Patent Publication Number: US-5523088-A

Title: Inactivated avian polyomavirus vaccine in psittacine birds

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to an avian polyomavirus vaccine and to a method of preventing avian polyomavirus infection in Psittaciformes. 
     2. Background Art 
     The first acute, generalized infection associated with avian polyomavirus was described in 1980 in young psittacine birds and was called budgerigar fledgling disease (Davis, R. B., et al., &#34;A viral disease of fledgling budgerigars,&#34; Avian Dis., 1981, 25:179-183; Bozeman, L. H., et al., &#34;Characterization of a papovavirus isolated from fledgling budgerigars,&#34; Avian Dis., 1981, 25:972-980; Bernier, G., et al., &#34;A generalized inclusion body disease in the budgerigar (Melopsittacus undulatus) caused by a papovavirus-like agent,&#34; Avian Dis., 1981, 25:1083-1092; Dykstra, M. J., et al., &#34;Investigations of budgerigar fledgling disease virus,&#34; Am. J. Vet. Res., 1984, 45:1883-1887; Lehn, H., Muller, H., &#34;Cloning and characterization of budgerigar fledgling disease virus (BFDV), an avian polyomavirus,&#34; Virology, 1986, 151:362-370). Since its discovery in 1980, avian polyomavirus has been associated with disease in a number of different species of companion and aviary birds including Budgerigars, caiques, macaws, Amazon parrots, conures, cockatoos, lovebirds, Splendid Parakeets, Pionus Parrots, African Grey Parrots, Eclectus Parrots, Cockatiels, finches and lories (Davis et al.,  1981; Bozeman et al., 1981; Bernier et al., 1981; Lehn and Muller, 1986; Jacobson, E. R., et al., &#34;Epornitic of papova-like virus-associated disease in a psittacine nursery,&#34; J. Am. Vet. Med. Assoc., 1984, 185:1337-1341; Clubb, S. L., Davis, R. B., &#34;Outbreak of papova-like viral infection in a psittacine nursery-a retrospective view,&#34; Proc. Assoc. Avian Vet., Toronto, 1984, 121-129; Graham, D. L., &#34;An update on selected pet bird virus infections,&#34; Proc. Assoc. Avian Vet., Toronto, 1984, 267-280; Gaskin, J. M., &#34;Psittacine viral disease: A perspective,&#34; J. Zoo. Wildl. Med., 1989, 20:249-264; Johnston, K. M., Riddell, C., &#34;Intranuclear inclusion bodies in finches,&#34; Can. Vet. J., 1986, 27:432-434; Marshall, R., &#34;Papova-like virus in a finch aviary,&#34; Proc. Assoc. Avian Vet., 1989, 203-207; Schmidt, R. E., et al., &#34;Morphologic identification of papovavirus in a Moluccan cockatoo (Cacatua moluceensis) with neurologic signs,&#34; Assoc. Avian Vet. Today, 1987, 1:107-108; Pass, D. A., et al., &#34;A papova-like virus infection of splendid parakeets (Neophema splendida),&#34; Avian Dis., 1987, 31:680-684; Pass, D. A., &#34;A papova-like virus infection of lovebirds (Agapornis sp.),&#34; Aust. Vet. J.; 1985, 82:318-319). 
     The type of clinical disease in Budgerigars, for example, depends upon the age and condition of the bird when exposure to the virus occurs. Neonates from infected flocks may develop normally for 10-15 days and then suddenly die with no premonitory signs. Other infected hatchlings may develop clinical signs that include abdominal distention, subcutaneous hemorrhage, tremors of the head and neck, ataxia and reduced formation of down and contour feathers (Hirai, K., et al., &#34;Isolation of a papova-like agent from young budgerigars with feather abnormalities,&#34; J. Vet. Sci. 46:577-587, 1984; Bernier et al., 1984; Clubb and Davis, 1984; Schmidt et al., 1987; Histopathology Reports #SC90-0637 and #SC90-0638, Schubot Exotic Bird Health Center, Texas A&amp;M University; Vernot, J., personal communication; Dykstra, M. J., Bozeman, L. H., &#34;A light and electron microscopic examination of budgerigar fledgling disease virus in tissue and in cell culture, Avian Pathol. 11:11-18, 1982). Infections have also been associated with decreased hatchability and embryonic death (Hudson, L., Hay, F. C., &#34;Isolation and structure of immunoglobulins,&#34;  Hudson, L., Hay, F. C. Ed., Practical Immunology, Boston, 1980, 156-202). Mortality rates can be as high as 100% in affected hatchlings. Surviving birds often exhibit dystrophic primary tail feathers, lack of down feathers on the back and abdomen, and lack of filoplumes on the head and neck. Additionally, surviving birds with primary feather abnormalities are usually unable to fly. 
     In larger psittacine birds, polyomavirus infections may cause peracute death with no premonitory signs, or acute death after development of clinical changes including depression, anorexia, weight loss, delayed crop-emptying, regurgitation, diarrhea, dehydration, subcutaneous hemorrhages, dyspnea, polyuria, and posterior paresis and paralysis (Pass et al., 1987; Johnston and Riddell, 1986; Mathey, W. J., Cho, B. R., &#34;Tremors of nestling budgerigars with budgerigar fledgling disease,&#34; Proc. 33rd West. Poult. Dis. Conf., 1984, 102; Woods, L., &#34;Papova-like virus in a purple finch,&#34; J. Zoo. Wild. Med., 1989, 218-219; Gaskin, J. M., &#34;The serodiagnosis of psittacine viral infections,&#34; Assoc. Avian Vet., Honolulu, 1988, 7-10). Characteristic lesions associated with a polyomavirus infection have been demonstrated in companion birds from the United States (Jacobson et al., 1984; Clubb and Davis, 1984; Graham, 1984), Canada (Gough, J. F., &#34;Outbreaks of budgerigar fledgling disease in three aviaries in Ontario,&#34; Can. Vet. J., 1989, 30:672-674, Bernier et al., 1984), Japan (Hirai et al., 1984), Italy (Pascucci, S., et al., &#34;Malattia da virus papova-simile nel papagallino ondulato (Melopsittacus undulatus), Clin. Med. (Milan), 1983, 106:38-41), Hungary (Sztojkov, V., et al., &#34;A hullamous papagaj (Melopsittacus Undulatus) papovavirus okozta megbetegedesenek hazai megallapitasa, Magy Allatorv Lapja 1985, 40:59-63), Germany (Krautwald, M-E, Kaleta, E. F., &#34;Relationship of French moult and early virus induced mortality in nestling budgerigars,&#34; Proc. 8th Intl. Cong. World Vet. Poult. Assoc., 1985, 115) and Australia (Pass et al., 1987; Pass, 1985). 
