In ovo vaccine composition against coccidiosis

Abstract

The present invention relates to compositions comprising Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella, administered intraovo into embryonated eggs of domesticated birds, compositions for use as in ovo vaccines and vaccines against coccidiosis. The present invention further relates to combination vaccines for use in the prevention and / or treatment of Eimeria infection and infectious bursal disease (Gumboro disease). The present invention further relates to kits for use as vaccines comprising said compositions.

Description

[Technical field]

[0001] This application claims the benefit of European Patent Application No. 22382216, filed March 8, 2022.

[0002] The present invention is in the field of chicken coccidiosis caused by Eimeria species. More particularly, the present invention relates to compositions in the prevention and / or treatment of Eimeria infection by in ovo injection, in ovo vaccines and compositions for use as vaccines against coccidiosis. The present invention further relates to said vaccines for use in generating an immunogenic and / or protective immune response against at least Eimeria infection, as well as kits comprising one or more doses of said vaccines. [Background technology]

[0003] Chicken coccidiosis is one of the most important poultry diseases, mainly caused by parasites of various different Eimeria species, which is recognized as an economically significant disease of chickens and has been associated with considerable economic losses to poultry farmers worldwide. Such large costs along with the growing demand for poultry highlight the importance of finding new ways to reduce the incidence of coccidiosis in chickens, as disclosed in Blake et al., "Re-calculating the cost of coccidiosis in chickens," Vet Res., 2020, 51, 115. It has also been demonstrated that Eimeria infection exacerbates the outcome of exposure to other pathogens, such as Clostridium perfringens, and in combination causes necrotic enteritis, as disclosed in Soutter et al., "Poultry coccidiosis: design and interpretation of vaccine studies," Frontiers Vet. Sci., 2020, 7, 101.

[0004] Coccidiosis has a complex monohost life cycle consisting of both asexual and sexual stages. The cycle is initiated when a bird ingests sporulated oocysts, which are generally associated with fecal material. These oocysts contain invasive asexual sporozoites that are released into the bird's digestive tract. The sporozoites then invade epithelial cells and develop into multinucleated structures called schizonts. Each schizont matures and releases numerous invasive asexual structures known as merozoites into the bird's digestive tract, where they then invade other epithelial cells. These multiple invasive asexual stages, involving both sporozoites and merozoites, cause the abnormal digestive tract lesions that characterize coccidiosis. The sexual stage of the coccidiosis life cycle is initiated when the merozoites differentiate into gametocytes. The gametocytes then fuse and the fertilized products, called oocysts, are released into the intestinal lumen and are excreted via the feces. The parasite life cycle is completed by the release of immature non-sporulated oocysts and their sporulation to form mature sporulated oocysts outside the host.

[0005] Although various species of Eimeria infect a wide range of hosts including mammals, seven species of Eimeria (E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella) are recognized to affect chickens, as disclosed in Acharya et al., "Alternatives to fight against coccidiosis: a review", Nepalese, Vet. J., 2017, 34, 152-167. Other species of Eimeria that have also been disclosed to affect chickens are E. mivati, E. lata, E. nagambiae and E. zaria. The life cycle of E. tenella (the representative species) in chickens is completed in about 7-9 days.

[0006] Several approaches to combating coccidiosis have been proposed in the art.

[0007] One approach consists of administering anticoccidial drugs, however this is accompanied by some degree of resistance to said drugs.

[0008] Another approach is the use of vaccines. Immunoprophylaxis designed to combat chicken coccidiosis appeared in the United States in the 1950s, when non-attenuated live parasite vaccines began to be used to combat Eimeria tenella. The main problem with this type of vaccine, which contains non-attenuated parasites, is the fact that the vaccination itself must be used in combination with an anticoccidial product to prevent the animal from contracting coccidiosis.

[0009] Vaccine efficacy can be assessed by parameters such as reduction in intestinal lesions, reduction in parasite load or replication, reduction in the occurrence of environmental oocysts and thus infection, reduction in clinical signs of disease and improved performance, including parameters such as feed conversion efficiency, body growth rate, flock uniformity and / or egg production.

[0010] Administration of a live vaccine aims to provide protective immunity by deliberately inducing infection in chickens by administration of small numbers of Eimeria parasites.

[0011] Available vaccines against coccidiosis are divided into two main groups: live non-attenuated and live attenuated vaccines.

[0012] Vaccination is generally carried out in the hatchery or on the farm on the day of the bird's birth, for example by administering live Eimeria vaccine directly to the bird by spraying or sprinkling it on the bird's feed and / or drinking water. The infective oocysts complete their life cycle in the bird's intestinal tract as described above, and finish by releasing a new generation of non-sporulated oocysts in 5-11 days depending on the Eimeria species. The non-sporulated oocysts shed with the bird's faeces then become infectious in the external environment, i.e. form spores, and re-infect the bird via host ingestion. After two or three such cycles, the bird becomes immune to coccidiosis. The acquired immunity wanes over a period of 3-4 months if there is no subsequent exposure to infective oocysts.

[0013] Wild-type Eimeria are generally isolated from clinical disease outbreaks in poultry flocks and can be propagated for use as virulent infectious strains. A typical non-attenuated vaccine consists of infectious oocysts from mildly to moderately virulent strains of different Eimeria species that have been maintained by laboratory passage. These non-attenuated Eimeria are capable of causing coccidiosis if ingested in large quantities. After the first dose, the vaccination process relies solely on reinfection via the host's ingestion of sporulated oocysts from the litter. A major concern with the use of wild-type vaccines is the need to administer anticoccidial agents to stop the replication of wild-type Eimeria strains.

[0014] The first commercial vaccines against coccidiosis were based either on the oral administration of Eimeria species in drinking water or on the addition of the vaccine to the animal feed. The first vaccines contained live non-attenuated Eimeria tenella oocysts, which were further developed by incorporating various Eimeria species, as disclosed in RB Williams, "Fifty years of anticoccidial vaccines for poultry (1952-2002)", Avian Dis., 2002, 46(4), 775-802. However, as disclosed in Soutter et al. op. cit., the use of wild-type and therefore fully virulent vaccines represents a significant risk, as large numbers of oocysts can rapidly accumulate in the poultry litter, causing high levels of exposure and clinical disease and even death, necessitating the use of anticoccidial drugs after vaccination.

[0015] International Patent Application No. 96 / 40233 and US Patent Application Publication No. 2002 / 0090378 disclose live in ovo vaccines containing an effective immunizing amount of live Eimeria sporocysts or oocysts, or a mixture thereof. The vaccines covered by said patent documents are, for example, the Inovocox EM1 vaccine, a non-attenuated coccidiosis vaccine containing live oocysts of E. acervulina, E. maxima and E. tenella, as disclosed in Sokale et al., "Effects of administration of an in ovo coccidiosis vaccine at different embryonic ages on vaccine cycling and performance of broiler chickens," Poultry Sci., 2020, 100:100914. However, Sokale et al., "Effects of coccidiosis vaccination administered by in ovo injection on the hatchability and hatching chick quality of broilers," Poult. Sci., 2017, 96, 541-7, disclose that in ovo vaccination with Inovocox EM1 vaccine results in a reduced hatchability of injected eggs compared to the non-injected treatment group.

[0016] International Patent Application No. WO 96 / 40234 discloses a live in ovo vaccine containing an effective immunising amount of live Eimeria sporozoites or merozoites, respectively, or a mixture thereof.

[0017] Attenuated vaccines are made of infectious oocysts with reduced virulence. Due to the strong correlation between attenuated virulence and having a shorter prepatent period, many attenuated strains are also precocious. Consistently, attenuated strains with a shorter prepatent period are also commonly referred to as "precocious strains".

[0018] Live attenuated vaccines have been prepared from Eimeria parasites, most of which have been selected for their shortened life cycle, also known as "precocity," and the attenuation method is described in TK Jeffers, "Attenuation of Eimeria tenella through selection for precocity." Precociousness), J. Parasitol., 1975, 61(6), 1083-1090. Precocious parasite strains show reduced virulence but retain their immunogenicity. However, the production of attenuated vaccines does not fully meet the needs of industrial poultry keeping. The production of this type of vaccine is complicated and its cost is high compared to anticoccidiostats and non-attenuated live parasite vaccines. Furthermore, these products have a very short shelf life which complicates transportation. Because it is difficult to attenuate all pathogenic Eimeria species and because the production is complicated and requires a lot of labor and animal handling, this type of product is scarce on the market and their use remains limited. The said live attenuated vaccines usually contain a mixture of sporulated oocysts of different Eimeria species. However, attenuated vaccines produce fewer oocysts than non-attenuated strains and the accumulation of infectious oocysts in the environment is slower, which results in a longer time required for immunity to be fully established.

[0019] International Patent Application No. 99 / 50387 discloses a vaccine comprising one or more attenuated strains of E. maxima ARI-73 / 97, E. acervulina ARI-77 / 97, ​​E. tenella ARI-ll / 98, E. necatrix MCK01 and / or E. necatrix ARIMEDNEC3+8 or antigens of said one or more strains in combination with a veterinarily acceptable carrier or excipient for administration to hatched chicks.

[0020] Vaccines may be administered in drinking water, food gels, feed sprays, animal sprays, intrayolk sac injections, or in ovo prior to hatching. In ovo vaccination in particular offers the potential for faster vaccination of large numbers of eggs, reduced labor costs, and reduced risk of human error, as disclosed in ED Pebbles, "In ovo applications in poultry: A review", Poult. Sci., 2018, 97, 2333-38.

[0021] However, in ovo vaccines still have different challenges to overcome in order to confer immunity efficiently. One challenge to be overcome by in ovo administration is related to the reduced hatchability with this particular route of administration due to egg breakage, damage to the embryo or supporting structures within the egg, and increased microbial infections that usually lead to later mortality and culling.

[0022] European Patent Application No. 0291173 discloses a method for controlling coccidiosis comprising administering to birds a non-replicating immunogen effective to induce immunity to the disease, where the immunogen is administered to the bird while the bird is an embryo contained within the egg, in a preferred embodiment the immunogen is an extract of sporulated Eimeria oocysts.

[0023] European Patent Application No. 0650733 discloses a live in-ovo vaccine for avian species comprising an attenuated or non-pathogenic microorganism expressing one or more antigenic epitopes from Eimeria species, preferably a specific Salmonella typhimurium strain.

[0024] International Patent Application No. 2009 / 148895 discloses a vaccine comprising a first strain of Eimeria spp. and a second strain of said Eimeria spp., wherein the first strain and the second strain have an asynchronous pre-patent period, and wherein the first strain is a non-attenuated strain and the second strain is a precocious strain.

[0025] Despite the various approaches available in the state of the art, unfortunately existing coccidiosis vaccines exhibit shortcomings and weaknesses that need to be addressed.

[0026] Thus, there remains a need in the art to develop and provide new vaccine compositions against coccidiosis as new advances in the control of chicken coccidiosis, for improved cost-effective manufacture and administration to embryonated eggs with optimal safety and efficacy when compared to compositions based on non-attenuated Eimeria spp.

[0027] Additionally, other diseases affecting avian species, such as Infectious Bursal Disease (IBD), also known as Gumboro disease, can affect hatcheries and farms along with Eimeria infections. Infectious Bursal Disease is an immunosuppressive viral infection caused by the Infectious Bursal Disease Virus (IBDV) that affects young chicks. IBD is widespread in poultry. IBDV can cause histopathological changes in both the bursa and thymus, affecting both humoral and cell-mediated responses. As disclosed in JJ Giambrone, "Effects of infectious bursal disease on the severity of Eimeria tenella infections in broiler chicks", Poult. Sci., 1977, 56(1), 243-6, studies have shown that prior exposure to IBDV can increase susceptibility to other important poultry diseases, including Eimeria infections.

[0028] Therefore, there is also a need in the art to provide new means to prevent and control both Eimeria spp. and IBDV infection in chicks from the first day of life. Summary of the Invention

[0029] An object of the present invention is a composition for use as a vaccine in the prevention and / or treatment of Eimeria infection in embryonated eggs of domesticated birds by in ovo injection.

[0030] Another aspect of the invention is said composition for use as a combination vaccine in the prevention and / or treatment of Eimeria infection and infectious bursal disease.

[0031] Another aspect of the invention is a kit for use as a vaccine.

[0032] Another aspect of the present invention is a composition comprising Eimeria sporulated oocysts from a precociously attenuated strain of E. acervulina and at least one precociously attenuated strain selected from E. maxima, E. plecox and E. tenella, wherein the amount of sporulated oocysts from E. acervulina is about 100-6000 per dose, the amount of sporulated oocysts from E. maxima is about 50-5000 per dose, the amount of sporulated oocysts from E. plecox is about 50-3000 per dose, and the amount of sporulated oocysts from E. tenella is about 50-3000 per dose, and the composition is suitable for preparation of an in ovo vaccine.

[0033] Another aspect of the present invention is a composition comprising Eimeria sporulated oocysts from a precociously attenuated strain of E. acervulina and at least one precociously attenuated strain selected from E. maxima, E. plecox and E. tenella, wherein the amount of sporulated oocysts from E. acervulina is about 100-6000 per dose, the amount of sporulated oocysts from E. maxima is about 50-5000 per dose, the amount of sporulated oocysts from E. plecox is about 50-3000 per dose and the amount of sporulated oocysts from E. tenella is about 50-3000 per dose, and the composition is suitable for the preparation of an in ovo vaccine, particularly with the proviso that the composition does not comprise E. mitis sporulated oocysts.

[0034] Another embodiment of the present invention is an in ovo vaccine comprising Eimeria sporulated oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella, wherein the amount of sporulated oocysts from E. acervulina is about 100-6000 per dose, the amount of sporulated oocysts from E. maxima is about 50-5000 per dose, the amount of sporulated oocysts from E. plecox is about 50-3000 per dose, and the amount of sporulated oocysts from E. tenella is about 50-3000 per dose.

[0035] Another embodiment of the present invention is an in ovo vaccine comprising Eimeria sporulated oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella, wherein the amount of sporulated oocysts from E. acervulina is about 100-6000 per dose, the amount of sporulated oocysts from E. maxima is about 50-5000 per dose, the amount of sporulated oocysts from E. plecox is about 50-3000 per dose and the amount of sporulated oocysts from E. tenella is about 50-3000 per dose, particularly with the proviso that the in ovo vaccine does not comprise E. mitis sporulated oocysts.

[0036] Another aspect of the invention is a kit comprising the composition or in ovo vaccine of the invention. [Brief description of the drawings]

[0037] [Figure 1] Figure 2 shows the efficacy results of a vaccine composition based on attenuated Eimeria oocysts administered by in ovo injection as disclosed in Example 2. The mean intestinal lesion scores of birds after experimental infection in vaccination groups 1 to 4 (G1 to G4) per Eimeria species for experimental infection (E. acervulina (A), E. plecox (P), E. maxima (M) and E. tenella (T)) are shown. Groups 1 to 3 (G1 to G3) correspond to birds vaccinated with an attenuated Eimeria vaccine containing different oocyst doses by the in ovo route and group 4 (G4) corresponds to birds vaccinated with a mock vaccine by the in ovo route. The mean intestinal lesion scores are represented on the y-axis. Groups 1 to 4 (G1 to G4) and Eimeria species are represented on the x-axis. [Diagram 2]Figure 2 shows the safety results of attenuated Eimeria oocyst-based vaccine composition administered by in ovo injection as disclosed in Example 3. The mean body weights (in grams) of birds of groups 1 to 3 (G1 to G3) at 0, 4.5, 6, 7 and 14 days after hatch are represented on the y-axis. Group 1 (G1) corresponds to birds vaccinated with a single dose of the attenuated vaccine composition by in ovo injection. Group 2 (G2) corresponds to birds vaccinated with a 10-fold dose of the attenuated vaccine composition by in ovo injection. Group 3 (G3) corresponds to birds vaccinated with a mock vaccine composition containing PBS only. The number of days after hatch is represented on the x-axis. [Diagram 3] Figure 3 shows the hatchability results obtained with attenuated Eimeria oocyst-based vaccine composition administered by the in ovo route as disclosed in Example 4. The hatchability (%) of each group (A: control group, B: vaccinated group) is represented on the ordinate (y-axis). Figure 3A shows the hatchability (%) obtained in hatchery A, Figure 3B shows the hatchability (%) obtained in hatchery B and Figure 3C shows the overall average hatchability (%) of the two different hatcheries included in the study. [Figure 4] Figure 1 shows the results for efficacy of a single dose of a combination attenuated Eimeria vaccine against chicken coccidiosis and a live attenuated vaccine against infectious bursal disease (IBD) by in ovo administration into embryonated chicken eggs against infection with highly virulent IBD virus (vvIBDv) as disclosed in Example 5. The mean acute functional histopathological lesion scores 6 days after experimental infection (day 30 of the study) are shown on the ordinate and each treatment group (group 1: vaccinated group, group 2: control group) is shown on the abscissa. [Diagram 5] Further results from Example 5 are shown, in which the mean mononuclear cell infiltration scores 6 days after experimental infection (day 30 of the study) are shown on the ordinate and each treatment group (group 1: vaccinated group, group 2: control group) is shown on the abscissa. [Figure 6]Further results from Example 5 are shown, in which the mean fold edema scores 6 days after experimental infection (day 30 of the study) are shown on the ordinate and each treatment group (Group 1: vaccinated group, Group 2: control group) is shown on the abscissa. [Figure 7] Further results from Example 5 are shown, in which the mean muscle wall edema scores 6 days after experimental infection (day 30 of the study) are shown on the ordinate and each treatment group (Group 1: vaccinated group, Group 2: control group) is shown on the abscissa. [Figure 8] Further results from Example 5 are shown, in which the mean serosal edema scores 6 days after experimental infection (day 30 of the study) are shown on the ordinate and each treatment group (Group 1: vaccinated group, Group 2: control group) is shown on the abscissa. [Figure 9] Results for efficacy of a single dose of a combination attenuated Eimeria vaccine against chicken coccidiosis and a live attenuated vaccine against infectious bursal disease (IBD) administered in ovo into embryonated eggs against infection with four different Eimeria species. The mean intestinal lesion scores of birds after experimental infection with each Eimeria species are shown on the ordinate, with each treatment group (group 1: vaccinated group, group 2: control group) and each Eimeria species (E. acervulina: A, E. maxima: B, E. plecox: C and E. tenella: D) shown on the abscissa. [Figure 10] For Example 7, the average Eimeria spp. oocyst counts (oocysts per gram of fresh feces) in fresh feces are shown on the ordinate from days 1-9 post-hatch, with each treatment group (Group 1 (solid line): live attenuated Eimeria spp. vaccine composition of the present invention administered in ovo, and Group 2 (dashed line): live attenuated Eimeria spp. vaccine administered by coarse spray post-hatch) shown on the abscissa. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] The object of the present invention is a composition, an in ovo vaccine and a composition for use as a vaccine in the prevention and / or treatment of Eimeria infection, the composition comprising Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella, and an effective immunizing amount of the vaccine is administered in ovo to an embryonated egg of a domestic bird, the composition for use as a vaccine. Another phrase for the object of the present invention is a composition, an in ovo vaccine and a composition for use as a vaccine in the prevention and / or treatment of Eimeria infection, the composition comprising Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella, and an effective immunizing amount of the vaccine is administered in ovo to an embryonated egg of a domestic bird, the composition for use as a vaccine.

