Abstract:
A multivalent poultry vaccine is provided having two or more live biological agents or microbial components. Each live biological agent or microbial component is effective in preventing or treating an avian disease, and the multivalent vaccine is safe and effective for immunizing poultry in ovo. Methods are also provided for vaccinating poultry by administering such a multivalent vaccine in ovo.

Description:
This application is a divisional of U.S. patent application Ser. No. 08/874,103, filed Jun. 12, 1997, now U.S. Pat. No. 6,048,535, which application is incorporated by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     Domestic Avian Diseases 
     Commercial chicken flocks in most parts of the world are routinely vaccinated to protect them against environmental exposure to pathogens. Some of the more common viruses that cause disease in domestic poultry include Marek&#39;s disease virus (MDV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian encephalomyelitis (AEV), chick anemia virus (CAV), Fowlpox virus (FPV), avian influenza virus (AIV), reovirus, avian leukosis virus (ALV), reticuloendotheliosis virus (REV), avian adenovirus and hemorrhagic enteritis virus (HEV). These diseases are of economic importance to the poultry industry. Currently, vaccines are available to protect commercial poultry against most of these diseases. ( Diseases of Poultry , 10th ed. (Calneck et al., eds.), Iowa State University Press, Ames, Iowa (1997)). 
     One of the most economically important diseases is Marek&#39;s disease (MD). MD is a lymphoproliferative disease that occurs naturally in chickens. The disease is caused by a herpesvirus that is extremely contagious, spreads horizontally, and has been responsible for major economic losses to the poultry industry. Chickens have sensitivity to this virus with no influence of lineage or sex, but it is reported that younger chickens have a higher sensitivity and suffer greater damage from the virus. The symptoms of MD appear widely in the nerves, genital organs, internal organs, eyes and skin of the infected birds causing motor trouble due to paralysis when the nerves have been affected, functional trouble of the internal organs due to tumors, and chronic undernourishment if the internal organs are attacked by the virus. The chickens usually die. MD is one of the leading causes of economic losses in the poultry industry. 
     Post-hatch Vaccination 
     In most commercial flocks, newly hatched chicks are given certain vaccines parenterally at hatch. Because exposure to pathogens often occurs at a very young age, they often need to be vaccinated before they are placed in rearing or brooder houses. Such a vaccination scheme requires handling of individual birds and involves the possible risk of accidental self-injection. Another problem with this vaccination method is that the vaccines are not always effective. The young chicks may become exposed to a virulent form of a disease too soon after vaccination, i.e., before they have the opportunity to develop adequate protective immunity. 
     In ovo Vaccination 
     It has been shown that certain live viral vaccines can be administered in eggs before the birds hatch. This procedure is called “in ovo vaccination.” The in ovo vaccinated birds develop resistance to the target disease. The exact mechanism by which embryonal vaccination results in increased resistance to challenge at hatch is not yet clear. The poultry industry in the U.S. and abroad has responded to the benefits of in ovo vaccination and this procedure is rapidly gaining popularity. Over seven billion birds receive vaccines yearly in the U.S. In 1994, about 30% of the U.S. commercial chicken population was vaccinated against MD by the in ovo procedure. In 1997, the figure has risen to over 75% or about 6.0 billion chickens. 
     It should be noted, that many vaccines used previously for hatched birds cannot be used for in ovo vaccination because the vaccine agents are pathogenic for the embryo. Late stage embryos are highly susceptible to infection with most vaccine viruses examined. Not all vaccine viruses that are non-pathogenic for newly hatched chicks are also safe for late-stage embryos. For instance, vaccine strains of IBV and NDV that are used routinely as neonatal vaccines in newly hatched chicks are lethal for embryos following in ovo inoculation. These viruses have been modified to render them safe for in ovo use. Currently only the MD vaccine is being administered in ovo. 
     Use of Inactivated vs. Live Vaccines 
     Oil-emulsion vaccines prepared with mineral oil are highly efficacious formulations used widely against poultry diseases in various monovalent and polyvalent forms. Mineral oil vaccines, however, cause excessive tissue reactions. Also, the oil persists too long, is practically non-digestible, and is considered carcinogenic. A 42 day holding period is required before slaughter of poultry if a mineral oil vaccine is administered. Animal and vegetable oil vaccines have been developed to replace mineral oil vaccines. However, these have also resulted in tissue reactions. 