     Immunodiffusion and virus neutralization techniques have been used to demonstrate anti-polyomavirus antibodies in psittacine birds (Jacobson et al., 1984; Clubb and Davis, 1984; Gaskin, 1989; Davis et al., 1981; Gaskin, 1988; Lynch, J., et al., &#34;Isolation and experimental chicken-embryo-inoculation studies with budgerigar papovavirus,&#34; Avian Dis. 1984, 28:1135-1139; Wainwright, P. O., et al., &#34;Serological evaluation of some psittaciformes for budgerigar fledgling disease virus,&#34; Avian Dis. 1987, 31:673-676). During epornitics in mixed psittacine bird collections, infected survivors and asymptomatic birds exposed to them have been shown to develop anti-polyomavirus neutralizing antibodies (Jacobson et al., 1984; Clubb and Davis, 1984; Wainwright et al., 1987). Seronegative young adult birds will seroconvert when housed adjacent to seropositive breeding adults, indicating that an antibody response does occur following natural exposure to the virus (Jacobson et al., 1984; Clubb and Davis, 1984; Wainwright et al., 1987; Davis, R. B., &#34;Budgerigar fledgling disease (BFD), 32nd West Poult. Dis. Conf., 1983, 104). However, prior to the present invention it had not been determined whether this antibody response could be induced through vaccination or whether the resulting immunologic response would be protective. 
     In the past, attempts at producing a vaccine against avian polyomavirus have been unsuccessful. The existence of subclinical infections and chronically infected carrier birds, coupled with a lack of understanding of the epidemiologic and pathophysiologic characteristics of infection have all contributed to the lack of success. 
     Consequently, avian polyomavirus infections continue to cause high levels of mortality in companion and aviary birds, resulting in psychological distress for clients and financial burdens for aviculturists and retail distributors despite discovery of the virus over 14 years ago. Therefore, there exists a long-felt need in the art for a safe and effective vaccine against avian polyomavirus which is cross-protective against the disease in multiple species of Psittaciformes. 
     Another problem associated with vaccine failure in Psittaciformes has been the lack of a suitable adjuvant. Two killed oil-adjuvanted herpesvirus (Pacheco&#39;s disease virus) vaccines that were conditionally licensed for use in Psittaciformes were found to cause unacceptable reactions in a number of vaccinates, particularly cockatoos (Davis et al., 1981; Bozeman et al., 1981; Bernier et al., 1981; Dykstra et al., 1984). These reactions were characterized by the formation of abscesses (subcutaneous inoculation) or muscle necrosis (IM inoculation). In some Psittaciformes, granulomatous or necrotizing lesions were not noted until several months after vaccination. In other birds, lesions were noted within several weeks of administration of a booster vaccination (Davis et al., 1981; Bozeman et al., 1981; Schmidt et al., 1987). In some cases, deaths have been associated with the use of oil-adjuvanted vaccines. 
     In general, the advantage of oil-adjuvanted vaccines in comparison to other immunization products is their ability to induce durable immunity when mixed with an inactivated antigen. However, the occurrence of adverse reactions in some Psittaciformes vaccinated with oil-adjuvanted vaccines created the need for an alternative adjuvant for use with inactivated antigens intended for administration in this order of birds (Davis et al., 1981; Bozeman et al., 1981; Schmidt et al., 1987). Therefore, there exists a need in the art for a suitable adjuvant for use in Psittaciformes which augments the immune response yet does not produce an adverse reaction in the vaccinate. 
     SUMMARY OF THE INVENTION 
     The present invention satisfies the long-felt need in the art for a safe and effective vaccine to protect psittacine birds against avian polyomavirus disease by providing a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier. 
     In one embodiment, the vaccine is derived from a strain of inactivated avian polyomavirus known as the L4 strain. In another embodiment, the invention provides a vaccine wherein immunogenic amount of the inactivated avian polyomavirus corresponds to a titer of between 10 4 .5 TCID 50  and 10 7  TCID 50  for the avian polyomavirus before inactivation, but especially about 10 5 .8 TCID 50 . 
     The present invention also satisfies the need for a suitable adjuvant for use in psittacine species by providing an adjuvant, e.g., a long chain polydispersed β (1,4) linked mannan polymer interspersed with O-acetylated groups such as ACEMANNAN, for use, not only in the vaccines of the present invention, but also in other psittacine vaccines. 
     Also provided is a composition which produces an anamnestic response against avian polyomavirus infection in a sensitized bird which is classified as being a member of the Psittaciformes order, comprising an anamnestic response inducing amount of a recombinant protein of avian polyomavirus and a pharmaceutically acceptable carrier. In one embodiment, the composition comprises a recombinant VP1 capsid protein of avian polyomavirus. 
     The present invention also provides a method of preventing avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising administering to the bird a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier. In one embodiment, the method further comprises administering at least one booster vaccine to the bird. 
     Further, the invention provides a method of preventing avian polyomavirus infection in a bird from a species which is classified as being a member of the Psittaciformes order, comprising administering to the bird a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus which infects a bird from a different species of the Psittaciformes order and a pharmaceutically acceptable carrier. The vaccines and compositions provided by the invention can be utilized in the methods provided herein. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. 
     The present invention provides a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier. The term &#34;immunogenic amount&#34; means an amount of an immunogen, i.e., the inactivated avian polyomavirus or a portion thereof, which is sufficient to induce an immune response in the vaccinated bird and which protects the bird against active infection with avian polyomavirus upon exposure thereto. 
     The birds which can be treated by the invention can be any of the various species of birds which are classified as being members of the Psittaciformes order. Examples of such birds include, but are not limited to, Budgerigars (Melopsittacus undulatus), caiques (e.g., Pionites leucogaster leucogaster), macaws (e.g., Ara ararauna), Amazon parrots (e.g., Amazona ochrocephala auropalliata, conures (e.g., Pyrrhara picta, Aratinga wagleri wagleri, Aratinga solstitialis, Aratinga guarouba, Aratinga holochlora rubritorquis or Aratinga acuticaudata acuticaudata), cockatoos (e.g., Cacatua moluccensis, Cacatua ducorps, Cacatua sulphura, Cacatua goffini or Cacatua alba), Splendid Parakeets (Neophema splendida), Pionus Parrots (Pionus maximillani), African Grey Parrots (Psittacus erithacus erithacus, Eclectus Parrots (Electus roratus), Cockatiels (Nymphicus hollandicus) and parakeets (e.g. Psittacula krameri krameri). Specifically exemplified by the invention is a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier wherein the bird is selected from the group consisting of a macaw, an Amazon parrot, a conure, a cockatoo, a Pionus Parrot, and an African Grey Parrot. 
     Given the surprising fact provided by the invention that avian polyomavirus can be prevented in multiple species of Psittaciformes utilizing a single strain of avian polyomavirus, it is contemplated that the vaccines of the present invention can be constructed from any isolated strain of avian polyomavirus which infects a member of the Psittaciformes order by utilizing the methods taught herein. For example, the subject avian polyomavirus can be isolated and cultured utilizing the method taught by Bozeman et al., (1981) or by other methods known in the art. Once isolated, the virus can be purified if desired, inactivated, the vaccine prepared and the immunogenic dose optimized by the methods taught herein. 
