Immunization of Broiler Chicks by In Ovo Injection
of Eimeria tenella Sporozoites, Sporocysts, or Oocysts
F. H. Weber*,1 and N. A. Evans†
*Pfizer Animal Health Group, Veterinary Medicine Research and Development, 301 Henrietta Street, Kalamazoo,
Michigan 49001; and †Pfizer Animal Health Group, 150 East 42nd Street, New York, New York 11201
ABSTRACT Immunization of chickens by in ovo injec-
tion of Eimeria tenella parasite stages was investigated.
Fertile Hubbard × Petersen broiler chicken eggs were
injected through the air cell on d 18 of incubation with
sporozoites, sporocysts, or oocysts of E. tenella. Injected
doses were in the range of 1 × 102 to 1 × 106 sporozoites,
2 × 105 to 2 × 107 sporocysts, or 1 × 102 to 5 × 106 oocysts
per egg. Hatch rates were generally unaffected. Hatched
chicks shed oocysts, with oocysts per gram of feces reach-
ing a maximum at 3 d posthatch for chicks injected with
(Key words: Eimeria tenella, immunization, in ovo, oocyst, sporozoite)
INTRODUCTION
Vaccination of chickens against coccidiosis with live
oocysts is an accepted method of disease control, espe-
cially for long-lived birds such as breeders. The appear-
ance of drug resistance among coccidia of domestic fowl
(Chapman, 1997) has prompted renewed interest in vacci-
nation as a means of controlling disease in birds with
shorter life spans, specifically broiler chickens. Growers
of these birds have historically relied on chemotherapy as
a means of preventing or controlling coccidiosis. Research
has focused on ways of administering live vaccines early
in the life of the bird to achieve rapid development of
immunity. Such methods include eye-spray application
to newly hatched chicks (Chapman and Cherry, 1997)
and incorporation of oocysts in gels that are added to
containers in which chicks are shipped to growout loca-
tions (Danforth et al., 1997). Other studies have investi-
gated application methods such as in feed or in drinking
water at several days of age (Bedrnik et al., 1989; Williams,
1994). Live commercial vaccines currently comprise un-
modified and attenuated strains of live coccidia (Shirley,
1992). Although research on nonreplicating, subunit vac-
cines and heterologous gene/vector systems continues,
no commercial products of this type are currently avail-
2003 Poultry Science Association, Inc.
Received for publication February 25, 2003.
Accepted for publication June 2, 2003.
1
To whom correspondence should be addressed: weberf@pfizer.com.
1701
sporozoites and at 7 d posthatch for chicks receiving oo-
cysts or sporocysts in ovo. After 2 wk in wire-floored
cages or 3 wk on litter, birds were challenged with 2.5 ×
104 sporulated oocysts of E. tenella. Chicks immunized
by in ovo injection of parasite stages had significantly
reduced lesion scores compared to their nonimmunized
counterparts. The results demonstrate the feasibility of
immunizing broiler chickens against E. tenella infection
by in ovo injection of sporozoites, sporocysts, or oocysts.
2003 Poultry Science 82:1701–1707
able (Lillehoj and Trout, 1993; Wallach and Vermeulen,
1996; Jenkins, 1998; Vermeulen, 1998; Pogonka et al.,
2003). The status and role of live vaccines in the control
of coccidiosis in poultry have recently been reviewed
(Chapman et al., 2002), as have advances in the immunob-
iology of the parasite and its host (Allen and Fetterer
2002; Yun et al., 2000).
Watkins et al. (1995) attempted to vaccinate broiler
chickens against Eimeria maxima by in ovo injection of live
oocysts or sporocysts at 17 to 18 d of embryo incubation.
Although they found evidence of infection in the newly
hatched chicks, these birds showed no protective immu-
nity when challenged at 10 d of age. Provaznikova and
Bedrnik (1997) reported successful immunization of broil-
ers with an egg-adapted strain of E. tenella by in ovo
administration of sporozoites, although this method was
unsuccessful for other species of coccidia tested.
The objectives of the present work were to determine
if in ovo injection of sporozoites, oocysts, or sporocysts
of an unmodified strain of E. tenella on d 18 of embryo
incubation results in infection of newly hatched chicks;
to characterize the infection; and to determine if infection
resulting from in ovo injection of E. tenella parasite stages
can also provide protective immunity.
MATERIALS AND METHODS
Design
Four experiments were conducted using fertilized
broiler eggs (Hubbard × Petersen) obtained from a com-
1702 WEBER AND EVANS
mercial hatchery.2 In all studies, eggs were injected on d
18 of incubation with saline or sporozoites, sporocysts,
or sporulated oocysts of a laboratory strain of Eimeria
tenella. Eggs were injected according to the procedure
described by Sharma (1985). Briefly, a small hole was
made in the shell at the large end of the egg using an 18-
ga needle. A 2.54-cm (1-inch), 20-ga needle was then used
to deliver 100 µL of test material through the air cell
membrane, targeting the amniotic fluid. Hatch rates were
calculated by dividing the number of live chicks obtained
by the number of viable embryos transferred from the
incubator to the hatcher and multiplying the result by
100 to obtain the percentage of hatch. At hatch, chicks
were weighed and placed in wire-floored battery cages
