coli ExpressingEimeria acervulina Merozoite Recombinant Antigen Induces

Partial Protection against CoccidiosisKI S. KIM,t MARK C. JENKINS,* AND HYUN S. LILLEHOJ

Protozoan Diseases Laboratory, Livestock and Polultry Sciences Instituite, Agricuiltlural Research Service,U.S. Department of Agricultutre, Beltsville, Maryland 20705

Received 30 December 1988/Accepted 10 May 1989

Inoculation of chickens with live Escherichia coli N6405 transformants containing a plasmid which encodesampicillin resistance and an immunodominant p250 surface antigen of Eimeria acervulina merozoites inducedpartial protection against challenge with live coccidia. The inoculation with E. coli transformants inducedantigen-specific immunoglobulin and cell-mediated immune responses. Challenge with infective oocysts ofEimeria acervulina enhanced both immune parameters, indicating that administration of live E. colitransformants served to prime the immune system for recognition of specific epitopes on the 250-kilodaltonprotein. Although the mechanism of antigen presentation is unclear, the data suggest that in vivo expression ofrecombinant merozoite antigen is operative. After administration, no E. coli N6405 transformants could berecovered from intestinal or fecal materials of inoculated chickens, as assessed by enumeration on selectivemedium. However, ampicillin-resistant E. coli originating from the normal flora and harboring the genesequences for both antibiotic resistance and Eimeria acervulina merozoite surface protein could be recoveredfrom these chickens. Furthermore, normal-flora E. coli transformants were capable of generating functional,-lactamase product, as evidenced by their resistance to ampicillin, and immunoreactive E. acervulinamerozoite recombinant antigen, as revealed by immunofluorescence staining with p250-specific antiserum.

Coccidiosis is a disease of major worldwide importance tochicken producers, inflicting an estimated loss of over $500million per year. Medication with several different anticoc-cidial drugs has been effective in preventing severe out-breaks of disease. However, the life of most of these drugs islimited because of the emergence of resistant strains ofcoccidia (3, 4, 9, 21, 23). Therefore, there is a need for analternative method of control, such as vaccination (22, 32).A moderate infection with any Eimeriia species that para-

sitizes chickens induces species-specific protection againstsubsequent challenge (6, 17, 29, 33). Several workers havebeen successful in preventing coccidiosis by immunizingchickens with nonviable extracts of infected tissues (24) andsporozoite antigen (26). Although it is unclear what immunemechanism is responsible for establishing protection, it isknown that both humoral (5, 17, 20, 33) and cell-mediated(14, 18, 25, 28, 30, 31) immune responses are involved, withthe latter playing a major role (18, 30, 34).

In previous studies, immunodominant surface antigens ofEimeria acervulina sporozoites and merozoites were identi-fied (11). cDNAs encoding several antigens of Eimer-iatenella and Eimeria acervulina have been cloned and ex-pressed in Escherichia coli (2, 12). Recent work in ourlaboratory has shown that one of these Eimeria acervulinaicDNAs encodes a portion of a p250 immunodominant mero-zoite surface protein and contains epitopes that are recog-nized by sera and T lymphocytes from immune chickens(12). This recombinant antigen also appears to be composedof tandem-repeated amino acid sequences (10) similar inmany respects to several immunogenic proteins in other

* Corresponding author.t This work was carried out during sabbatical leave from Rural

Development Administration, Veterinary Research Institute, An-Yang, Republic of South Korea.

related protozoans, such as Plasmodium (39), Leishmania(38), and Tiypanosoma (27) spp. Immunization of chickenswith this recombinant merozoite antigen induces both hu-moral (K. S. Kim, H. S. Lillehoj, and M. C. Jenkins, AvianDis., in press) and cellular (19) immune responses.The purpose of the present study was to evaluate admin-

istration of live E. coli transformants containing a recombi-nant plasmid that possesses the gene for an immunogenicregion of the 250-kilodalton (kDa) merozoite protein as ameans of delivering antigen to the chicken immune system.The basis for these studies is a recent finding that oralinoculation of mice with Salmonella typhimurium trans-formed with Plasmodiuim berghei circumsporozoite genecould induce protection against malaria (35).