     In one embodiment of the invention, the inactivated avian polyomavirus vaccine is derived from an isolated avian polyomavirus designated the &#34;L4&#34; strain. The L4 strain was isolated from an infected Budgerigar at the University of Georgia College of Veterinary Medicine in 1981 utilizing the method of Bozeman et al., 1981, and can be obtained from the Laboratory of Dr. Phil D. Lukert, College of Veterinary Medicine, University of Georgia, Athens, Ga. 30602. 
     One embodiment of the invention provides a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier, wherein the immunogenic amount of the inactivated avian polyomavirus corresponds to a tiler of between 10 4 .5 TCID 50  and 10 7  TCID 50  for the avian polyomavirus before inactivation. 
     In a presently preferred embodiment, the immunogenic amount of the inactivated avian polyomavirus corresponds to a titer of 10 5 .8 TCID 50  for the avian polyomavirus before inactivation. As used herein, the immunogenic amount is expressed in terms of &#34;TCID 50  &#34; titer which is given its common meaning in the art of a tissue culture infection dose which infects 50% of the cells of a tissue culture inoculum. Thus, the immunogenic amount of any particular strain of inactivated avian polyomavirus that is utilized to prepare the vaccines of the invention is based upon the tissue culture infectivity titer for that particular strain of virus before the virus is inactivated for vaccine preparation. Also, depending upon the species, size and condition of the bird being vaccinated, the immunogenic amount can be varied by the optimization procedures taught herein or by procedures known in the art. 
     The vaccines of the present invention can be used either alone or in combination with a suitable adjuvant. In one embodiment the invention provides a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising an immunogenic amount of an inactivated avian polyomavirus, a pharmaceutically acceptable carrier, and an adjuvant which is suitable for use in a bird which is classified as being a member of the Psittaciformes order. The term &#34;suitable&#34; is meant to include as an adjuvant, any substance which can be used in combination with the immunogen (e.g., inactivated avian polyomavirus or portion thereof) of the vaccine to augment the immune response without producing adverse side effects in the vaccinated bird. It is contemplated by the invention that the adjuvants described herein can be utilized in a vaccine against any psittacine pathogen. The adjuvants described herein can be utilized in any species which is a member of the Psittaciformes order including, but not limited to, the examples of Psittaciformes cited herein. 
     In one embodiment, the suitable adjuvant is a long chain polydispersed β (1,4) linked mannan polymer interspersed with O-acetylated groups. The presently preferred mannan polymer of the invention is ACEMANNAN. In another embodiment, the suitable adjuvant is a deproteinized highly purified cell wall extract derived from a non-pathogenic strain of Mycobacterium species. A presently preferred Mycobacterium extract is EQUIMUNE. 
     The vaccine protocol used to administer the immunogenic amount can vary depending upon the species, size, and condition of the bird. The vaccine of the invention is typically administered parenterally, either subcutaneously or intramuscularly by injection. Of course, the immunogenic amount can be given in divided doses or administered at multiple sites in the bird. Booster immunizations can be given utilizing vaccines containing whole inactivated avian polyomavirus or any immunogenic portion thereof. 
     In one embodiment, the invention specifically provides a composition which produces an anamnestic response against avian polyomavirus infection in a sensitized bird which is classified as being a member of the Psittaciformes order, comprising an anamnestic response inducing amount of a recombinant protein of avian polyomavirus and a pharmaceutically acceptable carrier. As used herein, the term &#34;anamnestic response&#34; means a secondary (booster) immune response in a sensitized bird. By &#34;sensitized bird&#34; is meant a bird which has been previously been in contact with avian polyomavirus antigen either by natural exposure to the virus or by vaccination (primary immunization) with avian polyomavirus or an antigenic portion thereof. 
     The invention also provides the discovery that the VP1 protein of avian polyomavirus can be utilized to booster the immune response to primary vaccination against avian polyomavirus in a safe and efficient manner and with minimal stress to the vaccinated bird. In a presently preferred embodiment, the present invention provides a composition which produces an anamnestic (secondary) response against avian polyomavirus infection in a sensitized bird which is classified as being a member of the Psittaciformes order, comprising an anamnestic response inducing amount of a recombinant avian polyomavirus VP1 capsid protein and a pharmaceutically acceptable carrier. Briefly, the recombinant VP1 protein in the presently preferred embodiment was produced in E. coli by cloning the gene that codes for this protein into the pFLAG expression vector (International Biotechnologies, New Haven, Conn.). The expressed protein was partially purified by affinity chromatography using an anti-FLAG monoclonal antibody and the composition prepared by adding the protein to sterile saline (Garcia, A. P., et al., &#34;Diagnosis of polyomavirus infection in seedcrackers using DNA in situ hybridization,&#34; J. Assoc. Avian Vet., 1993; In press; Gaskin, 1989). 
     The other recombinant proteins and polypeptide fragments from polyoma virus can be utilized as an immunogen. Such proteins, or fragments thereof, can, for example, be obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the antigenic polypeptide or fragments thereof. 
     Given the amino acid sequence of the avian polyomavirus antigen (see, e.g., R. Stoll et al., J. Gen Virology, 1993, 74:229-237), one can synthesize, using standard peptide synthesis techniques, peptide fragments chosen to be homologous to immunoreactive regions of the antigen and to modify these fragments by inclusion, deletion or modification of particular amino acid residues in the derived sequences. Thus, synthesis or purification of an extremely large number of peptides derived from the antigen is possible. Such peptides can be used to immunize a member of the Psittaciformes order. 
     The amino acid sequences of the present polypeptides can contain an immunoreactive portion of avian polyomavirus antigen attached to sequences designed to provide for some additional property, such as solubility. The amino acid sequences of an avian polyomavirus antigen can include sequences in which one or more amino acids have been substituted with another amino acid to provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, alter enzymatic activity, or alter interactions, e.g., at the injection site or with gastric acidity if an oral administration of the vaccine is used. In any case, the peptide must posses immunogenicity. 
     Recombinant viral proteins or protein fragments can be tested to determine their immunogenicity by the methods taught in the examples or by other methods known in the art. Briefly, various concentrations of a putative immunogenically specific fragment are prepared and administered to a bird and the immunological response (e.g., the production of antibodies or cell mediated immunity) of the bird to each concentration is determined. The amount of antigen administered will depend upon the species, size and condition of the bird. Thereafter an animal so inoculated with the antigen can be exposed to virulent avian polyomavirus to test the potential vaccine effect of the specific immunogenic fragment. The specificity of the putative immunogenic fragment can be ascertained by testing sera, and other fluids or lymphocytes from the inoculated bird, for cross-reactivity with other closely related avian polyomaviruses. Once the immunogenicity of a viral fragment is established, the immunogenic amount to be administered to a particular bird can be determined by optimization procedures as taught herein and known in the art. 