or in pens with wood shavings (litter) and given feed and
water ad libitum. The ration met the National Research
Council (1994) requirements for broiler chickens. Chicks
used in all experiments were straight-run.
In the first experiment, eggs were injected with saline
or 1 × 104, 5 × 104, 1 × 105, 5 × 105, or 1 × 106 sporozoites.
The study comprised one wire-floored pen of 10 birds
per treatment, with the exception of the high dose group,
which contained three pens. Feces were collected from
this group daily for the first 10 d of the study for determi-
nation of oocyst output. The birds were challenged at
14 d of age, and only one pen of the high dose group
was challenged.
Eggs used for the second experiment were injected with
saline or 1 × 102, 5 × 103, or 1 × 105 sporozoites, and the
chicks were placed on litter with three pens of 12 birds
each per treatment group. Litter samples for oocyst counts
were collected on d 7, 14, and 21 posthatch, and birds
were challenged on d 21 after transferring them to wire-
floored cages. Nonimmunized birds were housed to-
gether during the 21-d period prior to challenge, and were
separated into challenged and nonchallenged groups on
d 21. All prechallenge data collected from nonimmunized
birds therefore applies to challenged and nonchal-
lenged groups.
Birds were placed in wire-floored cages for the third
experiment, after hatching from eggs receiving 2 × 105, 4
× 105, 2 × 106, 4 × 106, or 2 × 107 sporocysts, or 5 × 104, 1
× 105, 5 × 105, 1 × 106, or 5 × 106 oocysts per egg. There
were two pens of 10 birds per treatment for the high dose
groups, and one pen of 10 birds for all other groups. Feces
were collected daily from the high dose groups for the
first 10 d of the study for determination of oocyst output.
The birds were challenged at 14 d of age, and only one
pen of the high dose group was challenged.
In the fourth experiment, eggs were injected with 1 ×
102, 1 × 103, 1 × 104, or 1 × 105 oocysts and the chicks
placed on litter with three pens of 12 birds per treatment
group. Litter samples for oocyst counts were collected on
d 7, 14, and 21, at which time birds were transferred to
2
Hoover’s Hatchery, Rudd, IA.
3
Sigma Chemical Co., St. Louis, MO.
4
Fisher Scientific, Fairlawn, NJ.
wire-floored cages and challenged. As before, nonimmu-
nized birds were housed together prior to challenge and
divided into challenged and nonchallenged groups on
d 21.
Oocyst, Sporocyst,
and Sporozoite Preparation
Oocysts were prepared by propagation of Eimeria ten-
ella in coccidia-free broiler chicks using standard meth-
ods. Oocyst preparations were sanitized by exposure to
50% household bleach (2.63% sodium hypochlorite) for
20 min, followed by repeated washing with water. To
prepare sporocysts, the required number of oocysts was
prewarmed to 41°C, mixed with glass beads, and vortexed
for 60 s to rupture the oocysts. This procedure resulted
in a preparation containing approximately 95% sporo-
cysts and 5% oocysts as determined by direct count using
a hemacytometer. To obtain sporozoites, sporocysts were
incubated at 41°C for 60 min in a saline solution con-
taining 0.25% (wt/vol) trypsin3 and 0.50% (wt/vol) tauro-
deoxycholic acid.3 The resulting sporozoite suspension
was centrifuged, resuspended in saline, and the total
number of each parasite stage was determined by direct
count using a hemacytometer. This procedure resulted
in 97 to 99% sporozoites, with the remaining material
containing small numbers of oocysts and sporocysts. The
preparations were then diluted with saline to obtain the
appropriate concentration of parasites for in ovo injection
with 100 µL per egg. Control eggs received 100 µL of
sterile saline.
Determination of Oocyst Output
For birds reared in wire cages, the entire fecal output
of each pen that was to be sampled was collected daily
for the first 10 d after hatch and weighed. Feces were
mixed with an amount of water equivalent to approxi-
mately six times the volume of feces and homogenized
using a hand-held blender. A single 1-mL sample of each
suspension was mixed with 9 ml of 30% (wt/vol) NaNO3.4
After being mixed, samples were loaded into duplicate
McMasters counting chambers, and the average number
of oocysts was determined. The number of oocysts per
gram of feces within each pen was calculated. Litter sam-
ples were taken by collecting a handful of litter from each
corner and the center of each pen on d 7, 14, and 21
posthatch. The combined samples were mixed thor-
oughly, and a 10-g sample was mixed with 100 mL of
tap water and allowed to stand overnight. The samples
were then homogenized in a mechanical blender and the
homogenate filtered through cheesecloth. A 15-mL ali-
quot of the filtrate was centrifuged and resuspended in
15 mL of 30% (wt/vol) NaNO3. The sample was then
loaded into duplicate McMasters chambers and the oo-
cysts counted. If necessary, dilutions were carried out in
30% (wt/vol) NaNO3.
 IN OVO IMMUNIZATION WITH LIVE EIMERIA TENELLA STAGES 1703
TABLE 1. Hatch rate and immunity to challenge infection among broiler chickens immunized in ovo
with Eimeria tenella sporozoites, reared on wire, and challenged at 14 d of age
with 2.5 × 104 sporulated E. tenella oocysts