MATERIALS AND METHODS

Chickens and parasites. One-day-old outbred Sexsal chick-ens were obtained from Moyer's Hatchery, Quakerstown,Pa. Embryonated inbred SC chicken eggs were obtainedfrom Hy-Line International Production Center, Dallas Cen-ter, Iowa. Inbred SC chickens were required in this study formeasuring T-lymphoblastogenic responses, since assayingT-cell proliferation in outbred strains is confounded by highlevels of background stimulation due to mixed lymphocyteresponses (i.e., lymphocytes responding to non-self majorhistocompatibility complex antigens). All birds were housedin clean wire-floor cages and provided feed and water adlibitum. Pure lines of Eimeria acervutlina (PDL strain 12)developed from a single oocyst and maintained at the Pro-tozoan Diseases Laboratory were used for coccidial chal-lenge. All experiments, including E. coli administrations,followed the recombinant DNA guidelines of the NationalInstitutes of Health and were approved by the BeltsvilleAgricultural Research Center Biosafety Committee.

Plasmid construction. Insert cDNA encoding a 35-kDa

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EcoRI

Hind Mi

r p c

Pstl

cMZ8

pCO5-cMZ8

5.9 kbp

Hind Imf

FIG. 1. Restriction map of pCO,-cMZ8 plasmid, indicating thelocation of the ampicillin resistance gene fragment and cll-p35merozoite surface antigen sequence. kbp, Kilobase pairs; pL. phagelambda promoter.

fragment of the Eimneiia acerilidina 250-kDa merozoite sur-

face protein was excised from recombinant pcMZ-8 plasmid(12) by EcoRI digestion and purified by electroelution froman agarose gel followed by passage over a NACS column(Bethesda Research Laboratories, Inc., Gaithersburg, Md.).Purified insert cDNA was treated with T4 DNA polymeraseto create blunt ends, ligated with HindII linkers (Pharmacia,Inc., Piscataway, N.J.) by using T4 DNA ligase and RNAligase, and subjected to a limited HinidIII digestion (to avoiddigesting the insert cDNA that contained an internal HinidIIIsite). The insert cDNA was phenol-chloroform extracted,isolated by passage over a NACS column, and ligated toHindIII-digested, alkaline phosphatase-treated pCO, plas-mid DNA (1) (kindly provided by Dante Zarlenga), using T4DNA ligase. pCOs was chosen as the expression vectorbecause exogenous genes cloned into the clI repressor-

coding sequence are under control of the temperature-inducible bacteriophage lambda promoter. The inductiontemperature of 42°C is similar to the ambient body temper-ature of chickens, thus allowing a comparison of in vitroversus in vivo induction of recombinant protein expression.

E. coli. E. coli N6405 was transformed with either pCO, or

pCO5-cMZ8 (Fig. 1) by standard procedures (8). E. ( 0/iN6405 transformed with pCO, (control strain 1) or pCOs-cMZ8 (vaccine strain 1) was identified by growth on Luriabroth (LB) agar containing ampicillin and, for pCO,-cMZ8transformants, by production of immunoreactive p42-46antigen. A nonpathogenic ampicillin-sensitive strain of E.coli (designated S548) was isolated from the normal intesti-nal floras of outbred chickens and was transformed withpCO,-cMZ8 by procedures similar to those described above.This normal-flora transformant (vaccine strain 2) and pCO,-transformed E. (oli S548 (control strain 2) were comparedfor production of immunoreactive p42-46 merozoite antigen.

Vaccination of chickens with E. coli transformants. E. (0olivaccine strains 1 and 2 and control strains 1 and 2 were

grown in LB medium containing ampicillin at 32°C to mid-logphase (optical density at 550 nm = 0.4 to 0.5). Culturesdesigned for in vitro induction were then grown at 42°C for 1

to 2 h. Induced and uninduced transformants were harvestedby centrifugation and suspended to 1010 cells per ml inphosphate-buffered saline (PBS). Outbred Sexsal and inbredSC chickens (n = 10 per subgroup; three subgroups pertreatment) were vaccinated per os with 10i vaccine strain 1or 2 or control strain 1 or 2 E. (/oli. Chickens of each groupwere challenged with 5 x 103 sporulated oocysts of Eimcieriiiaceil'u/iina 2 weeks after vaccination. Protection against

Eimneria a(ervllinla challenge was assessed by measuringoocyst output in the fecal material between days 5 and 9post-coccidial challenge. The level of protection was ex-pressed as the percent oocyst reduction compared with thatof unvaccinated controls, using the following formula: %oocyst reduction = 100 x r1 - (OOVG/OOCG)], whereOOVG equals total oocyst production in the vaccinatedgroup and OOCG equals total oocyst production in thenonvaccinated control group. Mean and standard errorswere calculated for each experimental group and comparedby analysis of variance.Serum collection and enzyme-linked immunosorbent assay

