In ovo vaccination against coccidiosis

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IN OVO VACCINATION AGAINST COCCIDIOSIS i

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5 IN OVO VACCINATION AGAINST COCCIDIOSIS

Background of the Invention

The present invention relates to a method of vaccinating domesticated birds against coccidiosis. In particular, the invention relates to the in ovo administration of live Eimeria spp sporozoites or merozoites, or mixtures thereof, into the developing 10 eggs of domesticated birds in order to immunize the hatched chicks against coccidiosis.

Coccidiosis is an enteric disease of domesticated birds caused by infection with intracellular protozoan parasites of the genus Eimeria. Coccidiosis is the most economically devastating parasitic disease of domesticated birds. It is estimated that 15 anticoccidial medications and losses due to coccidiosis cost the poultry industry hundreds of millions of dollars every year.

Various attempts to vaccinate domesticated birds against coccidiosis have been reported since the early 1950's. Current vaccination methods include administering live Eimeria oocysts to birds through feed or water. These methods, 20 however, are inconvenient and inefficient because not all birds get the intended oocyst dose and many are either unprotected by the vaccine or receive a pathogenic infection.

In J. M. Sharma and B. R. Burmester, Avian Dis. 26: 134-149, 1981, the authors reported that chickens vaccinated in ovo with herpesvirus of turkey 25 developed immunity against subsequent challenge with Marek's disease virus. In European patent publication no. 291173, an immunization process is referred to wherein a nonreplicating immunogen is administered in ovo. The immunogens specifically referred to in the European patent are a genetically engineered Eimeria antigen and an Eimeria oocyst extract. The European patent specifically excludes 30 live parasite stages such as those used in the vaccination method claimed herein.

The present vaccination method involves in ovo administration uf live Eimeria sporozoites or merozoites, or a mixture thereof, into the developing eggs of domesticated birds. The available literature suggests that such a vaccination method would be ineffective in ovo and should be applied post-hatch. In T.K. Jeffers and 35 G.E. Wagenbach, J. Parasit. 56(4): 656-662, 1970, the authors reported that in ovo injection of E. tenella sporozoites on day 10 of incubation provided no significant immunological protection against subsequent challenge with E. tenella oocysts. In

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-2- fact, they reported that chicks that received no treatment had a greater survival rate against subsequent challenge with E tenella oocysts than chicks that had been treated in ovo with sporozoites. In K.L. Watkins et al., Proc. VI th. International Coccidiosis Conf., Abstract E1-2, Ontario, Canada, 1993, the authors described in 5 ovo inoculation with live E. maxima sporocysts and sporulated oocysts and concluded that their study provided no evidence that in ovo exposure protects against subsequent coccidial challenge with E. maxima oocysts 10 days post-hatch. Watkins et al. further concluded that significant immunological protection is provided if inoculation is done soon after hatch rather than in ovo. Contrary to this teaching, 10 the in ovo vaccination method of the present invention provides unexpected immunity that protects the hatched birds against subsequent coccidial challenge.

Summary of the Invention

The present invention, also referred to herein as the "present vaccination 15 method", relates to a method of vaccinating a domesticated bird against coccidiosis comprising administering in ovo, during the final quarter of incubation, an effective immunizing dose of live Eimeria sporozoites or merozoites, or a mixture thereof.

The term "domesticated bird(s)", as used herein, unless otherwise indicated, includes chickens, turkeys, ducks, game birds (including, but not limited to, quail, 20 pheasants, and geese) and ratites (including, but not limited to, ostrich,.

The term "in ovo", as used herein, unless otherwise indicated, means into a domesticated bird egg containing a live, developing embryo.

The term "administering in ovo" or "in ovo administration", as used herein, unless otherwise indicated, means administering the vaccine described herein to a 25 domesticated bird egg containing a live, developing embryo by any means of penetrating the shell of the egg and introducing the vaccine. Such means of administration include, but &lt;ve not limited to, injection of the vaccine.

The term "final quarter of incubation", as used herein, unless otherwise indicated, means the final quarter of incubation of a developing egg of a 30 domesticated bird.

The term "Eimeria", as used herein, unless otherwise indicated, means one or more species of the genus Eimeria that infect domesticated birds. Such Eimeria species include those that are found in chicken, including E. tenella, E. acervulina, -3- E. maxima, E. necatrix, E. mitis, E. praecox, and E. brunetti, and also those that are found in turkeys, including E. meieagrimitis, E. adenoeides, E. gaiiopavonis, E. disperse, E. meieagridis, E. innocua, and E. subrotunda, and also Eimeria species that infect other domesticated birds as defined above. The term "Eimeria'' also includes all strains of the foregoing species of Eimeria, including, but not limited to, precocious strains, and attenuated strains, which includes strains that have been irradiated, or otherwise treated, so that they fail to complete development. The term Eimeria also includes any newly-discovered strains or species of Eimeria that infect domesticated birds as defined above.