     In addition to the E. coli expression vectors herein, there are numerous E. coli expression vectors known to one of ordinary skill in the art useful for the expression of antigenie avian polyomavirus proteins and polypeptide fragments. Other microbial hosts suitable for use include bacilli, such as Bacillus subtills, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary an amino terminal methionine can be provided by insertion of a Met codon 5&#39; and fused in-frame with the antigen. Also, the carboxy-terminal extension of the antigen can be removed using standard oligonucleotide mutagenesis procedures. 
     Additionally, yeast expression can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion system exhibits correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encoded by the MFα-1 gene) is routinely used to direct protein secretion from yeast. The leader region of pre-pro-alpha-factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence. The antigen coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter. The antigen coding sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the antigen coding sequences can be fused to a second protein coding sequence, such as Sj26 or β-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography. The insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast. 
     The DNA sequences can be expressed in hosts after the sequences have been operably linked to, i.e., positioned to ensure the functioning of an expression control sequence in an appropriate expression vector. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors can contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362). 
     Polynucleotides encoding a variant polypeptide may include sequences that facilitate transcription (expression sequences) and translation of the coding sequences such that the encoded polypeptide product is produced. Construction of such polynucleotides is well known in the art. For example, such polynucleotides can include a promoter, a transcription termination site (polyadenylation site in eukaryotic expression hosts), a ribosome binding site, and, optionally, an enhancer for use in eukaryotic expression hosts, and, optionally, sequences necessary for replication of a vector. 
     The vaccines and compositions of the invention can include, as noted above, an effective amount of inactivated avian polyomavirus in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. By &#34;pharmaceutically acceptable&#34; is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. Actual methods of preparing dosage forms are known, or will be apparent, to those skilled in this art; for example, see Martin, E. W., Ed., Remington&#39;s Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, Pa. 
     Parenteral administration is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795. 
     The present invention also provides a method of preventing avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising administering to the bird a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier. The subject bird of the methods of the invention can be any of the various species of birds which are classified as being members of the Psittaciformes order including, but not limited to, the examples cited herein. Specifically provided, however, is a method of preventing avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising administering to the bird a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier, wherein the bird is selected from the group consisting of a macaw, an Amazon parrot, a conure, a cockatoo, a Pionus Parrot, and an African Grey Parrot. 
     Also provided is a method of preventing avian polyomavirus infection in a bird from a species which is classified as being a member of the Psittaciformes order, comprising administering to the bird a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus which infects a bird from a different species of the Psittaciformes order and a pharmaceutically acceptable carrier. 
     In one embodiment, the vaccine utilized in the methods of the invention is derived from an isolated avian polyomavirus designated the L4 strain. Given the surprisingly broad species coverage of the L4 strain vaccine as provided herein, other strains of avian polyomavirus isolated from Psittaciformes bird can be utilized to produce the vaccines of the invention and utilized in the above methods to cross-protect multiple species of Psittaciformes with a single vaccine. 
     Any of the vaccines and compositions described herein can be utilized in the methods of the invention, where appropriate, to prevent infection with or booster immunity to avian polyomavirus in a subject bird. For example, the vaccine utilized in the methods of the invention can further comprise an adjuvant suitable for use in a bird which is classified as a member of the Psittaciformes order. The adjuvant can be a long chain polydispersed β (1,4) linked mannan polymer interspersed with O-acetylated groups such as, e.g., ACEMANNAN (Carrington Laboratories, Dallas, Tex.) or a deproteinized highly purified cell wall extract derived from a non-pathogenic strain of Mycobacterium species such as, e.g., EQUIMUNE (Vetrepharm Research Inc., Athens, Ga.). 
     In the methods described herein, the administering step is typically preformed by parenteral administration, i.e., subcutaneous or intramuscular injection of the vaccine into the subject bird. The immunogenic amount of vaccine utilized in the methods of the invention is the same as that provided for in the vaccines of the invention. Specifically, the immunogenic amount of the inactivated avian polyomavirus corresponds to a titer of between 10 4 .5 TCID 50  and 10 7  TCID 50  for the avian polyomavirus before inactivation but especially about 10 5 .8 TCID 50 . 
     The methods of the invention can further comprise the step of administering at least one booster vaccine to the bird. One or more booster inoculations are typically administered at bi-weekly intervals. The booster vaccine can be any of the vaccine preparations contemplated herein. However, a preferred embodiment of the invention provides a method of preventing avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, the composition comprising administering to the bird a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier. After the initial inoculation, at least one booster vaccine is administered to the bird. The booster vaccine is a composition which produces an anamnestic response against avian polyomavirus infection in a sensitized bird which is classified as being a member of the Psittaciformes order. The booster comprises an anamnestic response inducing amount of a recombinant protein of avian polyomavirus and a pharmaceutically acceptable carrier. The booster vaccine can be comprised of any recombinant protein derived from avian polyomavirus or an immunogenic polypeptide fragment thereof. In one embodiment, the recombinant protein is the VP1 capsid protein. 
     Briefly, the first booster vaccine can be administered to the subject bird about two weeks following primary inoculation. If desired, a second booster can be administered in about two weeks. 
     A recombinant protein such as the VP1 protein produces a specific antibody response in the animal to only a portion of the virus. Secondary response to a specific immunogenic protein greatly reduces the risks associated with booster vaccination. Reaction to the booster is therefore milder yet sufficiently immunogenic to boost the bird&#39;s primary immunity to the virus. In addition to being safer and less stressful, booster vaccines derived from recombinant proteins are more economical to manufacture. 
     Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. 
     EXAMPLES 
     Example I: Inactivated Avian Polyomavirus Vaccine 
     Material and Methods 
     Virus: A stock strain of avian polyomavirus (passage level 6), recovered originally from infected Budgerigars (Melopsittacus undulatus), was grown in chicken embryo fibroblasts in M199 tissue culture medium supplemented with 5% calf serum and was used for all the experiments described in this study. Flasks of infected cells were grown for 7 days and were then frozen and thawed three times. The suspended cells were homogenized using a glass bead homogenizer with 0.1 mm beads according to the manufacturer&#39;s recommendations (Bead Beater, Biospec Products, Bartlesville, Okla.). Beads and cellular debris were separated from the supernatant by centrifugation (300×g for 5 minutes). The virus-containing supernatant (50 ml) was serially diluted in a MICROTITER plate containing a 24-hour monolayer of primary chicken embryo fibroblasts. This preparation had an infectivity titer of 10 4 .3 TCID 50  per ml. 