(ELISA). Samples of blood were collected from each groupof chickens by wing vein puncture at 1 day prior to and 2weeks after E. (0/i vaccination and 2 weeks post-Eimeriaa(ervlll(lna challenge, using standard procedures (36). Seraobtained from each group of chickens were tested for thepresence of antibodies specific for recombinant merozoiteantigen, using standard procedures (Kim et al., in press). Inbrief, recombinant merozoite antigen was purified by pas-sage over a high-pressure liquid chromatography (HPLC)size-exclusion column as described elsewhere (Kim et al., inpress) and was absorbed to the surface of ELISA microdi-lution plates for 1 h at 37°C and then overnight at 4°C. Inorder to avoid measuring immune responses to clI repressorprotein sequences, the source of recombinant merozoiteantigen was a 3-galactosidase fusion protein described else-where (12) which shares the merozoite antigenic region withthe clI fusion protein. The ELISA plates were washed toremove unbound antigen and treated with PBS containing0.05% Tween 20 and 1% bovine serum albumin to blocknonspecific immunoglobulin binding in later incubationsteps. Individual sera from each experimental group ofchickens (O = 30) and positive (immune serum) and negative(prebleed serum) controls were diluted 1:1,600 and applied intriplicate to ELISA microdilution wells for 1 h at 37°C. Thisstep was followed by rabbit anti-chicken immunoglobulin,biotinylated goat anti-rabbit immunoglobulin G, avidin-per-oxidase, and o-phenylenediamine-H,O, substrate. Each pre-vious reagent solution was removed by washing the micro-dilution wells with PBS-Tween 20. The level of binding wasassessed by measurement on a microdilution plate reader at492 nm and expressed as titers relative to values for thenegative controls (Kim et al., in press).

In vitro T-cell proliferation assay. Nylon wool-nonadherentT-cell-enriched splenic lymphocytes (13) were preparedfrom each group of chickens at 2 weeks postvaccination and9 to 12 days post-Eitinerii a(cervudlina challenge. Proliferativeresponses to HPLC-purified recombinant merozoite antigenand control bacterial protein were assessed by previouslydescribed procedures (16). The stimulating merozoite anti-gen used in these proliferation assays was a P-galactosidasefusion protein (12), to avoid measuring responses to the cIlrepressor portion of the recombinant merozoite protein. ThecIl repressor fusion protein and the P-galactosidase fusionprotein share the merozoite antigen sequence. Controls for3-galactosidase were identical HPLC fractions from alambda gtll purification. The level of T-cell proliferation inthe presence of recombinant merozoite protein was ex-pressed as the amount of [3H]thymidine uptake (in countsper minute) corrected for uptake in the presence of culturemedium alone. Statistical comparisons between vaccinatedgroups in the level of T-cell activation were performed byusing Student's t test.

Isolation of ampicillin-resistant fecal E. coli from chickens.Rectal swabs from a subset of chickens in each experimental

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group were obtained 1 day prior to and 1 and 7 days after oralinoculation of vaccine strain 1 or control strain 1 E. coli.These samples were inoculated on MacConkey agar with orwithout ampicillin (400 pLg/ml), incubated overnight at 37°C,and examined for the presence of colonies typical of thefamily Enterobacteriaceae. Several ampicillin-sensitive and-resistant normal-flora E. coli were isolated and were iden-tified further by standard biochemical testing (7).

Immunoblotting and immunofluorescence assays. Vaccinestrain, control strain, and ampicillin-sensitive and -resistantE. coli were cultured at 32°C in Luria broth with or withoutampicillin until mid-log phase (optical density at 550 nm =

