Vol. 23, No. 1INFECTION AND IMMUNITY, Jan. 1979, p. 108-1140019-9567/79/01-0108/07$02.00/0

Effects of Acquired Resistance on Infection with Eimeriafalciformis var. pragensis in Mice

G. M. MESFINt AND J. E. C. BELLAMY*Department of Veterinary Pathology, Western College of Veterinary Medicine, University ofSaskatchewan,

Saskatoon, Saskatchewan S7N OWO Canada

Received for publication 2 August 1978

Mice immunized with infections of 500, 5,000, or 20,000 oocysts of E. falciformisvar. pragensis were reinfected with 20,000 and 100,000 oocysts at 20 and 38 days,respectively, after the initial infection. After the first challenge infection, none ofthe immunized mice showed clinical signs of coccidiosis; a few mice passed verylow numbers of oocysts, and oocyst discharge seemed to correlate negatively withimmunizing dose. None of the mice immunized twice passed oocysts after chal-lenge. Mice immunized with three infections were completely immune to chal-lenge for 4 months. The effect of the immune response on the life cycle of thecoccidium was determined by histological examination ofthe intestines ofimmuneand nonimmune mice infected with the parasite. In both the immune andnonimmune groups, sporozoites penetrated absorptive epithelial cells and mi-grated to crypt epithelial cells during the first 6 to 24 h postinfection. At 48 to 72h postinfection, the sporozoites developed into mature first-generation schizontsin the nonimmune mice, whereas the developing first-generation schizonts degen-erated within the crypt epithelial cells of the immune mice. In nonimmune mice,third-generation merozoites, inoculated intracecally, developed into maturefourth-generation schizonts, whereas in immune mice the developing fourth-generation schizonts degenerated before maturing. The possibility that a cell-mediated immune mechanism is responsible foy the arrest in schizogony isdiscussed.

Both humoral and cellular immunities arethought to play a role in resistance to coccidia(26), but the precise mechanisms involved arepoorly understood. Coccidia have a complex lifecycle. Sporozoites enter host cells and developinto successive generations of schizonts whichrelease merozoites that invade other host cells.The final generation of merozoites develops intogametes. Microgametes and macrogametes com-bine to form oocysts, which are passed from thehost into the environment. Knowledge of thespecific event in the pathogenesis or stage ofdevelopment which is affected by the immuneresponse could give some indication ofthe mech-anisms responsible for the immunity. Opinionsabout which stage is affected vary considerably.It has been reported that sporozoites of Eimerianieschulzi and E. tenella failed to penetrateintestinal epithelial cells of immune rats andchickens, respectively (22; S. A. Edgar, Ph.D.thesis, University of Wisconsin, Madison, 1944).Several studies with avian coccidia suggested

t Present address: Department of Pathology and Hygiene,School of Veterinary Medicine, University of Illinois, Urbana,IL 61801.

that sporozoites can penetrate epithelial cells ofimmune birds but fail to develop into mature 1stgeneration schizonts (1, 12, 18, 33). Similar re-sults were observed in rabbits infected with E.magna (25). The coccidial stages affected by theimmune response have not been determined forany of the murine coccidia.The aims of this study were to determine the

following in mice by using E. falciformis var.pragensis: (i) the effects of the immune responseon the life cycle of coccidia, (ii) the relativeimmunizing ability of different doses of oocysts,and (iii) the duration of acquired resistance tococcidia.

MATERIALS AND METHODSExperimental animals. Swiss white mice, ob-

tained from the Animal Resources Center, Universityof Saskatchewan, Saskatoon, Saskatchewan, Canada,were used in this study. Mice used to determine oocystdischarge and weight changes were kept in individualwire-netted cages. The others were kept in groups offour to five in plastic cages.

Oocysts and merozoites. E. falciformis var. pra-gensis (4), established from a single oocyst infection,was maintained by frequent passage through coccidia-

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free mice, sporulated in 2.5% potassium dichromate,and stored at 40C for not more than 8 weeks before itwas used. A suspension of third-generation merozoiteswas obtained from mice killed 5 days after oral inoc-ulation with 50,000 oocysts of E. falciformis var. pra-gensis. To reduce the intestinal bacterial population,the donor mice were given neomycin at 10 mg/kg ofbody weight for 4 days before they were killed. Afterthe colonic contents were washed away with water,the colonic mucosa was completely removed with ascalpel and suspended in phosphate-buffered saline(pH 7.2). The merozoites were released from the epi-thelial cells by gently crushing and teasing the mucosalscrapings. The suspension was filtered through a 1-mm wire mesh, and the filtrate was centrifuged. Thesediment was suspended in a small volume of phos-phate-buffered saline and maintained at 370C untilused within 1 to 3 h. The number of merozoites givento each mouse was estimated with a Fuchs-Rosenthalhemocytometer.