     The virus preparation was purified by isopycnic centrifugation. Initially, the cellular suspension was clarified by centrifugation (7000×g for 45 minutes). The clarified supernatant was layered over 45% (w/v) sucrose in phosphate-buffered saline solution (PBSS, pH 7.2) and centrifuged (140,000×g, 2 hours, 4° C.) to obtain crude viral pellets (Beckman L8-80M Ultracentrifuge, type 35 rotor, Beckman Instruments, Palo Alto, Calif.). The virus was resuspended in PBSS, adjusted to 1.34 g/ml with cesium chloride, and centrifuged to equilibrium (270,000×g, 16 hours, 20° C.) (Beckman L8-80M Ultracentrifuge, type 70.1 rotor, beckman Instruments, Palo Alto, Calif.). The gradient was collected with a density gradient fractionator (Density Gradient Fractionator, Isco, Lincoln, Nebr.). Fractions containing purified virus were dialyzed against PBSS and stored at -20° C. Fifty microliters of the virus suspension was serially diluted in a MICROTITER plate containing a 24-hour monolayer of primary chicken embryo fibroblasts. Fractions containing purified virus were dialyzed against PBSS and stored at - 20° C. The cesium chloride purified virus preparation had an infectivity titer of 10 5 .8 TCID 50  per ml. 
     Virus neutralizing antibody assay: Polyomavirus neutralizing antibody titers were determined according to published procedures (Lukert, P. D., &#34;Budgerigar fledgling disease,&#34; Purchase H. G., et al., Ed., A Laboratory Manual for the Isolation and Identification of Avian Pathogens, Kennet Square: Am. Assoc. Avian Pathol., 1989:106-107) with the exception that the chicken embryo fibroblasts were fixed with 95% ethanol and stained with crystal violet to detect CPE (cytopathic effect). Antibody titers were expressed as the reciprocal of the serum dilution that protected the chicken embryo fibroblasts from detectable cytopathic effects. 
     Vaccine preparation: The purified virus used to produce the vaccine had a titer of 10 5 .8 TCID 50  per ml before inactivation. The purified virus was inactivated by adding a sufficient quantity of g-propiolactone to create a solution with a final concentration of 0.1% β-propiolactone. The solution was incubated with constant mixing at 37° C. for 4 hours. The mixture was then placed in a refrigerator at 4° C. overnight. This preparation was evaluated to demonstrate that the virus had been inactivated by placing 50 μl of the β-propiolactone treated virus in a 24-hour monolayer of chicken embryo fibroblasts. A control vaccine was produced by making a PBSS solution that contained a final concentration of 0.1% β-propiolactone. 
     Vaccination protocol: Six 30-to 45-day-old Blue and Gold Macaw (Ara ararauna) chicks from an aviary with no previous history of clinical avian polyomavirus infections were used in this study. The chicks were divided randomly into 3 pairs and housed in groups of 2. The vaccinated and unvaccinated chicks were maintained in separate locations to reduce the possibility of inadvertent viral exposure in the unvaccinated chicks. Blood was collected by jugular venipuncture from all chicks on days 0, 10, 20, 30 and 37. On days 10, 20 and 30, two groups of chicks were vaccinated subcutaneously with 50 μl of inactivated polyomavirus suspension emulsified in 150 μl of an oil adjuvant. The third group of chicks was vaccinated on the same days subcutaneously with β-propiolactone mixed in 50 μl of PBSS emulsified in 150 μl of an oil adjuvant. The chicks were examined immediately after inoculation and four times each day to determine whether vaccination caused any immediate or delayed local or systemic reactions. 
     Challenge: All chicks were challenged with live virus on days 37, 39 and 52. The in vitro infectivity of the challenge inoculum was confirmed by demonstrating characteristic cytopathic effects when a 50-μl sample of the challenge inoculum was placed on a 24-hour monolayer of chicken embryo fibroblasts grown in MICROTITER plates as described above. On days 37 and 39, the chicks were administered 50 μl of virus suspension containing 10 3 .3 TCID 50  of live virus. On day 52, the chicks were given 10 4 .5 TCID 50  of live virus. On each day of challenge, two of the vaccinated chicks received 50 μl of virus preparation by the oral and intracloacal routes; another two 50 μl of virus preparation by intramuscular inoculation. One of the unvaccinated control birds received 50 μl of virus preparation by the oral and intracloacal routes; the other 50 μl of virus preparation by intramuscular inoculation. Blood was collected from the chicks by jugular venipuncture on days 37, 46, 59 and 68. The blood samples were allowed to clot, and the serum was collected and used to determine virus-neutralizing antibody titers as descried above. The chicks were examined four times a day for clinical changes suggestive of an active infection or adverse response to challenge. 
     DNA probe detection of polyomavirus nucleic acid: Whole blood samples collected in sodium heparin (20 μl heparin per ml of blood) were obtained on days 37, 46, 59 and 68. Samples were processed for detection of polyomavirus nucleic acid using amplification procedures and viral-specific DNA probes according to a modification of published procedures (Niagro, F. D., et al., &#34;Use of polymerase chain reaction for detection of BFD in suspect birds,&#34; Proc. Assoc. Avian Vet., Phoenix, 1990, 25-37). Cloacal swabs were collected from the chicks on days 10, 20, 37 and daily thereafter. The swabs were collected before feeding each morning. These swabs were processed for detection of polyomavirus nucleic acid, as described previously (Davis et al., 1981). 
     Results 
     None of the chicks used in this study had detectable levels of polyomavirus neutralizing (VN) antibodies at the beginning of the study. The virus used for vaccination did not induce detectable cytopathic effects in a monolayer of chicken embryo fibroblasts after 7 days of incubation confirming that it had been inactivated following the addition of β-propiolactone. The vaccinated chicks developed a transitory polyuria (24 hrs) the day after the first vaccination and were considered to be slightly lethargic for a 24-to-48 hour period starting two days after each vaccination. The VN antibody titers that developed in the chicks vaccinated with β-propiolactone-treated PBSS (controls) or with inactivated avian polyomavirus are listed in Table 1. The chicks inoculated with β-propiolactone-treated PBSS emulsified in an oil adjuvant remained seronegative throughout the prechallenge period. The first increase in VN titers in the chicks vaccinated with inactivated antigen was detected 20 days after the initial vaccination. By the 37th day after the initial vaccination, the VN titers ranged from 20 to 40, with a geometric mean titer of 28. 
     All of the chicks remained clinically normal following challenge with live virus. The unvaccinated chicks had a 6-to-7 fold increase in VN antibody titer, suggesting that an active infection had occurred. None of the vaccinated chicks had a significant increase (greater than 4 fold) in VN antibody titer following challenge (Table 2). All of the chicks vaccinated with oil-adjuvanted vaccine developed lesions at each site of subcutaneous inoculation. Two of the birds developed moderate reactions (subcutaneous mass&lt;0.5 cm in diameter) and two developed severe reactions (subcutaneous mass&gt;0.5 cm in diameter with skin necrosis). 