     Live, also called infectious, viral vaccines contain attenuated or naturally non-pathogenic isolates of viruses. The vaccines do not, by themselves, cause disease. The immune response stimulated by live vaccines protects against virulent target viruses present in the environment. Adjuvants such as mineral oil are not needed with live vaccines. 
     Use of Single vs. Combined Vaccines 
     Combined or multivalent vaccines offer a number of obvious advantages over monovalent vaccines. One advantage of a multivalent vaccine is that fewer vaccine inoculations are required. A single preparation can be administered in one inoculation and is effective against several diseases. As the range of potential vaccines increases, the combination of vaccines becomes even more mandatory in order to minimize the number of inoculations. The decreased number of inoculations needed when vaccines are combined would likely lead to an increased compliance to the vaccination schedule. This in turn would likely lead to a resulting increase in vaccine coverage, which would ultimately lead to better disease control. 
     Additional advantages of combined vaccines are the reduced costs of storage, transport and administration since there will be fewer vials, ampules, syringes and needles needed. Given the fact that on a worldwide basis more than 90% of the cost of vaccination is caused by such logistic costs, this advantage is certainly not negligible. Also, if several vaccines are combined, vaccine schedules and record keeping is simplified. 
     Problems with Combined Vaccines 
     An unexpected problem of combined vaccines is the recently identified negative influence that one vaccine may have on another in a combination vaccine. It has been found that when two existing vaccines are simply mixed, one or both may lose their potency (Andre, F. E., “Development of Combined Vaccines: Manufacturers&#39; Viewpoint,” Biologicals 22:317-321 (1994); Hadler, S. C., “Cost benefit of combining antigens,” Biologicals 22:415-418 (1994); Goldenthal, K, L., et al., “Overview—Combination Vaccines and Simultaneous Administration. Past, Present, and Future.” In:  Combined Vaccines and Simultaneous Administration. Current Issues and Perspectives  (Eds. Williams, J. C., et al.) The New York Academy of Sciences, New York, pp. 1 XI-XV (1995); Clemens, J., et al., “Interactions between PRP-T Vaccine against Hemophilus influenzae Type b and Conventional Infant Vaccines. Lessons for Future Studies of Simultaneous Immunization and Combined Vaccines,” In:  Combined Vaccines and Simultaneous Administration. Current Issues and Perspectives  (Eds. Williams, J. C., et al.) The New York Academy of Sciences, New York, pp. 255-266 (1995); Insel, R. A., “Potential Alterations in Immunogenicity by Combining or Simultaneously Administering Vaccine Component,”. In:  Combined Vaccines and Simultaneous Administration. Current Issues and Perspectives  (Eds. Williams, J. C., et al.) The New York Academy of Sciences, New York, pp. 35-47 (1995)). 
     Unfortunately, it cannot always be predicted by the use of currently established potency tests in the laboratory whether individual vaccine components will retain their potency. Developing a multivalent vaccine can be as difficult as developing a new monovalent vaccine. For example, several independent studies reported that when the Hib vaccine is combined with a whole cell pertussis vaccine there is no interference between the two vaccines, but when the Hib vaccine is combined with acellular pertussis vaccines, there is a substantial loss of the Hib immunogenicity. It was shown that when Hib is combined with DTaP, it maintains its immunogenicity if given at separate sites, while the immunogenicity is 5-15 times lower when the vaccines are administered combined at the same site. This unexpected result confirms that combining two existing vaccines is not simple and often gives very unpredictable results that are not detected during the initial studies, but only after extensive clinical testing. 
     Trials have confirmed that there may not be a direct correlation between antibody titers and protection. This finding indicates that the antibody titers that are measured may not correlate with protective efficacy of vaccines, and therefore that any modification to the vaccine (for instance, the addition of another component to make a combination vaccine), may influence vaccine efficacy without changing the antibody titers. 
     Another major difficulty in the development of combined vaccines is the unexpectedly large cost of developing these combined vaccines (Andre, F. E., “Development of Combined Vaccines: Manufacuters&#39; Viewpoint,” Biologicals 22:317-321 (1994)). While a few years ago it was believed that combining two already existing vaccines would be a simple and inexpensive operation, it has become obvious that the development of a combination requires a long time and a budget which is often similar to that necessary for the development of a new vaccine. 
     Thus, there is an unmet need for “multivalent” vaccines, i.e. vaccines containing multiple agents, that can be administered to poultry in ovo. 
     SUMMARY OF THE INVENTION 