Immunizing dose Hatch rate (%) Mean lesion Postchallenge

(sporozoites/egg) (n = 50) score1 gain per bird (g)1
0 100 3.4a 288
1 × 104 98 1.9b 306
5 × 104 98 2.5b 289
1 × 105 92 3.0ab 317
5 × 105 96 2.8ab 315
1 × 106 94 2.1b 287
0 (nonchallenged) 100 0c 283
SEM 0.16 5.74

Means with different superscripts within a column are significantly different from each other (P < 0.05;
a–c

ANOVA).
1
Means represent one pen per treatment, 10 birds per pen.

Challenge Infection
Birds reared on wire were challenged at 14 d of age,
and birds reared on litter were challenged at 21 d of age
after transfer to wire-floored cages. Each chick was leg-
banded, weighed, and challenged with 2.5 × 104 sporu-
lated oocysts of Eimeria tenella by oral gavage. This chal-
lenge dose produced a moderate level of disease in pre-
liminary dose titration studies conducted in birds of the
same breed and of similar age (data not shown). The
challenge strain was the same as that which with the
birds were immunized. Six days after challenge, birds
were weighed again and killed by cervical dislocation,
and cecal lesions were scored according to the method
of Johnson and Reid (1970).
Statistical Analysis
Body weights, weight gains, and lesion score data were
analyzed by the ANOVA procedure of Statview5 with
treatment as the main effect. Mean differences were deter-
mined using Fisher’s protected least significant difference
test. Hatchability data were analyzed by chi square.
were slightly lower than those of the nonimmunized con-
trols, the reduction was not significant. There were no
differences in postchallenge weight gain between any of
the groups.
Chicks, hatched from eggs injected with 1 × 106 sporo-
zoites and reared on wire, shed oocysts after hatching
(Figure 1). Oocyst shed was first detected at 2 d posthatch,
about 5 d after injection of sporozoites into embryos on
d 18 of incubation. Oocyst output peaked 3 d posthatch,
with a small secondary peak at 7 d posthatch. Chicks that
hatched from eggs receiving saline did not shed detect-
able numbers of oocysts (data not shown).
In ovo administration of sporozoites to 18-d-old em-
bryos did not significantly affect hatch rates in the second
experiment (Table 2). Small numbers of oocysts were ob-
served in the litter of immunized birds as early as 7 d

