Immunization Of Broiler Chicks By In Ovo Injection

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 injection 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 reaching a maximum at 3 d posthatch for chicks injected with

sporozoites and at 7 d posthatch for chicks receiving oocysts 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.

(Key words: Eimeria tenella, immunization, in ovo, oocyst, sporozoite) 2003 Poultry Science 82:1701–1707

INTRODUCTION Vaccination of chickens against coccidiosis with live oocysts is an accepted method of disease control, especially for long-lived birds such as breeders. The appearance of drug resistance among coccidia of domestic fowl (Chapman, 1997) has prompted renewed interest in vaccination 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 locations (Danforth et al., 1997). Other studies have investigated 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 unmodified and attenuated strains of live coccidia (Shirley, 1992). Although research on nonreplicating, subunit vaccines and heterologous gene/vector systems continues, no commercial products of this type are currently avail-

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 immunobiology 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 immunity when challenged at 10 d of age. Provaznikova and Bedrnik (1997) reported successful immunization of broilers 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 2003 Poultry Science Association, Inc. Received for publication February 25, 2003. Accepted for publication June 2, 2003. 1 To whom correspondence should be addressed: [email protected].

Design Four experiments were conducted using fertilized broiler eggs (Hubbard × Petersen) obtained from a com-

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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 18ga 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 determination 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 wirefloored cages. Nonimmunized birds were housed together 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 nonchallenged 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. Sigma Chemical Co., St. Louis, MO. Fisher Scientific, Fairlawn, NJ.

3 4

wire-floored cages and challenged. As before, nonimmunized 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 tenella in coccidia-free broiler chicks using standard methods. 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% sporocysts 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 containing 0.25% (wt/vol) trypsin3 and 0.50% (wt/vol) taurodeoxycholic 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 approximately 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 samples 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 thoroughly, 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 aliquot 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 oocysts counted. If necessary, dilutions were carried out in 30% (wt/vol) NaNO3.

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IN OVO IMMUNIZATION WITH LIVE EIMERIA TENELLA STAGES 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 (sporozoites/egg) 0 1 5 1 5 1 0

× 104 × 104 × 105 × 105 × 106 (nonchallenged)

Hatch rate (%) (n = 50)

Mean lesion score1

Postchallenge gain per bird (g)1

100 98 98 92 96 94 100

3.4a 1.9b 2.5b 3.0ab 2.8ab 2.1b 0c

288 306 289 317 315 287 283 5.74

SEM

0.16

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. a–c

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 legbanded, weighed, and challenged with 2.5 × 104 sporulated oocysts of Eimeria tenella by oral gavage. This challenge dose produced a moderate level of disease in preliminary 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).

were slightly lower than those of the nonimmunized controls, 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 sporozoites 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 detectable numbers of oocysts (data not shown). In ovo administration of sporozoites to 18-d-old embryos did not significantly affect hatch rates in the second experiment (Table 2). Small numbers of oocysts were observed 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 determined 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 sporozoites 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 significant. 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

5

SAS Institute, Cary, NC.

FIGURE 1. Posthatch oocysts shed by broiler chicks after in ovo immunization with 1 × 106 sporozoites of Eimeria tenella on d 18 of embryo incubation. Each value is the mean of three pens of 10 birds each.

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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

Treatment Nonimmunized, nonchallenged Nonimmunized, challenged 1 × 102 sporozoites/egg 5 × 103 sporozoites/egg 1 × 105 sporozoites/egg SEM

Oocysts per gram of litter2

Hatch rate (%)

Day 7

Day 14

Day 21

88 89 93 78

0 0 102 5.6 × 102,4

303 0 0 3.9 × 103

2.4 × 103,4 1.7 × 104 8.4 × 103 1.2 × 105

Mean lesion score2

Postchallenge gain/bird (g)2

0.0e 2.4a 2.0b 1.2c 0.5d 0.091

298b 302b 320ab 324ab 343a 4.69

Means with different superscripts within a column are significantly different (P < 0.05; ANOVA). 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. a–e 1

posthatch. The number of oocysts per gram of litter increased in all groups between d 7 and 21 posthatch, including the nonimmunized birds. Microscopic observation 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-contamination 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 shedding oocysts on d 7 provided a potential source of contamination. 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 immunized birds tended to be greater than among nonimmunized 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 nonchallenged controls.