     The present invention provides a multivalent poultry vaccine comprising two or more live biological agents or microbial components. For example, the vaccine may comprise two live biological agents, or it may comprise two microbial components, or it may comprise one of each. Each live biological agent or microbial component used in the multivalent vaccine is effective in preventing or treating a different avian disease. The multivalent vaccine is safe and effective for immunizing poultry in ovo. The dosage of the vaccine can be readily determined by a clinician or veterinarian employing animal models or other test systems that are well known to the art. 
     The live biological agent can be a virus, a bacterium, a fungus or a parasite. Preferably, the live biological agent is an attenuated virus. The microbial component can be derived from a microbe of viral, bacterial, fungal or parasitic origin. The microbial component, for example, can be a microbial protein or other subunit, or a recombinant polypeptide including a microbial polypeptide or a fusion polypeptide. The microbial component is able to produce active immunity. As used herein, the term “recombinant” is intended to mean that the polypeptide (or protein) of the microbial component is obtained by the techniques of genetic engineering. For example, a protein may be isolated from a microorganism, such as a bacterial cell, that has been transformed using an appropriate vector with foreign DNA fragments obtained from the genome of a viral strain of interest. As one skilled in the art will understand, it is not necessary to use the entire protein. A unique peptide corresponding to a portion of the full protein can be used. The polypeptide or a recombinant in which there is a vector containing immunogenic genes of other agents is present in the vaccine in an amount sufficient to induce an immune response against relevant antigens and thus protect animals against the disease of interest. 
     The attenuated viruses that may be present in the multivalent vaccine preferably include MDV, IBDV, NDV, IBV, ILTV, AEV, CAV, FPV, AIV, avian reovirus, ALV, REV, avian adenovirus, HEV, and recombinants thereof. More preferably, the viruses MDV serotypes 1, 2 and 3, IBDV, IBV and possibly also a pox vector containing F and HN genes of the NDV. 
     The vaccine of this invention may be administered to any avian animal, whether domestic or wild. In particular, it may be administered to those that are commercially reared for meat or egg production. Without limitation thereto, exemplary avians include chickens, turkeys, geese, ducks, pheasants, quail, pigeons, ostriches and the like. Preferably, the avian species is a chicken or turkey. Birds which are reared in high density brooder houses, such as broiler and layer chickens, are especially vulnerable to environmental exposure to infectious agents and would largely benefit from pre-hatch vaccination. 
     The present invention also provides a method of vaccinating poultry by administering in ovo an effective immunity-producing amount of a live multivalent vaccine. The multivalent vaccine can be administered during the final quarter of the embryonal incubation period. Preferably, the vaccine contains MDV serotypes 1, 2 and 3 and IBDV (such as commercially available vaccines like Bursine II (BurII)). In addition to MDV serotypes 1, 2 and 3, and BurII, the vaccine may contain non-pathogenic strain of NDV or recombinant vaccines containing immunogenic genes from NDV. Alternatively, the vaccine may comprise HVT containing NDV genes and MDV genes, FPV containing ILTV, influenza virus, IBDV, or vectors containing other protective genes. For most common avian diseases, the known vaccines designed for post-hatch administration would be used in accordance with the inventive method, adjusting the dosage as necessary. 
     DETAILED DESCRIPTION 
     As discussed previously, one of the most prevalent and economically destructive diseases of the poultry industry is Marek&#39;s disease (MD). It has been found that the turkey herpesvirus (HVT) vaccine can be effective in preventing MD. This vaccine has been routinely inoculated into newly hatched chicks prior to their being placed in brooder houses. Although HVT vaccine is generally quite effective, on occasion inoculated flocks experience heavy MD losses. Several factors may be responsible for vaccine failure. Because vaccine protection is mediated via immune response, exposure to virulent MDV before the vaccine has had time to generate adequate immunity may result in clinical MD. Also, quantitative studies have shown that the protective efficacy of HVT vaccine, especially if the vaccine is cell-free, is compromised if the recipient chickens have anti-HVT antibodies. Because of wide use of HVT vaccine in breeding flocks, most commercial chicks hatch with such maternally derived antibodies. 