The terms "sporozoites", "sporocysts", "oocysts", and "merozoites", as used herein, unless otherwise indicated, mean live Eimeria sporozoites, sporocysts, oocysts, and merozoites.

The term "effective immunizing dose", as used herein, unless otherwise indicated, means a number of sporozoites or merozoites, or, when mixed, a number of sporozoites and merozoites, sufficient to provide immunological protection in the hatched birds that is greater than the inherent immunity of non-immunized birds. As used herein, the terms "immunize" and "vaccinate" are synonymous and are used interchangeably. A preferred dose to be administered in accord with the method of the invention comprises 10 to 10s sporozoites or merozoites, or a mixture thereof wherein the total number of said sporozoites and merozoites ranges from 10 to 10e. A more preferred dose comprises 103 to 10® sporozoites or merozoites, or a mixture thereof wherein the total number of said sporozoites and merozoites ranges from 103 to 10®.

Another preferred dose comprises 102 to 10® sporozoites or merozoites, or a mixture thereof wherein the total number of said sporozoites and merozoites ranges from 10? to 105. A preferred domesticated bird to be vaccinated in accord with the method of the invention is a chicken. A preferred dose to be administered in ovo to chicken eggs comprises sporozoites or merozoites, or a mixture thereof, of two or more species of Eimeria selected from the group consisting of E. tenella, E. acervuiina, E. maxima, E. necatrix, E. mitis, E. praecox, and E. brunetti. The dose preferably includes from 10 -4- to 10® sporozoites or merozoites, or a mixture thereof, for each species that is included in the dose.

Another preferred domesticated bird to be vaccinated in accord with the method of the invention is a turkey. A preferred dose to be administered in ovo to turkey eggs comprises sporozoites or merozoites, or a mixture thereof, of two or more species of Eimeria selected from the group consisting of E. meieagrimitls, E. adenoeides, E. gallopavonis, E. disperse, E. meleagridis, E. innocua, and E. subrotunda. The dose preferably includes from 10 to 10® sporozoites or merozoites, or a mixture thereof, for each species that is included in the dose.

Other preferred domesticated birds to be vaccinated in accord with the method of the invention are game birds, ducks and ratites.

The method of the invention further comprises, in combination with the present vaccination method, administering in ovo an immune stimulant at any time during incubation. A preferred method of administering the immune stimulant is simultaneously with the in ovo administration of a dose of sporozoites or merozoites, or mixture of said sporozoites and merozoites, during the final quarter of incubation.

Detailed Description of the Invention

The present vaccination method involves the in ovo administration, during the final quarter of incubation, of live Eimeria sporozoites or merozoites, or a mixture thereof, into domesticated birds' eggs. In the case of chickens, in ovo administration is preferably done on days 15-20 of incubation, and most preferably on day 18 of incubation. In the case of turkeys, in ovo administration is preferably done on days 21-26 of incubation.

The present vaccination method can be performed using any suitable in ovo administration method. Preferably, the present vaccine is administered via injection. According to one method of injection, a hole is made in the egg shell at the large end of the egg using an 18 guage needle to expose the egg's air cell. A 1.0-1.5 inch 22 guage needle attached to a syringe of appropriate size (1-3 ml) can be inserted through the hole and through the membrane of the air cell. An appropriate number of sporozoites or merozoites, or, when mixed, an appropriate number of sporozoites 118490/2 -5- and merozoites, are suspended in a suitable liquid carrier, for instance 10-500//1 of phosphate-buffered saline, and then injected into the egg. The appropriate volume will depend on the size of the egg being treated, with ostrich eggs obviously being capable of taking more volume than chicken eggs. The site of injection can be within 5 any region of the egg or embryo. Preferably, injection is done axially through the center of the large end of the egg into the amnion.

Alternatively, an automated egg injection system can be used in the present ; vaccination method. Such systems are described in U.S. Patent nos. 4,681,063, corresponding to IL 83020, 4,040,388, 4,469,047, and 4,593,646, which are herein incorporated by reference. 10 Other appropriate methods of injection are known to those skilled in the art.