[0039] Another object of the present invention is an in ovo vaccine comprising Eimeria sporulated oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella, wherein the amount of sporulated oocysts from E. acervulina is about 100-6000 per dose, the amount of sporulated oocysts from E. maxima is about 50-5000 per dose, the amount of sporulated oocysts from E. plecox is about 50-3000 per dose and the amount of sporulated oocysts from E. tenella is about 50-3000 per dose, with the proviso that the in ovo vaccine does not contain E. mitis sporulated oocysts.

[0040] Another object of the present invention is a method for the prevention and / or treatment of Eimeria infection by in ovo administration to embryonated eggs of domesticated birds of a composition comprising an effective immunizing amount of a precociously attenuated strain of E. acervulina and Eimeria oocysts from at least one precociously attenuated strain selected from E. maxima, E. plecox and E. tenella, In one embodiment, said prevention and / or treatment results in an improvement compared to untreated controls of the same domesticated birds.

[0041] The authors of the present invention have surprisingly found a composition comprising oocysts from a defined attenuated species of the genus Eimeria that is capable of effectively inducing an immune response, in particular a protective immune response, against Eimeria infection by in ovo administration. Furthermore, the inventors have surprisingly observed an increase in the hatchability of eggs vaccinated in ovo, contrary to what the prior art discloses.

[0042] The authors also unexpectedly discovered that a combination vaccine comprising a composition comprising oocysts from an attenuated strain of Eimeria in combination with a composition comprising an IBDV (infectious bursal disease virus) antigen administered by the in ovo route is safe and simultaneously elicits effective protection against both Eimeria spp. and IBDV infection.

[0043] In this specification and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges defined by the terms "between" or "to" include the two endpoints. The term "about" refers to a deviation of ±10%, preferably ±5%.

[0044] Within the scope of the present invention, the terms "vaccine", "vaccine composition" and "composition for use as a vaccine" refer to a preparation administered to mimic the immune response of a subject against a particular infectious disease, in particular a preparation for the prevention and / or treatment of Eimeria infection. The terms "vaccine", "vaccine composition" and "composition for use as a vaccine" as used herein also refer to a preparation which upon administration to a subject elicits a cellular or antibody-mediated immunological response in the host, in particular a protective response. The preparation may contain different Eimeria species that infect domesticated birds. The vaccine and vaccine composition of the present invention are further intended to prevent and / or treat chicken coccidiosis.

[0045] The term "domesticated bird" as used herein includes chickens, turkeys, ducks, game birds (including but not limited to quails, pheasants and geese) and ratites (including but not limited to ostriches), unless otherwise specified. Preferably, the domesticated bird is a poultry, more preferably a chicken. In the context of the present invention, the term "domesticated bird" is also referred to as a host or subject.

[0046] As used herein, unless otherwise specified, the term "administration in ovo into embryonated eggs" means administration into a domesticated bird egg which contains a live, developing embryo.

[0047] As used herein, the terms "administration in ovo", "in ovo administration", "in ovo route" or "in ovo injection" refer to administration of the vaccine described herein to a domesticated avian egg containing a live developing embryo, unless otherwise specified, by penetrating the egg shell and introducing the vaccine. Such means of administration include, but are not limited to, injection of the vaccine. Thus, the term "in ovo vaccine" refers to a vaccine suitable for in ovo administration. In ovo administration encompasses administration of the composition for use as a vaccine of the present invention during the last quarter of the culture of the domesticated avian. In ovo administration in embryonated chicken eggs occurs between day 15 and day 20 of culture, preferably between day 17 and day 19.5 of culture, more preferably between day 18 and day 19.2 of culture, and even more preferably at about day 18.5 of culture. In ovo administration can be performed using any suitable in ovo administration method, preferably the vaccine is administered by injection. According to one injection method, an 18 gauge needle is used to pierce the egg shell at the large end of the egg to expose the air cell of the egg. A 2.54-3.81 cm (1.0-1.5 inch) 22 gauge needle attached to an appropriately sized syringe can be inserted through the hole and through the membrane of the air cell. The injection site may be within any area of ​​the egg, preferably the injection is made axially through the center of the large end of the egg into the amniotic membrane. Alternatively, automated egg injection systems such as Embrex® Inovoject Systems (Zoetis), Egginject® In ovo Systems (Ceva Animal Health), Ovo-Jector® (Boehringer Ingelheim), Select Inject™ (Vinovo Poultry Health Solutions), Ultimate In-ovo Egg Injection (Innovatec Hatchery Automation BV, Sanovo vax (Sanovo Technology Group) can also be used. Other suitable injection methods are known to those of skill in the art.

[0048] The term "oocysts" as used herein, unless otherwise specified, means live Coccidiosis oocysts, and in particular live Eimeria sporulated oocysts or a mixture of sporulated and non-sporulated oocysts.

[0049] The term "Eimeria" as used herein, unless otherwise specified, refers to one or more species of the genus Eimeria that infect domestic birds. Eimeria species include those present in chickens, in particular E. tenella, E. acervulina, E. maxima, E. necatrix, E. mitis, E. plecox, E. mibatii and E. brunetti, and those present in turkeys, in particular E. meleagrimitis, E. adenoeides, E. gallopavonis, E. dispersa, E. meleagridis, E. innocua and E. subrotunda, and also Eimeria species infecting other domestic birds as defined above. The term "Eimeria" also includes any newly discovered strains or species of Eimeria that infect domestic birds as defined above.

[0050] The term "attenuated" strain or "attenuated Eimeria strain" as used herein means, unless otherwise specified, a strain selected for its reduced pathogenicity in a host. Attenuation of a virulent or wild-type strain of an Eimeria species can be achieved by many means, such as attenuation by repeated passage in chicken embryos to produce an embryo-adapted strain, attenuation by selection for early maturation of oocysts during serial passage (such as serial chick or embryo passage) to produce a precocious strain, or other means known to those skilled in the art, such as, but not limited to, chemical mutagenesis and irradiation methods. Preferably, the Eimeria strain is attenuated by performing many serial embryo passages.

[0051] As used herein, unless otherwise specified, the term "precocious" or "precocious attenuated strain" refers to an Eimeria strain that has a reduced prepatent period compared to a non-attenuated strain of the same Eimeria species.

[0052] The term "wild-type" as used herein, unless otherwise specified, refers to an Eimeria wild isolate that has not been altered or modified by attenuation passage or any other attenuating treatment. Wild-type Eimeria strains may have different degrees of pathogenicity.

[0053] The term "strain" or "Eimeria strain" as used herein, unless otherwise specified, refers to a subpopulation of a Coccidia species that can be distinguished from the general population of that species by one or more characteristics such as genomic structure and organization, pathogenicity, immunogenicity, geographic distribution, prepatent period, and / or populations resulting from the expansion of a single oocyst.

[0054] The term "parental strain" as used herein refers to a subpopulation of Coccidia species derived from a population obtained from wild-type oocysts, in particular a subpopulation of Eimeria species derived from wild-type oocysts. The parental strain is a wild-type strain, which may therefore have different degrees of pathogenicity. When preparing an attenuated strain, the parental strain is considered as the original strain from which the attenuation pressure begins to ultimately obtain an attenuated strain. As described above, attenuation can be achieved by many means, including serial passaging of the parental strain (such as serial chick or embryo passaging).

[0055] The term "effective immunizing amount" or "immunologically effective amount" as used herein, unless otherwise specified, means an amount that induces an immune response in a vaccinated animal, and in particular a sufficient number of live oocysts to elicit an immune response in a vaccinated animal and further confer immunological protection to the hatched birds that is greater than the genetic immunity of non-immunized and / or untreated controls of the same domesticated bird. As used herein, the terms "immunize" or "immunization" and "vaccinate" or "vaccination" are synonymous and are used interchangeably. An effective immunizing amount may be administered either as a single dose or as part of a series of doses and is effective in the treatment and / or prevention of Eimeria spp. infection and / or coccidiosis. The effective immunizing amount varies depending on the health and physical condition of the treated subject, age, the capacity of the subject's immune system to produce antibodies from any of the immune classes such as immunoglobulin A, D, E, G or M, proliferation of B and T lymphocytes, provision of activation, proliferation and differentiation signals to immune cells, expansion of helper T cells, suppressor T cells and / or cytotoxic T cells and / or gamma, delta-T cell populations, the degree of protection desired, the formulation of the vaccine, veterinary evaluation of the medical situation and other relevant factors. For a given vaccine, the skilled artisan can know the effective immunizing amount according to the definition by using routine methods well known in the art without resorting to any of the techniques of the present invention, such as the method disclosed in Example 2 below in particular.

[0056] The term "immunological response" or "immune response" as used herein, unless otherwise specified, refers to an immune reaction against an antigen or composition that results in an innate, humoral and / or cellular immune response in the vaccinated subject against the antigen present in the composition of interest. The response thus causes a corresponding increase in antibody titers and / or an increase in activation of cell-mediated immunity. Preferably, the immune response elicited confers protective immunity that prevents and / or reduces clinical disease signs of coccidiosis. Preferably, the immune response prevents and / or reduces clinical signs of coccidiosis, such as weight loss, morbidity and / or mortality, in the vaccinated subject, i.e., a domesticated bird as defined above, when naturally or experimentally infected with a toxic dose of Eimeria spp. The prevention and / or reduction of clinical signs of coccidiosis provides an improvement compared to an infected non-immunized or untreated control group of the same domesticated bird. Preferably, the improvement comprises a vaccine efficacy parameter selected from the group consisting of reduced weight loss, reduced Eimeria oocyst excretion, reduced Eimeria oocyst load, reduced Eimeria oocyst shedding, reduced Eimeria infection, depression, ruffled feathers, arched body position, diarrhea, changes in faeces including blood or mucus content, dehydration, intestinal lesions, morbidity and / or mortality.

[0057] The term "combination" or "combination vaccine" as used herein means that the vaccine contains various antigens in a single formulation, unless otherwise stated. Thus, the composition of the present invention, the in ovo vaccine or the composition for use as a vaccine can also be administered in combination with other vaccine compositions. The vaccines to be combined are mixed into a single formulation, preferably before in ovo administration. Combination vaccines also include vaccines that are not mixed but are administered simultaneously to domesticated birds. Thus, combination vaccines can be in a single formulation or in different formulations. Combination vaccines can be in the same container or in different containers.

[0058] The term "treatment" as used herein, unless otherwise stated, refers to any kind of treatment with the immunogenic composition or composition for use as a vaccine of the present invention, aimed at terminating, preventing, ameliorating or reducing a clinical condition or susceptibility to an existing disease as described herein, including complete cure of the disease as well as amelioration or reduction of said disease. In a preferred embodiment, the term "treatment" relates to prophylactic treatment of a disease or condition as defined herein (i.e., treatment to prevent or reduce susceptibility to a clinical condition). Thus, "treatment", "treating" and their equivalent terms refer to obtaining a desired pharmacological or physiological effect, including any treatment of an abnormal condition or disease in a subject, including domesticated birds. The effect may be prophylactic in that it completely or partially prevents the disease or symptoms, and / or therapeutic in that it partially or completely cures the disease and / or the adverse effects caused by the disease. That is, "treatment" includes (1) preventing the onset or recurrence of a disease in a subject; (2) inhibiting a disease, such as by arresting its occurrence; (3) halting or terminating the disease or at least its associated clinical signs, e.g., by restoring or repairing a loss, defect or deficiency of function or stimulating an inefficient process, resulting in reversal of the disease or its clinical signs, so that the host is no longer afflicted by the disease or its clinical signs; or (4) alleviating, reducing or ameliorating the disease or its associated clinical signs (wherein amelioration is used broadly to refer to at least a decrease in the magnitude of a clinical sign parameter, a decrease in pathogen load, pathogen shedding, or a decrease in pathogen transmission).

[0059] The terms "prevention" or "reduction" or "preventing" or "reducing" as used herein each refer to, but are not limited to, a prophylactic process in which a subject is exposed to an immunogenic composition or composition for use as a vaccine of the present invention prior to induction or onset of a disease process, and which, when administered to the subject, elicits or can elicit an immune response in the subject against Eimeria infection. In essence, such treatment results in the prevention or reduction of clinical signs of chicken coccidiosis or clinical signs associated with chicken coccidiosis, respectively, or a reduction in Eimeria oocyst shedding. More specifically, the terms "prevention" or "preventing" as used herein generally refer to a prophylactic process in which a domesticated bird is exposed to an immunogenic composition or composition for use as a vaccine of the present invention prior to induction or onset of chicken coccidiosis.

[0060] As used herein, "reduction of clinical signs associated with chicken coccidiosis or Eimeria infection" means, but is not limited to, reducing the number of infected subjects in a flock, reducing or eliminating the number of subjects exhibiting clinical signs of Eimeria infection, or reducing the severity of any clinical signs present in a subject, compared to either wild-type infected subjects or experimentally infected non-immune or untreated subjects. For example, it refers to any reduction in Eimeria oocyst shedding, reduction in Eimeria oocyst infection, or reduction in any clinical signs of chicken coccidiosis. Preferably, these clinical signs are reduced by at least 10 percent (%) in subjects receiving a composition for use as a vaccine of the present invention, compared to subjects who have not received the composition and who may become infected. More preferably, the clinical signs are reduced by at least 20 percent (%), preferably at least 30 percent (%), more preferably at least 40 percent (%), and even more preferably at least 50 percent (%) in subjects receiving a composition for use as a vaccine of the present invention, compared to subjects who have not received the composition and who may become infected. The clinical signs referred to herein may be selected from the group consisting of depression, ruffled feathers, hunched body position, diarrhea, changes in the faeces including blood or mucus content, dehydration, intestinal lesions, weight loss and Eimeria oocyst excretion.

[0061] Herein, "reduction of clinical signs associated with IBDV, i.e., infectious bursal disease virus or Gumboro disease" means, but is not limited to, reducing the number of infected subjects in a population, reducing or eliminating the number of subjects showing clinical signs of IBDV infection, or reducing the severity of any clinical signs present in a subject compared to either wild-type infected subjects or experimentally infected non-immune or untreated subjects. For example, it may refer to any reduction in clinical signs selected from the group consisting of mean acute histological functional score, inflammatory infiltrate, fold edema, and absence of serosal edema and muscle wall edema. Preferably, these clinical signs are reduced by at least 10 percent (%) in subjects receiving a composition for use as a vaccine of the present invention compared to subjects who have not received the composition of the present invention and who may become infected. More preferably, the clinical signs are reduced by at least 20 percent (%), even more preferably at least 30 percent (%), even more preferably at least 40 percent (%), and even more preferably at least 50 percent (%) compared to subjects who have not received the composition of the present invention and who may become infected.

[0062] Vaccine Compositions In a first embodiment, the composition for use as a vaccine confers at least an immunogenic and / or protective immune response against Eimeria infection.

[0063] In a further embodiment, the composition for use as a vaccine provides an immunogenic and / or protective immune response against infection caused by at least one Eimeria species.

[0064] In a further embodiment, the composition for use as a vaccine reduces or prevents one or more clinical signs associated with chicken coccidiosis in hatched birds.

[0065] In a further embodiment, the composition for use as a vaccine reduces or prevents one or more clinical signs associated with chicken coccidiosis caused by Eimeria infection in hatched birds.

[0066] In further embodiments, the clinical signs are selected from the group consisting of intestinal lesions, weight loss, oocyst excretion, oocyst burden, oocyst shedding, diarrhea, dehydration, presence of blood in the faeces, presence of mucus in the faeces, Eimeria infection, depression, ruffled feathers, hunched body position, morbidity and mortality.

[0067] In a further embodiment, the clinical signs are selected from the group consisting of intestinal lesions, weight loss, oocyst excretion, diarrhea, dehydration, presence of blood in the stool and presence of mucus in the stool.

[0068] In a further embodiment, the composition for use as a vaccine increases hatch rate compared to a non-vaccinated control group, hi one embodiment, the control group is administered PBS.

[0069] In a further embodiment, the composition for use as a vaccine of the present invention reduces oocyst shedding in hatched birds compared to hatched birds inoculated in ovo with a composition comprising a non-attenuated Eimeria strain.

[0070] In a further embodiment, an effective immunizing amount of a composition for use as a vaccine of the present invention reduces oocyst shedding in hatched birds compared to hatched birds inoculated in ovo with a composition comprising a non-attenuated Eimeria strain.

[0071] In a further embodiment, the domesticated bird is selected from chickens, turkeys, ducks, game birds and ratites. Preferably, the domesticated bird is a chicken.

[0072] Eimeria infection In one embodiment, the Eimeria infection causes coccidiosis, hi one embodiment, the coccidiosis is caused by an Eimeria species selected from the group consisting of Eimeria acervulina, Eimeria maxima, Eimeria plecox, and Eimeria tenella.

[0073] In a further embodiment, the composition for use as a vaccine is for the prevention of Eimeria infection.

[0074] In a further embodiment, the composition for use as a vaccine is for the treatment of Eimeria infection.

[0075] In a further embodiment, the composition for use as a vaccine is for the prevention of chicken coccidiosis.

[0076] In a further embodiment, the composition for use as a vaccine is for the treatment of chicken coccidiosis.

[0077] Eimeria species The composition of the invention, an in ovo vaccine or a composition for use as a vaccine, comprises, consists of or consists essentially of Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella.

[0078] In one embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine consists or consists essentially of Eimeria oocysts from a precocious attenuated strain of E. acervulina and E. maxima.

[0079] In one embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine consists or consists essentially of Eimeria oocysts from precocious attenuated strains of Eimeria acervulina and Eimeria plecox.

[0080] In one embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine consists or consists essentially of Eimeria oocysts from a precocious attenuated strain of E. acervulina and E. tenella.

[0081] In a preferred embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine comprises, consists of, or consists essentially of Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least two precocious attenuated strains selected from E. maxima, E. plecox and E. tenella. In one embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine consists of, or consists essentially of Eimeria oocysts from a precocious attenuated strain of E. acervulina, E. maxima and E. plecox. In one embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine consists of, or consists essentially of Eimeria oocysts from a precocious attenuated strain of E. acervulina, E. maxima and E. tenella. In one embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine consists or consists essentially of Eimeria oocysts from precocious attenuated strains of E. acervulina, E. plecox and E. tenella.

[0082] In another preferred embodiment, the composition of the invention, or composition for use as an in ovo vaccine or vaccine, comprises, consists of, or consists essentially of a mixture of sporulated oocysts from precocious attenuated strains of E. acervulina, E. maxima, E. plecox and E. tenella.

[0083] In another embodiment, the composition of the invention, an in ovo vaccine or a composition for use as a vaccine comprises Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox, and E. tenella. In another embodiment, the composition of the invention, an in ovo vaccine or a composition for use as a vaccine comprises Eimeria oocysts from a precocious attenuated strain of E. acervulina, E. maxima, E. plecox, and E. tenella.

[0084] In a further embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine in the prevention and / or treatment of Eimeria infection, provided that it does not contain Eimeria mitis sporulated oocysts.

[0085] In a further embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine in the prevention and / or treatment of Eimeria infection comprises Eimeria oocysts selected from a precocious attenuated strain of Eimeria acervulina having reduced virulence and reduced oocyst output or shedding compared to the parent Eimeria acervulina strain over a 14 day period.

[0086] In a further embodiment, when the precocious attenuated strain of Eimeria acervulina used in the compositions, in ovo vaccines or compositions for use as a vaccine of the present invention is administered to a host, the host excretes about 30% to about 75% fewer, preferably about 35% to about 65% fewer, more preferably about 40% to about 60% fewer, and even more preferably about 45% to about 55% fewer oocysts during a 14 day period compared to the oocysts excreted by a host that received the parent Eimeria acervulina strain.