0.4 to 0.5). The cultures were induced for recombinantantigen production by shifting the incubation temperature to42°C and grown at this elevated temperature for 1 to 2 h in ashaking water bath. The cells were harvested by centrifuga-tion at 2,000 x g for 10 min and suspended either insonication buffer (0.1 M Tris [pH 7.5], 0.05 M MgCl2)containing protease inhibitors for immunoblotting analysisor in PBS (0.1 M Na,P04, 0.15 M NaCI) for immunofluores-cence assays. For the former purpose, the cell suspensionwas lysed by freezing and thawing and then treated with 10pLg each of DNase I per ml and RNase A for 10 min on ice.A sample of the E. coli extract was diluted in an equalvolume of sample buffer (15), heated in a boiling water bath,and subjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), using a 4% stacking gel and a10% resolving gel. After electrophoresis, the gels weretransferred to nitrocellulose by standard procedures (37).Nitrocellulose membranes containing SDS-PAGE-separatedE. coli proteins were probed with mouse antiserum specificfor recombinant merozoite protein. Polyclonal monospecificmouse antiserum was obtained by immunizing mice via thefootpads three times at 2-week intervals with HPLC-purifiedrecombinant 3-galactosidase-merozoite fusion protein. Theimmunoblots were developed with biotinylated anti-mouseimmunoglobulin G (Vector) and avidin-peroxidase (SigmaChemical Co., St. Louis, Mo.), followed by substrate (4-chloro-1-naphthol-0.01% H,02 in PBS).

Immunofluorescence assays were performed on intact E.(oli cells that were either untreated or subjected to a 3%butanol fixation prior to being dried on microdilution slides.Bacteria were treated with PBS containing 1% bovine serumalbumin (dilution buffer) for 1 h to block nonspecific immu-noglobulin binding. After pretreatment, the cells were im-munostained with a 1:100 dilution of mouse anti-merozoiterecombinant protein serum or control mouse serum at 37°Cfor 30 min. The cells were washed three times with PBS andthen subjected to fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (ICN Immunochemicals, Irvine,Calif.) for 30 min at 37°C. The cells were washed three timesto remove unbound fluorescein isothiocyanate-conjugatedimmunoglobulin and then examined under an epifluores-cence microscope.

RESULTS

E. coli vaccine strains 1 and 2 were analyzed for produc-tion of immunoreactive recombinant merozoite antigen byreacting nitrocellulose membranes containing protein ex-tracts from these bacteria with immune serum. Extractsfrom both vaccine strains, unlike those from transformantcontrol strains, contained the p42-46 recombinant protein(Fig. 2). This molecular mass estimate is 6 to 10 kDa greaterthan that predicted for the fusion of the 35-kDa merozoiteprotein and 1-kDa clI lambda repressor protein (i.e., 36

kDa 1 2 3 4

214 -

111 -

68 -

*f.@46f. 42

24 -

FIG. 2. Immunostaining of nitrocellulose membrane containingSDS-PAGE-separated E. coli vaccine strain 1 (lane 1), control strain1 (lane 2), vaccine strain 2 (lane 3), and control strain 2 (lane 4)cellular protein with mouse antiserum specific for the 35-kDarecombinant Eirneria aceriulina merozoite antigen. Molecularmasses are indicated in kilodaltons.

kDa). However, several researchers have noted that recom-binant proteins containing proline-rich tandem-repeatedamino acid sequences electrophorese at molecular masseshigher than predicted estimates (Robert Brey, personalcommunication). The p42-46 recombinant antigen repre-sented about 5 to 10% of the total cell protein, as evidencedby SDS-PAGE analysis (data not shown). It is unclear whya doublet at 42 and 46 kDa is present rather than a single-molecular-mass protein. One of several possible explana-tions is that a site-specific proteolytic enzyme recognizesand cleaves the recombinant antigen into two constituents,each containing the immunoreactive B-cell epitope.

In order to ascertain the vaccine potential of live E. colicontaining gene sequences of Eimeria merozoites, threegroups of outbred Sexsal chickens (n = 30) were inoculatedper os with 10' vaccine strain 1 or control strain 1 E. (oli andchallenged with 5 x 103 sporulated Eimne)ia acerv'l/inaoocysts. Unlike chickens inoculated with the control strainand uninoculated controls, chickens inoculated with thevaccine strain exhibited a significant (P < 0.05) anti-recom-binant merozoite immunoglobulin response prior to Eineriitac-ervid/ina challenge (Fig. 3). After challenge, all threegroups of chickens had similar anti-merozoite immunoglob-ulin titers (P > 0.05; Fig. 3). Furthermore, only chickensinoculated with E. (coli vaccine strain 1 exhibited a significantreduction (P < 0.05) in oocyst output compared with controlstrain-inoculated and uninoculated chickens (Fig. 3), sug-gesting that the vaccine strain conferred some partial pro-tection against coccidiosis.Given the importance of cell-mediated immune responses

in establishing protection against coccidiosis, inbred SCstrain chickens were vaccinated with either E. (0oli vaccinestrain 1 or 2 or control strain 1 or 2. A small number (n = 3)of SC chickens from each group were sacrificed to obtainsplenic T cells to assess in vitro proliferative responses toHPLC-purified recombinant merozoite antigen and controlE. coli extract. Whereas concanavalin A responses weresimilar for each group, T cells obtained from chickensinoculated with vaccine strain 1 or 2 exhibited a three- tofivefold-greater response (P < 0.05) in the presence of