RESULTS
Immunization with Sporozoites
Hatchability was unaffected by in ovo injection of spo-
rozoites of E. tenella on d 18 of embryo incubation in the
first experiment (Table 1). Although hatchability among
eggs receiving sporozoites was slightly lower than among
controls receiving saline, the differences were not signifi-
cant. After challenge with the homologous, immunizing
strain of E. tenella, lesion scores among chicks that hatched
from eggs receiving 1 × 104, 5 × 104, or 1 × 106 sporozoites
were significantly reduced compared to nonimmunized,
challenged controls. Although lesion scores among
groups immunized with 1 × 105 or 5 × 105 sporozoites

FIGURE 1. Posthatch oocysts shed by broiler chicks after in ovo

immunization with 1 × 106 sporozoites of Eimeria tenella on d 18 of
5
SAS Institute, Cary, NC. embryo incubation. Each value is the mean of three pens of 10 birds each.
1704 WEBER AND EVANS
TABLE 2. Hatch rate, oocyst presence in litter, and immunity to challenge infection among broiler
chickens immunized in ovo with Eimeria tenella sporozoites1

Oocysts per gram of litter2

Hatch Mean lesion Postchallenge
Treatment rate (%) Day 7 Day 14 Day 21 score2 gain/bird (g)2

Nonimmunized, nonchallenged 0.0e 298b

Nonimmunized, challenged 88 0 303 2.4 × 103,4 2.4a 302b
1 × 102 sporozoites/egg 89 0 0 1.7 × 104 2.0b 320ab
5 × 103 sporozoites/egg 93 102 0 8.4 × 103 1.2c 324ab
1 × 105 sporozoites/egg 78 5.6 × 102,4 3.9 × 103 1.2 × 105 0.5d 343a
SEM 0.091 4.69
a–e
Means with different superscripts within a column are significantly different (P < 0.05; ANOVA).
1
Birds were reared on litter for the first 21 d, then placed in wire-floored cages prior to challenge with 2.5 ×
104 sporulated E. tenella oocysts. Data from the first 21 d for nonimmunized, challenged birds also applies to
nonimmunized, nonchallenged birds, because these groups were housed concurrently for the first 21 d.
2
Means represent three pens of 12 birds per treatment.
3
Oocysts detected in one of three pens.
4
Oocysts detected in two of three pens.