[0087] In a further embodiment, when the precocious attenuated strain of E. acervulina used in the composition, in ovo vaccine or composition for use as a vaccine of the invention is administered to a host, the host excretes a similar number of oocysts in a 14 day period as the number of oocysts excreted in a 14 day period by a host that received E. acervulina strain 120718P5. Preferably, when the precocious attenuated strain of E. acervulina used in the composition, in ovo vaccine or composition for use as a vaccine of the present invention is administered to a host, the host excretes about ±75% of the oocysts excreted by a host that has received E. acervulina strain 120718P5, preferably about ±70%, preferably about ±60%, preferably about ±50%, preferably about ±40%, preferably about ±30%, more preferably about ±25%, more preferably about ±20%, more preferably about ±15%, and even more preferably about ±10% of the oocysts excreted by a host that has received E. acervulina strain 120718P5 over a 14 day period.

[0088] Oocyst excretion and enumeration can be performed using any of the methods disclosed in the art, for example, in Shirley et al., COST 89 / 820: Biotechnology: guidelines on techniques in coccidiosis research, 1995, Luxembourg: Office for Official Publications of the European Communities.

[0089] In one embodiment, the E. acervulina precocious strain used in the composition, in ovo vaccine or composition for use as a vaccine of the invention is attenuated by repeated passaging from a parent strain selected for precocious maturation. In a further embodiment, the repeated passaging is repeated laboratory passaging. In a particular embodiment, the E. acervulina precocious strain is attenuated after at least 3, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or at least 11 passages from a parent strain selected for precocious maturation. In a further embodiment, the E. acervulina precocious strain is attenuated after at least 5 passages from a parent strain selected for precocious maturation.

[0090] In one embodiment, the E. acervulina precocious attenuated strain used in the composition, in ovo vaccine or composition for use as a vaccine of the invention is E. acervulina strain 120718P5 deposited at the Culture Collection of Algae & Protozoa (Scottish Marine Institute, OBAN, Argyl, PA37 1QA, Scotland, UK) by HIPRA SCIENTIFIC, SLU (Avda La Selva 135, 17170 Amer, Girona, Spain) under the Budapest Treaty on 06.10.2021 under the accession number CCAP2016 / 1. This strain has a reduced pathogenicity and when administered to a host, the host excretes about 50% fewer live oocysts compared to the oocysts excreted by a host that received the parent E. acervulina strain during a 14-day period. This strain is obtained by laboratory passage, preferably after five repeated passages of one selected for precocious maturity from a parent strain, using methods such as those disclosed in Jeffers et al., supra.

[0091] Oocysts from precocious attenuated strains of E. maxima, E. plecox and E. tenella are well known in the art and the skilled artisan knows how to obtain them. Furthermore, there are several commercially available vaccines on the market that contain said precocious attenuated strains, for example, oocysts may be isolated from commercial compositions such as EVANT® (Laboratorios Hipra, SA, Avda. La Selva 135, 17170 Amer, Girona, Spain) and PARACOX8® (Merck Sharp&Dohme Animal Health, SL, 37008 Carbajosa de la Sagrada, Salamanca, Spain), which contain a combination of Eimeria oocysts, including E. maxima, E. plecox and E. tenella oocysts, among other sources. Other sources of oocysts from precocious attenuated strains of E. maxima and E. tenella are EVALON® (Laboratorios Hipra, SA, Avda. La Selva 135, 17170 Amer, Girona, Spain), PARACOX5® (Merck Sharp&Dohme Animal Health, SL, 37008 Carbajosa de la Sagrada, Salamanca, Spain), HATCHPAK® COCCI III (Boehringer Ingelheim Animal Health USA Inc., 3239 Duluth, USA) and HuveGuard MMAT® (Huvepharma NV., 2600 Antwerp, Belgium).

[0092] Oocysts Oocysts of precocious attenuated strains of Eimeria used in accordance with the present invention can be prepared by any of a number of methods known to those of skill in the art (eg, Shirley et al., supra).

[0093] Attenuation of the virulence of each Eimeria species is achieved by repeated passage in chickens by selecting for early appearance of oocysts, as disclosed by Jeffers, op. cit. Selection for early maturity allows for the selection of populations with an important reduction in the prepatent period (i.e., the time from ingestion of sporulated oocysts to appearance of oocysts in the feces) and a significant reduction in virulence.

[0094] For the attenuation of each Eimeria species, the purified parent strain is inoculated into coccidiosis-free specific pathogen-free (SPF) chicks to obtain the initial passage (P0). The first oocysts removed from the feces are then re-inoculated into new coccidiosis-free SPF chicks to obtain the second passage. Many similar successive repeated passages are performed until a population is obtained with oocysts that have a greatly reduced prepatent period and reduced pathogenicity compared to the parent strain.

[0095] To obtain Eimeria oocysts for the preparation of the composition of the invention, the in ovo vaccine or the composition for use as a vaccine, coccidiosis-free SPF chicks are inoculated with a suspension of sporulated oocysts for producing each Eimeria species. After a certain period of time, their feces are collected and the oocysts are collected. They are then sporulated and the obtained oocysts are re-inoculated into a new group of coccidiosis-free SPF chicks. This process is repeated several times, in particular three more times to collect oocysts of each Eimeria species. The collected oocysts are finally purified and sporulated.

[0096] As known in the prior art, the patency pattern of each Eimeria species may vary for each Eimeria species and strain. Completion of the life cycle, indicated by the appearance of non-sporulated oocysts in the host feces, is known as patency. Thus, the recovery period of oocysts from bird feces may vary, and those skilled in the art will know the optimal time to recover oocysts from each Eimeria species and strain, as disclosed in Shirley et al., supra.

[0097] The collected oocysts for each Eimeria species may be sporulated by any means known to those skilled in the art, such as potassium dichromate solution, potassium sorbate solution, or compositions containing peroxygen and organic acids, among others. The oocysts are allowed to sporulate by incubation at 29°C for 48 hours, preferably with a 2-3% potassium dichromate solution, with aeration. After sporulation, the potassium dichromate solution is removed by centrifugation. The sporulated oocysts are disinfected with sodium hypochlorite, and once removed, they are resuspended in PBS (phosphate buffered saline).

[0098] The sporulated oocysts of the different Eimeria species are then counted for each bulk suspension and the volume of oocyst suspensions of each Eimeria species to be mixed is calculated. The final suspension is obtained by mixing the different Eimeria oocyst suspensions and adjusting the final volume with PBS.

[0099] Sporocysts obtained from the disinfected sporulated oocysts may be frozen in liquid nitrogen (cryopreserved) for storage according to methods known in the art, such as those disclosed in Shirley et al., supra.

[0100] The oocysts of the present invention can be used to prepare a composition for the prevention and / or treatment of Eimeria infection, a composition for use as an in ovo vaccine or a vaccine. In one embodiment, the oocysts of the present invention are present in a concentration of 10 to 10 per embryonated egg. 8 10-10 oocysts per embryonated egg 7 10 oocysts per embryonated egg 2 ~10 7 10 oocysts per embryonated egg 2 ~10 6 10 oocysts or 1 embryonated egg 2 ~10 5 More preferably, the composition, in ovo vaccine or composition for use as a vaccine of the present invention is used to prepare a composition or in ovo vaccine at a dose containing 10 oocysts per embryonated egg.2 ~10 4 Contains a dose of oocysts.

[0101] Excipients In one embodiment, the in ovo vaccine or composition for use as a vaccine of the invention comprises at least a pharma- ceutical or veterinarily acceptable carrier or vehicle or excipient or adjuvant.

[0102] The pharma- ceutically or veterinarily acceptable carrier or vehicle or excipient may be any compound or combination of compounds that facilitates administration and / or delivery of the composition or the in ovo vaccine. Among other compounds, it may include, for example, wetting agents, dispersing agents, emulsifying agents, buffers (e.g., phosphate buffers), carbohydrates (e.g., glucose, sucrose, mannitol, sorbitol, starch or dextran), or stabilizers such as proteins (e.g., albumin, casein, bovine serum or skim milk).

[0103] Pharmaceutically or veterinarily acceptable carriers or vehicles are well known to those of skill in the art and include, among other components, water, saline, buffered saline such as phosphate buffered saline, or any other physiologically acceptable vehicle.

[0104] In one embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine comprises wetting agents, dispersing agents, emulsifying agents, buffers, salts, stabilizers, proteins, water, saline (e.g., sodium chloride and / or potassium chloride solutions), buffered saline (e.g., phosphate buffer), physiologically acceptable vehicles and mixtures thereof.

[0105] Suitable excipients for the composition or in ovo vaccine of the invention are described for example in the book "Handbook of pharmaceutical excipients", edited by RC Rowe, PJ Sheskey and ME Quinn, Pharmaceutical Press, 6th edition, 2009.

[0106] In one embodiment, the pharma- ceutically or veterinarily acceptable carrier or vehicle or excipient can be selected from the group of sodium or potassium chloride, phosphate buffer (e.g., a combination of disodium phosphate and potassium dihydrogen phosphate), surfactants, preferably non-ionic surfactants (e.g., polysorbates which are ethoxylated sorbitan fatty acid esters), and mixtures thereof.

[0107] In one embodiment, the composition, in ovo vaccine or composition for use as a vaccine of the invention is an aqueous suspension, hi a further embodiment, the composition, in ovo vaccine or composition for use as a vaccine comprises a combination of disodium phosphate, potassium dihydrogen phosphate, polysorbate, potassium chloride, sodium chloride and water as vehicles and excipients.

[0108] In further embodiments, the composition of the invention, the in ovo vaccine or the composition for use as a vaccine comprises polysorbate at a concentration in the range of about 0.001% to about 1% (v / v), about 0.005% to about 0.5% (v / v), about 0.006% to about 0.3% (v / v), about 0.007% to about 0.1% (v / v), about 0.005% to about 0.05% (v / v). Preferably, the composition of the invention, the in ovo vaccine or the composition for use as a vaccine comprises polysorbate at a concentration of about 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1% (v / v). More preferably, the composition of the invention, the in ovo vaccine or the composition for use as a vaccine, comprises polysorbate at a concentration of about 0.01% and 0.02% (v / v), even more preferably about 0.01% (v / v).

[0109] In a further embodiment, the composition of the invention, the in ovo vaccine or the composition for use as a vaccine comprises a polysorbate selected from the group consisting of polysorbate 20 (polyoxyethylene 20 sorbitan monolaurate), polysorbate 40 (polyoxyethylene 20 sorbitan monopalmitate), polysorbate 60 (polyoxyethylene 20 sorbitan monostearate), and polysorbate 80 (polyoxyethylene 20 sorbita monooleate). Preferably, the composition of the invention, the in ovo vaccine or the composition for use as a vaccine comprises polysorbate 80.

[0110] The compositions of the invention, in ovo vaccines or compositions for use as vaccines may comprise or consist essentially of one or more adjuvants. Adjuvants suitable for use in the practice of the invention are: (1) acrylic or methacrylic acid polymers, maleic anhydride and alkenyl derivative polymers, (2) immunostimulatory sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more unmethylated CpG units, (3) oil-in-water emulsions, (4) cationic lipids containing quaternary ammonium salts, (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, or (7) any combination or mixture thereof.

[0111] (1) Among the several types of adjuvant polymers, particularly preferred are crosslinked acrylic or methacrylic acid polymers, especially those crosslinked with polyalkenyl ethers of sugars or polyalcohols. These compounds are known as carbomers (products sold under the name Carbopol (BF Goodrich, Ohio, USA)).

[0112] Advantageously the adjuvant mixture is formed extemporaneously, preferably simultaneously with or immediately prior to administration of the formulation, and advantageously the vaccine and adjuvant mixture is formed immediately prior to administration, for example allowing sufficient time for the mixture to form a complex prior to administration, for example about 10 to about 60 minutes prior to administration, preferably about 30 minutes prior to administration.

[0113] Administration The composition for use as a vaccine according to the invention is administered in ovo during the last quarter of the culture of the domesticated bird. In one embodiment, the composition for use as a vaccine is administered in ovo between day 15 and day 20 of culture. In a further embodiment, the composition for use as a vaccine is administered in ovo between day 17 and day 19.5 of culture. In a further embodiment, the composition for use as a vaccine is administered in ovo between day 18 and day 19.2 of culture. In a further embodiment, the composition for use as a vaccine is administered in ovo at about day 18.5 of culture.

[0114] In one embodiment the composition, the in ovo vaccine or the composition for use as a vaccine is an aqueous suspension.

[0115] In one embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine comprises about 100-6000 sporulated oocysts of E. acervulina per dose, preferably about 200-5000 sporulated oocysts of E. acervulina per dose, more preferably about 300-2000 sporulated oocysts of E. acervulina per dose, more preferably about 400-1000 sporulated oocysts of E. acervulina per dose, and even more preferably about 598-809 sporulated oocysts of E. acervulina per dose.

[0116] In a further embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine comprises about 50-5000 E. maxima sporulated oocysts per dose, preferably about 100-4000 E. maxima sporulated oocysts per dose, more preferably about 150-3000 E. maxima sporulated oocysts per dose, more preferably about 200-1000 E. maxima sporulated oocysts per dose, more preferably about 300-600 E. maxima sporulated oocysts per dose, and even more preferably about 352-476 E. maxima sporulated oocysts per dose.

[0117] In a further embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine comprises about 50-3000 sporulated oocysts of Eimeria plecox per dose, preferably about 100-2000 sporulated oocysts of Eimeria plecox per dose, more preferably about 200-1000 sporulated oocysts of Eimeria plecox per dose, more preferably about 200-500 sporulated oocysts of Eimeria plecox per dose, and even more preferably about 235-317 sporulated oocysts of Eimeria plecox per dose.

[0118] In a further embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine comprises about 50-3000 sporulated oocysts of E. tenella per dose, preferably about 100-2000 sporulated oocysts of E. tenella per dose, more preferably about 200-1000 sporulated oocysts of E. tenella per dose, more preferably about 200-500 sporulated oocysts of E. tenella per dose, and even more preferably about 221-299 sporulated oocysts of E. tenella per dose.

[0119] In one embodiment, the amount of sporulated oocysts per dose of each Eimeria species is the same as listed above when different combinations of Eimeria species of the invention are selected, i.e., E. acervulina and E. maxima, E. acervulina and E. plecox, E. acervulina and E. tenella, E. acervulina, E. maxima and E. plecox, E. acervulina, E. maxima and E. tenella, E. acervulina, E. plecox and E. tenella.

[0120] In a preferred embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine contains about 100-6000 sporulated oocysts of E. acervulina per dose, preferably about 200-5000 sporulated oocysts of E. acervulina per dose, more preferably about 300-2000 sporulated oocysts of E. acervulina per dose, more preferably about 400-1000 sporulated oocysts of E. acervulina per dose, and even more preferably about 59-6000 sporulated oocysts of E. acervulina per dose. 8-809 sporulated oocysts of E. acervulina per dose, about 50-5000 sporulated oocysts of E. maxima per dose, preferably about 100-4000 sporulated oocysts of E. maxima per dose, more preferably about 150-3000 sporulated oocysts of E. maxima per dose, more preferably about 200-1000 sporulated oocysts of E. maxima per dose, more preferably about 300-600 sporulated oocysts of E. maxima per dose, More preferably, about 352-476 sporulated oocysts of E. maxima per dose, about 50-3000 sporulated oocysts of E. plecox per dose, preferably about 100-2000 sporulated oocysts of E. plecox per dose, more preferably about 200-1000 sporulated oocysts of E. plecox per dose, more preferably about 200-500 sporulated oocysts of E. plecox per dose, and even more preferably about 235-476 sporulated oocysts of E. maxima per dose, about 50-3000 sporulated oocysts of E. plecox per dose, more preferably about 100-2000 sporulated oocysts of E. plecox per dose, more preferably about 200-1000 sporulated oocysts of E. plecox per dose, more preferably about 200-500 sporulated oocysts of E. plecox per dose, and even more preferably about 235-476 sporulated oocysts of E. maxima per dose, and about 50-3000 sporulated oocysts of E. plecox per dose. 317 sporulated oocysts of Eimeria tenella and about 50-3000 sporulated oocysts of Eimeria tenella per dose, preferably about 100-2000 sporulated oocysts of Eimeria tenella per dose, more preferably about 200-1000 sporulated oocysts of Eimeria tenella per dose, more preferably about 200-500 sporulated oocysts of Eimeria tenella per dose, and even more preferably about 221-299 sporulated oocysts of Eimeria tenella per dose.

[0121] In a preferred embodiment, the composition, the in ovo vaccine or the composition for use as a vaccine comprises about 598-809 sporulated oocysts of E. acervulina per dose, about 352-476 sporulated oocysts of E. maxima per dose, about 235-317 sporulated oocysts of E. plecox per dose, and about 221-299 sporulated oocysts of E. tenella per dose.

[0122] In one embodiment of the composition or in ovo vaccine of the present invention, the amount of sporulated oocysts from E. acervulina is about 200-5000 per dose, more preferably about 300-2000 per dose, more preferably about 400-1000 per dose, even more preferably about 598-809 per dose, and the amount of sporulated oocysts from E. maxima is about 100-4000 per dose, more preferably about 150-3000 per dose, more preferably about 200-1000 per dose, more preferably about 300-600 per dose, even more preferably about 598-809 per dose. more preferably about 352-476 sporulated oocysts per dose, the amount of sporulated oocysts from Eimeria plecox is about 100-2000 per dose, more preferably about 200-1000 per dose, more preferably about 200-500 per dose, even more preferably about 235-317 per dose, and the amount of sporulated oocysts from Eimeria tenella is about 100-2000 per dose, more preferably about 200-1000 per dose, more preferably about 200-500 per dose, even more preferably about 221-299 per dose.

[0123] As shown in Example 7, an unexpected earlier (day 3) and higher (70,000 oocyst shedding per gram of fresh feces) peak in the oocyst clearance profile was observed for the in ovo live-attenuated Eimeria spp. vaccine composition of the present invention compared to the coarse spray administration (40,000 oocyst shedding per gram of fresh feces on day 6), indicating that the in ovo vaccine composition of the present invention can elicit an earlier immune response and is protective against Eimeria spp. infection in vaccinated subjects.

[0124] Dose volumes are discussed in the overview of immunization and vaccination methods and can be determined by one of skill in the art from the present disclosure taken together with general knowledge in the art without any undue experimentation.

[0125] In one embodiment, the composition for use as a vaccine of the present invention is furthermore conveniently diluted in a medium before in ovo administration. The medium may be a diluent or solvent. The diluent or solvent is an aqueous solution. The diluent or solvent may be selected from any physiological medium, preferably an aqueous solution containing a salt selected from disodium phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride and mixtures thereof to have the above oocyst content per dose, or an aqueous solution containing a sugar compound such as sucrose or saccharose, a nitrogen source such as peptone (e.g. NZ amine) or a casein hydrolysate (e.g. Hy-case amino) to have the above oocyst content per dose.

[0126] In one embodiment, compositions for use as vaccines of the invention are diluted in a ratio of about 0.05 / 10, 0.1 / 10, 0.5 / 10, 1 / 10, 1.1 / 10, 1.2 / 10, 1.3 / 10, 1.31 / 10, 1.32 / 10, 1.33 / 10, 1.34 / 10, 1.35 / 10, 1.36 / 10, 1.37 / 10, 1.38 / 10, 1.39 / 10, 1.4 / 10, 1.5 / 10, 1.6 / 10, 1.7 / 10, 1.8 / 10, 1.9 / 10, 2 / 10, 3 / 10 composition of the invention to diluent or solvent (v / v).