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^I I -l_QOocyst -60It I ReductionO 2 I1 0

D 0 0

oek I Wes I WekEoi I Ec I

COTO* VCN UINOUAEFIG.3 nt-3 Ig Inegnioyttesb LS n

I I ~~~~~~~~~~~~~~~C0 I~~~~~~~~~~~~4

I I~~~~~~~~2

7 00 24 104 1024

Weeks Weeks I Weeksin.ac I E.col ICONTROL VACCINE UNINOCULATEDSTRAIN 1 STRAIN 1

FIG. 3. Anti-p35 IgG and IgM antibody titers by ELISA andpercent oocyst reduction in Sexsal chickens inoculated with tem-perature-induced vaccine strain 1 or control strain 1 E. co/i followedby challenge inoculation with E.i(eoia acerlina. ELISA andoocyst reduction data are expressed as mean ± standard error(at30).

recombinant Eimier-ia a-eri'iu/ina merozoite antigen com-pared with the respective control strain-inoculated groups(Fig. 4). This proliferative response to recombinant merozo-ite protein was enhanced after Eirneriii a(eru/ina challengein chickens inoculated with E. (oli vaccine strains 1 and 2(Fig. 5). Although the level of activation was lower than thatof E. -o/i vaccine strain-inoculated chickens, significant (P <0.05) proliferation of T cells from the control strain-inocu-lated groups was observed after Eimiei-it acerlvu/ina chal-lenge (due in part to the presence of native p250 merozoiteantigen). There was a measurable degree of protectionagainst Eimerie acer-vulina challenge in chickens inoculatedwith E. coli vaccine strains 1 and 2, compared with that ofcontrols (Fig. 5).

43x10

0.

n

104

cx

I-3

m 3x10

0

-J

3-

10

E.coliControlStrain 1

CConA ;1LLambda

cmz-s L

E.coliControlStrain 2

E.coliVaccineStrain 1

E.coliVaccineStrain 2

FIG. 4. T-cell proliferative response to HPLC-purified recombi-nant merozoite and control lambda antigens and concanavalin A inSC chickens 7 days postvaccination with temperature-induced vac-

cine strain 1 or 2 control strain 1 or 2 E. coli. Antigen concentra-tions: recombinant merozoite antigen and lambda gtll. 5 ,g/mlIconcanavalin A, 12.5 ,ug/ml. Background stimulation: E. coli controlstrain 1, 2.200 cpm; control strain 2, 7,100 cpm; vaccine strain 1.5,200 cpm; vaccine strain 2. 5,400 cpm. l3H]thymidine (3H-TdR)uptake is expressed as mean + standard error (n = 3).

E. coli I E.coli E.coli i E.colil I _CONTROL I CONTROL I VACCINE I VACCINESTRAIN 1 i STRAIN 2 I STRAIN 1 I STRAIN 2

FIG. 5. T-cell proliferative response to recombinant merozoiteantigen p35 and percent oocyst reduction in SC chickens inoculatedwith temperature-induced E. (oli vaccine strain 1 or 2 or controlstrain 1 or 2. followed by challenge inoculation with EimnelriaaCe(rl'vidinaO. Antigen concentrations: concanavalin A, 12.5 p.g/ml;lambda gtll. 5 pg/mI; recombinant merozoite antigen. 5 pg/ml.Background culture medium stimulation: E. co/i control strain 1, 434cpm; E. coli control strain 2, 398 cpm; E. (o/i vaccine strain 1, 513cpm; E. coli vaccine strain 2, 2,642 cpm. [3H]thymidine (3H-TdR)uptake is expressed as mean ± standard error (n = 3).