     Furthermore, the commonly used HVT vaccine has been found to offer poor protection against strains of MDV that are highly virulent and may be responsible for some of the MD loses in vaccinated flocks (Sharma, J. M., “Embryo Vaccination Against Marek&#39;s Disease with Serotypes 1, 2 and 3 Vaccines Administered Singly or in Combination,” Avian Dis. 27:453-463 (1983); Calnek, B. W. and R. L. Witter, “Marek&#39;s Disease.” In:  Diseases of Poultry , 10th ed. (Calnek, B. W. et al. eds.), Iowa State University Press, Ames Iowa, USA. (1997)). Polyvalent vaccines containing two or three serotypes of the virus offer better protection against very virulent MDVs than the monovalent vaccines containing only HVT (Sharma, J. M., “Embryo Vaccination Against Marek&#39;s Disease with Serotypes 1, 2 and 3 Vaccines Administered Singly or in Combination,” Avian Dis. 27:453-463 (1983); Calnek, B. W. and R. L. Witter, “Marek&#39;s Disease.” In:  Diseases of Poultry , 10th ed. (Calnek, B. W. et al. eds.), Iowa State University Press, Ames Iowa, USA. (1997)). It has been shown that MD vaccines can be injected into eggs during later stages of embryonation, i.e., at embryonation day (ED) 17-18. 
     Insofar as the mechanism for protection is dependent upon an immune response, the development of immunologic competence in the embryo is one of the critical determinative factors relating to the time of inoculation. As a general rule, this competence develops in the final quarter of the incubation period, before which the embryos are highly susceptible to harmful effects of the infectious agents. It has been found that if vaccination occurs prior to this stage, not only is the extent of protection in the neonate reduced, but also the vaccine may induce lesions in the embryo and/or extraembryonic membranes. For instance, prenatal chickens injected in earlier stages of development with HVT manifested an apparent immunosuppression and developed pathological lesions. While vaccination may be given anytime during the final 25% of the incubation period, it can be appreciated that the immunologic response is not immediate. For optimum protection of the hatchling, eggs should therefore be inoculated at least about 3-4 days prior to hatch. In the chicken, for example, this translates to injection by the seventeenth or eighteenth day of the 21 -day incubation, corresponding to the time when embryonated eggs are routinely transferred to hatching trays. Injection could be conveniently combined with the transfer step. 
     Another critical consideration relating to the time frame for inoculation is the receptiveness of the inner egg structure to efficacious inoculation. The site of injection must either be within the region defined by the amnion, to include the amniotic or allantoic fluid, or else in the yolk sac. By the beginning of the fourth quarter of incubation, the amnion is sufficiently enlarged that penetration thereof is assured nearly all of the time when the injection is made from the center of the large end of the egg along the longitudinal axis. With a chicken egg in its eighteenth day of incubation, injection midway along, and perpendicular to, the longitudinal axis results in an amnion penetration frequency of about 80%, versus about 20% for the yolk sac or the embryo itself. 
     Also, by the final quarter, the embryo is sufficiently developed and differentiated that it can tolerate the inherent randomization in the actual site of injection with no significant adverse effect on the rate of hatchability or on vital functions. Moreover, at this stage of incubation, the embryo is consistently positioned in the egg such that entry from the center of the large end will predictably result in injection in the upper dorsal region of the prenatal chick. Insofar as the embryo is bathed in the amniotic fluid and proceeds to ingest it during the final few days of incubation, vaccinal infection is readily initiated when the amniotic fluid receives the injection. Similarly, vaccine injected into the yolk sac may infect the embryo during the yolk absorption process prior to hatch. Generally, the allantoic or amniotic region is the preferred site of injection for the reason that the yolk may carry maternal antibodies that would have the effect of partially neutralizing non-cell associated vaccines. 
     The mechanism of injection is not particularly critical provided that it does not unduly damage the tissues and organs. of the embryo or the extraembryonic membranes surrounding it. A hypodermic syringe fitted with a needle of about #22 gauge is suitable for the purpose. A one to 1¼ inch needle when fully inserted from the center of the large end will penetrate the shell, the outer and inner shell membranes enclosing the air cell, and the amnion. Depending on the precise stage of development and position of the embryo, a needle of this length will terminate either in the fluid above the chick or in the chick itself. A pilot hole may be punched or drilled through the shell in order to prevent damaging or dulling of the needle. In an automated system, it is envisioned that a penetrating device, such as that taught by Miller (U.S. Pat. No. 4,040,388), would be effective. While it would not be desirable to apply heat to the needle as suggested therein to the extent of inactivating the vaccine or cooking any portion of the egg&#39;s interior, sterilization between injections would be beneficial in preventing cross-contamination. Cross-contamination can be avoided by high pressure jet injection as known in the art of en masse human inoculation. It is usually unnecessary to reseal the hole after injection, though paraffin or the like would be suitable for the purpose. An automated system suitable for the injection procedure is currently commercially available (INOVOJECT®, Embrex Inc.). 