[0127] In one embodiment, compositions for use as vaccines of the invention are diluted in a ratio of about 0.05 / 10, 0.1 / 10, 0.2 / 10, 1 / 10, 0.3 / 10, 0.4 / 10, 0.5 / 10, 0.6 / 10, 0.61 / 10, 0.62 / 10, 0.63 / 10, 0.64 / 10, 0.65 / 10, 0.66 / 10, 0.67 / 10, 0.68 / 10, 0.69 / 10, 0.7 / 10, 0.8 / 10, 0.9 / 10, 1 / 10, 2 / 10, 3 / 10 composition of the invention to diluent or solvent (v / v).

[0128] In one embodiment, the composition for use as a vaccine of the present invention is administered in a dose comprising between 0.050 ml and 0.20 ml.

[0129] In one embodiment, the composition for use as a vaccine of the present invention is administered in a dose comprising between 0.050 ml and 0.10 ml.

[0130] In one embodiment, a composition for use as a vaccine according to the invention is administered in a dose of 0.050 ml.

[0131] In one embodiment, the composition for use as a vaccine of the present invention is administered in a dose of 0.10 ml.

[0132] Preventing and / or Treating Eimeria Infections The present invention encompasses compositions for use as vaccines in the prevention and / or treatment of Eimeria infection. As shown in Example 2, a significant reduction in intestinal lesions is clearly observed in all groups receiving a vaccine containing oocysts from a precocious attenuated Eimeria strain compared to birds in the control group (group 4) when administered by in ovo administration, regardless of the oocyst dose of E. acervulina they received. This significant reduction is observed after experimental infection for all Eimeria species (Figure 1).

[0133] A composition for use as a vaccine of the present invention comprising oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella can confer protection against coccidial infection when administered by the in ovo route.

[0134] The composition for use as a vaccine of the present invention is suitable for active immunization of embryonated eggs to prevent and / or reduce clinical signs, intestinal lesions and oocyst shedding associated with coccidiosis caused by Eimeria strains, preferably caused by any one of E. acervulina, E. maxima, E. plecox and E. tenella.

[0135] As shown in Example 3, unexpectedly, the composition for use as a vaccine of the present invention administered in ovo was found to be safe, since no weight difference was observed between animals from the vaccinated group receiving the composition of the present invention compared to animals from the control group. Even more surprisingly, it was found that animals vaccinated with a dose of the composition of the present invention ten times higher than the standard dose had similar weights as animals in the control group (Figure 2). These results therefore confirm that a vaccine comprising oocysts from a precocious attenuated strain of Eimeria is exceptionally safe when administered in ovo.

[0136] No changes in fecal appearance and general clinical signs were detected in the safety studies, therefore these results also confirm the good safety profile of the attenuated vaccine of the present invention when administered in ovo.

[0137] Furthermore, administration of the compositions of the present invention to embryonated chicken eggs unexpectedly shows increased hatch rates in groups vaccinated with the vaccine of the present invention compared to an unvaccinated control group that received a sham vaccine based only on phosphate buffered saline (PBS).

[0138] As shown in Figure 3C, the overall mean hatch rate is increased by 1.15% in animals that received a composition for use as a vaccine of the present invention (Group B) compared to the control group (Group A). ​​Considering the large volumes of eggs that are processed in industrial and commercial hatcheries, this increase in hatch rate (%) confers a surprising and significant advantage over current in ovo non-attenuated Eimeria vaccines.

[0139] In one embodiment of the present invention, the composition for use as a vaccine increases the hatch rate of vaccinated embryonated eggs compared to non-vaccinated embryonated eggs that receive a fake vaccine, i.e., a control group.

[0140] Combination vaccines The composition of the present invention, an in ovo vaccine or a composition for use as a vaccine, may also comprise additional active ingredients such as a pathogen or an immunogen, antigen or epitope of that pathogen and at least one immunogen, antigen or epitope of another avian pathogen, avian parasite, avian bacterium or avian virus, i.e. the coccidiosis vaccine may be combined with an additional avian vaccine composition.

[0141] In one embodiment, the composition of the invention, the in ovo vaccine or the composition for use as a vaccine further comprises an additional avian vaccine comprising at least one immunogen, antigen or epitope of another avian pathogen.

[0142] Such immunogens, antigens or epitopes may be derived from bacteria, parasites or viruses and may be inactivated or attenuated forms of such bacteria, parasites or viruses.

[0143] The additional avian vaccine comprises at least one immunogen, antigen or epitope of another avian pathogen, which may be, but is not limited to, a pathogen in an inactivated or attenuated form. In the case of the avian multivalent immunogenic composition, combination vaccine and multivalent vaccine, the additional avian pathogen may be Marek's Disease Virus (MDV) (e.g., serotypes 1 and 2, advantageously 1), Herpesvirus of Turkeys (HVT), Newcastle Disease Virus (NDV), non-Newcastle Disease paramyxoviruses (PMV2-PMV7), Infectious Bronchitis Virus (IBV), Infectious Anemia Virus or Chicken Anemia Virus (CAV), Infectious Laryngotracheitis Virus (ILTV), Infectious Lymphoma ... Bursal disease virus (IBDV, Gumboro disease), encephalomyelitis virus or avian encephalomyelitis virus (AEV or avian leukosis virus ALV), turkey hemorrhagic enteritis virus (HEV), pneumovirus (TRTV), fowl plague virus (avian influenza), chicken pericarditis virus, avian reovirus, coccidia, egg drop syndrome virus (EDS), fowl pox, inclusion body hepatitis (adenovirus), lymphoproliferative disease virus (LDV), reticuloendotheliosis virus (REV), rotavirus enteritis and turkey rhinotracheitis virus, Clostridium species, Escherichia coli, Mycoplasma gallinarum gallinarum, Mycoplasma gallisepticum, Haemophilus avium, Pasteurella gallinarum, Pasteurella multocida gallicida, Salmonella spp. and mixtures thereof.

[0144] In one embodiment, the additional avian vaccine comprises at least one immunogen, antigen or epitope of the avian pathogen Infectious Bursal Disease Virus (IBDV, Gumboro Disease).

[0145] The safety and efficacy data further demonstrate that the composition, in ovo vaccine or composition for use as a vaccine of the present invention can be mixed with an additional vaccine comprising at least one immunogen, antigen or epitope of another avian pathogen for the prevention and / or treatment of chicken coccidiosis and for the prevention and / or treatment of at least another avian pathogen. The composition, in ovo vaccine or composition for use as a vaccine of the present invention can be combined with an additional vaccine comprising, for example, an infectious bursal disease virus (IBDV, Gumboro disease) antigen before use and administered simultaneously by the in ovo route. In one embodiment, the composition, in ovo vaccine or composition for use as a vaccine of the present invention is a combination vaccine for use in the prevention and / or treatment of chicken coccidiosis and at least another avian pathogen by in ovo administration.

[0146] In one embodiment, the composition of the invention is a combination vaccine for use in the prevention and / or treatment of chicken coccidiosis and infectious bursal disease (Gumboro disease).

[0147] In one embodiment, the composition for use as a vaccine of the present invention is for the prevention and / or treatment of Eimeria and Infectious Bursal Disease Virus (IBDV, Gumboro disease) infections. In one embodiment, the composition for use as a vaccine provides an immunogenic and / or protective immune response against Eimeria and IBDV infections.

[0148] In a further embodiment, the composition for use as a vaccine provides an immunogenic and / or protective immune response against infection caused by at least one Eimeria species and IBDV.

[0149] In a further embodiment, the composition for use as a vaccine reduces or prevents one or more clinical signs associated with chicken coccidiosis and gumboro disease in hatched birds.

[0150] In one embodiment, the IBDV antigen is selected from inactivated IBDV, live attenuated IBDV and antigenic subunits of IBDV. Preferably, the IBDV antigen is live attenuated IBDV, more preferably, the IBDV antigen is an immune complex comprising live attenuated IBDV and an IBDV-specific antibody solution.

[0151] In one embodiment, the additional avian vaccine comprises about 10 1 ~10 10 EID 50 / ml (50% embryo infectious dose / ml) of live attenuated IBDV, preferably about 10 2 ~10 9 EID 50 / ml, more preferably about 10 3 ~10 8 EID 50 / ml, more preferably about 10 4 ~10 7 EID 50 / ml, and even more preferably about 10 5 ~10 6.5 EID 50 / mL of live attenuated IBDV.

[0152] In one embodiment, the additional avian vaccine comprising live attenuated IBDV further comprises an IBDV-specific antibody that forms an immune complex with an IBDV antigen. The IBDV-specific antibody has a titer that neutralizes at least 50% of the live attenuated IBDV. Preferably, the IBDV-specific antibody has a titer that neutralizes at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and even more preferably at least 99% of the live attenuated IBDV. In a further embodiment, the IBDV-specific antibody has a titer that neutralizes 100% of the live attenuated IBDV.

[0153] In one embodiment, the combination vaccine comprises about 10 1 ~10 10 EID 50 / ml (50% embryo infectious dose / ml) of live attenuated IBDV, preferably about 10 2 ~10 9 EID 50 / ml, more preferably about 10 3 ~10 8 EID 50 / ml, more preferably about 10 4 ~10 7 EID 50 / ml, and even more preferably about 10 5 ~10 6.5 EID 50 / mL of live attenuated IBDV.

[0154] In one embodiment, the combination vaccine comprising live attenuated IBDV further comprises an IBDV-specific antibody that forms an immune complex with an IBDV antigen. The IBDV-specific antibody has a titer that neutralizes at least 50% of the live attenuated IBDV. Preferably, the IBDV-specific antibody has a titer that neutralizes at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and even more preferably at least 99% of the live attenuated IBDV. In a further embodiment, the IBDV-specific antibody has a titer that neutralizes 100% of the live attenuated IBDV.

[0155] Immunoconjugates of IBDV antigens, including live attenuated IBDV and IBDV-specific antibodies, are known in the prior art and commercially available. Said immunoconjugate IBDV antigens may be obtained through commercial vaccine compositions, such as GUMBOHATCH® (Laboratorios HIPRA SA, Avda. La Selva 135, 17170 Amer, Girona, Spain) or CEVAC TRANSUME® (Ceva Animal Health, SA, 08028 Barcelona, ​​Spain).

[0156] When used in combination, the onset and duration of immunity of the Eimeria spp. contained in a composition for use as a combination vaccine of the present invention is equivalent to that determined for said composition when used without the additional avian vaccine composition.

[0157] In one embodiment, the composition of the invention, the in ovo vaccine or the composition for use as a vaccine and the additional avian vaccine are contained in separate containers, optionally in the same package, optionally together with instructions for mixing and / or administration.

[0158] In one embodiment, the composition of the invention comprising an IBDV antigen, an in ovo vaccine or a composition for use as a vaccine and an additional avian vaccine are contained in separate containers, optionally in the same package, optionally with mixing and / or instructions for administration. In another embodiment, the composition of the invention comprising an in ovo vaccine or a composition for use as a vaccine and an additional avian vaccine comprising an IBDV antigen are contained in separate containers, optionally with mixing and / or instructions for administration.

[0159] In one embodiment, the composition for use as a vaccine of the present invention is mixed with additional avian vaccines prior to use and administered simultaneously in ovo into embryonated eggs of domesticated birds.

[0160] In one embodiment, the composition for use as a vaccine of the present invention is mixed with additional avian vaccines prior to use and administered in ovo in a single dose.

[0161] In one embodiment, the composition for use as a vaccine of the present invention is mixed with an additional avian vaccine comprising an IBDV antigen prior to use and simultaneously administered in ovo into embryonated eggs of domesticated birds.

[0162] In one embodiment, the composition for use as a vaccine of the present invention is mixed with an additional avian vaccine comprising an IBDV antigen prior to use and administered in ovo in a single dose.

[0163] In one embodiment, the composition for use as a vaccine of the present invention and the additional avian vaccine are administered separately and substantially simultaneously in ovo.

[0164] In one embodiment, the composition for use as a vaccine according to the present invention and an additional avian vaccine comprising an IBDV antigen are administered separately and substantially simultaneously in ovo.

[0165] In one embodiment, the composition for use as a vaccine of the present invention and the additional avian vaccine comprising an IBDV antigen are combined in a dosage ratio (also referred to as "vaccine of the present invention":"additional avian vaccine comprising an IBDV antigen") selected from the dosage ratios of 2:1, 3:2, 1:1, 2:3 and 1:2. In a further embodiment, the composition for use as a vaccine of the present invention and the additional avian vaccine comprising an IBDV antigen are combined in a dosage ratio of 1:1.

[0166] A composition according to the invention or a composition for use as an in ovo vaccine or vaccine which also comprises such an additional immunogenic component (additional immunogen, antigen or epitope) has the advantage of simultaneously eliciting an immune response or protection against several infections or diseases or their causative agents.

[0167] An embodiment of the present invention is a composition for use as a combination vaccine in the prevention and / or treatment of Eimeria infection and infectious bursal disease, the composition comprising Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella, the vaccine being administered in ovo into embryonated eggs of domesticated birds in combination with an additional avian vaccine comprising at least one immunogen, antigen or epitope of the avian pathogen Infectious Bursal Disease Virus (IBDV, Gumboro Disease).

[0168] Another phrase for this aspect of the invention is a composition for use as a combination vaccine in the prevention and / or treatment of Eimeria infection and infectious bursal disease, the composition comprising Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella, wherein an effective immunizing amount of the vaccine is administered in ovo into embryonated eggs of domesticated birds in combination with an additional avian vaccine comprising at least one immunogen, antigen or epitope of the avian pathogen Infectious Bursal Disease Virus (IBDV, Gumboro Disease).

[0169] In one embodiment, the combination of Eimeria oocysts present in the composition for use as a combination vaccine consists or consists essentially of Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox, and E. tenella.

[0170] In one embodiment, the combination of Eimeria oocysts present in the composition for use as a combination vaccine consists or consists essentially of Eimeria oocysts from a precocious attenuated strain of E. acervulina and E. maxima.

[0171] In one embodiment, the combination of Eimeria oocysts present in the composition for use as a combination vaccine consists or consists essentially of Eimeria oocysts from a precocious attenuated strain of E. acervulina and E. plecox.

[0172] In one embodiment, the combination of Eimeria oocysts present in the composition for use as a combination vaccine consists or consists essentially of Eimeria oocysts from a precocious attenuated strain of E. acervulina and E. tenella.

[0173] In a preferred embodiment, the combination of Eimeria oocysts present in the composition for use as a combination vaccine comprises, consists of, or consists essentially of Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least two precocious attenuated strains selected from E. maxima, E. plecox, and E. tenella. In one embodiment, the composition for use as a combination vaccine consists of, or consists essentially of Eimeria oocysts from precocious attenuated strains of E. acervulina, E. maxima, and E. plecox. In one embodiment, the composition for use as a combination vaccine consists of, or consists essentially of Eimeria oocysts from precocious attenuated strains of E. acervulina, E. maxima, and E. tenella. In one embodiment, the composition consists or consists essentially of Eimeria oocysts from precocious attenuated strains of E. acervulina, E. plecox, and E. tenella.

[0174] In another preferred embodiment, the combination of Eimeria oocysts present in said composition for use as a combination vaccine of the invention comprises, consists of or consists essentially of a mixture of sporulated oocysts from precocious attenuated strains of E. acervulina, E. maxima, E. plecox and E. tenella, i.e. in a preferred embodiment, the composition for use as a combination vaccine in the prevention and / or treatment of Eimeria infection and infectious bursal disease comprises Eimeria oocysts from a precocious attenuated strain selected from E. acervulina, E. maxima, E. plecox and E. tenella, which is administered in ovo into embryonated eggs of domesticated birds in combination with an additional avian vaccine comprising at least one immunogen, antigen or epitope of the avian pathogen Infectious Bursal Disease Virus (IBDV, Gumboro Disease).

[0175] In another preferred embodiment, the combination of Eimeria oocysts present in said composition for use as a combination vaccine of the invention comprises, consists of or consists essentially of a mixture of sporulated oocysts from precocious attenuated strains of E. acervulina, E. maxima, E. plecox and E. tenella, i.e. in a preferred embodiment, the composition for use as a combination vaccine in the prevention and / or treatment of Eimeria infection and infectious bursal disease comprises Eimeria oocysts from a precocious attenuated strain selected from E. acervulina, E. maxima, E. plecox and E. tenella, and an effective immunizing amount of the vaccine is administered in ovo into embryonated eggs of domesticated birds in combination with an additional avian vaccine comprising at least one immunogen, antigen or epitope of the avian pathogen Infectious Bursal Disease Virus (IBDV, Gumboro Disease).

[0176] Specific embodiments of the combination vaccine refer to the characteristics of the precocious attenuated strain of Eimeria acervulina, the excipients contained therein, the date of administration, the form of administration, the composition of sporulated oocysts, the volume of the dose of the vaccine, the number of doses, the IBDV antigen, and the ratio between the two vaccines as described above disclosing the characteristics of the composition for use as a vaccine in the prevention and / or treatment of Eimeria infection, as well as the characteristics of an additional avian vaccine against infectious bursal disease (Gumboro disease).

[0177] In one embodiment, the additional vaccine comprising an Infectious Bursal Disease Virus (IBDV, Gumboro disease) avian pathogen IBDV immunogen, antigen or epitope is lyophilized.

[0178] In one embodiment, a lyophilized booster vaccine comprising an Infectious Bursal Disease Virus (IBDV, Gumboro Disease) immunogen, antigen or epitope of the avian pathogen is diluted in a suitable vehicle, such as PBS, prior to in ovo administration.

[0179] In one embodiment, the lyophilized additional vaccine containing an infectious bursal disease virus (IBDV, Gumboro disease) avian pathogen IBDV immunogen, antigen or epitope and Eimeria vaccine are mixed in the same vehicle before in ovo administration. The dose ratio of the combination vaccine of the present invention is described above. In a specific embodiment, the combination vaccine of the present invention is mixed in a dose ratio of 1:1.

[0180] The efficacy of this combination vaccine against infectious bursal disease is effectively demonstrated in Example 5, which confirms the absence of adverse effects on the efficacy of the IBDV vaccine by the combination of an attenuated Eimeria vaccine administered by the in ovo route with an IBDV vaccine, and also confirms that this combination vaccine is equally effective against IBDV infection.

[0181] The efficacy of this combination vaccine against Eimeria infection is effectively shown in Example 6, where a combination of a live attenuated Eimeria vaccine against chicken coccidiosis and a live attenuated vaccine against infectious bursal disease was administered in ovo into embryonated chicken eggs against infection with four different Eimeria species. The results of the study demonstrate the efficacy of a single dose of a combination vaccine of a live attenuated vaccine against chicken infectious bursal disease and a live attenuated vaccine against chicken coccidiosis when mixed prior to in ovo administration into 18-day-old embryonated chicken eggs against chicken coccidiosis caused by Eimeria infection.

[0182] In one embodiment, the combination vaccine of the present invention is for immunization of embryonated eggs to prevent and / or reduce clinical signs associated with Gumboro disease or IBDV infection. In a particular embodiment, the combination vaccine of the present invention prevents and / or reduces at least one clinical sign associated with Gumboro disease or IBDV infection selected from the group consisting of absence of mean acute histological functional score, gross lesions, mononuclear cell infiltrate edema, inflammatory infiltrate, fold edema, serosal edema, and muscle wall edema.

[0183] kit In one embodiment the composition of the invention, an in ovo vaccine or a composition for use as a vaccine, is included in a kit.