To investigate the mechanism by which live E. coli con-taining a plasmid that encodes an Eiineria aCerv'u/lina mero-zoite surface protein could induce both humoral and cell-mediated immune responses and protect against coccidialchallenge, we sought to determine the residence time ofvaccine strain 1 bacteria in the guts of inoculated chickens.Previous experiments had shown that ampicillin-resistant E.c oli could not be recovered from the intestinal tracts or fecalmaterials of uninoculated chickens (unpublished observa-tions). Separate groups of outbred Sexsal chickens, un-treated or treated with ampicillin, were inoculated with 108uninduced (32°C) or induced (42°C) vaccine strain 1 orcontrol strain 1 E. (0oli. At 1 day before and 1 and 7 days afterE. co/li inoculation, rectal swabs were made from all chick-ens in each group and analyzed by culturing on MacConkeyagar containing ampicillin. Ampicillin-resistant E. (0oli couldnot be recovered from any groups of chickens prior to E. coliinoculation or from control strain-inoculated groups at 1 or 7days postadministration (Table 1). However, ampicillin-resistant E. (/oli could be recovered from all vaccine strain1-inoculated chickens at day 1 postadministration and onlyfrom temperature-induced vaccine strain-inoculated chick-ens on day 7 (Table 1). Quite perplexing was the finding thatthe ampicillin-resistant E. (oli recovered from inoculatedchickens were different in several characteristics (e.g., col-ony morphology and lactose fermentation ability) from theparent strain N6405. Biochemical analysis (7) of parentstrain and ampicillin-resistant and -sensitive normal-flora E.coli revealed that inoculated and recovered ampicillin-resis-tant E. (oli were distinct strains. Furthermore, ampicillin-sensitive and -resistant E. (oli isolated from the intestinaltracts of inoculated chickens were identical with respect tobiochemical characteristics.Attempts were made to generate recombinant merozoite

p42-46 antigen in vitro by inducing ampicillin-resistant E.(oli recovered from chickens inoculated with vaccine strain

.9hialL4.

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TABLE 1. Isolation of ampicillin-resistant E. (oli from Sexsalchickens orally inoculated with vaccine strain 1 or control

strain 1 E. coli

Temp of Administration Growth of E.E. co/i inoculum Chicken culture of ampicillin (o/i" on day:

no. (°C) (60 mg/chicken) -1 1 7

Control strain 1 547 32 Yes - - -530 - - -

513 32 No - - -535 - - -

Vaccine strain 1 557 32 Yes - +++ -

558 - + -

529 32 No - + -549 - + -

527 42 Yes - +++ ++538 - +++ +

503 42 No - + + +531 - + +

None 537 No - -548 -- -

"All E. coli colonies were grown on MacConkey agar plates with 400 ,ug ofampicillin per ml and were lactose positive smooth, different from thelactose-negative rough colonies of E. co/i N6405 transformant inoculated intochickens. -, No colony of E. coli; +, 1 to 30 colonies; + +, 31 to 200 colonies;+ + +, more than 200 colonies.

1 bacteria. The cellular protein extracts were analyzed byimmunoblotting, using immune serum specific for the 35-kDarecombinant merozoite antigen. Although these normal-floraE. coli transformants contained plasmid sequences, as indi-cated by their resistance to ampicillin, no immunoreactiveantigen in any molecular mass range could be detected byusing this assay system. These induced E. coli were alsosubjected to immunofluorescence staining using immuneserum and fluorescein-conjugated anti-immunoglobulin. Incontrast to the immunoblotting results, E. c oli containing themerozoite cDNA sequence exhibited positive fluorescencecompared with control serum (Fig. 6E and F). Ampicillin-sensitive normal-flora E. coli exhibited negligible stainingwith immune serum (Fig. 6D). As expected, E. coli vaccinestrains 1 and 2 (Fig. 6A and C) exhibited measurableimmunofluorescence compared with control strains 1 and 2(Fig. 6B and D). There was no measurable difference in thestaining patterns between unfixed and butanol-fixed E. c oli,and control mouse sera exhibited negligible immunofluores-cence staining of any E. coli examined (data not shown).