     Using DNA probes, viral nucleic acid could not be demonstrated in cloacal swabs collected from any of the chicks prior to challenge (day 37). Viral nucleic acid was detected in cloacal swabs from the unvaccinated chicks exposed to live virus by the oral and intracloacal routes on days 1 and 2 after challenge. Viral nucleic acid was detected in cloacal swabs from the unvaccinated chicks exposed to live virus by the intramuscular route on days 2 and 3 after challenge. Using the same detection technique, viral nucleic acid could not be demonstrated in cloacal swabs from the vaccinated chicks at any time during the study. The DNA probes failed to demonstrate the presence of viral nucleic acid in the amplified products from any of the whole blood samples tested. 
     Discussion 
     The vaccine of the present invention elicited polyomavirus neutralizing antibodies in all the vaccinates. The induced immunologic response protected the vaccinates from subsequent challenge with live virus. Although the sample size was small, all of the vaccinated chicks were resistant to infection, whereas the unvaccinated chicks became infected, demonstrating that the inactivated vaccine used in this study was effective. 
     Viral replication did not appear to occur in the vaccinated chicks because there was no significant change (greater than 4 fold increase) in the VN antibody titers in these birds following challenge. An increase in titers would have indicated an active infection with an associated anamnestic response. Although the detectable immunologic response in the form of VN antibodies was low (geometric mean titer, 28), the vaccinated chicks were resistant to challenge demonstrating that a protective immunogenic response occurred following vaccination. 
     Example II: A Suitable Adjuvant for Psittaciformes 
     Four adjuvants (mineral oil, E-3, EQUIMUNE, and ACEMANNAN) were evaluated to determine their effect on the immunogenicity of inactivated polyomavirus and the degree of local reactions that they induced when injected into a group of mixed species of Psittaciformes. All four adjuvant-antigen combinations were found to stimulate the production of anti-polyomavirus neutralizing antibodies. All of the birds vaccinated with the oil-adjuvanted vaccine developed abscesses or open draining lesions at the sites of the subcutaneous inoculation. Three of the eight birds vaccinated with E-3 adjuvanted antigen died following the second vaccination. The EQUIMUNE-adjuvanted antigen induced small ulcerated lesions with subsequent scab formation in one of the three vaccinates. The ACEMANNAN adjuvanted antigen was associated with the mildest reaction which was limited to hyperemia at the site of initial subcutaneous inoculation in one of the seven vaccinates. 
     A group of adult psittacine birds with representatives from 8 different genera was used to determine whether any of four commercially available adjuvants mixed with inactivated avian polyomavirus would induce the formation of anti-polyomavirus neutralizing antibodies without causing unacceptable adverse systemic or local reactions. 
     Materials and Methods 
     Birds: The study population included 26 adult psittacine birds: 2 Umbrella Cockatoos (Cacatua alba), 4 African Grey Parrots (Psittacus erithacus erithacus), 4 Ducorp&#39;s Cockatoos (Cacatua ducorps), 1 Sulphur-crested Cockatoo (Cacatua sulphurea), 1 Goffin&#39;s Cockatoo (Cacatua goffini), 1 Red-fronted Conure (Aratinga wagleri wagleri), 1 Pionus Parrot (Pionus maximillani), 1 Sun Conure (Aratinga solstitialis), 1 Yellow-Naped Amazon Parrot (Amazona ochrocephala auropalliata), 5 White-bellied Caiques (Pionites leucogaster leucogaster), 1 Golden Conure (Aratinga guarouba), 1 Green Conure (Aratinga holochlora rubritorquis), 1 Blue-crowned Conure (Aratinga acuticaudata acuticaudata), 1 Ringed-neck Parakeet (Psittacula krameri krameri) and 1 Blue and Gold Macaw (Ara ararauna). Nine of the birds were known to be carriers of avian polyomavirus because of intermittent detection of viral nucleic acid in their excrement using DNA probes and the detection of sustained high polyomavirus-neutralizing antibody titers for over one year (Niagro, F. D., et al., &#34;Avian polyomavirus: Discordance between neutralizing antibody titers and viral shedding in an aviary,&#34; Proc. Assoc. Avian Vet., 1991, 22-26; Gaskin, 1988). 
     Virus: A stock strain of Budgerigar fledgling disease virus, isolated originally from infected Budgerigars (Melopsittacus undulatus), was grown in chicken embryo fibroblasts in M199 with 5% calf serum. Infected cells were incubated 5  to 7 days. The culture flasks were frozen and thawed three times, and the contents were pooled. Freeze-thawed suspensions were centrifuged (500×g for 10 minutes) at 4° C. to remove cellular debris and the supernatant was used as the stock virus. The infectivity titer of the stock virus was determined by making ten-fold serial dilutions in M199 with fetal calf serum. Fifty microliters of each dilution (10 -3  through 10 -8 ) was inoculated in replicates of eight per dilution into flat bottom 96-well MICROTITER plates. To each well, 200 ul of packed chicken embryo fibroblasts (in M199 with 5% fetal calf serum) was then added in a ratio of 1:200, respectively. Plates were incubated for 6 days, fixed with 95% ethanol, stained with crystal violet, and evaluated for cytopathic effects (CPE). The Spearman-Karber method (Villegas, P., Purchase, G. H., &#34;Titration of biological suspensions,&#34; Hitchner, S. B., et al., Ed., Isolation and Identification of Avian Pathogens, Endwell: Creative Publishing Co., 1980) was used to calculate the TCID 50  of the stock virus preparation. The infectivity titer of the virus preparation used for vaccination on days 0 and 14 was approximately 10 7  TCID 50  per ml and the infectivity tiler of the virus preparation used for all other vaccinations was approximately 10 9  TCID 50  per ml. 
     Vaccine preparation: The virus used for vaccination was produced as described above except that the growth media was changed to M199 without calf serum 24 hours after the cell cultures were inoculated. The virus was inactivated by adding β -propiolactone. After 1 ml of β-propiolactone (95% solution) was added to 500 ml of virus preparation, the pH was adjusted to 7.0 by the addition of 1N sodium hydroxide (NaOH). This mixture was stirred constantly at room temperature overnight. One microliter of β-propiolactone (95% solution) was then added to 1000 ml of virus preparation and the pH adjusted to 7.0 by the addition of 1N NaOH. This mixture was maintained at room temperature and stirred constantly for 2 hours. The β-propiolactone was then neutralized by the addition of 1 ml of 0.2M sodium thiosulfate per 100 ml of virus suspension. The mixture was stored at 4° C. until used. This preparation was assayed for infectivity to insure complete virus inactivation by placing  50 μl of the β-propiolactone treated virus preparation on chicken embryo fibroblasts as described above. 
