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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- sporozoites and at 7 d posthatch for chicks receiving oo-
tion of Eimeria tenella parasite stages was investigated. cysts or sporocysts in ovo. After 2 wk in wire-floored
Fertile Hubbard × Petersen broiler chicken eggs were cages or 3 wk on litter, birds were challenged with 2.5 ×
injected through the air cell on d 18 of incubation with 104 sporulated oocysts of E. tenella. Chicks immunized
sporozoites, sporocysts, or oocysts of E. tenella. Injected by in ovo injection of parasite stages had significantly
doses were in the range of 1 × 102 to 1 × 106 sporozoites, reduced lesion scores compared to their nonimmunized
2 × 105 to 2 × 107 sporocysts, or 1 × 102 to 5 × 106 oocysts counterparts. The results demonstrate the feasibility of
per egg. Hatch rates were generally unaffected. Hatched immunizing broiler chickens against E. tenella infection
chicks shed oocysts, with oocysts per gram of feces reach- by in ovo injection of sporozoites, sporocysts, or oocysts.
ing a maximum at 3 d posthatch for chicks injected with
(Key words: Eimeria tenella, immunization, in ovo, oocyst, sporozoite)
2003 Poultry Science 82:1701–1707

INTRODUCTION able (Lillehoj and Trout, 1993; Wallach and Vermeulen,


1996; Jenkins, 1998; Vermeulen, 1998; Pogonka et al.,
Vaccination of chickens against coccidiosis with live 2003). The status and role of live vaccines in the control
oocysts is an accepted method of disease control, espe- of coccidiosis in poultry have recently been reviewed
cially for long-lived birds such as breeders. The appear- (Chapman et al., 2002), as have advances in the immunob-
ance of drug resistance among coccidia of domestic fowl iology of the parasite and its host (Allen and Fetterer
(Chapman, 1997) has prompted renewed interest in vacci- 2002; Yun et al., 2000).
nation as a means of controlling disease in birds with Watkins et al. (1995) attempted to vaccinate broiler
shorter life spans, specifically broiler chickens. Growers chickens against Eimeria maxima by in ovo injection of live
of these birds have historically relied on chemotherapy as oocysts or sporocysts at 17 to 18 d of embryo incubation.
a means of preventing or controlling coccidiosis. Research Although they found evidence of infection in the newly
has focused on ways of administering live vaccines early hatched chicks, these birds showed no protective immu-
in the life of the bird to achieve rapid development of nity when challenged at 10 d of age. Provaznikova and
immunity. Such methods include eye-spray application Bedrnik (1997) reported successful immunization of broil-
to newly hatched chicks (Chapman and Cherry, 1997) ers with an egg-adapted strain of E. tenella by in ovo
and incorporation of oocysts in gels that are added to administration of sporozoites, although this method was
containers in which chicks are shipped to growout loca- unsuccessful for other species of coccidia tested.
tions (Danforth et al., 1997). Other studies have investi- The objectives of the present work were to determine
gated application methods such as in feed or in drinking if in ovo injection of sporozoites, oocysts, or sporocysts
water at several days of age (Bedrnik et al., 1989; Williams, of an unmodified strain of E. tenella on d 18 of embryo
1994). Live commercial vaccines currently comprise un- incubation results in infection of newly hatched chicks;
modified and attenuated strains of live coccidia (Shirley, to characterize the infection; and to determine if infection
1992). Although research on nonreplicating, subunit vac- resulting from in ovo injection of E. tenella parasite stages
cines and heterologous gene/vector systems continues, can also provide protective immunity.
no commercial products of this type are currently avail-
MATERIALS AND METHODS

2003 Poultry Science Association, Inc.


Design
Received for publication February 25, 2003.
Accepted for publication June 2, 2003. Four experiments were conducted using fertilized
1
To whom correspondence should be addressed: weberf@pfizer.com. broiler eggs (Hubbard × Petersen) obtained from a com-

1701
1702 WEBER AND EVANS

mercial hatchery.2 In all studies, eggs were injected on d wire-floored cages and challenged. As before, nonimmu-
18 of incubation with saline or sporozoites, sporocysts, nized birds were housed together prior to challenge and
or sporulated oocysts of a laboratory strain of Eimeria divided into challenged and nonchallenged groups on
tenella. Eggs were injected according to the procedure d 21.