[0184] In one embodiment, the composition, in ovo vaccine or composition for use as a vaccine of the invention comprises: a) a container containing a suspension of Eimeria oocysts from a precocious attenuated strain of coccidia in an aqueous medium comprising disodium phosphate, potassium dihydrogen phosphate, a polysorbate, preferably polysorbate 80, potassium chloride, sodium chloride and water; and b) a container containing an aqueous solution containing disodium phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride and water; Included in the kit is:

[0185] In one embodiment, the kit or kit for use of the vaccine composition of the invention further comprises a container containing an additional vaccine comprising at least one immunogen, antigen or epitope of another avian pathogen against infection of the birds described above.

[0186] In another embodiment, the composition, in ovo vaccine or composition for use as a vaccine of the present invention comprises: a) a container containing a suspension of Eimeria oocysts from a precocious attenuated strain of coccidia in an aqueous medium comprising disodium phosphate, potassium dihydrogen phosphate, a polysorbate, preferably polysorbate 80, potassium chloride, sodium chloride and water; and b) a container containing an aqueous solution comprising disodium phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride and water; and c) a container containing an additional vaccine comprising at least one immunogen or antigen of another avian pathogen against infection of birds; Included in the kit is:

[0187] In another embodiment, the composition, in ovo vaccine or composition for use as a vaccine of the present invention comprises: a) a container containing a suspension of Eimeria oocysts from a precocious attenuated strain of coccidia in an aqueous medium comprising disodium phosphate, potassium dihydrogen phosphate, a polysorbate, preferably polysorbate 80, potassium chloride, sodium chloride and water; and b) a container containing an aqueous solution comprising disodium phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride and water; and c) a container containing an additional vaccine containing at least one immunogen or antigen of IBDV (Gumboro disease) against infection of birds; Included in the kit is:

[0188] In one embodiment, the kit may comprise one or more containers or vials of a composition or vaccine of the invention, or one or more containers or vials of a composition or vaccine of the invention together with instructions for administration to birds at risk of Eimeria infection and / or at risk of Eimeria and IBDV infection (Gumboro disease).

[0189] Another aspect of the invention is a kit for use as a vaccine in the prevention and / or treatment of Eimeria infection, the vaccine being administered in ovo into embryonated eggs of a domesticated bird, the kit comprising a composition for use as a vaccine of the invention, in particular Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella.

[0190] Another aspect of the invention is a kit for use as a vaccine comprising the composition for use as a vaccine of the invention, preferably comprising Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella.

[0191] The present invention includes the following embodiments.

[0192] 1. A composition for use as a vaccine in the prevention and / or treatment of Eimeria infection, said composition comprising Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella, said vaccine being administered in ovo into embryonated eggs of a domesticated bird, preferably an effective immunizing amount of said vaccine being administered in ovo into embryonated eggs of a domesticated bird.

[0193] 2. A composition for use as a vaccine according to embodiment 1, wherein said prevention and / or treatment results in an improvement compared to an untreated control group of the same domesticated bird.

[0194] 3. A composition for use as a vaccine according to embodiment 1 or embodiment 2, which provides an immunogenic and / or protective immune response against at least Eimeria infection.

[0195] 4. A composition for use as a vaccine according to embodiment 1 or embodiment 2, which provides an immunogenic and / or protective immune response against infection caused by at least one Eimeria species.

[0196] 5. A composition for use as a vaccine according to any one of embodiments 1 to 4, which reduces or prevents one or more clinical signs associated with chicken coccidiosis in hatched birds.

[0197] 6. The composition for use as a vaccine according to embodiment 5, wherein chicken coccidiosis is caused by Eimeria infection.

[0198] 7. The composition for use as a vaccine according to embodiment 5 or embodiment 6, wherein the clinical signs are selected from the group consisting of intestinal lesions, weight loss, oocyst excretion, oocyst burden, oocyst shedding, diarrhea, dehydration, presence of blood in the faeces, presence of mucus in the faeces, Eimeria infection, depression, ruffled feathers, arched body position, morbidity and mortality.

[0199] 8. A composition for use as a vaccine according to any one of embodiments 1 to 7, which increases hatchability compared to a non-vaccinated control group.

[0200] 9. A composition for use as a vaccine according to embodiment 8, wherein the control group is administered PBS.

[0201] 10. A composition for use as a vaccine according to any one of embodiments 1 to 9, wherein the composition reduces oocyst shedding in hatched birds compared to hatched birds inoculated in ovo with the composition comprising a non-attenuated Eimeria strain.

[0202] 11. The composition for use as a vaccine according to any one of embodiments 1 to 10, wherein the domesticated bird is selected from chickens, turkeys, ducks, game birds and ratites, preferably the domesticated bird is a chicken.

[0203] 12. A composition for use as a vaccine according to any one of embodiments 1 to 11, for the prevention of Eimeria infection.

[0204] 13. A composition for use as a vaccine according to any one of embodiments 1 to 11, for the treatment of an Eimeria infection.

[0205] 14. A composition for use as a vaccine according to any one of embodiments 1 to 11, for the prevention of chicken coccidiosis.

[0206] 15. A composition for use as a vaccine according to any one of embodiments 1 to 11, for the treatment of chicken coccidiosis.

[0207] 16. A composition for use as a vaccine according to any one of embodiments 1 to 15, consisting or consisting essentially of Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella.

[0208] 17. A composition for use as a vaccine according to embodiment 16, consisting or consisting essentially of Eimeria oocysts from precocious attenuated strains of E. acervulina and E. maxima.

[0209] 18. A composition for use as a vaccine according to embodiment 16, consisting or consisting essentially of Eimeria oocysts from precocious attenuated strains of Eimeria acervulina and Eimeria plecox.

[0210] 19. A composition for use as a vaccine according to embodiment 16, consisting or consisting essentially of Eimeria oocysts from precocious attenuated strains of E. acervulina and E. tenella.

[0211] 20. A composition for use as a vaccine according to any one of embodiments 1 to 15, comprising, consisting of, or consisting essentially of Eimeria oocysts from a precocious attenuated strain of Eimeria acervulina and at least two precocious attenuated strains selected from Eimeria maxima, Eimeria plecox and Eimeria tenella.

[0212] 21. A composition for use as a vaccine according to embodiment 20, consisting or consisting essentially of Eimeria oocysts from precocious attenuated strains of E. acervulina, E. maxima and E. plecox.

[0213] 22. A composition for use as a vaccine according to embodiment 20, consisting or consisting essentially of Eimeria oocysts from precocious attenuated strains of E. acervulina, E. maxima and E. tenella.

[0214] 23. A composition for use as a vaccine according to embodiment 20, consisting or consisting essentially of Eimeria oocysts from precocious attenuated strains of E. acervulina, E. plecox and E. tenella.

[0215] 24. A composition for use as a vaccine according to any one of embodiments 1 to 15, comprising, consisting of or consisting essentially of a mixture of sporulated oocysts from precocious attenuated strains of E. acervulina, E. maxima, E. plecox and E. tenella.

[0216] 25. A composition for use as a vaccine according to any one of embodiments 1 to 24, comprising Eimeria oocysts selected from an Eimeria acervulina precocious attenuated strain having reduced virulence and reduced oocyst shedding compared to the parent Eimeria acervulina strain over a 14 day period.

[0217] 26. A composition for use as a vaccine according to embodiment 25, wherein when the precocious attenuated strain of Eimeria acervulina used therein is administered to a host, the host excretes about 30% to about 75% fewer, preferably about 35% to about 65% fewer, more preferably about 40% to about 60% fewer, and even more preferably about 45% to about 55% fewer oocysts during a 14 day period compared to the oocysts excreted by a host that received the parent Eimeria acervulina strain.

[0218] 27. A composition for use as a vaccine according to any one of embodiments 1 to 24, wherein when the precocious attenuated strain of Eimeria acervulina used therein is administered to a host, the host excretes a similar number of oocysts in a 14 day period as the number of oocysts excreted in a 14 day period by a host that has received Eimeria acervulina strain 120718P5.

[0219] 28. A composition for use as a vaccine according to any one of embodiments 1-24, wherein when the precocious attenuated strain of Eimeria acervulina used therein is administered to a host, the host excretes about ±75% of the oocysts excreted by a host that has received E. acervulina strain 120718P5, preferably about ±70%, preferably about ±60%, preferably about ±50%, preferably about ±40%, preferably about ±30%, more preferably about ±25%, more preferably about ±20%, more preferably about ±15%, and even more preferably about ±10% of the oocysts excreted by a host that has received E. acervulina strain 120718P5 over a period of 14 days.

[0220] 29. A composition for use as a vaccine according to any one of embodiments 1 to 28, wherein the precocious Eimeria acervulina strain used therein has been attenuated by repeated passaging of the precocious strain selected for precocious maturation from the parent strain, preferably after at least 3 passages, at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages or at least 11 passages.

[0221] 30. A composition for use as a vaccine according to embodiment 29, wherein the precocious strain of Eimeria acervulina has been attenuated after at least five passages from a parent strain selected for precocious maturation.

[0222] 31. The composition for use as a vaccine according to any one of embodiments 1 to 30, wherein the Eimeria acervulina precocious attenuated strain used therein is Eimeria acervulina strain 120718P5 deposited at the Culture Collection of Algae & Protozoa (Scottish Marine Institute, OBAN, Argyl, PA37 1QA, Scotland, UK) by HIPRA SCIENTIFIC, SLU (Avda La Selva 135, 17170 Amer, Girona, Spain) under the Budapest Treaty on 06.10.2021 under the accession number CCAP2016 / 1.

[0223] 32. A composition for use as a vaccine according to any one of embodiments 1 to 31, comprising at least a pharma- ceutically or veterinarily acceptable carrier or vehicle or excipient or adjuvant.

[0224] 33. The composition for use as a vaccine according to embodiment 32, wherein the pharma- ceutically or veterinarily acceptable carrier or vehicle or excipient includes wetting agents, dispersing agents, emulsifying agents, buffers, salts, stabilizers, proteins, water, saline, buffered saline, physiologically acceptable vehicles and mixtures thereof.

[0225] 34. The composition for use as a vaccine according to embodiment 33, wherein the pharma- ceutically or veterinarily acceptable carrier or vehicle or excipient is selected from the group of sodium chloride, potassium chloride, phosphate buffer, surfactants and mixtures thereof.

[0226] 35. A composition for use as a vaccine according to embodiment 34, comprising as vehicles and excipients a combination of disodium phosphate, potassium dihydrogen phosphate, polysorbate, potassium chloride, sodium chloride and water.

[0227] 36. A composition for use as a vaccine according to embodiment 35, comprising polysorbate at a concentration in the range of about 0.001% to about 1% (v / v), about 0.005% to about 0.5% (v / v), about 0.006% to about 0.3% (v / v), about 0.007% to about 0.1% (v / v), or about 0.005% to about 0.05% (v / v).

[0228] 37. A composition for use as a vaccine according to any one of embodiments 33 to 36, comprising or consisting essentially of one or more adjuvants selected from polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, immunostimulatory sequences (ISS), oil-in-water emulsions, cationic lipids comprising quaternary ammonium salts, cytokines, aluminum hydroxide, aluminum phosphate and mixtures thereof.

[0229] 38. A composition for use as a vaccine according to any one of embodiments 1 to 37, administered in ovo during the last quarter of the culture of the domesticated bird, preferably between day 15 and 20 of culture, preferably between day 17 and 19.5 of culture, preferably between day 18 and 19.2 of culture, more preferably around day 18.5 of culture.

[0230] 39. A composition for use as a vaccine according to any one of embodiments 1 to 38, which is an aqueous suspension.

[0231] 40. A composition for use as a vaccine according to any one of embodiments 1-39, comprising about 100-6000 sporulated oocysts of E. acervulina per dose, preferably about 200-5000 sporulated oocysts of E. acervulina per dose, more preferably about 300-2000 sporulated oocysts of E. acervulina per dose, more preferably about 400-1000 sporulated oocysts of E. acervulina per dose, and even more preferably about 598-809 sporulated oocysts of E. acervulina per dose.

[0232] 41. A composition for use as a vaccine according to any one of embodiments 1-40, comprising about 50-5000 sporulated oocysts of E. maxima per dose, preferably about 100-4000 sporulated oocysts of E. maxima per dose, more preferably about 150-3000 sporulated oocysts of E. maxima per dose, more preferably about 200-1000 sporulated oocysts of E. maxima per dose, more preferably about 300-600 sporulated oocysts of E. maxima per dose, and even more preferably about 352-476 sporulated oocysts of E. maxima per dose.

[0233] 42. A composition for use as a vaccine according to any one of embodiments 1-41, comprising about 50-3000 sporulated oocysts of E. plecox per dose, preferably about 100-2000 sporulated oocysts of E. plecox per dose, more preferably about 200-1000 sporulated oocysts of E. plecox per dose, more preferably about 200-500 sporulated oocysts of E. plecox per dose, and even more preferably about 235-317 sporulated oocysts of E. plecox per dose.

[0234] 43. A composition for use as a vaccine according to any one of embodiments 1 to 42, comprising about 50 to 3000 sporulated oocysts of E. tenella per dose, preferably about 100 to 2000 sporulated oocysts of E. tenella per dose, more preferably about 200 to 1000 sporulated oocysts of E. tenella per dose, more preferably about 200 to 500 sporulated oocysts of E. tenella per dose, and even more preferably about 221 to 299 sporulated oocysts of E. tenella per dose.

[0235] 44. A composition for use as a vaccine according to any one of embodiments 1-43, comprising about 598-809 sporulated oocysts of E. acervulina per dose, about 352-476 sporulated oocysts of E. maxima per dose, about 235-317 sporulated oocysts of E. plecox per dose, and about 221-299 sporulated oocysts of E. tenella per dose.

[0236] 45. A composition for use as a vaccine according to any one of embodiments 1 to 44, which is further diluted with a vehicle that is an aqueous solution prior to in ovo administration.

[0237] 46. ​​A composition for use as a vaccine according to embodiment 45, wherein the aqueous solution comprises a) a salt selected from disodium phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride and mixtures thereof, b) a sugar compound, or c) a nitrogen source.

[0238] 47. A composition for use as a vaccine according to any one of the preceding embodiments, administered in a dose comprised between 0.050 ml and 0.20 ml, preferably between 0.050 ml and 0.10 ml.

[0239] 48. A composition for use as a vaccine according to embodiment 47, administered in a dose of 0.050 ml.

[0240] 49. A composition for use as a vaccine according to embodiment 47, administered in a dose of 0.10 ml.

[0241] 50. A composition for use as a vaccine according to any one of embodiments 1 to 49, further comprising an additional avian vaccine comprising at least one immunogen, antigen or epitope of another avian pathogen.

[0242] 51. Additional avian pathogens include Marek's disease virus (MDV), herpesvirus of turkeys (HVT), Newcastle disease virus (NDV), non-Newcastle disease paramyxoviruses (PMV2-PMV7), infectious bronchitis virus (IBV), infectious anemia virus or chicken anemia virus (CAV), infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBDV, Gumboro disease), encephalomyelitis virus or avian encephalomyelitis virus (AEV or avian leukosis virus ALV), turkey hemorrhagic enteritis virus (HEV), pneumovirus (TRTV), and fowl plague virus. 51. The composition for use as a vaccine according to embodiment 50, wherein the virus is selected from the group of influenza (avian influenza), chicken pericarditis virus, avian reovirus, coccidia, egg-laying drop syndrome virus (EDS), fowl pox, inclusion body hepatitis (adenovirus), lymphoproliferative disease virus (LDV), reticuloendotheliosis virus (REV), rotavirus enteritis and turkey rhinotracheitis virus, Clostridium spp., Escherichia coli, Mycoplasma gallinarum, Mycoplasma gallisepticum, Haemophilus avium, Pasteurella gallinarum, Pasteurella multocida gallicida, Salmonella spp. and mixtures thereof.

[0243] 52. The composition for use as a vaccine according to embodiment 50 or 51, wherein the additional avian vaccine comprises at least one immunogen, antigen or epitope of the avian pathogen Infectious Bursal Disease Virus (IBDV, Gumboro Disease).

[0244] 53. A composition for use as a vaccine according to any one of embodiments 50 to 52, for the prevention and / or treatment of Eimeria infection and Infectious Bursal Disease Virus (IBDV, Gumboro disease) infection, in particular for the prevention and / or treatment of chicken coccidiosis and Infectious Bursal Disease (Gumboro disease).

[0245] 54. A composition for use as a vaccine according to embodiment 52, providing an immunogenic and / or protective immune response against Eimeria and IBDV infection.

[0246] 55. A composition for use as a vaccine according to embodiment 52, providing an immunogenic and / or protective immune response against infection caused by at least one Eimeria species and against IBDV.

[0247] 56. A composition for use as a vaccine according to embodiment 52, which reduces or prevents one or more clinical signs associated with chicken coccidiosis and gumboro disease in hatched birds.

[0248] 57. A composition for use as a vaccine according to any one of embodiments 52 to 56, wherein the IBDV antigen is selected from inactivated IBDV, live attenuated IBDV and an antigenic subunit of IBDV, preferably the IBDV antigen is a live attenuated IBDV, more preferably the IBDV antigen is an immune complex comprising live attenuated IBDV and an IBDV-specific antibody solution.

[0249] 58. Additional avian vaccines are available at approximately 10 1 ~10 10 EID 50 / ml (50% embryo infectious dose / ml) of live attenuated IBDV, preferably about 10 2 ~10 9 EID 50 / ml, more preferably about 10 3 ~10 8 EID 50 / ml, more preferably about 10 4 ~10 7 EID 50 / ml, and even more preferably about 10 5 ~10 6.5 EID 50 58. A composition for use as a vaccine according to embodiment 57, comprising:

[0250] 59. A composition for use as a vaccine according to any one of embodiments 50 to 58, wherein the composition and the additional avian vaccine are contained in separate containers, optionally with instructions for mixing and / or administration.

[0251] 60. A composition for use as a vaccine according to embodiment 59, wherein said composition is mixed with an additional avian vaccine prior to use and simultaneously administered in ovo into embryonated eggs of domesticated birds.

[0252] 61. The composition for use as a vaccine according to embodiment 60, wherein the composition is administered in a single dose.

[0253] 62. The composition for use as a vaccine according to embodiment 59, wherein the composition and the additional avian vaccine are administered separately and substantially simultaneously in ovo.

[0254] 63. A composition for use as a vaccine according to any one of embodiments 1 to 62, contained in a kit.

[0255] 64. The kit comprises: a) a container containing a suspension of Eimeria oocysts from a precocious attenuated strain of coccidia in an aqueous medium comprising disodium phosphate, potassium dihydrogen phosphate, a polysorbate, preferably polysorbate 80, potassium chloride, sodium chloride and water; and b) a container containing an aqueous solution containing disodium phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride and water; 64. A composition for use as a vaccine according to embodiment 63, comprising:

[0256] 65. The kit comprises: a) a container containing a suspension of Eimeria oocysts from a precocious attenuated strain of coccidia in an aqueous medium comprising disodium phosphate, potassium dihydrogen phosphate, a polysorbate, preferably polysorbate 80, potassium chloride, sodium chloride and water; and b) a container containing an aqueous solution comprising disodium phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride and water; and c) a container containing an additional vaccine comprising at least one immunogen or antigen of another avian pathogen against infection of birds; 64. A composition for use as a vaccine or combination vaccine according to embodiment 63, comprising:

[0257] 66. The kit comprises: a) a container containing a suspension of Eimeria oocysts from a precocious attenuated strain of coccidia in an aqueous medium comprising disodium phosphate, potassium dihydrogen phosphate, a polysorbate, preferably polysorbate 80, potassium chloride, sodium chloride and water; and b) a container containing an aqueous solution comprising disodium phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride and water; and c) a container containing an additional vaccine comprising at least one immunogen or antigen of IBDV (Gumboro disease) 64. A composition for use as a vaccine or combination vaccine according to embodiment 63, comprising:

[0258] 67. A composition for use as a vaccine according to any one of embodiments 63 to 66, wherein the kit comprises one or more containers or vials of the vaccine or comprises one or more containers or vials of the vaccine together with instructions for administration to birds at risk of Eimeria infection and / or at risk of Eimeria and IBDV infection (Gumboro disease).