Infection. Mice, randomly assigned to three testgroups, were infected orally with 500, 5,000 or 20,000oocysts. On the 20th day postinfection (p.i.), previouslyinfected mice and uninfected mice were infected with20,000 oocysts. Mice immunized with two oral infec-tions of 20,000 oocysts at 18- to 20-day intervals andchallenged with 106 oocysts were used to study the lifecycle of the coccidium in immune mice. The thirdimmunizing dose of 100,000 oocysts was given on the18th day after the second infection. Mice were infectedwith merozoites by inoculating approximately 4 x 10'third-generation merozoites into the cecum exposedsurgically. Mice were kept under halothane anesthesiaduring the surgery.

Clinical observations and oocyst counting pro-cedures. Feed intake and consistency of feces werevisually evaluated daily. The day of inoculation wasdesignated as day 0 and weights were recorded on days0, 3, 6, 7, 8, 9, 10, 12, 14, and 18 during the firstchallenge and on days 0, 3, 6, 8, 10, 12, and 16 duringthe second challenge to compare weight changes be-tween test and control groups. Daily oocyst outputwas determined by the method of Long and Rowell(20). If there were too few oocysts to count using aMcMaster chamber, feces were qualitatively examinedfor oocysts by the sugar flotation method.Determination of duration of immunity. The

immune status of mice immunized with 3 infections ofE. falciformis var. pragensis was evaluated by deter-mining the oocyst discharge in mice infected with100,000 oocysts at 2, 3, 4, 5 and 6 months after the lastimmunizing dose. Test mice were killed on the 15thday after the challenge infection and the cecum andcolon were examined histologically.

Histological methods. Three pieces of colon atapproximately 2, 4, and 5 cm distal to the cecocolicjunction were obtained from mice killed by atlantooc-cipital dislocation. Tissues were fixed in Bouin's fluidfor 24 h, postfixed in 70% alcohol, dehydrated, paraffinembedded, sectioned at 5 to 6 pm, and stained withhematoxylin-eosin. Selected tissues were stainedwith Heidenhain's iron hematoxylin and Warthin-Faulkner silver stain. The comparative numbers ofcoccidial stages developing in the colonic mucosa of

IMMUNITY TO MURINE COCCIDIOSIS 109

immune and nonimmune mice were determined bycounting all of the sporozoites, trophozoites, and schi-zonts, in one histological section (6 ALm) of colon fromeach mouse taken at each of 6, 12, 24, 48, 72, 96, and120 h p.i.

Statistical methods. The t test was used to com-pare the total number of coccidial stages found in theimmune and nonimmune mice. The mean weights ofmice immunized with different doses of the coccidiumwere compared by using an analysis of variance andDuncan's multiple range test (32).

RESULTSEffects of immunity on clinical signs and

oocyst discharge. For convenience of expres-sion, the term "immune" will be used to refer tomice that have been previously infected with thecoccidia and have subsequently recovered fromthe disease.The clinical signs exhibited by the mice after

the primary infection with the different doses ofoocysts were similar to those observed previ-ously (G. M. Mesfin, J. E. C. Bellamy, and P. H.G. Stockdale, Can. J. Comp. Med., in press),namely, dehydration, weight loss, diarrhea, anddysentery. These signs were not seen in immunemice; the feces of a few of the immune mice wereless well formed for 1 or 2 days, but diarrhea wasnever evident. The weight changes observed inthe immune and nonimmune mice after chal-lenge with 20,000 oocysts are shown in Fig. 1.Significant weight loss occurred in the suscepti-

DAYS POST INFECTION

FIG. 1. Weight changes (in grams) in mice immu-nized with different doses of E. falciformis var. pra-gensis and challenged with 20,000 oocysts 20 daysafter the primary infection. Symbols: 4 immunizedwith 500 oocysts; 0, 5,000 oocysts; X, 20,000 oocysts;0, uninfected control; 0, nonimmune control. Eachpoint represents the mean ± standard error for sixmice.