Oocysts can be prepared by any of several methods known to those skilled in the art. Such methods include those described in J.F. Ryley et al., Parasitology 73:311-326, 1976 and P.L. Long et al.. Folia Veterinaria Latina Vl#3, 201-217, 1976, which are herein incorporated by reference. According to one method, 1 5 commercial broiler chickens, approximately 2 weeks old, are infected with the Eimera species of interest by oral gavage of an appropriate dose of sporulated oocysts. For example, a typical dose used for E. tenella is 200,000 sporulated oocysts/bird. Well known procedures for collection and purification of oocysts from infected birds are then followed. For most species of Eimeria, feces are collected from infected birds 20 5-7 days post-infection, blended and filtered to remove debris, then centrifuged at a speed sufficient to pellet the remaining fecal material. For E. tenella, a similar procedure is used except that cecal cores are taken at 6 days post-infection. The pellet is resuspended in q saturated salt solution, in which the oocysts float and most of the contaminating debris can be removed by centrifugation. The oocyst 25 suspension is then diluted to lower the salt concentration. The oocysts are washed repeatedly to remove the salt and resuspended in potassium dichromate solution (2.5% w/v). The oocyst suspension is incubated at 29°C with shaking (e.g., 140 rpm) for approximately 72 hours to induce spoliation of the oocysts. Alternatively, the oocysts can be treated with sodium hypochlorite and then sporulated. The 30 number of sporulated oocysts/ml is determined by direct count using a hemocytometer, and the culture is stored, preferably under refrigeration until needed. -6-

To prepare sporocysts, the potassium dichromate is removed from the oocyst suspension described above by repeated washing of the oocysts, which involves collection of oocysts by centrifugation and resuspending in deionized or distilled water. When the dichromate has been removed as judged by the lack of yellowish-orange coloration, the oocyst suspension is mixed with an equal volume of sodium hypochlorite (bleach) and incubated at room temperature for 15 minutes. The bleach is then removed by repeated washings, and the oocysts are resuspended in physiological saline or deionized water. Oocysts can be broken to release sporocysts using a variety of known techniques. For example, oocysts can be broken to release sporocysts by mixing the oocysts with glass beads of 1 -4 mm diameter and shaking by hand, vortex mixer, or shaking incubator, or using a hand-held homogenizer. Unbroken oocysts and oocysts walls can be separated from the released sporocysts by differential centrifugation in 50% Percoll™ (sold by Pharmacia Biotech, or 1 M sucrose as described in Dulski et al., Avian Diseases, 32: 235-239, 1988.

To prepare sporozoites or a sporozoite-rich preparation to be used in accord with the present invention sporozoites are excysted from the sporocyst preparation described above. In one procedure, sporocysts prepared as described above are pelleted by centrifugation, resuspended inexcystation buffer (0.5% taurodeoxycholic acid and 0.25% trypsin in phosphate buffered saline, pH 8.0, and incubated with shaking for one hour at 41 °C. A sample of the resulting suspension is counted to determine the number of sporozoites, the sporozoites are washed once to remove the excystation buffer, and resuspended in phosphate-buffered saline at the desired concentration for in ovo injection. This preparation contains sporozoites, sporocysts and oocysts, and, without further purification, can be used in accord with the present vaccination method. Purified sporozoites, removed from sporocysts and oocysts, can be prepared by DE-52 anion exchange chromatography as described in D.M. Schmatz etal., J. Protozool. 31: 181-183, 1984, which is herein incorporated by reference. The dose of sporozoites to be used in the present vaccination method will vary according to the type of domesticated bird being vaccinated and the species of Eimeria being used in the vaccine. In general, the dose can range from 10 to 106 sporozoites per egg. Preferably, the dose ranges from 10 to 105 sporozoites per egg, and, more preferably, the dose ranges from 102 to 105 sporozoites per egg.