[0259] 68. A composition for use as a combination vaccine in the prevention and / or treatment of Eimeria infection and infectious bursal disease, wherein the composition comprises Eimeria oocysts from a precocious attenuated strain of Eimeria acervulina and at least one precocious attenuated strain selected from Eimeria maxima, Eimeria plecox and Eimeria tenella, the vaccine being administered in ovo into embryonated eggs of domestic birds in combination with an additional avian vaccine comprising at least one immunogen, antigen or epitope of the avian pathogen Infectious Bursal Disease Virus (IBDV, Gumboro Disease).

[0260] 69. A composition for use as a combination vaccine according to embodiment 68, wherein the combination of Eimeria oocysts present in the composition is selected according to any one of embodiments 16 to 24.

[0261] 70. A composition for use as a combination vaccine according to embodiment 68 or embodiment 69, wherein the additional vaccine comprising an infectious bursal disease virus (IBDV, Gumboro disease) avian pathogen IBDV immunogen, antigen or epitope is lyophilized.

[0262] 71. A composition for use as a combination vaccine according to embodiment 70, wherein the lyophilized additional vaccine is diluted in a suitable vehicle before in ovo administration.

[0263] 72. A composition for use as a combination vaccine according to embodiment 71, wherein the lyophilized additional vaccine comprising an IBDV immunogen, antigen or epitope of the avian pathogen Infectious Bursal Disease Virus (IBDV, Gumboro disease) and the Eimeria vaccine are mixed in the same vehicle before in ovo administration.

[0264] 73. A composition for use as a combination vaccine according to any one of embodiments 68 to 72, for immunization of embryonated eggs to prevent and / or reduce clinical signs associated with Gumboro disease or IBDV infection.

[0265] 74. A composition for use as a combination vaccine according to embodiment 73, which prevents and / or reduces at least one of the clinical signs associated with Gumboro disease or IBDV infection selected from the group consisting of absence of mean acute histological functional score, gross lesions, mononuclear cell infiltrate edema, inflammatory infiltrate, fold edema, serosal edema and muscle wall edema.

[0266] 75. A kit for use as a vaccine comprising the composition for use as a vaccine according to any one of embodiments 1 to 62.

[0267] 76. A kit for use as a vaccine in the prevention and / or treatment of Eimeria infection, wherein the vaccine is administered in ovo into embryonated eggs of a domesticated bird, and the kit comprises the composition for use as a vaccine according to any one of embodiments 1 to 62.

[0268] 77. A composition comprising Eimeria sporulated oocysts from a precocious attenuated strain of Eimeria acervulina and at least one precocious attenuated strain selected from Eimeria maxima, Eimeria plecox and Eimeria tenella, wherein the amount of sporulated oocysts from E. acervulina is about 100-6000 per dose, the amount of sporulated oocysts from E. maxima is about 50-5000 per dose, the amount of sporulated oocysts from E. plecox is about 50-3000 per dose, and the amount of sporulated oocysts from E. tenella is about 50-3000 per dose.

[0269] 78. A composition comprising Eimeria sporulated oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella, wherein the amount of sporulated oocysts from E. acervulina is about 100-6000 per dose, the amount of sporulated oocysts from E. maxima is about 50-5000 per dose, the amount of sporulated oocysts from E. plecox is about 50-3000 per dose, and the amount of sporulated oocysts from E. tenella is about 50-3000 per dose, provided that the composition does not contain E. mitis sporulated oocysts.

[0270] 79. A composition comprising Eimeria sporulated oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella, wherein the amount of sporulated oocysts from E. acervulina is about 100-6000 per dose, the amount of sporulated oocysts from E. maxima is about 50-5000 per dose, the amount of sporulated oocysts from E. plecox is about 50-3000 per dose, and the amount of sporulated oocysts from E. tenella is about 50-3000 per dose, and the composition is suitable for preparing an in ovo vaccine.

[0271] 80. A composition comprising Eimeria sporulated oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox, and E. tenella, wherein the amount of sporulated oocysts from E. acervulina is about 100-6000 per dose, the amount of sporulated oocysts from E. maxima is about 50-5000 per dose, the amount of sporulated oocysts from E. plecox is about 50-3000 per dose, and the amount of sporulated oocysts from E. tenella is about 50-3000 per dose, provided that the composition does not contain E. mitis sporulated oocysts, and the composition is suitable for the preparation of an in ovo vaccine.

[0272] 81. The composition according to any one of embodiments 77 to 80, which is an immunogenic composition.

[0273] 82. The composition of any one of embodiments 77 to 81, comprising, consisting of, or consisting essentially of sporulated oocysts from precocious attenuated strains of Eimeria acervulina, Eimeria maxima, Eimeria plecox and Eimeria tenella.

[0274] 83. The composition according to any one of embodiments 77 to 81, comprising, consisting of, or consisting essentially of sporulated oocysts from at least two precocious attenuated strains selected from E. acervulina and E. maxima, E. acervulina and E. plecox, E. acervulina and E. tenella, precocious attenuated strains of E. acervulina, and E. maxima, E. plecox and E. tenella, E. acervulina, E. maxima and E. plecox, E. acervulina, E. maxima and E. tenella, or E. acervulina, E. plecox and E. tenella.

[0275] 84. The composition of any one of embodiments 77 to 83, comprising Eimeria oocysts selected from an Eimeria acervulina precocious attenuated strain having reduced virulence and reduced oocyst shedding compared to a parent Eimeria acervulina strain over a 14 day period.

[0276] 85. A composition according to any one of embodiments 77 to 84, wherein when the precocious attenuated strain of Eimeria acervulina is administered to a host, the host excretes about 30% to about 75% fewer, preferably about 35% to about 65% fewer, more preferably about 40% to about 60% fewer, and even more preferably about 45% to about 55% fewer oocysts during a 14 day period compared to the oocysts excreted by a host that received the parent Eimeria acervulina strain.

[0277] 86. A composition according to any one of embodiments 77 to 85, wherein when the precocious attenuated strain of Eimeria acervulina is administered to a host, the host excretes a similar number of oocysts in a 14 day period as the number of oocysts excreted in a 14 day period by a host that received Eimeria acervulina strain 120718P5.

[0278] 87. The composition according to any one of embodiments 77 to 86, wherein the precocious attenuated strain of Eimeria acervulina, when administered to a host, excretes about ±75% of the oocysts excreted by a host that has received Eimeria acervulina strain 120718P5, preferably about ±70%, preferably about ±60%, preferably about ±50%, preferably about ±40%, preferably about ±30%, more preferably about ±25%, more preferably about ±20%, more preferably about ±15%, and even more preferably about ±10% of the oocysts excreted by a host that has received Eimeria acervulina strain 120718P5, over a period of 14 days.

[0279] 88. The composition of any one of embodiments 77 to 87, wherein the precocious Eimeria acervulina strain has been weakened by repeated passaging of the strain selected for precocious maturation from the parent strain, preferably after at least 3 passages, at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages or at least 11 passages.

[0280] 89. The composition of embodiment 88, wherein the precocious Eimeria acervulina strain is attenuated after at least five passages from a parent strain selected for precocious maturation.

[0281] 90. The composition according to any one of embodiments 77 to 89, wherein the precocious attenuated strain of Eimeria acervulina is Eimeria acervulina strain 120718P5 deposited at the Culture Collection of Algae & Protozoa (Scottish Marine Institute, OBAN, Argyl, PA37 1QA, Scotland, UK) by HIPRA SCIENTIFIC, SLU (Avda La Selva 135, 17170 Amer, Girona, Spain) under the Budapest Treaty on 06.10.2021 under the accession number CCAP2016 / 1.

[0282] 91. The composition of any one of embodiments 77 to 90, wherein the amount of sporulated oocysts from Eimeria acervulina is about 200 to 5000 per dose, more preferably about 300 to 2000 per dose, more preferably about 400 to 1000 per dose, and even more preferably about 598 to 809 per dose.

[0283] 92. The composition according to any one of embodiments 77 to 91, wherein the amount of sporulated oocysts from Eimeria maxima is about 100 to 4000 per dose, more preferably about 150 to 3000 per dose, more preferably about 200 to 1000 per dose, more preferably about 300 to 600 per dose, and even more preferably about 352 to 476 per dose.

[0284] 93. The composition of any one of embodiments 77 to 92, wherein the amount of sporulated oocysts from Eimeria plecox is about 100 to 2000 per dose, more preferably about 200 to 1000 per dose, more preferably about 200 to 500 per dose, and even more preferably about 235 to 317 per dose.

[0285] 94. The composition according to any one of embodiments 77 to 93, wherein the amount of sporulated oocysts from Eimeria tenella is about 100 to 2000 per dose, more preferably about 200 to 1000 per dose, more preferably about 200 to 500 per dose, and even more preferably about 221 to 299 per dose.

[0286] 95. The composition of any one of embodiments 77-94, comprising about 598-809 sporulated oocysts of E. acervulina per dose, about 352-476 sporulated oocysts of E. maxima per dose, about 235-317 sporulated oocysts of E. plecox per dose, and about 221-299 sporulated oocysts of E. tenella per dose.

[0287] 96. An in ovo vaccine comprising the composition defined in any one of embodiments 77 to 95.

[0288] 97. An in ovo vaccine comprising the composition defined in any one of embodiments 77 to 95, provided that it does not contain Eimeria mitis sporulated oocysts.

[0289] 98. The in ovo vaccine according to embodiment 96 or 97, further comprising at least a pharma- ceutically or veterinarily acceptable carrier or vehicle or excipient or adjuvant.

[0290] 99. An in ovo vaccine according to embodiment 98, wherein at least the pharma- ceutically or veterinarily acceptable carrier or vehicle or excipient or adjuvant is suitable for in ovo use.

[0291] 100. The in ovo vaccine according to any one of embodiments 96-99, further comprising an additional avian vaccine comprising at least one immunogen, antigen or epitope of another avian pathogen.

[0292] 101. Additional avian pathogens include Marek's disease virus (MDV), herpesvirus of turkeys (HVT), Newcastle disease virus (NDV), non-Newcastle disease paramyxoviruses (PMV2-PMV7), infectious bronchitis virus (IBV), infectious anemia virus or chicken anemia virus (CAV), infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBDV, Gumboro disease), encephalomyelitis virus or avian encephalomyelitis virus (AEV or avian leukosis virus ALV), turkey hemorrhagic enteritis virus (HEV), pneumovirus (TRTV), and poultry pemphigus virus (PPV). 100. The in ovo vaccine according to embodiment 100, wherein the virus is selected from the group of: Avian influenza, Chicken pericarditis virus, Avian reovirus, Coccidia, Egg laying drop syndrome virus (EDS), Fowl pox, Inclusion body hepatitis (adenovirus), Lymphoproliferative disease virus (LDV), Reticuloendotheliosis virus (REV), Rotavirus enteritis and Turkey rhinotracheitis virus, Clostridium spp., Escherichia coli, Mycoplasma gallinarum, Mycoplasma gallisepticum, Haemophilus avium, Pasteurella gallinarum, Pasteurella multocida gallicida, Salmonella spp. and mixtures thereof.

[0293] 102. The in ovo vaccine according to embodiment 101, wherein the additional avian vaccine comprises at least one immunogen, antigen or epitope of the avian pathogen Infectious Bursal Disease Virus (IBDV, Gumboro Disease).

[0294] 103. An in ovo vaccine according to embodiment 102, wherein the IBDV antigen is selected from inactivated IBDV, live attenuated IBDV and an antigenic subunit of IBDV, preferably the IBDV antigen is live attenuated IBDV, more preferably the IBDV antigen is an immune complex comprising live attenuated IBDV and an IBDV-specific antibody solution.

[0295] 104. Additional avian vaccines are available at approximately 1 ~10 10 EID 50 / ml (50% embryo infectious dose / ml) of live attenuated IBDV, preferably about 10 2 ~10 9EID 50 / ml, more preferably about 10 3 ~10 8 EID 50 / ml, more preferably about 10 4 ~10 7 EID 50 / ml, and even more preferably about 10 5 ~10 6.5 EID 50 / mL of live attenuated IBDV.

[0296] 105. The in ovo vaccine according to any one of embodiments 100 to 104, wherein the composition and the additional avian vaccine are contained in separate containers, optionally with instructions for mixing and / or administration.

[0297] 106. An in ovo vaccine according to any one of embodiments 98 to 105, comprising a combination of disodium phosphate, potassium dihydrogen phosphate, polysorbate, potassium chloride, sodium chloride and water as vehicles and excipients.

[0298] 107. The in ovo vaccine of any one of embodiments 98 to 106, comprising polysorbate at a concentration in the range of about 0.001% to about 1% (v / v), about 0.005% to about 0.5% (v / v), about 0.006% to about 0.3% (v / v), about 0.007% to about 0.1% (v / v), or about 0.005% to about 0.05% (v / v).

[0299] 108. The in ovo vaccine according to any one of embodiments 98 to 107, comprising or consisting essentially of one or more adjuvants selected from polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, immunostimulatory sequences (ISS), oil-in-water emulsions, cationic lipid cytokines comprising quaternary ammonium salts, aluminum hydroxide, aluminum phosphate and mixtures thereof.

[0300] 109. A kit comprising a composition according to any one of embodiments 77-95 or an in ovo vaccine according to any one of embodiments 96-108.

[0301] The following examples are provided to illustrate different embodiments of the present invention and should be understood as non-limiting. EXAMPLES

[0302] Example 1: Obtaining Eimeria acervulina strain 120718P5 The parent of the attenuated Eimeria acervulina strain 120718P5 was isolated from a clinical case of chicken coccidiosis diagnosed by duodenal scraping of a 30-day-old chick on a Spanish poultry farm in January 2017.

[0303] These chicks showed mild lesions characteristic of E. acervulina infection in the duodenum as described by Johnson et al., "Anticoccidial drugs: lesion scoring technics in battery and floor-pen experiments with chickens," Exp. Parasitol., 1970, 28:30-36.

[0304] Immature oocysts were obtained directly from duodenal mucosal scrapings homogenized with 2.5% potassium dichromate and allowed to sporulate for 48 h at 28.5°C (suspension 1).

[0305] The sporulated oocysts of suspension 1 were then inoculated into five "coccidioid-free" SPF chicks for propagation passage. Feces were collected 4-6 days after inoculation. All birds survived to different passages. At the end of the serial passages, the animals were necropsied. At necropsy, the birds showed mild E. acervulina lesions in the duodenum. No other gross lesions were detected in the whole intestine or in other tissues. The oocysts were homogenized in a 2.5% potassium dichromate solution and then allowed to sporulate for 48 h at 28.5°C (suspension 2).

[0306] To avoid any contamination with other Eimeria species infecting chickens, the population of oocysts was obtained from a single oocyst of suspension 2. Using a 0.1 ml microcapillary pipette with a suction tube, the selected oocyst was isolated from the rest of the oocysts present in suspension 2. Oocysts of only one species were inoculated into each bird (3 weeks old).

[0307] Oocysts from the inoculated birds were recovered from the faeces of one "coccidiofree" SPF chick and allowed to sporulate (suspension 3). Due to the low number of oocysts recovered, these were inoculated into another group of five "coccidiofree" SPF chicks in order to obtain a large number of oocysts (suspension 4).

[0308] The suspension of sporulated oocysts obtained from this final propagation passage was designated Eimeria acervulina 120718 parent strain.

[0309] The E. acervulina 120718 parent strain was subsequently attenuated for precocious maturity after laboratory passage in "coccidia-free" SPF chicks. The parent 120718 E. acervulina strain was subjected to five passages selected for precocious maturity. The final strain obtained after five passages was designated E. acervulina 120718P5.

[0310] Eimeria acervulina strain 120718P5 was deposited at the Culture Collection of Algae & Protozoa (Scottish Marine Institute, OBAN, Argyl, PA37 1QA, Scotland, UK) by HIPRA SCIENTIFIC, SLU (Avda La Selva 135, 17170 Amer, Girona, Spain) under the Budapest Treaty on 06.10.2021 under the accession number CCAP2016 / 1.

[0311] This strain, 120718P5, exhibits reduced virulence and, when administered to a host, the host excretes approximately 50% fewer live oocysts over a 14-day period compared to hosts that received the parent Eimeria acervulina strain.

[0312] Example 2: Efficacy study of a live vaccine containing precocious attenuated Eimeria oocysts administered in ovo A total of 160 embryonated poultry eggs were selected for this study. The embryonated eggs were randomly assigned to four groups with 40 eggs in each group. On day 18 of incubation, the embryonated eggs were injected with 0.1 mL of different experimental vaccine compositions by the in ovo route according to group assignment.

[0313] Group 1 (high dose E. acervulina): This group received a vaccine composition containing 978 sporulated oocysts from attenuated E. acervulina strain 120718P5, 414 sporulated oocysts from attenuated E. maxima strain 013, 276 sporulated oocysts from attenuated E. plecox strain 007 and 260 sporulated oocysts from attenuated E. tenella strain 004 in 0.006 mL of phosphate buffered saline (PBS) containing 0.01% (v / v) polysorbate 80 diluted in 0.094 mL of PBS. A total volume of 0.1 mL of this vaccine composition was injected into each egg.

[0314] Group 2 (medium dose E. acervulina): This group received a vaccine composition containing 782 sporulated oocysts from attenuated E. acervulina strain 120718P5, 414 sporulated oocysts from attenuated E. maxima strain 013, 276 sporulated oocysts from attenuated E. plecox strain 007 and 260 sporulated oocysts from attenuated E. tenella strain 004 in 0.006 mL of phosphate buffered saline (PBS) containing 0.01% (v / v) polysorbate 80 diluted in 0.094 mL of PBS. A total volume of 0.1 mL of this vaccine composition was injected into each egg.

[0315] Group 3 (low dose E. acervulina): This group received a vaccine composition containing 587 sporulated oocysts from attenuated E. acervulina strain 120718P5, 414 sporulated oocysts from attenuated E. maxima strain 013, 276 sporulated oocysts from attenuated E. plecox strain 007 and 260 sporulated oocysts from attenuated E. tenella strain 004 in 0.006 mL of phosphate buffered saline (PBS) containing 0.01% (v / v) polysorbate 80 diluted in 0.094 mL of PBS. A total volume of 0.1 mL of this vaccine composition was injected into each egg.

[0316] Group 4 (control group): This group served as a positive control. The birds received a sham vaccine containing only PBS.

[0317] All oocysts included in the vaccine composition were derived from precocious attenuated Eimeria strains. The precocious attenuated strain of E. acervulina used in this study was obtained in Example 1. The precocious attenuated strains of E. maxima, E. plecox and E. tenella used in the vaccine composition of the study were obtained from the commercial vaccine EVANT® (Laboratorios HIPRA, SA, Avda. La Selva, 135, 17170 Amer, Girona, Spain), which is available in the European Union by marketing authorization issued by the EMA (EMEA / V / C / 004902) on 05 / 02 / 2019. It is also available in other non-European countries by corresponding national licenses.