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110 MESFIN AND BELLAMY

ble (nonimmune) group, especially from 8 to 10days p.i. The mice in this group showed a com-pensatory weight gain from 12 to 16 days p.i. Noweight loss occurred in the immune mice; their-growth curve was similar to that of the unin-fected controls. Similarly, when the groups werechallenged again with 100,000 oocysts, only thenonimmune mice lost weight.The effects of immunity on oocyst discharge

are indicated in Table 1. Immunization greatlyreduced oocyst production after a challenge in-fection with 20,000 oocysts. Every mouse in thenonimmune group passed more than a total of5 x 106 oocysts, whereas none of the immunemice passed even one-tenth that number. In theimmune group, the numbers of dischargedoocysts were so low that usually the counts couldnot be done with a McMaster chamber. Only29% of the mice immunized with 20,000 oocysts

TABLE 1. Total oocyst discharge from immune andnonimmune mice infected with 20,000 oocysts of E.

falciformis var. pragensisImmuniz- Oocyst discharge (x104) in mouse no.:ing dose(oocysts) 1 2 3 4 5 6 7

500 4 0 4 1 <la <1 05,000 1 <1 0 2 3 0 0

20,000 0 48 1 0 0 0 00 593 1,186 616 888 561 NDb ND

a When there were too few oocysts to count with aMcMaster chamber, oocyst production was assessedqualitatively with the sugar flotation method and, ifdetected, they were assigned the value <1 x 104.

b ND, Not determined.

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passed oocysts after the challenge infection,whereas 57 and 72% of the two groups immu-nized with fewer oocysts passed oocysts afterchallenge. The prepatent period of the infectionwas extended by 24 h in some of the immunizedmice. In the same immune mice, the patentperiod was shortened by 3 to 5 days. Immunemice did not pass any oocysts when rechallengedwith 100,000 oocysts.

Effects of immunity on the life cycle andpathological changes. The effects of immu-nity on the total number of coccidia that devel-oped in specific areas of the colonic mucosa aresummarized in Table 2. The number and loca-tion of sporozoites during the first 24 h of theinfection were similar in both the immune andnonimmune mice. Sporozoites penetrated theabsorptive epithelium and migrated to the cryptepithelium apparently between the basementmembrane and the basal edge of the epithelialcells.The pathological changes observed at 6 and

12 h were similar in both the immune and non-

immune mice. They consisted of submucosaledema and neutrophil migration through sub-mucosal venules. At 24 h p.i., the sporozoitesand trophozoites in the nonimmune group ap-peared normal. Most of the trophozoites in theimmune mice were normal as well, but a fewappeared degenerate (Fig. 2A). Sporozoites wereoccasionally found in the lamina propria of bothimmune and nonimmune mice, and some ap-

peared degenerate. Desquamation of epithelialcells into the crypt lumen was more prominentin the immune group, but coccidial stages were

TABLE 2. Number and location of coccidial stages in the colonic mucosa of immune and nonimmune miceinfected with 106 oocysts of E. falciformis var. pragensis

No. of coccidial stagesa

Time (h Nonimmune Immune

p.i) Toa ptei i rpaTtl~CryptAbsorptive Crypt epithe- Lamina Absorptive pithe- LaminaTtl epithelium lium propria Ttl epithelium elPiutm propria

6 81±28 58±28 14±9 11±6 72±34 51±22 12±6 9±712 63±32 17±12 42±18 9±6 52±27 15±7 36±24 5±324 67±30 3±3 60±27 4±4 57±29 5±3 47±24 5±448 161±44 5±4 152±44 4±4 47±41 2±2 43±39 2±272 473±121 14±12 457±119 3±2 22±28c 0 21±27 1±196 500 6±6 1±1 5±4 1±1120 500 0 0 0 0a Values represent the total number of sporozoites, trophozoites, and immature and mature schizonts found

in a complete histological transverse section (6 Lm) at one specific site in the colon. The values are expressed asthe mean ± standard deviation for 5 mice.