posthatch. The number of oocysts per gram of litter in-
creased in all groups between d 7 and 21 posthatch, in-
cluding the nonimmunized birds. Microscopic observa-
tion of the size of the oocysts (approximately 18 × 21
microns) indicated that all were E. tenella. Although it is
possible that the birds became infected by an external
source of E. tenella, it seems more likely that cross-contam-
ination occurred from pens of immunized chicks to pens
of nonimmunized chicks. Because all pens had open tops
and were in the same room, transmission of E. tenella
from pen to pen by insects or animal care personnel seems
more probable than infection from an outside source,
especially as the two immunized groups that were shed-
ding oocysts on d 7 provided a potential source of contam-
ination. When chicks that hatched from eggs injected with
sporozoites were reared on litter and challenged at 21 d
of age, all immunized groups had significantly reduced
lesion scores compared with nonimmunized, challenged
controls (Table 2). Postchallenge weight gain among im-
munized birds tended to be greater than among nonim-
munized birds, the difference being significant for the
group immunized with the largest sporozoite dose (1 ×
105 per egg). Challenge of the nonimmunized birds did
not suppress weight gain compared to the nonchal-
lenged controls.
Immunization with Oocysts or Sporocysts
Hatchability in the third experiment was generally un-
affected by in ovo injection of oocysts or sporocysts of E.
tenella (Table 3). When these chicks were reared on wire
and challenged with E. tenella, lesion scores at all but the
two lowest doses of sporocysts were significantly reduced
compared to the nonimmunized, challenged chicks. There
was no significant treatment effect on weight gain among
these birds. Chicks shed oocysts after hatching when em-
bryos were injected with 5 × 106 oocysts or 2 × 107 sporo-
cysts. In both cases, peak oocyst output occurred at 7 d
posthatch (Figure 2).
In the fourth experiment (Table 4), in ovo injection of
the highest and lowest oocyst doses decreased hatchabil-
ity significantly, however these were the only groups
affected. Chicks that hatched from eggs receiving oocysts
and reared on litter shed oocysts, as shown by the pres-
ence of oocysts in the litter during the 21-d growth period.
When these birds were challenged with E. tenella, lesion
scores were significantly reduced compared to the nonim-
munized, challenged control, although this group had
lower lesion scores than expected based on a preliminary
challenge dose titration (data not shown). Although non-
immunized birds did not shed detectable numbers of
oocysts during the prechallenge phase of the study, there
was evidence of a low level of E. tenella infection in the
nonimmunized birds in the form of mild cecal lesions in
a few of the birds and relatively poor weight gain com-
pared to other groups (Table 4). Immunity resulting from
such an infection may also account for the low lesion
score and lack of weight gain suppression in the nonim-
munized, challenged group. This infection may also ac-
count for the lower weight gain in the nonimmunized,
nonchallenged controls. There were no significant differ-
ences, however, in postchallenge weight gain between
treatment groups.
DISCUSSION
The peak in oocyst output at 3 d posthatch following
in ovo injection of sporozoites suggests that sporozoites
initiated their life cycle in the embryo near the time of in
ovo injection (i.e., 3 d prior to hatching). The minimum
prepatent period for E. tenella is about 115 h, or roughly
5 d (Conway and McKenzie, 1991). Thus, initial detection
of oocyst output on d 2 posthatch implied that infection
occurred 5 d earlier, or 3 d prior to hatching. In contrast,
the kinetics of oocyst output when oocysts or sporocysts
were injected in ovo suggested that the parasite life cycle
was initiated around the time of hatching. When birds
are infected by oral gavage with oocysts of E. tenella, peak
oocyst output occurs around 7 d postinfection (Ryley et
al., 1976). Peak oocyst output after in ovo injection of
oocysts or sporocysts was observed at 7 d posthatch,
indicating that unless there were significant changes in
 IN OVO IMMUNIZATION WITH LIVE EIMERIA TENELLA STAGES 1705
TABLE 3. Hatch rate and immunity to challenge infection among broiler chickens immunized in ovo with
Eimeria tenella oocysts or sporocysts, reared on wire, and challenged at 14 d of age
with 2.5 × 104 sporulated E. tenella oocysts

Immunizing dose Hatch rate (%) Postchallenge

Method (infective stages/egg) (n = 25) Mean lesion score1 gain per bird (g)1
0 96 2.4a 279
Oocysts 5 × 104 89 1.8bc 316
1 × 105 81 1.8bc 309
5 × 105 81 1.1de 288
1 × 106 96 0.6ef 310
5 × 106 78 0.4f 315
Sporocysts 2 × 105 81 2.0ab 314
4 × 105 100 1.9ab 314
2 × 106 96 1.4bcd 320
4 × 106 92 1.1de 302
2 × 107 81 1.3cd 283
0 (nonchallenged) 88 0f 316
SEM 0.076 3.48
a–f
Means with different superscripts within a column are significantly different from each other (P < 0.05;
ANOVA).
1
Means represent one pen per treatment, 10 birds per pen.