[0318] At 21 days after hatching, 10 birds per group were randomly assigned to four different groups. Each group was experimentally infected with a different Eimeria species to evaluate the efficacy of an attenuated vaccine composition administered with different doses of sporulated Eimeria oocysts when administered by the in ovo route. Birds were experimentally infected by oral gavage administering 1 ml per bird of a formulation containing oocysts from a virulent Eimeria strain using an insulin syringe without a needle. The first group was infected with a formulation containing 250.000 oocysts / ml from the virulent strain Eimeria acervulina Houghton. The second group was infected with a formulation containing 100.000 oocysts / ml from the virulent strain Eimeria maxima Weybridge. The third group was infected with a formulation containing 1.000.000 oocysts / ml from the virulent strain Eimeria plecox Houghton. The fourth group was infected with a preparation containing 30.000 oocysts / ml from the virulent strain Eimeria tenella Houghton, the distribution of which is given in Table I. [Table 1]

[0319] To evaluate the efficacy of E. plecox strain 007 present in the present vaccine composition administered by the in ovo route, birds were ethically euthanized 4.5 days after experimental infection with E. plecox strain Houghton and intestinal lesions following infection were determined.

[0320] To evaluate the efficacy of E. acervulina strain 120718P5 and E. maxima strain 013 present in the present vaccine composition administered by the in ovo route, birds were ethically euthanized 6 days after experimental infection with E. acervulina strain Houghton and E. maxima strain Weybridge and post-infection intestinal lesions were determined.

[0321] To evaluate the efficacy of E. tenella strain 004 present in the present vaccine composition administered by the in ovo route, birds were ethically euthanized 7 days after experimental infection with E. tenella strain Houghton and intestinal lesions following infection were determined.

[0322] Intestinal lesions were evaluated for E. acervulina, E. maxima and E. tenella according to Johnson et al. op. cit. For E. praecox, the evaluation was performed as disclosed by Williams et al., "Pathogenesis of Eimeria praecox in chickens: virulence of field strains compared with laboratory strains of E. praecox and Eimeria acervulina", Avian Pathol., 2009, 38(5), 359-366.

[0323] The mean intestinal lesion scores per vaccine and experimentally infected group assignment were then calculated.

[0324] As shown in Figure 1, a significant reduction in intestinal lesions was clearly observed in all groups receiving the present vaccine composition containing oocysts from a precocious attenuated Eimeria strain by in ovo administration, regardless of the E. acervulina oocyst dose (high, medium or low) they received, when compared to birds in the unvaccinated control group (group 4). This significant reduction was observed after experimental infection for all Eimeria species.

[0325] Thus, a vaccine containing oocysts from precocious attenuated strains selected from E. acervulina, E. maxima, E. plecox, and E. tenella is safe and capable of generating an effective immune response against Eimeria infection, indicating that the vaccine composition confers protection against chicken coccidiosis when administered by the in ovo route.

[0326] Example 3: Safety study of a live vaccine containing precocious attenuated Eimeria oocysts administered by the in ovo route This was a 14-day study with two treatment groups and one control group to evaluate the safety of a single dose (1x) and an overdose (10x) of an attenuated Eimeria oocyst vaccine composition when administered in ovo into embryonated chicken eggs.

[0327] One hundred fifty embryonated chicken eggs were selected for the study. These eggs were randomly assigned to three different groups of 50 eggs in each group. On day 18 of incubation, the eggs were injected with a single dose (group 1) and a ten-fold (10x) overdose (group 2) of an experimental attenuated vaccine against coccidiosis by in ovo administration. Group 3 was left unvaccinated and served as the control group.

[0328] Group 1 (1x) embryonated eggs received 0.1 ml of a vaccine composition containing 0.0069 mL of attenuated E. acervulina strain 120718P5, 414 sporulated oocysts from attenuated E. maxima strain 013, 276 sporulated oocysts from attenuated E. plecox strain 007, and 260 sporulated oocysts from attenuated E. tenella strain 004 in phosphate buffered saline (PBS) containing 0.01% (v / v) polysorbate 80 diluted in 0.0931 mL of PBS. A total volume of 0.1 mL of this vaccine composition was injected into each egg.

[0329] Group 2 (10x) embryonated eggs received 0.1 ml of a vaccine composition containing 7030 sporulated oocysts from attenuated E. acervulina strain 120718P5, 4140 sporulated oocysts from attenuated E. maxima strain 013, 2760 sporulated oocysts from attenuated E. plecox strain 007, and 2600 sporulated oocysts from attenuated E. tenella strain 004 in 0.069 ml of phosphate buffered saline (PBS) containing 0.01% (v / v) polysorbate 80 diluted in 0.031 ml of PBS. A total volume of 0.1 ml of this vaccine composition was injected into each egg.

[0330] Group 3 embryonated eggs received 0.1 ml of a composition containing a mock vaccine based solely on phosphate buffered saline (PBS).

[0331] All oocysts included in the vaccine formulation were derived from precocious attenuated Eimeria strains. The precocious attenuated strain of E. acervulina used in this study was obtained in Example 1. The precocious attenuated strains of E. maxima, E. plecox and E. tenella used in the vaccine composition of the study were obtained from the commercial vaccine EVANT® (Laboratorios HIPRA, SA, Avda. La Selva, 135, 17170 Amer, Girona, Spain), which is available in the European Union by marketing authorization issued by the EMA (EMEA / V / C / 004902) on 05 / 02 / 2019. It is also available in other non-European countries by corresponding national licenses.

[0332] To determine the safety of the vaccine composition, bird weights were recorded at 0, 4.5, 6, 7 and 14 days after hatch.

[0333] The study showed that there was no difference in body weight between animals from the vaccinated groups, i.e., groups 1 and 2, compared to animals from the control group (group 3). Surprisingly, animals from group 2, vaccinated with an oocyst dose 10 times higher than the standard dose, had similar body weight to animals from the control group, i.e., group 3 (Figure 2). These results therefore confirm that vaccines containing oocysts from a precocious attenuated strain of Eimeria are safe when administered in ovo, even at doses exceeding the recommended standard doses.

[0334] Clinical signs were also monitored during the study. No changes in fecal appearance and general clinical signs were detected in the vaccinated groups. These results therefore also confirm the optimal safety profile of the attenuated vaccine when it is administered in ovo.

[0335] Example 4: Hatchability study of a live vaccine containing precocious attenuated Eimeria oocysts administered in ovo As disclosed in the prior art, in ovo vaccination can negatively affect hatchability of embryonated eggs. This can be due to the type of vaccine administered to the embryonated eggs. For example, in ovo vaccination with Inovocox EM1 vaccine, a vaccine containing live sporulated oocysts from a non-attenuated Eimeria strain, reduced hatchability by 1-2.59% compared to diluent-injected groups (Sokale et al., 2017, op. cit. and Sokale et al., 2021, op. cit.).

[0336] This study evaluates the safety, in terms of hatchability, of a vaccine containing sporulated oocysts from a precocious attenuated Eimeria strain when it is administered by the in ovo route under field conditions.

[0337] Two commercial hatcheries were included in the study. Embryonated eggs from homogenous batches (hens from the same or similar age group) from each hatchery were distributed into two groups (groups A and B). Each group received a different vaccine as described below. All vaccinations were performed at the hatchery after random distribution of the eggs into the two groups. Allocation of the vaccine to each group was performed randomly at the hatchery. On day 18 of incubation, embryonated eggs were injected with 0.05 ml / egg of the vaccine by in ovo administration.

[0338] Group A: This group served as a positive control group. Animals in this group received a sham vaccine based on phosphate-buffered saline (PBS).

[0339] Group B: Animals in this group received a vaccine containing 0.006 mL of a mixture of 598-809 sporulated oocysts from attenuated E. acervulina strain 120718, 352-476 sporulated oocysts from attenuated E. maxima strain 013, 235-317 sporulated oocysts from attenuated E. plecox strain 007, and 221-299 sporulated oocysts from attenuated E. tenella strain 004 in phosphate buffered saline (PBS) containing 0.01% (v / v) polysorbate 80 diluted in 0.044 mL of diluent PBS. A total volume of 0.05 mL of this vaccine composition was injected into each egg.

[0340] All oocysts included in the vaccine formulation were derived from precocious attenuated Eimeria strains. The precocious attenuated strain of E. acervulina used in this study was obtained in Example 1. The precocious attenuated strains of E. maxima, E. plecox and E. tenella used in the vaccine composition of the study were obtained from the commercial vaccine EVANT® (Laboratorios HIPRA, SA, Avda. La Selva, 135, 17170 Amer, Girona, Spain), which is available in the European Union by marketing authorization issued by the EMA (EMEA / V / C / 004902) on 05 / 02 / 2019. It is also available in other non-European countries by corresponding national licenses.

[0341] The total number of embryonated eggs per group varied by hatchery. In the first hatchery (Hatchery A), a total of 219,996 embryonated eggs were included in the study, with 108,471 embryonated eggs allocated to group B and 111,525 embryonated eggs allocated to group A (control group). In the second hatchery (Hatchery B), a total of 144,476 embryonated eggs were included in the study, with 72,187 embryonated eggs allocated to group B and 72,289 embryonated eggs allocated to group A (control group).

[0342] Hatchability was calculated by dividing the number of hatched eggs by the total number of fertilized eggs in each group. Hatchability was compared between groups for each batch of eggs and chickens included in the study. Additionally, the overall mean hatchability obtained for each group across all batches of eggs and chickens included in the study was compared between groups.

[0343] The results unexpectedly show an increase in hatchability in the group vaccinated with the vaccine of the present invention (Group B) compared to the control group (Group A) in two different hatcheries tested (Figures 3A and 3B). The overall mean hatchability is increased by 1.15% compared to the control group (Figure 3C). Considering the large volume of eggs processed in commercial hatcheries, this observed increase in hatchability (%) provides a surprising and significant advantage over current in ovo non-attenuated Eimeria vaccines.

[0344] Example 5: Efficacy of a single dose of a live attenuated Eimeria vaccine against chicken coccidiosis combined with a live attenuated vaccine against infectious bursal disease (IBD) administered in ovo into embryonated chicken eggs against infection with highly virulent IBD virus (vvIBDv) This study evaluated the efficacy of a single dose vaccine based on the combined administration of a live attenuated vaccine against infectious bursal disease virus (IBDV) and a live attenuated Eimeria vaccine when mixed prior to administration in embryonated chicken eggs by the in ovo route against infection with a highly virulent IBDV strain on the 24th day of life.

[0345] A total of 28 embryonated chicken eggs were selected for this study from a commercial hatchery that did not use anticoccidial drugs. These eggs were randomly assigned to two different groups of 14 embryonated eggs each. On day 18 of incubation, the embryonated eggs were injected by the in ovo route with 0.05 ml / egg of the following vaccines according to group assignment:

[0346] Group 1: 14 18-day-old embryonated chicken eggs received a combination vaccine based on a combination of a live attenuated Eimeria vaccine of the present invention with a live attenuated infectious bursal disease vaccine. The Eimeria vaccine contained a mixture of attenuated E. acervulina, E. maxima, E. plecox and E. tenella oocysts, i.e. 598-809 sporulated oocysts from E. acervulina strain 120718P5, 352-476 sporulated oocysts from E. maxima strain 013, 235-317 sporulated oocysts from E. plecox strain 007 and 221-299 sporulated oocysts from E. tenella strain 004 per dose in phosphate buffered saline (PBS) containing 0.01% (v / v) polysorbate 80. The live infectious bursal disease (IBD) vaccine was GUMBOHATCH® (Laboratorios HIPRA SA, Avda. La Selva 135, 17170 Amer, Girona, Spain).

[0347] GUMBOHATCH® is a minimum dose of 10 1.48 PU (potency units) of live attenuated IBDV strain 1052 and 2.7 × 10 6 It is a live immunoconjugate vaccine containing an IBDV-specific antibody solution in VNU (virus neutralizing units).

[0348] GUMBOHATCH® is available in the European Union through a marketing authorization issued by the EMA (EMEA / V / C / 004967) on 12 / 11 / 2019. It is available in other non-European countries through corresponding national licenses. Another IBD vaccine based on live immunoconjugates is CEVAC TRANSMUNE® (Ceva Animal Health, SA, 08028 Barcelona, ​​Spain), which is also available in the EU market, among other markets.

[0349] The combination vaccine of the invention was prepared as follows: Prior to the in ovo vaccination, one vial of 2000 doses of freeze-dried IBD immunoconjugate vaccine (GUMBOHATCH®, Laboratorios HIPRA, SA, Avda. La Selva 135, 17170 Amer, Girona, Spain) was reconstituted with 4 mL of sterile PBS, i.e. the diluent was injected into the freeze-dried vaccine fraction. After 5 minutes, the composition was stirred to completely dissolve the product. Once reconstituted, 0.103 mL of IBD immunoconjugate vaccine was mixed with 8.877 mL of PBS and 1.020 mL of attenuated Eimeria vaccine previously mixed. The vial was shaken again before use.

[0350] From this final volume of 10 ml, a single dose of 0.05 mL of combination vaccine (Eimeria vaccine + IBD vaccine) was administered to each embryonated egg 2 hours prior to reconstitution.

[0351] Group 2: Fourteen 18-day-old embryonated chicken eggs served as the control group. Animals in this group received a sham vaccine containing only PBS.

[0352] During the vaccination period, embryonated eggs and hatched chicks of vaccinated and control hens were handled similarly except for exposure to the products. Vaccinated and control groups were clearly identified to avoid cross-group contamination. All manipulations were performed first in the control group (group 2) and then in the vaccinated group (group 1). Clinical signs were monitored at flock level. Oocyst counts were also performed in litter and fresh faeces.

[0353] Prior to infection, blood samples were taken from the animals to check antibody levels against IBDV to confirm correct vaccination with GUMBOHATCH® and to demonstrate that the control group remained seronegative.

[0354] To evaluate the efficacy against IBDV of the combination of IBDV vaccine and attenuated Eimeria vaccine administered in ovo, all hatched chicks from both groups 1 and 2 were vaccinated on the 24th day of age by oculonasal route with 0.2 mL of a highly virulent pathogenic strain of IBDV, i.e., 10 6.4 EID 50 The mice were experimentally infected with strain VG-248 at a titer of 100 / ml.

[0355] On day 6 post-infection (day 30 of the study), when the acute phase of IBDv infection is expected to occur, the animals were necropsied and the outcome of infection was compared between groups. To assess protection, the bursa of Fabricius (BF) and spleen of the animals were examined macroscopically. A complete histopathological analysis of the BF was performed according to Sharma et al., "Comparative pathogenesis of serotype 1 and variant serotype 1 isolates of an infectious bursal disease virus and their effect on humoral and cellular immune competence of specific-pathogen-free chickens," Avian dis., 1989, 33:112-124.

[0356] An acute histological and functional lesion score was calculated for each animal and the average for each group is presented in Figure 4. The acute histological and functional lesion score is a summary score obtained by summing the individual scores for the clinical parameters indicative of acute lesions: pseudoeosinophilic and mononuclear cell infiltration, hemorrhage, luminal exudate, fold edema, muscle wall edema, serosal edema, necrotic cysts and lymphocytic necrosis.

[0357] A significant reduction in the mean acute histological functional score is observed in vaccinated birds (group 1) compared to the control group (group 2). These results indicate that the combination vaccine is able to reduce acute functional lesions in the bursa of infected animals.

[0358] Furthermore, bursae from the vaccinated group (group 1) showed significantly less inflammatory infiltrate and numerically less rugae edema than animals from the control group (group 2). A complete absence of serosal and muscle wall edema was also observed in vaccinated group 1. This inflammatory response represents the high virulence of the infecting strain, and its absence in vaccinated animals means that the vaccine is not only able to induce an immune response but also to protect against highly virulent IBDV strains such as those used in this study (Figures 5-8).

[0359] The percentage of presence of external edema in the bursa of Fabricius for both groups was also analyzed. The presence of external edema in the bursa of Fabricius is considered a clear sign of IBDv infection. As shown in Table II, external edema was observed in 85.71% of animals in the non-vaccinated control group (Group 2), while vaccinated animals in Group 1 did not show any clinical signs related to external edema. [Table 2]

[0360] All the differences observed between the vaccinated group (group 1) and the unvaccinated group (group 2, control group) demonstrate that the combination vaccine clearly overcomes the effects of vvIBDV and protects the animals against vvIBDV infection and Gumboro disease.

[0361] Efficacy is demonstrated by the fact that the appearance of serosal edema, muscle wall edema and external edema in the bursa of Fabricius after infection is completely prevented in vaccinated animals. Furthermore, vaccinated animals showed a reduction in the mean acute histological functional score, inflammatory infiltrate and fold edema compared to non-vaccinated animals.

[0362] Overall, the results obtained in this study surprisingly show that the combination of an attenuated Eimeria vaccine administered by the in ovo route with an IBDV vaccine has no adverse effect on the efficacy of the IBDV vaccine and that the combination vaccine is similarly effective against IBDV infection and therefore protects against Gumboro disease.

[0363] Example 6: Efficacy of a single dose of a live attenuated Eimeria vaccine against chicken coccidiosis combined with a live attenuated vaccine against infectious bursal disease (IBD) administered in ovo into embryonated chicken eggs against infection with four different Eimeria species This study evaluated the efficacy of a single dose vaccine based on the combined administration of a live attenuated Eimeria vaccine when mixed with live attenuated infectious bursal disease virus (IBDv) prior to administration by the in ovo route into embryonated chicken eggs after infection with four different Eimeria species at 21 days of age.

[0364] A total of 240 18-day-old embryonated SPF eggs were included in the study. These eggs were randomly assigned to two different groups with 120 embryonated eggs in each group. On day 18 of incubation, the embryonated eggs were injected by in ovo route with 0.05 ml / egg of the following vaccines according to group assignment:

[0365] Group 1: 120 18-day-old embryonated SPF chicken eggs received a vaccine based on a combination of a live attenuated Eimeria vaccine and a live attenuated Infectious Bursal Disease vaccine of the present invention. The Eimeria vaccine contained a mixture of attenuated E. acervulina, E. maxima, E. plecox and E. tenella oocysts, i.e., 598–809 sporulated oocysts from E. acervulina strain 120718P5, 352–476 sporulated oocysts from E. maxima strain 013, 235–317 sporulated oocysts from E. plecox strain 007 and 221–299 sporulated oocysts from E. tenella strain 004 per dose in phosphate-buffered saline (PBS) containing 0.01% (v / v) polysorbate 80. The live infectious bursal disease (IBD) vaccine was GUMBOHATCH® (Laboratorios HIPRA SA, Avda. La Selva 135, 17170 Amer, Girona, Spain) as previously disclosed in Example 5. IBD disease is commonly known as Gumboro disease. The IBD virus becomes a permanent fixture in poultry houses and a resident pathogen infecting successive flocks, making it a continuous threat.

[0366] The combination vaccine used in this study was prepared as disclosed in Example 5. One vial of 2000 doses of freeze-dried IBD immunoconjugate vaccine (GUMBOHATCH®, Laboratorios HIPRA, SA, Avda. La Selva 135, 17170 Amer, Girona, Spain) was reconstituted with 4 mL of sterile PBS, i.e. the diluent was injected into the freeze-dried vaccine fraction. After 5 minutes, the composition was stirred to completely dissolve the product. Once reconstituted, 0.103 mL of IBD immunoconjugate vaccine was mixed with 8.877 mL of PBS and 1.020 mL of attenuated Eimeria vaccine previously mixed. The vial was shaken again before use. From this final volume of 10 ml, a single dose of 0.05 mL of combination vaccine (Eimeria vaccine + IBD vaccine) was administered to each embryonated egg 2 hours before reconstitution.