     Two birds were vaccinated with 500 μl of β-propiolactone treated virus (Table 5). Twenty-two birds were vaccinated with β-propiolactone treated virus mixed with one of four adjuvants. The adjuvants used included: ACEMANNAN, a long chain polydispersed β (1,4) linked mannan polymer interspersed with O-acetylated groups (Carrington Laboratories, Dallas, Tex.); EQUIMUNE, a deproteinized highly purified cell wall extract derived from non-pathogenic strains of Mycobacterium species (Vetrepharm Research Inc., Athens, Ga.) and E3, an immunomodulator derived from the cell wall of E. coli (Immvac Inc., Colombia, Miss.). The β-propiolactone treated virus was mixed with each of the adjuvants so that the final suspension for inoculation contained 500 μl of inactivated virus. For the oil-adjuvanted vaccine, each dose represented a volume of 300 μl of mineral oil that had been mixed with 500 μl of β-propiolactone treated virus. For the ACEMANNAN and E3 vaccines, 100 μl of each adjuvant was mixed with 500 μl of β-propiolactone treated virus. For the EQUIMUNE vaccine, 50 μ l of adjuvant was mixed with 500 μl of β-propiolactone treated virus. 
     Two birds were vaccinated with recombinant VP1 protein emulsified in mineral oil. Recombinant VP1 protein was produced in E. coli by cloning the gene that codes for this protein into the pFLAG expression vector (International Biotechnologies, New Haven, Conn.). The expressed protein was partially purified by affinity chromatography using an anti-FLAG monoclonal antibody (Lin, K. H., Cheng, S. Y., &#34;An efficient method to purify active eukaryotic proteins from the inclusion bodies in E. coli,&#34; Biotechniques, 1991, 11:748-753). Twenty-five milligrams of protein (as determined using the Bradford reagent) (Bradford, M. M., &#34;A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding,&#34; Anal. Biochem. 1976, 72:248-254) was added to 500 μl of sterile saline solution and mixed with 300 μl of mineral oil. 
     Vaccination protocol: All injections and blood collections were performed with the birds under isoflurane anesthesia. Blood was collected by jugular venipuncture from each vaccinate on days 0, 14, 28, 42, 49 and 63. After blood collection, contour feathers were removed from the caudal proventer region and the birds were inoculated subcutaneously on days 0, 14 and 28. Alternating sides of the proventer region were used for each inoculation to help distinguish which of the injections was associated with a reaction. 
     The birds were divided into two groups on day 42 to determine the gross or clinically detectable systemic affects of repeated exposure and intramuscular inoculation with the two most promising adjuvants. Half the birds were vaccinated with ACEMANNAN-antigen and half were vaccinated with EQUIMUNE-antigen by deep intramuscular injection into the pectoral muscle on days 42 and 49. Preparation of the vaccine for intramuscular inoculation was the same as described above. 
     Grading reactions: The birds were examined immediately after inoculation and daily to determine whether the vaccine caused any immediate or delayed local or systemic reactions. The injection sites were observed and palpated at each booster time for the presence of hyperemia, discoloration of the skin, swelling, thickening of the skin, necrosis, and abscess or scab formation. The size and location of each reaction was recorded according to the scheme: 0=no reaction; 1=slight reaction (hyperemia, skin discoloration); 2=mild reaction (small scab formation, thickening of the skin); 3=moderate reaction (subcutaneous mass&lt;0.5 cm in diameter or&lt;0.5 cm area of necrosis); 4=severe reaction (&gt;0.5 cm subcutaneous mass, or&gt;0.5 cm area of necrosis). 
     Virus neutralizing antibody assay: To detect virus neutralizing antibodies, blood collected from each bird was placed in sodium EDTA, allowed to settle, and the plasma was separated by centrifugation. The plasma was stored at -20° C. until assayed. Polyomavirus neutralizing antibody titers were determined as previously described except that the chicken embryo fibroblasts were fixed with 95% ethanol and stained with crystal violet to detect CPE.1 Antibody titers were expressed as the reciprocal of the serum dilution that protected the chicken embryo fibroblasts from detectable cytopathic effects as detected by reduced staining with crystal violet. Rabbit anti-polyomavirus antibodies with a VN titer of 1:1024 and normal chicken serum with a VN titer of&lt;1:2 were used as positive and negative controls, respectively. 
     Results 
     Cytopathic effects (CPE) were not observed in chicken embryo fibroblasts inoculated with β-propiolactone treated virus indicating that the virus used for vaccination in this study had been inactivated. Eighty-six percent (18 of 21) of the birds vaccinated with inactivated avian polyomavirus seroconverted (greater than four fold increase in VN antibody liter with the resulting titer&gt;1:10) by two weeks after the third vaccination (day 42, Table 3). Three birds (#1, #22 and #23) died following the second booster inoculation with the E3-adjuvanted vaccine and were unavailable for titer calculations after day 28. Two other birds (#20 and #25) that did not seroconvert following subcutaneous vaccination were from the E3 adjuvanted vaccine group and did not receive a third booster vaccination. However, these latter birds also failed to seroconvert when vaccinated by the intramuscular route with antigen mixed with either ACEMANNAN or EQUIMUNE (Table 4). One-hundred percent (13 of 13) of the birds vaccinated with antigen alone or antigen mixed with mineral oil or ACEMANNAN seroconverted by the second week after the third inoculation (day 42, Table 3). 
     For each vaccinate, the highest VN antibody tiler detected by the second week after the third vaccination (day 42) in the oil-adjuvanted group ranged from 1:16 to 1:131,072 (geometric mean titer GMT=724), in the antigen only group ranged from 1:16 to 1:1024 (GMT=128), in the E3 group ranged from 1:4 to 1:16,384 (GMT=54), in the ACEMANNAN group ranged from 1:16 to 1:8192 (GMT=256) and in the EQUIMUNE group ranged from 1:8 to 1:131,072 (GMT=2580). One of two birds that were seropositive at the start of the study (#10 and #14) did seroconvert following vaccination with recombinant VP1 (rVP1) mixed in an oil adjuvant. These birds were inoculated with this vaccine to determine whether the rVP1 protein was immunogenic. 
     Of the vaccinates that were seropositive (VN antibody titer&gt;1:10) prior to vaccination, 100% (7 of 7, bird #1 died following the second vaccination and is not included) seroconverted by two weeks after the second vaccination (day 28, Table 5). Of the birds that were seronegative (VN antibody titer&lt;1:10) prior to vaccination, 71% (10 of 14) of these initially seronegative birds seroconverted by two weeks after the second vaccination (day 28, Table 5). 
     The use of E3 as an adjuvant was discontinued when three vaccinates died within two days of each other after the second inoculation. In these birds, necropsy findings considered relative to the vaccination procedure included: focal severe necrotizing dermatitis and myositis at the site of vaccination (all three birds); myeloid hyperplasia in the bone marrow suggestive of a heterophilic response to tissue necrosis (1 bird); Shwartzman reaction in the kidney (1 bird); and diffuse Gram-negative septicemia (1 bird). 
     Birds at the beginning of the study that had positive antibody titers developed higher VN antibody titers by the second week after the third subcutaneous vaccination than those birds that were considered seronegative at the beginning of the study (Table 5). However, the relative increase in titers was virtually the same, with a 4.2-fold increase in antibody titer in the seronegative group and a 4.1-fold increase in antibody titer in the seropositive group. The highest titers measured by the second week after the third subcutaneous vaccination (day 42) in the seropositive group ranged from 1:512 to 1:131,072 (GMT=8192, bird #1 not included) while the highest titers in the seronegative group ranged from 1:4 to 1:1024 (GMT=35, birds #22 and #23 not included). 