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Merozoites can be prepared by various methods known to those skilled in the art. In one method, sporozoites are infected into primary chick kidney cells (PCK) that are grown in culture as cell aggregates, using a modification of the method described in D. J. Doran, J. Parasit. 57: 891-900, 1971, which is herein 5 incorporated by reference. PCK cells are grown at 40°C in 3% CO2 in modified RK2 medium - DMEM/F12 with L-glutamine and 15 mM HEPES, supplemented with fetal bovine serum, penicillin-streptomycin, 15 mM sodium bicarbonate, 10 ng/ml epidermal growth factor, 5//g/ml insulin, 5//g/ml transferrin, 5 ng/ml selenious acid and 0.01 μΜ hydrocortisone HCL as described in S.D. Chung et al., J. Cell Biol. 95: 10 118-126, 1982. PCK cells are prepared from kidneys of 2 to 3 week old chicks by mincing the kidney, and then treating the tissue at 37 °C with several changes of 0.2 mg/ml collagenase (sold by Worthington Biochemical Corp., Freehold, NJ) in phosphate-buffered saline solution. The cellular aggregates in the supernatant are washed, resuspended in modified RK2 medium containing 5% fetal bovine serum and 15 used to seed tissue cultures flasks at a density of 105 aggregates per cm2. The PCK cells are incubated for 18 hours at 40°C in 3% CO2 and then infected with 4 x 105 sporozoites/cm2. Infected cultures are grown in modified RK2 medium containing 2% fetal bovine serum. After 24 hours of incubation, to allow invasion, uninvaded sporozoites are removed by agitating the flask and removing the culture medium. 20 The cell layer is washed once with modified RK2 medium containing 2% fetal bovine serum and the culture medium is again discarded.,Fresh RK2 medium is added, and the cultures are incubated for another 48 to 54 hours until merozoites are released into the culture medium.

Purification of the merozoites to remove host cell debris can be done by 25 various methods known to those skilled in the art. In accord with one method, as described in J.A. Olson, Antiinicrob. Agents Chemother. 34: 1435-1439, 1990, the culture medium containing the released merozoites is collected and spun at 450 X g for 10 minutes to concentrate the merozoites. The pellet containing the merozoites and the host cell debris is suspended in 0.1 M NaCI-0.05 M KCI-20% 30 bovine serum albumin and applied to a DE-52 anion exchange column equilibrated in 75 mM Tris-40 mM NaH2PO4-86 mM NaCI-100 mM glucose at pH 8.2. Merozoites flow through the column. Merozoites collected from the column can be tested for purity by electron microscopy as described in A. Kilejian, J. Biol. Chem. 249: 4650- -8- 4655, 1974. In general, the merozoite dose can range from 10 to 10® merozoites per egg. Preferably, the dose ranges from 10 to 10s merozoites per egg, and, more preferably, the dose ranges from 102 to 105 merozoites per egg. When sporozoites and merozoites are mixed, in general, the dose comprising the total number of merozoites and sporozoites can range from 10 to 10® per egg. Preferably, the dose ranges from 10 to 105 merozoites and sporozoites per egg, and, more preferably, the dose ranges from 102 to 105 merozoites and sporozoites per egg.

The sporozoites or merozoites, or mixture thereof, can be injected in ovo in any physiologically suitable medium. Preferably, they are suspended in physiologically balanced saline such as phosphate-buffered saline. The selected medium can optionally include one or more suspending agents including physiologically suitable gels, gelatins, hydrosols, cellulose, or polysaccharide gums.

Preferably, in the present vaccination method, sporozoites or merozoites, or a mixture thereof, of two or more Eimeria species are injected in ovo at the same time. In accord with the present vaccination method, sporozoites or merozoites, or a mixture thereof, of all identified species of Eimeria that infect a specific domesticated bird, such as chicken, can be injected in ovo at the same time, or in series, at appropriate doses to provide immunological protection against all species.

Immune stimulants can be used in conjunction with the present vaccination method. Suitable immune stimulants include, but are not limited to, cytokines, growth factors, chemokines, supernatants from cell cultures of lymphocytes, monocytes, or cells from lymphoid organs, cell preparations or cell extracts (e.g. Staphylococcus aureus or lipopolysaccharide preparations), mitogens, or adjuvants including low molecular weight pharmaceuticals. An immune stimulant can be administered in ovo at any time during incubation. Preferably, an immune stimulant is administered in ovo in the medium containing the dose of Eimeria sporozoites or merozoites, or mixture thereof.

The efficacy of the present invention in vaccinating against coccidiosis is illustrated in the following examples. Each dose was injected in ovo in physiologically-acceptable saline as described above. The effectiveness of a particular preparation was determined by monitoring its effect on hatch rate and hatch weight of the chicks, and, following challenge infection, oocyst production, weight gain, and pathogenicity (lesion score). Lesion scores were assigned -9-

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according to the protocol described in J.K. Johnson and W.M. Reid, Exp. Parasitol. 28: 30-36, 1970, according to which a value of 0 represents no disease and a value of 4 represents maximum pathology. 5 EXAMPLE 1

Chicken eggs were injected on day 18 of incubation with a preparation containing 105 E. tenella sporozoites per egg. The preparation was not purified to remove sporocysts and oocysts. Each dose also contained approximately 104 E. tenella sporocysts and approximately 104 E. tenella oocysts. As a control, eggs were 10 injected only with phosphate-buffered saline solution. In the sporozoite-treated population of birds the mean oocyst shed at 7 days post-hatch was 1.1 x 10® oocysts/bird. Non-immunized birds and the sporozoite-treated birds were challenged with various doses of sporulated oocysts of E. tenella, administered by oral gavage, on days 7, 14 or 21 post-hatch. The data appear in Table 1. 15