Immunization with Oocysts or Sporocysts Hatchability in the third experiment was generally unaffected 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 embryos were injected with 5 × 106 oocysts or 2 × 107 sporocysts. 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 presence 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 nonimmunized, challenged control, although this group had lower lesion scores than expected based on a preliminary challenge dose titration (data not shown). Although nonimmunized 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 compared 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 nonimmunized, challenged group. This infection may also account for the lower weight gain in the nonimmunized, nonchallenged controls. There were no significant differences, 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

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IN OVO IMMUNIZATION WITH LIVE EIMERIA TENELLA STAGES 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 Method Oocysts

Sporocysts

Immunizing dose (infective stages/egg) 5 1 5 1 5 2 4 2 4 2

0 × 104 × 105 × 105 × 106 × 106 × 105 × 105 × 106 × 106 × 107

0 (nonchallenged)

Hatch rate (%) (n = 25) 96 89 81 81 96 78 81 100 96 92 81 88

SEM

Mean lesion score1

Postchallenge gain per bird (g)1

2.4a

279

1.8bc 1.8bc 1.1de 0.6ef 0.4f 2.0ab 1.9ab 1.4bcd 1.1de 1.3cd 0f

316 309 288 310 315 314 314 320 302 283

0.076

316 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 (Weber 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 (Romanoff, 1960). The small secondary peak in oocyst shedding observed on d 7 when eggs were injected with sporozoites (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 hatchability in two groups in the fourth study, the lack of a dose response of hatchability to oocyst dose in this study implies 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 discrepancy, 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 contributing 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 injection 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 nonimmunized controls. Although challenge of naive birds produced 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 suppression is a characteristic of infection with E. tenella. Although there is no clear explanation for the lack of weight

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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

Treatment Nonimmunized, nonchallenged Nonimmunized, challenged 1 × 102 oocysts/egg 1 × 103 oocysts/egg 1 × 104 oocysts/egg 1 × 105 oocysts/egg SEM

Oocysts per gram of litter2

Hatch rate (%)

Day 7

Day 14

Day 21

96 60* 93 91 84*

0 0 1.3 × 103 1.5 × 105 9.7 × 104

0 0 0 4.4 × 103 8.0 × 103

0 × × × ×

1.6 4.9 3.1 3.2

4

10 104 104 104

Mean lesion score2

Postchallenge gain per bird2 (g)

0.1c 1.7a 0.8b 0.8b 0.8b 0.7b 0.05

352 380 387 359 368 367 4.42

Means with different superscripts within a column are significantly different (P < 0.05; ANOVA). 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). a–c 1

gain suppression, relevant factors include the challenge dose, virulence of the challenge strain, diet, breed of bird, and housing conditions. In some cases, cross-contamination 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 parasite 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 pronounced when birds immunized with sporozoites were reared on litter but was absent when birds were immunized with oocysts. Oocysts appeared to accumulate in the litter more rapidly when birds were immunized with oocysts than when birds were immunized with sporozoites. 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 sporozoites, contained 1.5 × 105 oocysts/g (Table 4). Thus, although 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 sporozoites. 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-contamination, 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 earlier after in ovo injection of oocysts than after in ovo injection of sporozoites is surprising given our observation 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 infection 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 injection of oocysts, allowing more time for oocyst cycling and thus greater immunity. Although results of our studies with birds on litter do not support this notion, definitive 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 sporulation would slow the accumulation of oocysts in the litter compared to an environment in which conditions of litter moisture and temperature facilitated sporulation. Differences 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 sporozoites. These data also demonstrate that protective immunity can be established by in ovo injection of sporocysts or oocysts. As expected, lower doses seemed to be required 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 maxima 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 immunizing infection in newly hatched chicks that will protect the birds from challenge infection. This result is consistent with findings that injection with Eimeria acervulina oocysts, sporocysts, or sporozoites in ovo resulted in infection of newly hatched chicks that then had measurable immunity to challenge infection (Doelling et al., 2001). More recent studies, reviewed by Williams (2002), confirm our finding that in ovo delivery of live Eimeria oocysts 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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