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 Oocyst, Sporocyst,
to deliver 100 µL of test material through the air cell and Sporozoite Preparation
membrane, targeting the amniotic fluid. Hatch rates were
calculated by dividing the number of live chicks obtained Oocysts were prepared by propagation of Eimeria ten-
by the number of viable embryos transferred from the ella in coccidia-free broiler chicks using standard meth-
incubator to the hatcher and multiplying the result by ods. Oocyst preparations were sanitized by exposure to
100 to obtain the percentage of hatch. At hatch, chicks 50% household bleach (2.63% sodium hypochlorite) for
were weighed and placed in wire-floored battery cages 20 min, followed by repeated washing with water. To
or in pens with wood shavings (litter) and given feed and prepare sporocysts, the required number of oocysts was
water ad libitum. The ration met the National Research prewarmed to 41°C, mixed with glass beads, and vortexed
Council (1994) requirements for broiler chickens. Chicks for 60 s to rupture the oocysts. This procedure resulted
used in all experiments were straight-run. in a preparation containing approximately 95% sporo-
In the first experiment, eggs were injected with saline cysts and 5% oocysts as determined by direct count using
or 1 × 104, 5 × 104, 1 × 105, 5 × 105, or 1 × 106 sporozoites. a hemacytometer. To obtain sporozoites, sporocysts were
The study comprised one wire-floored pen of 10 birds incubated at 41°C for 60 min in a saline solution con-
per treatment, with the exception of the high dose group, taining 0.25% (wt/vol) trypsin3 and 0.50% (wt/vol) tauro-
which contained three pens. Feces were collected from deoxycholic acid.3 The resulting sporozoite suspension
this group daily for the first 10 d of the study for determi- was centrifuged, resuspended in saline, and the total
nation of oocyst output. The birds were challenged at number of each parasite stage was determined by direct
14 d of age, and only one pen of the high dose group count using a hemacytometer. This procedure resulted
was challenged. in 97 to 99% sporozoites, with the remaining material
Eggs used for the second experiment were injected with containing small numbers of oocysts and sporocysts. The
saline or 1 × 102, 5 × 103, or 1 × 105 sporozoites, and the preparations were then diluted with saline to obtain the
chicks were placed on litter with three pens of 12 birds appropriate concentration of parasites for in ovo injection
each per treatment group. Litter samples for oocyst counts with 100 µL per egg. Control eggs received 100 µL of
were collected on d 7, 14, and 21 posthatch, and birds sterile saline.
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 Determination of Oocyst Output
separated into challenged and nonchallenged groups on
d 21. All prechallenge data collected from nonimmunized For birds reared in wire cages, the entire fecal output
birds therefore applies to challenged and nonchal- of each pen that was to be sampled was collected daily
lenged groups. for the first 10 d after hatch and weighed. Feces were
Birds were placed in wire-floored cages for the third mixed with an amount of water equivalent to approxi-
experiment, after hatching from eggs receiving 2 × 105, 4 mately six times the volume of feces and homogenized
× 105, 2 × 106, 4 × 106, or 2 × 107 sporocysts, or 5 × 104, 1 using a hand-held blender. A single 1-mL sample of each
× 105, 5 × 105, 1 × 106, or 5 × 106 oocysts per egg. There suspension was mixed with 9 ml of 30% (wt/vol) NaNO3.4
were two pens of 10 birds per treatment for the high dose After being mixed, samples were loaded into duplicate
groups, and one pen of 10 birds for all other groups. Feces McMasters counting chambers, and the average number
were collected daily from the high dose groups for the of oocysts was determined. The number of oocysts per
first 10 d of the study for determination of oocyst output. gram of feces within each pen was calculated. Litter sam-
The birds were challenged at 14 d of age, and only one ples were taken by collecting a handful of litter from each
pen of the high dose group was challenged. corner and the center of each pen on d 7, 14, and 21
In the fourth experiment, eggs were injected with 1 × posthatch. The combined samples were mixed thor-
102, 1 × 103, 1 × 104, or 1 × 105 oocysts and the chicks oughly, and a 10-g sample was mixed with 100 mL of
placed on litter with three pens of 12 birds per treatment tap water and allowed to stand overnight. The samples
group. Litter samples for oocyst counts were collected on were then homogenized in a mechanical blender and the
d 7, 14, and 21, at which time birds were transferred to 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
2
loaded into duplicate McMasters chambers and the oo-
Hoover’s Hatchery, Rudd, IA.