' Significantly fewer than in the corresponding nonimmune mice (P < 0.01).c Significantly fewer than in the corresponding nonimmune mice (P < 0.001).d Not counted; but since almost every crypt epithelial cell contained one or more schizonts, the total count

would be greater than 500.

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IMMUNITY TO MURINE COCCIDIOSIS 111

not seen in any of the exfoliated cells. Thesubmucosal edema that was prominent at 12 hp.i. was not very apparent in either group ofmice at 24 h.By 48 h p.i., obvious differences became ap-

parent between the immune and nonimmunemice. The number of coccidia developing in thecolonic mucosa of the immune mice was signifi-cantly lower than in the nonimmune group (Ta-ble 2). Numerous immature and a few maturefirst-generation schizonts were observed withinthe epithelial cells of the nonimmune mice. Incontrast, the coccidial stages in the immune micehad not matured and were degenerate (Fig. 3A).Most of the degenerate immature schizonts hadcoarse granular cytoplasm and poor nuclear dif-ferentiation, which rendered their appearancedistinctly different from the normal schizontsseen in the nonimmune mice. Often, the degen-erate coccidial stages could be positively identi-fied by the remains of their refractile globules.Many degenerate cells that could not be identi-

FIG. 2. Trophozoites (arrows) in the crypt epithe-lial cells of mice 24 h after infection with E. falcifor-mis var. pragensis. The trophozoite in the immunemouse (A) has degenerated, compared to the devel-oping trophozoite with four nuclei in the nonimmunemouse (B). Stained with hematoxylin-eosin; x930.

x,... I t,.-vi

FIG. 3. Immature schizonts (arrows) in the cryptepithelial cells of mice 48 h after infection with E.falciformis var. pragensis. The schizont in the im-mune mouse (A) is shrunken and lacks nuclear dif-ferentiation, compared to the well differentiated mul-tinucleated schizont in the nonimmune mouse (B).Stained with hematoxylin-eosin; x930.

fied as coccidial remnants or degenerate inflam-matory cells were frequently observed withincrypt epithelial cells. A few similar degeneratecells were also found in the nonimmune mice.Occasionally, unidentified degenerate eosino-philic globules, similar to those in the epithe-hum, were observed in the lamina propria. Lym-phocytes, neutrophils, and globule leukocyteswere randomly scattered throughout the cryptepithelium of the colon in both the immune andnonimmune mice. The morphology of epithelialcells that contained coccidial stages was similarin both the immune and nonimmune mice, re-gardless ofwhether the coccidia were degenerateor not. The host cell nucleus was often indentedand usually displaced to one edge of the cell;otherwise, the cell appeared normal.At 72 h p.i., the number of parasites seen in

the nonimmune group greatly exceeded thenumber seen in the immune group (Table 2).The mature schizonts seen in the nonimmunemice did not develop in the immunized mice;with the rare exception, only degenerate coccid-ial stages were observed in the immune group.By 96 h p.i., even the shrunken remnants ofcoccidia were few in number in the immunemice. A second phase of submucosal edema wasagain observed but only in the nonimmunegroup. By 120 h p.i., the crypt epithelial cells ofthe nonimmune group were filled with nonnallydeveloping schizonts whereas, in the immunemice, coccidial stages were almost completelyabsent. The coccidia had failed to develop in theimmunized mice. The pathological changes seenin the nonimmune mice were similar but moremarked than those described previously (Mesfinet al., in press). In the immune mice, the colonicmucosa appeared normal. A few retainedoocysts, and accompanying focal granulomatoustissue, as a result of the primary infection, wereseen in all immune mice.Mice infected intracecally with mero-

zoites. Mature fourth-generation schizonts, im-mature gamonts, and oocysts were found in twoof the three nonimmune mice killed at 24, 48,and 72 h p.i., respectively. Coccidial stages werenot seen in any of the immune mice killed at 12,48, and 72 h p.i.; however, at 24 h a few degen-erate and developmentally arrested fourth-gen-eration schizonts were found (Fig. 4A). Mostmice in both groups had a mild localized peri-tonitis due to the surgery.Duration of immnity. Mice immunized

with three infections of E. falciformis var. pra-gensis had complete immunity (as measured byoocyst discharge) for 4 months. By 5 and 6months after immunization, immunity began towane in some of the mice. However, although

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./* Z3A l Ad

FIG. 4. Fourth-generation schizonts in the cryptepithelial cells ofmice 24 h after infection with third-generation merozoites of E. falciformis var. pragen-sis. The schizont in the immune mouse (A) has a fewmarginated nuclear remnants compared to the ma-

ture schizont with numerous merozoites in the non-immune mouse (B). Stained with hematoxylin-eosin;x930.

four of the seven mice challenged 6 months afterimmunization did pass a few oocysts, they didnot become clinically ill. Histological examina-tion showed that the number of oocysts retainedin the colonic mucosa from the primary infectionprogressively decreased from the second to thesixth month after immunization.