     Pre-hatch vaccine administration does not adversely affect hatchability or survival of chicks. It effectively initiates early protective immunity while chicks are still in the eggs. It has been demonstrated that protection against single infectious diseases could be induced by injecting vaccines into late stage embryonated eggs (Sharma, J. M., “Embryo vaccination with infectious bursal disease virus alone or in combination with Marek&#39;s disease vaccine,” Avian Dis. 29:1155-1169 (1985); Wakenell, P. S., and J. M. Sharma, “Chicken embryonal vaccination with avian infectious bronchitis virus,” Am. J. Vet. Res. 47:933-938 (1986); Wakenell, P. S.,et al., “Embryo vaccination of chickens with infectious bronchitis virus: Histologic and ultrastructural lesion response and immunologic response to vaccination,” Avian Dis. 39:752-765 (1995); Ahmad, J., and Sharma, J. M., “Evaluation of a modified-live virus vaccine administered in ovo to protect chickens against Newcastle disease,” Am. J. Vet. Res. 53:1999-2004 (1992); Ahmad, J., and Sharma, J. M., “Protection against hemorrhagic enteritis and Newcastle disease in turkeys by embryo vaccination with monovalent and bivalent vaccines,” Avian Dis. 37:485-491 (1993)). 
     Exposure of embryonated eggs at ED16-18 with non-pathogenic strains of IBDV, NDV, and IBV resulted in active replication of the viruses in various embryonic tissues. Some viruses produced transient pathological lesions in the embryonic tissues. Cell culture attenuated IBV caused embryonic lesions (Wakenell, P. S., et al., “Embryo vaccination of chickens with infectious bronchitis virus: Histologic and ultrastructural lesion response and immunologic response to vaccination,” Avian Dis. 39:752-765 (1995)). 
     The responses of embryos to HVT and MDV, two serologically related herpes viruses, varied. Following exposure to HVT at ED 17, the virus replicated extensively in the embryo, first reaching high titers in the embryonic lung and then spreading to other tissues (Sharma, J. M., et al., “Comparative viral, immunologic, and pathologic responses of chickens inoculated with herpes virus of turkeys as embryos or at hatch,” Am. J. Vet. Res. 45:1619-123 (1984)). When single cell suspensions of lung tissue at the peak of infection were cultured in vitro, viral antigen could be visualized in sporadic adherent cells (Sharma, J. M., Embryo vaccination of chickens with turkey herpes virus: Characteristics of the target cell of early viral replication in embryonic lung,” Avian Pathol. 16:567-579 (1987)). 
     The response of the late-stage embryo to MDV was quite different from the response to HVT. When MDV was injected in ovo at ED17, there was no evidence that the virus replicated in embryonic tissues. Extensive efforts to isolate the virus from embryos by cocultivation with permissive monolayers were unsuccessful (Sharma, J. M., “Delayed replication of Marek&#39;s disease virus following in ovo inoculation during late-stage of embryonal development,” Avian Dis. 31:570-576 (1987)). Although it became undetectable, the virus persisted in the embryo because chicks hatching from virus injected eggs subsequently developed clinical MD and a majority died due to progressive tumors (Sharma, J. M., “Delayed replication of Marek&#39;s disease virus following in ovo inoculation during late-stage of embryonal development,” Avian Dis. 31:570-576 (1987)). 
     The mechanism by which vaccines induce protection following in ovo inoculation is not known. Because most vaccine viruses replicate in the embryo, the viruses likely stimulate the immune system of the embryo and initiate a protective response before the chick hatches. When 18-day-old embryos were exposed to NDV, IgM-bearing cells proliferated in the spleen and cecal tonsils and some of these cells allowed specific surface binding of biotin-labeled virus thus indicating that the cells were committed to a NDV-specific immune response (Sharma, J. M., and Ahmad, J., “Response of 18-day-old chicken embryos to an in ovo Newcastle disease vaccine,” Advances in Avian immunology Research, (Eds. Davison, T. F., et al.) Carfex International Periodical Publishers, pp. 273-277 (1994)). The kinetics of this response have not been carefully examined, nor is it proven that an embryonic immune response is a prerequisite in all cases for the protection of the hatched chicken. 
     Embryonal vaccination under the aforementioned conditions is characterized by a hatch rate comparable to untreated eggs. Any improvement in protection rate of prenatally inoculated chicks over post-hatch-inoculated chicks accordingly represents a positive improvement over the prior art. Resistance against MD of young birds from 18-day-vaccinated embryos challenged on the first 3 days post-hatch is up to about four times or more than that of birds vaccinated on the first day. The effect for later challenge is less dramatic in that the immune response in the 4-to 8-day-old chicks inoculated at hatch is roughly equivalent to that of the 1-day-old chick inoculated 3 days pre-hatch. 
    