3
Sigma Chemical Co., St. Louis, MO. cysts counted. If necessary, dilutions were carried out in
4
Fisher Scientific, Fairlawn, NJ. 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 were slightly lower than those of the nonimmunized con-
trols, the reduction was not significant. There were no
Birds reared on wire were challenged at 14 d of age, differences in postchallenge weight gain between any of
and birds reared on litter were challenged at 21 d of age the groups.
after transfer to wire-floored cages. Each chick was leg- Chicks, hatched from eggs injected with 1 × 106 sporo-
banded, weighed, and challenged with 2.5 × 104 sporu- zoites and reared on wire, shed oocysts after hatching
lated oocysts of Eimeria tenella by oral gavage. This chal- (Figure 1). Oocyst shed was first detected at 2 d posthatch,
lenge dose produced a moderate level of disease in pre- about 5 d after injection of sporozoites into embryos on
liminary dose titration studies conducted in birds of the d 18 of incubation. Oocyst output peaked 3 d posthatch,
same breed and of similar age (data not shown). The with a small secondary peak at 7 d posthatch. Chicks that
challenge strain was the same as that which with the hatched from eggs receiving saline did not shed detect-
birds were immunized. Six days after challenge, birds able numbers of oocysts (data not shown).
were weighed again and killed by cervical dislocation, In ovo administration of sporozoites to 18-d-old em-
and cecal lesions were scored according to the method bryos did not significantly affect hatch rates in the second
of Johnson and Reid (1970). experiment (Table 2). Small numbers of oocysts were ob-
served in the litter of immunized birds as early as 7 d
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.

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- ity significantly, however these were the only groups
creased in all groups between d 7 and 21 posthatch, in- affected. Chicks that hatched from eggs receiving oocysts
cluding the nonimmunized birds. Microscopic observa- and reared on litter shed oocysts, as shown by the pres-
tion of the size of the oocysts (approximately 18 × 21 ence of oocysts in the litter during the 21-d growth period.
microns) indicated that all were E. tenella. Although it is When these birds were challenged with E. tenella, lesion
possible that the birds became infected by an external scores were significantly reduced compared to the nonim-
source of E. tenella, it seems more likely that cross-contam- munized, challenged control, although this group had
ination occurred from pens of immunized chicks to pens lower lesion scores than expected based on a preliminary
of nonimmunized chicks. Because all pens had open tops challenge dose titration (data not shown). Although non-
and were in the same room, transmission of E. tenella immunized birds did not shed detectable numbers of
from pen to pen by insects or animal care personnel seems oocysts during the prechallenge phase of the study, there
more probable than infection from an outside source, was evidence of a low level of E. tenella infection in the
especially as the two immunized groups that were shed- nonimmunized birds in the form of mild cecal lesions in
ding oocysts on d 7 provided a potential source of contam- a few of the birds and relatively poor weight gain com-
ination. When chicks that hatched from eggs injected with pared to other groups (Table 4). Immunity resulting from
sporozoites were reared on litter and challenged at 21 d such an infection may also account for the low lesion
of age, all immunized groups had significantly reduced score and lack of weight gain suppression in the nonim-
lesion scores compared with nonimmunized, challenged munized, challenged group. This infection may also ac-
controls (Table 2). Postchallenge weight gain among im- count for the lower weight gain in the nonimmunized,
munized birds tended to be greater than among nonim- nonchallenged controls. There were no significant differ-
munized birds, the difference being significant for the ences, however, in postchallenge weight gain between
group immunized with the largest sporozoite dose (1 × treatment groups.
105 per egg). Challenge of the nonimmunized birds did
not suppress weight gain compared to the nonchal- DISCUSSION
lenged controls.