DISCUSSIONInfection with E. falciformis var. pragensis

induced good immunity in mice. The number ofmice completely immune increased in propor-tion with the immunizing dose, suggesting thatthe degree of immunity was at least partly de-pendent on the immunizing dose. Every mousethat was immunized twice failed to dischargeany oocysts after a challenge. Immunity to E.falciformis var. pragensis, therefore, may relateto both the concentration of antigen and theimmunizing schedule. Dose-dependent resist-ance to coccidia has been reported with E. bovisin cattle (9, 31) and E. maxima and E. acervu-

lina in chickens (11).Complete immunity to E. falciformis var.pra-

gensis lasted for 4 months after immunization.Mice immunized with one or two infections ofE. pragensis (E. falciformis var. pragensis)were resistant to reinfection at 3 months afterimmunization (3). In the present study, althoughsome of the mice challenged at 6 months afterimmunization passed a few oocysts, clinical signsof coccidiosis were not seen in any of the mice.Immunity to coccidia has been reported to lastfrom a few weeks to up to 4 months, presumablyin the absence of reinfection (26). It is difficultto reach a general consensus as to the durationof immunity to coccidial parasites because of thedifferences in the mode of immunization and thecriteria used to assess immunity in the different

studies. The complete resistance of rabbits to E.steidae infection for up to 2 years was suggestedto have been due to persistent antigenic stimulusprovided by oocysts retained within the liver ofrecovered rabbits (13). Such an association be-tween oocyst retention and duration of immu-nity was not apparent in the present study. Micedischarged a few oocysts when challenged at 5and 6 months after immunization despite thepresence of oocysts retained within the mucosaof the colon.The immune response to E. falciformis var.

pragensis did not prevent penetration of theabsorptive epithelium by sporozoites, nor did itinterfere with sporozoite migration to crypt ep-ithelium, but it prevented the complete devel-opment of sporozoites into mature first-genera-tion schizonts. To determine whether developingfirst-generation schizonts were unique in thisrespect, immune mice were infected intracecallywith third-generation merozoites of the coc-cidium. The result was similar to that in oocyst-infected immune mice. It seems that the immuneresponse to E. falciformis var. pragensis acts tospecifically block merozoite production at everystage in merogony. In the natural situation, theimmune response probably blocks the matura-tion of most first-generation merozoites.

Local mucosal antibodies, which play such animportant role in immunity to viral and bacterialenteric infections by blocking adherence to orpenetration of the epithelium (2), seem to havelittle influence on the motile stages of the coc-cidium. Both sporozoites and merozoites wereable to penetrate intestinal epithelial cells ofimmune mice. Although this result is at variancewith that of Morehouse (22) and Edgar (Ph.D.thesis), it is consistent with the observations ofa number of investigators (1, 12, 13, 18, 33)working with other avian and mammnalian coc-cidia. Although a limited degree of protectionhas been conferred through passive transfer ofimmune serum (26, 27; W. Wittchow, doctoraldissertation, University of Berlin, Berlin, WestGermany, 1972), there is no evidence as yet tosupport the idea that local antibodies actingwithin the intestinal lumen have any influenceon the infection of the epithelium by the motileforms ofthe parasite. Because intestinal mucosalantibody is transported primarily through cryptepithelial cells (34) in which degeneration of thedeveloping schizonts occurred, it is conceivablethat local antibody could act within the epithe-lial cells to somehow mediate the effect. How-ever, the isolation of infective sporozoites of E.tenella and E. maxima after exposure to the"immune intestine" for up to 24 and 88 h, re-spectively (18, 29), does not support such amechanism, unless immunity in birds operates