the parasite life cycle under these conditions, the oocysts
and sporocysts remained dormant in the embryo after
injection until around the time of hatching.
Intact E. maxima oocysts have been observed in the
lumen of the embryo gut throughout the period between
FIGURE 2. Posthatch oocysts shed by broiler chicks after in ovo
immunization with 5 × 106 oocysts (OOC) or 2 × 107 sporocysts (SPC)
of Eimeria tenella on d 18 of embryo incubation. Each value is the mean
of two pens of 10 birds each.
injection and hatching, further supporting this idea (We-
ber et al., 2001). It is reasonable to speculate that oocysts
are delivered to the gut by ingestion of amniotic fluid by
the embryo during the last quarter of incubation (Ro-
manoff, 1960). The small secondary peak in oocyst shed-
ding observed on d 7 when eggs were injected with sporo-
zoites (Figure 1) might have resulted from the low level
of contaminating oocysts in the sporozoite preparation.
Watkins et al. (1995) found that in ovo injection of E.
maxima sporocysts into the amnion produced a significant
reduction in hatch rate. In the present work, hatchability
was generally unaffected by in ovo injection of E. tenella
infective stages. Although there was decreased hatchabil-
ity in two groups in the fourth study, the lack of a dose
response of hatchability to oocyst dose in this study im-
plies that the lower hatchability observed in these groups
might have been due to factors other than oocyst dose.
Although there is no ready explanation for this discrep-
ancy, the method by which the oocysts were purified, a
difference in the toleration of embryos to injection with
E. tenella versus E. maxima, or the difference between the
sites of infection of these two species could be contribut-
ing factors. In addition, because small numbers of eggs
were used in our studies, small changes in hatch rate
resulting from in ovo administration of parasites would
not have been statistically significant. Larger trials are
necessary to definitively evaluate the effect of in ovo injec-
tion of E. tenella on hatchability.
In ovo injection of parasite stages resulted in infection
of the newly hatched chicks and subsequent immunity
to experimental challenge as indicated by reduced lesion
scores among immunized birds compared to nonimmu-
nized controls. Although challenge of naive birds pro-
duced cecal lesions in the 2 to 3 range, little or no weight
gain suppression was induced in these studies. This result
was somewhat surprising because weight gain suppres-
sion is a characteristic of infection with E. tenella. Al-
though there is no clear explanation for the lack of weight
1706 WEBER AND EVANS
TABLE 4. Hatch rate, oocyst presence in litter, and immunity to challenge infection among
broiler chickens immunized in ovo with Eimeria tenella oocysts1

Oocysts per gram of litter2

Hatch Mean lesion Postchallenge
Treatment rate (%) Day 7 Day 14 Day 21 score2 gain per bird2 (g)

Nonimmunized, nonchallenged 0.1c 352

Nonimmunized, challenged 96 0 0 0 1.7a 380
1 × 102 oocysts/egg 60* 0 0 1.6 × 104
0.8b 387
1 × 103 oocysts/egg 93 1.3 × 103 0 4.9 × 104 0.8b 359
1 × 104 oocysts/egg 91 1.5 × 105 4.4 × 103 3.1 × 104 0.8b 368
1 × 105 oocysts/egg 84* 9.7 × 104 8.0 × 103 3.2 × 104 0.7b 367
SEM 0.05 4.42
a–c
Means with different superscripts within a column are significantly different (P < 0.05; ANOVA).
1
Birds were reared on litter for the first 21 d, then placed in wire-floored cages prior to challenge with 2.5 ×
104 sporulated E. tenella oocysts. Data from the first 21 d for nonimmunized, challenged birds also applies to
nonimmunized, nonchallenged birds, because these groups were housed concurrently for the first 21 d.
2
Means represent three pens of 12 birds per treatment.
*Significantly different from nonimmunized control (P < 0.05; chi square).