The peak in oocyst output at 3 d posthatch following
Immunization with Oocysts or Sporocysts in ovo injection of sporozoites suggests that sporozoites
initiated their life cycle in the embryo near the time of in
Hatchability in the third experiment was generally un- ovo injection (i.e., 3 d prior to hatching). The minimum
affected by in ovo injection of oocysts or sporocysts of E. prepatent period for E. tenella is about 115 h, or roughly
tenella (Table 3). When these chicks were reared on wire 5 d (Conway and McKenzie, 1991). Thus, initial detection
and challenged with E. tenella, lesion scores at all but the of oocyst output on d 2 posthatch implied that infection
two lowest doses of sporocysts were significantly reduced occurred 5 d earlier, or 3 d prior to hatching. In contrast,
compared to the nonimmunized, challenged chicks. There the kinetics of oocyst output when oocysts or sporocysts
was no significant treatment effect on weight gain among were injected in ovo suggested that the parasite life cycle
these birds. Chicks shed oocysts after hatching when em- was initiated around the time of hatching. When birds
bryos were injected with 5 × 106 oocysts or 2 × 107 sporo- are infected by oral gavage with oocysts of E. tenella, peak
cysts. In both cases, peak oocyst output occurred at 7 d oocyst output occurs around 7 d postinfection (Ryley et
posthatch (Figure 2). al., 1976). Peak oocyst output after in ovo injection of
In the fourth experiment (Table 4), in ovo injection of oocysts or sporocysts was observed at 7 d posthatch,
the highest and lowest oocyst doses decreased hatchabil- 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 injection and hatching, further supporting this idea (We-
and sporocysts remained dormant in the embryo after ber et al., 2001). It is reasonable to speculate that oocysts
injection until around the time of hatching. are delivered to the gut by ingestion of amniotic fluid by
Intact E. maxima oocysts have been observed in the the embryo during the last quarter of incubation (Ro-
lumen of the embryo gut throughout the period between 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
FIGURE 2. Posthatch oocysts shed by broiler chicks after in ovo was somewhat surprising because weight gain suppres-
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 sion is a characteristic of infection with E. tenella. Al-
of two pens of 10 birds each. 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 ovo may account for the lack of a dose response in this
dose, virulence of the challenge strain, diet, breed of bird, study. The relatively large number of oocysts produced
and housing conditions. In some cases, cross-contamina- during the first week provided ample opportunity for
tion with E. tenella from pens of immunized birds to reinfection and oocyst cycling, resulting in a high level
nonimmunized birds might have imparted a degree of of immunity after 21 d, even among groups receiving
immunity to nonimmunized controls. Even under these lower oocyst doses. Because the challenge in this study
circumstances, immunized birds were measurably more was mild, possibly due to a low level of immunity in
resistant to infection than were nonimmunized birds in nonimmunized controls resulting from cross-contamina-
most cases. tion, even the group receiving the lowest immunizing
There was little or no correlation between in ovo para- oocyst dose might have had sufficient immunity to almost
site dose and the extent of lesion control after challenge completely control the infection.