DISCUSSION

The present study demonstrates that inoculation of chick-ens with live E. coli containing gene sequences encoding anEimerica surface protein can elicit antigen-specific humoraland cellular immune responses against this protozoan. Fur-thermore, this inoculation confers partial protection (>50%)reduction in oocyst output) against a subsequent challengewith live coccidia. While immunoglobulin and T-cell re-sponses directed against the recombinant Eitneriai merozoiteantigen could be detected 7 days after E. co/i vaccine strainadministration, no such response was detected in controlstrain-inoculated chickens. After Eime/ii challenge, both E.coli control and vaccine strain-inoculated chickens exhibited

humoral and cellular responses against the recombinantantigen due to the presence of immunoreactive T- and B-cellepitopes on the respective 250-kDa merozoite surface pro-tein. A greater T-cell response was observed for vaccinestrain-inoculated chickens after Eineriia challenge comparedwith that of controls, suggesting that the E. coli inoculationserved to prime the cellular immune system for recognitionof the 250-kDa merozoite surface protein. No such anam-nestic immunoglobulin response was seen, suggesting thateither antibodies are not critical to protection or the mero-zoite p250 antigen is not the major target of a humoralresponse to this protozoan.One intriguing finding of this study was that gene se-

quences encoding both ampicillin resistance and merozoiterecombinant protein could be transferred to normal-florabacteria. Although in vitro expression of ampicillin resis-tance and recombinant merozoite protein was demonstrated,we are uncertain whether these processes occur in vivo.Recent observations indicate that in vivo expression isoperative, since attempts to immunize chickens with nonliv-ing E. coli transformants or recombinant merozoite proteinby subcutaneous injection have not yielded this level ofprotection (unpublished observations). Also, immunoreac-tive recombinant merozoite protein must undergo a differentform of processing in normal-flora transformants comparedwith the strain N6405 bacteria. The basis for this speculationis that B-cell epitopes on the 42- to 46-kDa protein that reactwith immune serum after denaturing SDS-PAGE and elec-trophoretic transfer are present when this recombinant anti-gen is produced in the N6405 strain but not when producedin the normal-flora bacterial transformants. One possibleexplanation is that transcription or translation of the p42-46gene sequence and subsequent intracellular processing (e.g.,membrane transport) occur by two different mechanisms inthe two E. coli strains. Recent experimental data supportingthis hypothesis indicate that the recombinant plasmid, afterbeing transferred to bacteria of the normal flora, is notretained as an extrachromosomal element but becomesassociated with the chromosomal procaryotic DNA. What-ever the mechanism, at least one conformation-dependentB-cell epitope is retained on the recombinant protein pro-duced in the normal-flora transformants, since positive im-munofluorescence was observed when these bacteria wereair dried or butanol fixed and treated with immune serum.Given the enhanced serum immunoglobulin and splenicT-lymphocyte responses, it is obvious that epitopes crucialfor recognition of both the recombinant antigen and therespective parasite protein were generated by the normal-flora transformants. Furthermore, these epitopes must playsome role in protection, because a significant reduction inthe level of oocyst output was observed in chickens inocu-lated with vaccine strain E. coli.

In our system it remains uncertain how plasmid DNA istransferred to normal-flora bacteria. Numerous attempts tomediate the transfer of recombinant plasmid from vaccinestrain 1 E. coli to normal-flora bacteria by conjugation invitro were unsuccessful (unpublished observations), sug-gesting that the microenvironment of the intestine playssome crucial role in this process. Whatever the mechanismof plasmid transfer, the present work demonstrates thatadministration of live bacteria expressing recombinant pro-tein is a potential means of delivering antigen to the hostimmune system. However, steps should be taken to avoidthe transfer of undesirable traits (e.g., antibiotic resistance)to bacteria in the normal flora.

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(I D

F

FIG. 6. Indirect immunofluorescence staining of butanol-fixed E. coli vaccine strains 1 and 2, control strains 1 and 2, and normal-floratransformants with mouse antiserum specific for the 35-kDa merozoite recombinant protein. (A) Vaccine strain 1. (B) Control strain 1. (C)Vaccine strain 2. (D) Control strain 2. (E) Ampicillin-resistant normal-flora transformant type 1. (F) Ampicillin-resistant normal-floratransformant type 2.

LITERATURE CITED1. Androphy, E. J., D. R. Lowy, and J. T. Schiller. 1987. Bovine

papilloma virus E2 trans-activating gene product binds to spe-cific sites in papillomavirus DNA. Nature (London) 324:70-73.

2. Brothers, V. M., I. Kuhn, L. S. Paul, J. D. Gabe, W. H.Andrews, S. M. Sias, M. T. McCanan, E. A. Dragon, and J. G.Files. 1988. Characterization of a surface antigen of Eineriatenella sporozoites and synthesis from a cloned cDNA inEscherichia coli. Mol. Biochem. Parasitol. 28:235-248.

3. Chapman, H. D. 1984. Drug resistance in avian coccidia(review). Vet. Parasitol. 15:11-27.

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