gain suppression, relevant factors include the challenge
dose, virulence of the challenge strain, diet, breed of bird,
and housing conditions. In some cases, cross-contamina-
tion with E. tenella from pens of immunized birds to
nonimmunized birds might have imparted a degree of
immunity to nonimmunized controls. Even under these
circumstances, immunized birds were measurably more
resistant to infection than were nonimmunized birds in
most cases.
There was little or no correlation between in ovo para-
site dose and the extent of lesion control after challenge
when birds were reared on wire, but it should be noted
that birds reared on wire have little exposure to feces and
thus oocyst cycling is limited. Multiple infections due
to cycling of oocysts have been shown to enhance the
development of immunity when birds are vaccinated
with live oocysts (Joyner and Norton, 1973). The greater
effectiveness of immunization observed in the current
study when birds were reared on litter compared to wire
cages was likely a result of reinfection from oocysts shed
in the feces, although allowing 21 d for immunity to
develop, rather than 14 d, might have played a role.
The dose response in lesion score reduction was pro-
nounced when birds immunized with sporozoites were
reared on litter but was absent when birds were immu-
nized with oocysts. Oocysts appeared to accumulate in
the litter more rapidly when birds were immunized with
oocysts than when birds were immunized with sporozo-
ites. On d 7 posthatch, litter from birds immunized in
ovo with 1 × 105 sporozoites per egg contained 5.6 × 102
oocysts per g (Table 2), yet litter from birds immunized
with 1 × 104 oocysts per egg, equivalent to 8 × 104 sporozo-
ites, contained 1.5 × 105 oocysts/g (Table 4). Thus, al-
though the two groups received similar doses in terms of
sporozoites, the group receiving oocysts in ovo produced
about 270-fold more oocysts per gram of litter after 7 d
than the group receiving sporozoites. Lower doses of
oocysts also produced higher levels of oocysts in the litter
on d 7 than were found in groups immunized with sporo-
zoites. The higher number of oocysts produced during
the first week of life among birds receiving oocysts in
ovo may account for the lack of a dose response in this
study. The relatively large number of oocysts produced
during the first week provided ample opportunity for
reinfection and oocyst cycling, resulting in a high level
of immunity after 21 d, even among groups receiving
lower oocyst doses. Because the challenge in this study
was mild, possibly due to a low level of immunity in
nonimmunized controls resulting from cross-contamina-
tion, even the group receiving the lowest immunizing
oocyst dose might have had sufficient immunity to almost
completely control the infection.
The observation that oocysts accumulated in litter ear-
lier after in ovo injection of oocysts than after in ovo
injection of sporozoites is surprising given our observa-
tion that peak oocyst output occurred at 3 d posthatch
when sporozoites were administered in ovo, and after 7
d posthatch when oocysts were given in ovo. Since infec-
tion in the embryo seemed to occur near the time when
sporozoites are injected in ovo, it might be expected that
in ovo administration of sporozoites would result in an
earlier infection and earlier oocyst shed compared to in-
jection of oocysts, allowing more time for oocyst cycling
and thus greater immunity. Although results of our stud-
ies with birds on litter do not support this notion, defini-
tive conclusions regarding oocyst output after hatch and
rearing on litter cannot be made because in both cases
there appeared to be cross-contamination with E. tenella
between pens, which complicated interpretation of oocyst
output data. In addition, conditions for sporulation of
oocysts in the litter, a necessary step for reinfection and
oocyst cycling to occur, might have differed between
studies. Conditions that were poorly conducive to sporu-
lation would slow the accumulation of oocysts in the litter
compared to an environment in which conditions of litter
moisture and temperature facilitated sporulation. Differ-
ences in oocyst cycling between studies could result in
differences in the level of immunity developed.
Provaznikova and Bedrnik (1997) showed that it was
possible to immunize chicks against E. tenella infection
by in ovo injection of sporozoites. In the present study, we
confirm these results and further characterize the primary
 IN OVO IMMUNIZATION WITH LIVE EIMERIA TENELLA STAGES
infection resulting from exposure of the embryo to sporo-
zoites. These data also demonstrate that protective immu-
nity can be established by in ovo injection of sporocysts
or oocysts. As expected, lower doses seemed to be re-
quired for birds reared on litter than for birds reared on
wire due to exposure to reinfection in the former instance.
Watkins et al. (1995) found evidence that Eimeria max-
ima completes its life cycle when oocysts or sporocysts
are injected in ovo but found no evidence of immunity
to challenge. In contrast, we have shown that in ovo
delivery of E. tenella parasite stages results in an immuniz-
ing infection in newly hatched chicks that will protect
the birds from challenge infection. This result is consistent
with findings that injection with Eimeria acervulina oo-
cysts, sporocysts, or sporozoites in ovo resulted in infec-
tion of newly hatched chicks that then had measurable
immunity to challenge infection (Doelling et al., 2001).
More recent studies, reviewed by Williams (2002), con-
firm our finding that in ovo delivery of live Eimeria oo-
cysts is a practical method of vaccination. Thus, despite
the results of earlier studies, it is clear that in ovo delivery
of live oocysts has potential as a means of vaccinating
broiler chicks to protect them from coccidiosis.
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