[0367] Group 2: 120 18-day-old SPF embryonated chicken eggs served as the control group. Animals in this group received a sham vaccine containing only PBS.

[0368] During the vaccination period, embryonated eggs and hatched chicks of vaccinated and control hens were handled similarly except for exposure to the products. Vaccinated and control groups were clearly identified to avoid cross-group contamination. All manipulations were performed first in the control group (group 2) and then in the vaccinated group (group 1). Clinical signs were monitored at flock level. Oocyst counts were performed in litter and fresh faeces.

[0369] Prior to infection, blood samples were taken from the animals to check antibody levels against IBDV to confirm correct vaccination with GUMBOHATCH® and to demonstrate that the control group remained seronegative.

[0370] The efficacy of both vaccine combinations was then determined using a chicken coccidiosis infection model against each Eimeria species included in the Eimeria vaccine of the present invention, in which each Eimeria infection was performed in a separate sub-study with the appropriate heterologous infecting Eimeria strain. Vaccinated and control birds were experimentally infected by the oral route on day 21 after hatch according to the European Pharmacopoeia requirements.

[0371] For each Eimeria species, chickens were observed at least daily for 14 days after infection. The following parameters were monitored: clinical signs (including bird appearance and fecal appearance), mortality, intestinal lesions, oocyst counts, body weight and food consumption. During this period, chickens were allocated to the same room and caged per Eimeria species. All procedures during the infection period were performed blinded to treatment.

[0372] Lesions were scored in samples from each group of animals on days 4.5, 6 and 7 post-infection, which are the optimal times for performing macroscopic intestinal lesion analysis for E. plecox (4.5), E. acervulina and E. maxima (6) and E. tenella (7), respectively. Lesions were scored according to the standard method of Johnson et al., op. cit., which is in line with the instructions contained in the European Pharmacopoeia monograph. For E. plecox, gross lesions were evaluated according to the bibliographic references of Williams et al., op. cit. Microscopic lesions were further evaluated by performing histological studies for E. plecox infection tests.

[0373] All procedures were approved by the ethical committee and were in accordance with the European regulations for animal welfare (2010 / 63 / EU).

[0374] At 21 days after hatching, 30 birds per group were randomly assigned to four different infection groups. Each group was experimentally infected with a different Eimeria species to evaluate the efficacy of the combination vaccine composition administered by the in ovo route against chicken coccidiosis as described above. Birds were experimentally infected by oral gavage, administering 1 ml per bird of a formulation containing oocysts from virulent Eimeria strains using an insulin syringe without a needle. The first group was infected with a formulation containing 250,000 oocysts / ml from the virulent E. acervulina Houghton strain. The second group was infected with a formulation containing 100,000 oocysts / ml from the virulent E. maxima Weybridge strain. The third group was infected with a formulation containing 1,000,000 oocysts / ml from the virulent E. praecox Houghton strain. The fourth group was infected with a preparation containing 30,000 oocysts / ml from the virulent Eimeria tenella Houghton strain, the distribution of which is given in Table III. [Table 3]

[0375] To evaluate the efficacy of E. plecox strain 007 present in this combination vaccine composition administered by the in ovo route, 18 birds per group were ethically euthanized 4.5 days after experimental infection with E. plecox strain Houghton and intestinal lesions following infection were determined.

[0376] To evaluate the efficacy of E. acervulina strain 120718P5 and E. maxima strain 013 present in the present combination vaccine composition administered by the in ovo route, 18 birds per group were ethically euthanized 6 days after experimental infection with E. acervulina strain Houghton and E. maxima strain Weybridge and post-infection intestinal lesions were determined.

[0377] To evaluate the efficacy of E. tenella strain 004 present in this combination vaccine composition administered by the in ovo route, 18 birds per group were ethically euthanized 7 days after experimental infection with E. tenella strain Houghton and intestinal lesions following infection were determined.

[0378] For E. acervulina, E. maxima, and E. tenella, intestinal lesions were assessed according to Johnson et al., supra. For E. plecox, lesions were assessed according to Williams et al., supra.

[0379] The mean intestinal lesion score per group and experimental infection group assignment was then calculated. Figure 9 shows the mean intestinal lesion score per group and experimental infection. Necropsy and lesion scoring were performed for each Eimeria species.

[0380] A significant reduction in intestinal lesions was observed in all groups receiving the combination vaccine composition containing attenuated Eimeria strains and sporulated oocysts from live attenuated IBDV (Group 1) when compared to the non-vaccinated control group (Group 2). Furthermore, no intestinal lesions were observed with the combination vaccine, providing protection against intestinal lesions caused by E. acervulina infection.

[0381] The number of oocysts excreted in fresh feces per bird after experimental infection in each group was also assessed. Fresh feces were collected from the trays on days 3-14 post-infection and the average number of oocysts excreted in fresh feces per bird was determined. The results are shown in Table IV. [Table 4]

[0382] Vaccine group 1, which received a combination of Eimeria and IBDv vaccines, shows that in ovo administration of this combination vaccine into embryonated chicken eggs significantly reduces oocyst shedding after experimental infection for all the different Eimeria species tested.

[0383] The percentage of oocysts excreted during the infection period was reduced by approximately 98.0% for E. acervulina, 96.2% for E. plecox, 92.4% for E. tenella, and 87.3% for E. maxima in the vaccinated group compared to the control group, and the reduction in the percentage of oocyst counts was significant for E. maxima.

[0384] Furthermore, 100% of vaccinated animals in Group 1 seroconverted to IBDv on day 20, indicating that the animals were successfully vaccinated with the combination vaccine. Control animals remained negative for IBDV throughout the entire vaccination period.

[0385] Thus, a combination vaccine comprising oocysts from a precocious attenuated strain selected from E. acervulina, E. maxima, E. plecox and E. tenella in combination with a live attenuated IBDV can produce an effective response against Eimeria infection and chicken coccidiosis when administered by the in ovo route into embryonated eggs. The effective response obtained by the combination vaccine of the present invention reduces and / or prevents intestinal lesions and reduces oocyst shedding caused by Eimeria infection.

[0386] Overall, the results of the study demonstrate the efficacy of a single dose of a live vaccine against infectious bursal disease combined with a live attenuated vaccine against chicken coccidiosis caused by Eimeria infection when mixed prior to in ovo administration into 18-day-old embryonated eggs.

[0387] Moreover, the parameters monitored during the vaccination period before experimental challenge were comparable between the vaccinated group (group 1) and the control group (group 2), thus further confirming the optimal safety of this combination vaccine administered in ovo into embryonated chicken eggs.

[0388] Example 7: Oocyst Removal Profile The primary objective of this study was to evaluate the clearance profile of Eimeria spp. oocysts following administration of a live attenuated Eimeria spp. vaccine composition administered by the in ovo route when compared to administration of a live attenuated Eimeria spp. vaccine composition administered by coarse spray. The study was performed with a combination administration of additional antigens other than Eimeria spp. that were mixed prior to in ovo administration into embryonated chicken eggs.

[0389] The Eimeria spp. vaccine composition used in this study is a live vaccine against chicken coccidiosis containing attenuated E. acervulina, E. maxima, E. plecox and E. tenella oocysts. Vaccines against Marek's disease (MD), Newcastle disease (ND) and infectious bursal disease (IBD) are routinely used in poultry farms. Because in ovo vaccination is performed simultaneously with other vaccines, interference may occur when the Eimeria spp. vaccine composition is used simultaneously with other vaccines. Therefore, we also evaluated whether these vaccines cause antigenic interference with the Eimeria spp. vaccine composition.

[0390] The Vectormune® ND vaccine is a recombinant Herpesvirus of Turkeys (HVT) vector vaccine that uses the Herpesvirus of Turkeys (HVT) as a vector and has the fusion (F) gene of Newcastle Disease Virus (NDV) inserted into its genome. The Vectormune® ND vaccine is indicated for in ovo administration to 18-day-old embryonated chicken eggs or 1-day-old chicks.

[0391] GUMBOHATCH® is a live immunoconjugate vaccine indicated against infectious bursal disease (IBD). The vaccine is intended to protect chicks against the highly virulent IBD virus (vvIBDV) when maternal antibody levels begin to decline. The GUMBOHATCH® vaccine is also indicated for in ovo administration in 18-day-old embryonated eggs.

[0392] A total of 214 18-day-old embryonated chicken eggs were included in the study. The embryonated eggs were randomly assigned into two different groups with 107 eggs in each group.

[0393] The first group of embryonated eggs (Group 1) received an in ovo injection of 0.05 mL / egg of a vaccine based on a combination of a live attenuated Eimeria spp. vaccine composition, a live attenuated infectious bursal disease vaccine and a recombinant Newcastle Disease Virus vaccine on day 18 of culture. The Eimeria spp. vaccine composition contained a mixture of live attenuated E. acervulina, E. maxima, E. plecox and E. tenella oocysts per dose, i.e., 598-809 sporulated oocysts from E. acervulina strain 120718P5, 352-476 sporulated oocysts from E. maxima strain 013, 235-317 sporulated oocysts from E. plecox strain 007 and 221-299 sporulated oocysts from E. tenella strain 004 in phosphate buffered saline (PBS) containing 0.01% (v / v) polysorbate 80.

[0394] The live infectious bursal disease (IBD) vaccine was GUMBOHATCH® (Laboratorios HIPRA SA, Avda. La Selva 135, 17170 Amer, Girona, Spain) previously disclosed in Examples 5 and 6. The recombinant Newcastle Disease Virus (NDV) vaccine was Vectormune® ND (CEVA-Phylaxia Co. Ltd., 1107 Budapest, Szallas u.5, Hungary). Vectormune® ND is a live recombinant vaccine containing a recombinant Herpesvirus of Turkeys at a dose of 2500-8000 PFU (plaque forming units) expressing the fusion protein of NDV. Vectormune® ND is available in the European Union by marketing authorization issued by the EMA (EMEA / V / C / 003829) on 08 / 09 / 2015.

[0395] The combination vaccine was prepared as follows: a 1 mL ampoule of Vectormune® ND was thawed and diluted in 42 mL of diluent according to the vaccine manufacturer's instructions. In this study, the diluent was an aqueous solution containing sucrose, casein hydrolysate, sorbitol, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, phenol red and water for injection as described according to the EU marketing authorization. Then, 6 mL of the live attenuated Eimeria spp. in ovo vaccine composition of the present invention was added slowly and gently. Then, one vial of GUMBOHATCH® lyophilized vaccine was reconstituted with 4 mL of diluent. Finally, 1 mL of the reconstituted GUMBOHATCH® vaccine was added to the 49 mL mixture of Vectormune® ND and live attenuated Eimeria spp. vaccine. A single dose of 0.05 mL of the combination vaccine was administered in ovo per embryonated egg.

[0396] The second group of embryonated eggs (Group 2) received an in ovo injection of 0.05 mL / egg of a vaccine based on a combination of a live attenuated IBD vaccine (GUMBOHATCH®) and a recombinant NDV vaccine (Vectormune® ND) on day 18 of culture. The combination vaccine was prepared as follows: one ampoule of 2 mL of Vectormune® ND was thawed according to the vaccine manufacturer's instructions and diluted in 96 mL of diluent. 2 mL of reconstituted GUMBOHATCH® vaccine was then added. A single dose of 0.05 mL of the combination vaccine was administered per egg.

[0397] Group 2 also received one standard dose of live attenuated Eimeria spp. vaccine, i.e. EVANT® (Laboratorios HIPRA, SA, Avda. La Selva, 135, 17170 Amer, Girona, Spain) by coarse spray on the day of hatch (1 day of age). EVANT® is a live attenuated Eimeria spp. vaccine containing 332-450 sporulated oocysts from E. acervulina strain 003, 196-265 sporulated oocysts from E. maxima strain 013, 293-397 sporulated oocysts from E. mitis strain 006, 293-397 sporulated oocysts from E. plecox strain 007 and 276-374 sporulated oocysts from E. tenella strain 004 per dose. EVANT® is available in the European Union with marketing authorization issued by the EMA (EMEA / V / C / 004902) on 05 / 02 / 2019.

[0398] At hatching, hatchability was recorded and birds were monitored for clinical signs and flock-level mortality. Additionally, litter and fresh feces oocyst counts were performed during the study to track vaccine clearance profiles. Blood samples were saved for serological studies of antibody responses to IBD and ND as well as qPCR testing for HVT. Finally, animal weights were also monitored during the trial. All procedures during the study were blinded to treatment.

[0399] Serial necropsies were performed to investigate safety parameters. This included examination of damage to the bursa of Fabricius and evaluation of intestinal lesions after administration of the vaccine. Regular necropsies were performed on days 21, 28, and 45 of the study. Twelve animals per group and time point were necropsied, thus a total of 36 chickens per group were necropsied for safety. Blood samples were also taken at each time point for qPCR testing against HVT. Body weights of all animals were also recorded.

[0400] During the vaccination period, embryonated eggs and hatched chicks of vaccinated and control hens were handled identically except for exposure to the products. Vaccinated and control groups were clearly identified to avoid cross-group contamination. All manipulations were performed first in the control group and then in the vaccinated group.

[0401] To assess Eimeria spp. oocyst removal, fresh fecal samples were collected from each group on days 1-9 post-hatch and counted for oocyst shedding. The mean Eimeria spp. oocyst counts in fresh feces of each group on different study days are shown in Figure 10.

[0402] The results of this study demonstrate that the oocyst clearance profile obtained after vaccinating a subject with a live attenuated Eimeria spp. vaccine composition by the in ovo route is different from the oocyst clearance profile obtained after vaccinating a subject with a live attenuated Eimeria spp. vaccine administered by coarse spray.

[0403] Because the mechanism of live attenuated Eimeria spp. vaccines requires completion of the Eimeria spp. life cycle within the host in order to adequately immunize the vaccinated subject, clearance of oocysts by the vaccinated subject is an important parameter for determining the protective performance of a live attenuated Eimeria spp. vaccine.

[0404] The study showed an unexpected earlier (day 3) and higher peak in the oocyst clearance profile (70,000 oocysts / g fresh faeces) of the in ovo live attenuated Eimeria spp. vaccine composition compared to coarse spray administration (40,000 oocysts / g fresh faeces at day 6), demonstrating that the in ovo Eimeria spp. vaccine of the present invention can initiate its life cycle earlier than the live attenuated Eimeria spp. vaccine administered by coarse spray.

[0405] Therefore, the faster the Eimeria spp. oocysts from the live attenuated vaccine are cleared, the faster an immune response will be elicited in the vaccinated subject.

[0406] Thus, it has been confirmed that the in ovo live-attenuated Eimeria spp. vaccine composition of the present invention is capable of eliciting an earlier immune response, which is also protective against Eimeria spp. infection in vaccinated subjects.

[0407] Furthermore, in ovo vaccination with an Eimeria spp. vaccine composition in combination with currently commercially available Marek's Disease, Newcastle Disease and Gumboro Disease vaccines did not affect either the safety or immunogenicity of the different antigens contained in the vaccine composition, such that no antigenic interference was observed when combining such vaccines.

[0408] Similar results regarding the oocyst clearance profile were also observed when the in ovo live-attenuated Eimeria spp. vaccine composition was administered alone without combining it with additional avian antigens such as Marek's disease, Newcastle disease and Gumboro disease antigens, confirming the earlier oocyst clearance profile of the in ovo live-attenuated Eimeria spp. vaccine composition than the Eimeria spp. vaccine administered by the coarse spray route.

Claims

1. 1. A composition for use as a vaccine in the prevention and / or treatment of Eimeria infection, said composition comprising Eimeria oocysts from a precocious attenuated strain of E. acervulina and at least one precocious attenuated strain selected from E. maxima, E. plecox and E. tenella, and said vaccine being administered in ovo into embryonated eggs of a domestic bird.

2. 2. The composition for use as a vaccine of claim 1, which reduces or prevents one or more clinical signs associated with chicken coccidiosis in hatched birds selected from the group consisting of intestinal lesions, weight loss, oocyst excretion, oocyst burden, oocyst shedding, diarrhea, dehydration, presence of blood in the faeces, presence of mucus in the faeces, Eimeria infection, depression, ruffled feathers, arched body position, morbidity and mortality.

3. 2. The composition for use as a vaccine according to claim 1, for the prevention of chicken coccidiosis.

4. 2. A composition for use as a vaccine according to claim 1, comprising a mixture of sporulated oocysts from precocious attenuated strains of E. acervulina, E. maxima, E. plecox and E. tenella.

5. 5. The composition for use as a vaccine of claim 4, wherein when the precocious attenuated strain of E. acervulina used therein is administered to a host, the host excretes about 30% to about 75% fewer oocysts over a 14 day period compared to the number of oocysts excreted by a host that received the parent E. acervulina strain.

6. 2. The composition for use as a vaccine according to claim 1, wherein the precocious attenuated strain of E. acervulina used therein is E. acervulina strain 120718P5 deposited at the Culture Collection of Algae & Protozoa (Scottish Marine Institute, OBAN, Argyl, PA37 1QA, Scotland, UK) by HIPRA SCIENTIFIC, S.L.U. (Avda La Selva 135, 17170 Amer, Girona, Spain) under the Budapest Treaty on 06.10.2021 with accession number CCAP2016 / 1.

7. 2. The composition for use as a vaccine according to claim 1, comprising about 100 to 6000 sporulated oocysts of Eimeria acervulina per dose.

8. 2. The composition for use as a vaccine according to claim 1, administered in a dose comprising between 0.050 ml and 0.20 ml.

9. 10. The composition for use as a vaccine of claim 1, further comprising an additional avian vaccine comprising at least one immunogen, antigen or epitope of another avian pathogen.

10. 10. The composition for use as a vaccine according to claim 9, for the prevention and / or treatment of Eimeria infection and infectious bursal disease virus (IBDV, Gumboro disease) infection.

11. 10. The composition for use as a vaccine according to claim 9, wherein said composition is mixed with an additional avian vaccine prior to use and simultaneously administered in ovo into embryonated eggs of domesticated birds.

12. 10. The composition for use as a vaccine of claim 9, wherein the additional avian vaccine comprises at least one immunogen, antigen or epitope of the avian pathogen Infectious Bursal Disease Virus (IBDV, Gumboro disease).

13. 13. The composition for use as a vaccine of claim 12, which prevents and / or reduces at least one of the clinical signs associated with Gumboro disease or IBDV infection selected from the group consisting of absence of mean acute histological functional score, gross lesions, mononuclear cell infiltrate edema, inflammatory infiltrate, fold edema, serosal edema and muscle wall edema.

14. 14. A kit for use as a vaccine in the prevention and / or treatment of Eimeria infection, wherein the vaccine is administered in ovo into embryonated eggs of a domesticated bird, and the kit comprises a composition for use as a vaccine according to any one of claims 1 to 13.

15. An in ovo vaccine comprising sporulated Eimeria oocysts from a precociously attenuated strain of Eimeria acervulina and at least one precociously attenuated strain selected from Eimeria maxima, Eimeria plecox, and Eimeria tenella, wherein the amount of sporulated oocysts from Eimeria acervulina is about 100-6,000 per dose, the amount of sporulated oocysts from Eimeria maxima is about 50-5,000 per dose, the amount of sporulated oocysts from Eimeria plecox is about 50-3,000 per dose, and the amount of sporulated oocysts from Eimeria tenella is about 50-3,000 per dose, provided that the in ovo vaccine does not contain Eimeria mitis sporulated oocysts.

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