when birds were reared on wire, but it should be noted The observation that oocysts accumulated in litter ear-
that birds reared on wire have little exposure to feces and lier after in ovo injection of oocysts than after in ovo
thus oocyst cycling is limited. Multiple infections due injection of sporozoites is surprising given our observa-
to cycling of oocysts have been shown to enhance the tion that peak oocyst output occurred at 3 d posthatch
development of immunity when birds are vaccinated when sporozoites were administered in ovo, and after 7
with live oocysts (Joyner and Norton, 1973). The greater d posthatch when oocysts were given in ovo. Since infec-
effectiveness of immunization observed in the current tion in the embryo seemed to occur near the time when
study when birds were reared on litter compared to wire sporozoites are injected in ovo, it might be expected that
cages was likely a result of reinfection from oocysts shed in ovo administration of sporozoites would result in an
in the feces, although allowing 21 d for immunity to earlier infection and earlier oocyst shed compared to in-
develop, rather than 14 d, might have played a role. jection of oocysts, allowing more time for oocyst cycling
The dose response in lesion score reduction was pro- and thus greater immunity. Although results of our stud-
nounced when birds immunized with sporozoites were ies with birds on litter do not support this notion, defini-
reared on litter but was absent when birds were immu- tive conclusions regarding oocyst output after hatch and
nized with oocysts. Oocysts appeared to accumulate in rearing on litter cannot be made because in both cases
the litter more rapidly when birds were immunized with there appeared to be cross-contamination with E. tenella
oocysts than when birds were immunized with sporozo- between pens, which complicated interpretation of oocyst
ites. On d 7 posthatch, litter from birds immunized in output data. In addition, conditions for sporulation of
ovo with 1 × 105 sporozoites per egg contained 5.6 × 102 oocysts in the litter, a necessary step for reinfection and
oocysts per g (Table 2), yet litter from birds immunized oocyst cycling to occur, might have differed between
with 1 × 104 oocysts per egg, equivalent to 8 × 104 sporozo- studies. Conditions that were poorly conducive to sporu-
ites, contained 1.5 × 105 oocysts/g (Table 4). Thus, al- lation would slow the accumulation of oocysts in the litter
though the two groups received similar doses in terms of compared to an environment in which conditions of litter
sporozoites, the group receiving oocysts in ovo produced moisture and temperature facilitated sporulation. Differ-
about 270-fold more oocysts per gram of litter after 7 d ences in oocyst cycling between studies could result in
than the group receiving sporozoites. Lower doses of differences in the level of immunity developed.
oocysts also produced higher levels of oocysts in the litter Provaznikova and Bedrnik (1997) showed that it was
on d 7 than were found in groups immunized with sporo- possible to immunize chicks against E. tenella infection
zoites. The higher number of oocysts produced during by in ovo injection of sporozoites. In the present study, we
the first week of life among birds receiving oocysts in confirm these results and further characterize the primary
IN OVO IMMUNIZATION WITH LIVE EIMERIA TENELLA STAGES 1707
infection resulting from exposure of the embryo to sporo- Jenkins, M. C. 1998. Progress on developing a recombinant coc-
zoites. These data also demonstrate that protective immu- cidiosis vaccine. Int. J. for Parasitol. 28:1111–1119.
Johnson, J. K., and W. M. Reid. 1970. Anticoccidial drugs: lesion
nity can be established by in ovo injection of sporocysts scoring techniques in battery and floor-pen experiments with
or oocysts. As expected, lower doses seemed to be re- chickens. Exp. Parasitol. 28:30–36.
quired for birds reared on litter than for birds reared on Joyner, L. P., and C. C. Norton. 1973. The immunity arising
wire due to exposure to reinfection in the former instance. from continuous low-level infection with Eimeria tenella. Par-
Watkins et al. (1995) found evidence that Eimeria max- asitology 67:333–340.
Lillehoj, H. S., and J. M. Trout. 1993. Coccidia: a review of recent
ima completes its life cycle when oocysts or sporocysts advances on immunity and vaccine development. Avian Pa-
are injected in ovo but found no evidence of immunity thol. 22:3–31.
to challenge. In contrast, we have shown that in ovo NRC, 1994. Nutrient Requirements of Poultry. 9th rev. ed. Na-
delivery of E. tenella parasite stages results in an immuniz- tional Research Council, National Academy of Science,
ing infection in newly hatched chicks that will protect Washington, DC.
the birds from challenge infection. This result is consistent Pogonka, T., C. Klotz, F. Kovacs, and R. Lucius. 2003. A single
dose of recombinant Salmonella typhimurium induces specific
with findings that injection with Eimeria acervulina oo- humoral immune responses against heterologous Eimeria ten-
cysts, sporocysts, or sporozoites in ovo resulted in infec- ella antigens in chicken. Int. J. Parasitol. 33:81–88.
tion of newly hatched chicks that then had measurable Provaznikova, M., and P. Bedrnik. 1997. Vaccination of chickens
immunity to challenge infection (Doelling et al., 2001). against coccidiosis during embryonal development. Pages
More recent studies, reviewed by Williams (2002), con- 100–101 in Control of coccidiosis into the next millenium.
VIIth International Coccidiosis Conference and COST820
firm our finding that in ovo delivery of live Eimeria oo-
Workshop. Keble College, Oxford, UK.
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