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Immunized Versus Non-lmmunized Birds: Responses To Different Challenge Doses At Different Times Post-Hatch TABLE 1 I Lesion score on sixth day after challenge j i Weight gain (grams) per bird over | six day period after challenge Group1 Challenge dose (sporulated oocysts per bird) Non-immunized Immunized In Ovo (105 sporozoites on day 18 of incubation) I Non-9 immunized Immunized In Ovo (105 sporozoites on day 18 of incubation) Challenge on day 7 0 i 0 0 ! 188 167 post-hatch 2.5 χ 103 j 3.0 0.3’ 1 154 153 5 χ 103 I 3.1 1.2* I 153 167 1 χ 104 I 37 1.4· I 123 165* Challenge on day 0 1 0 0 I 266 278 14 post-hatch 2.5 χ 103 I 2·4 0.4* J 260 269 5 χ 103 J 2.3 1.1 * | 244 265 1 χ 104 1 33 1.6· i 235 259 Challenge on day 0 S θ·1 0 ί 361 357 21 post-hatch 2.5 χ 103 i 2.3 0.7* 3 375 358 5 χ 103 3 2-1 0.8* | 332 344 1 χ 104 I 2.3 1.4* j 337 395* 1 5 1 Each group was subjected to e single challenge of sporulated oocysts on daye 7, 14, or 21 post-hatch. For example, birds challenged on day 21 post-hatch were not challenged on days 7 or 14 post-hatch. * Significantly different from non-immunized birds (p&lt;0.05, ANOVA)

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The data in Table 1 clearly show that the immunized birds were less susceptible to infection than their non-immunized hatch mates as indicated by the reduced lesion scores and improved weight gains in the immunized birds. The data also demonstrate that the method of the invention imparts immunity to the chicks at a relatively early age (within seven days post-hatch). Furthermore, the data show that the immunity continues as the chicks grow and develop. Imparting immunity to chicks at an early age provides a significant advantage in the broiler chickqn industry because broilers routinely reach market by 6 weeks of age. EXAMPLE 2

Chicken eggs were injected on day 18 of incubation with saline (control) or preparations containing different doses of sporozoites of E. tenella. as indicated in Table 2 which provides the pre-challenge results. The sporozoite preparation used for each dose contained 62% sporozoites, 9% sporocysts, and 29% oocysts. Each dose containing 105 sporozoites included a total of 1.6 X 105 parasite stages (sporozoites, sporocysts, and oocysts).

Effect Of In Ovo Vaccination On Hatch Rate And Weight TABLE 2

Sporozoites injected per egg on day 18 of incubation Hatch rate (%) Hatch weight (grams) 0 94 48.2 0 94 48.0 103 100 49.4 104 97 . 47.0 105 94 49.1

The data in Table 2 show that chicks hatched from eggs injected with live sporozoites were substantially identical to their non-immunized hatch mates in terms of hatch rate and hatch weight. The chicks were then challenged on day 14 post-

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-12- hatch with 1.25 X 10* sporulated oocysts of E. tenella per bird, administered by oral gavage. The post-challenge results are provided in Table 3. -13-

Response To Challenge Infection Of Non-lmmunized Birds Versus Birds Treated In Ovo With Different Doses Of Sporozoites TABLE 3

Sporozoites injected per egg on day 18 of incubation Weight Gain per bird over six day period after challenge Lesion Score on day 6 after challenge Oocysts shed per bird (x 106) on day 6 after challenge 0 278 3.2 12.3 0 (Unchalleng -ed control) 321 0 0.003* 103 289 2.6* 11.2 104 291 2.7 12.2 105 304* 1.4* 1.4*

Significantly different from non-immunized group (received saline) that was subjected to challenge (p&lt;0.05, ANOVA)

The data in Table 3 show that for every parameter (weight gain, lesion score, oocyst shed) chicks hatched from eggs injected with different doses of the sporozoite preparation showed evidence of immunity. In comparison to the control birds that were treated only with saline and were subjected to challenge infection, the birds immunized in ovo with the sporozoite preparation showed greater weight gain and reduced lesion scores. In addition, the birds immunized in ovo with the sporozoite preparation passed fewer oocysts than the control birds after challenge infection, indicating that the infection was less severe in the immunized birds.