    
     The following example is intended to illustrate but not limit the invention. 
     EXAMPLE 
     Multivalent Vaccine Containing Three Serotypes of Marek&#39;s Disease Virus and Infectious Bursal Disease Virus 
     A multivalent vaccine (MV) was developed by combining four viruses into one preparation. The vaccine contained one isolate each of serotypes 1, 2, and 3 of Marek&#39;s disease virus (MDV) and an infectious bursal disease virus (IBDV). The viruses used were as follows: 
     MDV serotype 1: CV1988 
     MDV serotype 2: 301B 
     MDV serotype 3: HVT 
     IBDV: Bursine II 
     These viruses were provided as a gift from Solvay Animal Health. These viruses are also commercially available from other sources. 
     Commercially available specific-pathogen-free white Leghorn chicken eggs were used for in ovo vaccination. The vaccines were injected manually in eggs at about the 18th day of embryonation. A hole was punched on the large end of the eggs and the inoculum was deposited by inserting a #22 gauge, 1¼ inch needle through the hole. Vaccine-injected and diluent-injected eggs were placed in hatching trays. Hatched chicks were wing-banded and placed in isolators. 
     For conventional vaccination, each chick at hatching was inoculated with 0.2 ml of the vaccine subcutaneously at the back of the neck. The dose in plaque forming units (PFU) of vaccine was the same for both methods of vaccination. The dose per egg or chick was 1000 PFU of each of the Marek&#39;s disease virus serotypes and 1000 50% tissue culture infectious doses (TCID 50 ) of IBDV. 
     The hatchability of eggs in each group was recorded. Chicks in different groups were held in separate isolators through the duration of the experiments. MD challenge virus (strain RB1B, 500 PFU per bird) was inoculated intra-peritoneally (I/P) at 7 days of age. The challenge was terminated at 7 weeks of age. IBDV challenge virus (strain IM, 500 ELD 50  per bird) was inoculated intra-ocularly (I/O) at 3 weeks of age. The challenge was termninated at 4 weeks of age. Embryonated chicken eggs injected with MV during late states of embryonation hatched normally. See Tables 1-3 below. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Hatchability Results from Project Number 115-1 
               