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IMMUNITY TO MURINE COCCIDIOSIS 113

in a manner different from that in mammals.Acquired resistance to a variety of obligate

and facultative intracellular pathogens is de-pendent on cell-mediated immunity (21, 24).Cell-mediated immunity to Eimeria has beendemonstrated by using transfer factor (14, 15,19), the macrophage migration inhibition test(23), the delayed hypersensitivity reaction (15,16, 28), and in vitro lymphocyte blastogenesis(16). Since the immune response to E. falcifor-mis var. pragensis and to several other coccidiaacts to cause degeneration of schizonts that aredeveloping within an epithelial cell, a cell-me-diated immune mechanism would appear to bea likely mediator of immunity.The possibility that cell-mediated cytotoxicity

of coccidia-bearing cells is responsible for im-munity deserves consideration. Because epithe-lial desquamation was more apparent in immunebirds than in nonimmune birds challenged withE. acervulina, Kouwenhovan and Van DerHorst (17) suggested that immunity to the coc-cidium was directed against the parasitized epi-thelial cells. Rose et al. (30), however, consideredthat the epithelial loss was due to inflammationresulting from a hypersensitivity reaction ratherthan as a result of an immune response actingspecifically against parasitized epithelial cells.Neither in the previous (17) nor in the presentstudy were parasitic stages seen within the des-quamated epithelial cells to suggest that thecoccidial stages were expelled in this manner.Injured parasite-containing epithelial cells, anindicator of cell-mediated cytotoxicity (5) werenot observed in immune mice infected with E.falciformis var. pragensis.The possibility that mediators released from

immune T cells mediate degeneration of thedeveloping coccidial stages warrants investiga-tion. Toxoplasma gondii and Besnoitia jelli-soni, parasites closely related to the Eimeria(iO), failed to proliferate in fibroblasts and kid-ney cells treated with mediators released fromantigen-stimulated immune lymphocytes (6). Anon-antibody-soluble substance released fromsensitized lymphocytes was also considered tohave mediated intra-erythrocytic degenerationof Babesia microti (7, 8).

ACKNOWLEDGMENTSWe are grateful to E. Bueckert, I. Shirley, G. Appl, G.

Green, and J. McKnight for their technical assistance.This study was conducted while G. M. Mesfin was a scholar

of the Canadian International Development Agency and wassupported by the Canadian Department of Agriculture.

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11. Hein, H. 1976. Eimeria acervulina, E. brunetti, E. max-ima, and E. necatrix: low doses of oocysts to immunizeyoung chickens. Exp. Parasitol. 40:250-260. 1976.

12. Horton-Smith, C., P. L Long, and A. E. Pierce. 1963.Behavior of invasive stages of Eimeria tenella in theimmune fowl (Gallus domesticus). Exp. Parasitol. 14:66-74.

13. Horton-Smith, C., P. L. Long, A. E. Pierce, and M. E.Rose. 1963. Immunity to coccidia in domestic animals.p. 273-295. In P. C. C. Garnham, A. E. Pierce, and I.Roitt (ed.), Immunity to protozoa. Blackwell ScientificPublishers, Oxford.

14. Klesius, P. H., T. T. Kramer, D. Burger, and A.Malley. 1975. Passive transfer of coccidian oocyst an-tigen and diphtheria toxoid hypersensitivity in calvesacross species barriers. Transplant. Proc. 7:449-452.

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22. Morehouse, N. F. 1938. The reaction of the immuneintestinal epithelium of the rat to reinfection with Ei-meria nieschulzi. J. Parasitol. 24:311-317.

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gration inhibition test of splenic cells of chickens in-fected with Eimeria tenella. J. Parasitol. 59:199-200.

24. Nelson, D. S. 1972. Macrophages as effectors of cellmediated immunity. Crit. Rev. Microbiol. 1:353-360.

25. Niilo, L. 1967. Acquired resistance to reinfection of rabbitswith Eimeria magna. Can. Vet. J. 8:201-208.

26. Rose, M. E. 1973. Immunity, p. 295-341. In D. M. Ham-mond and P. L. Long (ed.), The coccidia. UniversityPark Press, Baltimore.

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28. Rose, M. E. 1977. Eimeria tenella: skin hypersensitivityto injected antigen in the fowl. Exp. Parasitol. 42:129-141.

29. Rose, M. E., and P. Hesketh. 1976. Immunity to cocci-diosis: stages of the life cycle of Eimeria maxima which

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