             
          
           
               
                   
                 Inoculum at 
                   
                   
               
               
                   
                 ED18 
                 n 
                 % hatchability 
               
               
                   
                   
               
             
          
           
               
                   
                 MDV 1, 2, 3 a   
                 22 
                 100 
               
               
                   
                 Bur II b   
                 20 
                 90 
               
               
                   
                 MDV 1, 2, 3 + 
                 40 
                 95 
               
               
                   
                 Bur II 
               
               
                   
                 None 
                 47 
                 96 
               
               
                   
                   
               
               
                   
                 ED = embryonation day  
               
               
                   
                   a 1000 PFU of each virus  
               
               
                   
                   b 1000 TCID 50   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Hatchability Results from Project Number 115-2 
               
             
          
           
               
                   
                 Inoculum at 
                   
                   
               
               
                   
                 ED18 
                 n 
                 % hatchability 
               
               
                   
                   
               
               
                   
                 MDV 1, 2, 3 
                 24 
                 96 
               
               
                   
                 BurII 
                 24 
                 96 
               
               
                   
                 MDV 1, 2, 3 + 
                 41 
                 78 
               
               
                   
                 BurII 
               
               
                   
                 None 
                 50 
                 96 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Hatchability Results from Project Number 115-3 
               
             
          
           
               
                   
                 Inoculum at 
                   
                   
               
               
                   
                 ED18 
                 n 
                 % hatchability 
               
               
                   
                   
               
               
                   
                 MDV 1, 2, 3 
                 36 
                 92 
               
               
                   
                 Bur II 
                 19 
                 79 
               
               
                   
                 MDV 1, 2, 3 + 
                 52 
                 85 
               
               
                   
                 BurII 
               
               
                   
                 None 
                 52 
                 96 
               
               
                   
                   
               
             
          
         
       
     
     The chicks hatching from eggs injected with the multivalent vaccine were resistant to challenge with virulent MDV and IBDV. See Tables 4-6 below. Antibody levels against MDV and IBDV were comparable between groups receiving these viruses singly or in combination. The antibody data showed that the immune systems of the birds hatching from eggs given multivalent vaccines were intact. 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Efficacy of multivalent vaccine containing MDV serotypes 1, 2 and 3 
               
               
                 and IBDV 
               
               
                 Proj. No.: 115-1 
               
             
          
           
               
                   
                 IBDV 
                 MDV 
               
               
                   
                 challenge 
                 challenge 
               
               
                   
                 response 
                 response 
               
             
          
           
               
                 Inocula- 
                   
                 Bur. 
                   
                   
                   
                   
                   
               
               
                 tion at 
                   
                 Atr. in 
                 BI of 
                   
                   
                 Dead + 
                 % 
               
               
                 ED18 
                 Dead 
                 surv. 
                 surv. 
                 % IBD 
                 Dead 
                 Lesions 
                 MD 
               
               
                   
               
               
                 MDV 
                 ND 
                 ND 
                 ND 
                 ND 
                 0/16 
                 0/16 
                  0 
               
               
                 1, 2, 3 
               
               
                 BurII 
                 0/20 
                 3/20 
                 4.1 
                  15 
                 ND 
                 ND 
                 ND 
               
               
                 MDV 
                 0/20 
                 5/20 
                 4.1 
                  25 
                 0/15 
                 0/15 
                  0 
               
               
                 1, 2, 3 + 
               
               
                 BurII 
               
               
                 None 
                 15/23  
                 8/8  
                 2.4 
                 100 
                 18/19  
                 19/19  
                 100 
               
               
                   
               
               
                 ED = embryonation day  
               
               
                 Bur. Atr. in surv. = gross bursal atrophy in survivors  
               
               
                 BI of surv. = bursal index of survivors (bursa weight/body weight × 1000)  
               
               
                 ND = not done  
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Efficacy of multivalent vaccine containing MDV serotypes 1, 2 and 3 
               
               
                 and IBDV 
               
               
                 Proj. No.: 115-2 
               
             
          
           
               
                   
                 IBDV 
                 MDV 
               
               
                   
                 challenge 
                 challenge 
               
               
                   
                 response 
                 response 
               
             
          
           
               
                 Inocula- 
                   
                 Bur. 
                   
                   
                   
                   
                   
               
               
                 tion at 
                   
                 Atr. in 
                 BI of 
                   
                   
                 Dead + 
                 % 
               
               
                 ED18 
                 Dead 
                 surv. 
                 surv. 
                 % IBD 
                 Dead 
                 Lesions 
                 MD 
               
               
                   
               
               
                 MDV 
                 ND 
                 ND 
                 ND 
                 ND 
                 1/18* 
                 1/18 
                 5 
               
               
                 1, 2, 3 
               
               
                 BurII 
                 0/20 
                 8/22 
                 5.37 
                  36 
                 ND 
                 ND 
                 ND 
               
               
                 MDV 
                 0/20 
                 1/20 
                 4.83 
                  5 
                 1/18  
                 1/18 
                 5 
               
               
                 1, 2, 3 + 
               
               
                 BurII 
               
               
                 None 
                 12/24  
                 12/12  
                 4.62 
                 100 
                 14/20  
                 17/20  
                 85  
               
               
                   
               
               
                 ED = embryonation day  
               
               
                 Bur. Atr. in surv. = gross bursal atrophy in survivors  
               
               
                 BI of surv. bursal index of survivors (bursa weight/body weight × 1000)  
               
               
                 ND = not done  
               
               
                 *Died at week 6. Diagnosis based on gross lesions.  
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Efficacy of multivalent vaccine containing MDV serotypes 1, 2 and 3 
               
               
                 and IBDV 
               
               
                 Proj. No.: 115-3 
               
             
          
           
               
                 IBDV 
                   
               
               
                 challenge 
                 MDV challenge 
               
             
          
           
               
                 Inocula- 
                   
                 Bur. 
                   
                   
                   
                 Dead 
                   
               
               
                 tion at 18 
                   
                 atrop at 
                 BI of 
                 % 
                   
                 + 
                 % 
               
               
                 ED 
                 Dead 
                 surv. 
                 surv. 
                 IBD 
                 Dead 
                 lesions 
                 MD 
               
               
                   
               
               
                 MDV 
                 ND 
                 ND 
                 ND 
                 ND 
                 — 
                 1/10 
                 — 
               
               
                 1, 2, 3 
               
               
                 Bur II 
                 0/15 
                 4/15 
                 5.0 
                  26.6 
                 ND 
                 ND 
                 ND 
               
               
                 MDV 
                 0/23 
                 2/23 
                 5.7 
                  8.6 
                 1/12 
                 1/12 
                  8.3 
               
               
                 1, 2, 3 + 
               
               
                 Bur II 
               
               
                 None 
                 0/15 
                 15/15  
                 3.0 
                 100   
                 13/13  
                 13/13  
                 100   
               
               
                   
               
               
                 ED = embryonation day  
               
               
                 Bur. Atr. in surv. = gross bursal atrophy in survivors  
               
               
                 BI of surv. = bursal index of survivors (bursa weight/body weight × 1000)  
               
               
                 ND = not done  
               
             
          
         
       
     
     Although multivalent MDV+IBDV vaccines have been previously available for post-hatch use, the present invention is the first effective multivalent vaccine that is suitable for in ovo use. Further, the multivalent vaccine of the present invention contains all three serotypes of MDV, giving it a wider protective ability than a vaccine that has one or two of the serotypes missing. 
     All publications are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the scope of the invention.