     All vaccination-induced lesions were allowed to resolve without treatment to observe the natural progression of the lesions (Table 6). None of the birds developed lesions that were considered to be inducing undue distress or pain. The appetites and attitudes of the birds remained normal throughout the study except for one caique (#14) that exhibited a 3-day period of depression and lethargy after the second vaccination. This bird recovered uneventfully. 
     All of the birds (6 of 6) vaccinated with oil-adjuvanted vaccine developed some type of lesion after subcutaneous inoculation. These reactions were classified as moderate to severe. Half of the birds in this group that developed lesions were seronegative at the start of the study and half of the birds were seropositive. One of the birds (#21) in the non-adjuvanted vaccine group developed a mild reaction characterized by hyperemia and thickening of the skin. Reactions in the surviving members of the E3 group were considered mild. The cutaneous and subcutaneous lesions in the birds that died were considered clinically mild, but histologically severe. The reactions in the EQUIMUNE group were considered mild. ACEMANNAN was considered the least reactive adjuvant, with one vaccinate developing a slight reaction (hyperemia) following the initial vaccination. None of the birds vaccinated by the intramuscular route with ACEMANNAN or EQUIMUNE developed visible or palpable reactions. No difference was noted with respect to reactions in the birds that were either seropositive or seronegative at the start of the study. 
     Discussion 
     Some of the birds used in this study were considered to be polyomavirus carriers for two reasons; the demonstration of viral nucleic acid in cloacal swabs using DNA amplification procedures and demonstration of sustained high antibody titers (over one year of sustained high titers) (Niagro et al., 1991; Gaskin, 1988). Birds that were seropositive were used in this study to determine whether an adjuvanted inactivated avian polyomavirus vaccine would cause any adverse reactions in birds that already had a VN titer to polyomavirus from a previous or sustained infection. The initially seropositive birds used in this study developed no observable adverse reactions following vaccination that were not attributable to the adjuvant. Given the seroprevalence of the disease in companion birds and the frequency of subclinical carriers (Niagro et al., 1991; Gaskin, 1988; Jacobson et al., 1984; Clubb and Davis, 1984; Wainwright et al., 1987), it is noteworthy that an inactivated vaccine intended for commercial release did not cause adverse reactions in vaccinates that were seropositive prior to vaccination. 
     The birds in this study were initially vaccinated by the subcutaneous route, and several adjuvants were used. After the initial series of subcutaneous vaccinations, the birds were vaccinated by the intramuscular route using either ACEMANNAN or EQUIMUNE as adjuvants. Repeated exposure to these vaccines was intended to determine whether unacceptable local or clinically detectable systemic reactions might occur following frequent vaccination with the most promising adjuvants. 
     Reactions at the injection site varied with the type of adjuvant. The oil-adjuvanted reactions were moderate to severe, whereas the reactions with the other adjuvants were slight, mild, or undetectable. The oil-adjuvanted vaccine caused lesions that included severe granuloma formation and necrosis. In all, each bird was exposed to a minimum of 5 injections with an adjuvanted antigen (except for the E3 group that received 4 inoculations) over a 7-week period. The ACEMANNAN and EQUIMUNE adjuvants did not cause unacceptable reactions even following this high frequency of exposure to the adjuvant antigen mixture. Considering the ease of administration and the mild post-vaccinational reactions, a vaccine that contains an alternative adjuvant to oil would appear to be the most efficacious for use with avian polyomavirus in psittacine birds. 
     Although the present process has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims. 
     
                       TABLE 1                                                     
______________________________________                                    
Virus-neutralizing antibody titers in a group of chicks                   
on days 10, 20 and 30 after vaccination with                              
β-propiolactone-inactivated avian polyomavirus                       
β-propiolactone                                                      
treated PBSS                                                              
(virus-free    Inactivated avian                                          
inoculum)      polyomavirus vaccine                                       
Day   Bird 1  Bird 2   Bird 3                                             
                             Bird 4 Bird 5                                
                                          Bird 6                          
______________________________________                                    
0     0       0        0     0      0     0                               
10    0       0        0     0      0     0                               
20    0       0        0     0      0     0                               
30    0       0        10    0      10    0                               
37    0       0        40    20     20    40                              
______________________________________                                    
 
    
     
                       TABLE 2                                                     
______________________________________                                    
Virus-neutralizing antibody titers in a group                             
of chicks overtime after viral challenge                                  
with viable avian polyomavirus on days 37, 39 and 52                      
β-propiolactone                                                      
treated PBSS                                                              
(virus-free                                                               
inoculum)                                                                 
Bird 1   Bird 2  Inactivated avian polyomavirus vaccine                   
     oral    IM      Bird 3 Bird 4 Bird 5 Bird 6                          
     chal-   chal-   oral   IM     oral   IM                              
Day  lenge   lenge   challenge                                            
                            challenge                                     
                                   challenge                              
                                          challenge                       
______________________________________                                    
37   0       0       40     20     20     40                              
46   0       20      40     20     10     40                              
59   640     320     80     40     20     20                              
68   640     640     80     40     20     20                              
______________________________________                                    
 
    
     
                                           TABLE 3                                 
__________________________________________________________________________
Summary of reciprocal VN antibody titers                                  
in psittacine birds vaccinated by the subcutaneous                        
route with various antigen-adjuvant combinations                          
              Bird        Days Post Initial Vaccination                   
Species       #   Adjuvant                                                
                          0   14   28    42                               
__________________________________________________________________________
Umbrella Cockatoo                                                         
              8   Oil     &lt;2  128  256   1024                             
Ducorp&#39;s Cockatoo                                                         
              11  Oil     &lt;2  4    4     16                               
Green Conure  16  Oil     --  2048 32,768                                 
                                         131,072                          
African Grey Parrot                                                       
              19  Oil     &lt;2  128  128   64                               
Sulphur-crested Cockatoo                                                  
              9   Antigen Only                                            
                          &lt;2  1024 512   256                              
African Grey Parrot                                                       
              21  Antigen Only                                            
                          &lt;2  16   16    8                                
Caique        1   E3      512 1024 *     *                                
Sun Conure    4   E3      512 16,384                                      
                                   16,384                                 
                                         16,384                           
Amazon Parrot 18  E3      2   4    16    32                               
Ducorp&#39;s Cockatoo                                                         
              20  E3      2   4    4     2                                
African Grey Parrot                                                       
              22  E3      &lt;2  2    *     *                                
Golden Conure 23  E3      &lt;2  64   *     *                                
Goffin&#39;s Cockatoo                                                         
              24  E3      &lt;2  64   32    64                               
Pionus Parrot 25  E3      &lt;2  2    4     4                                
Red-fronted Conure                                                        
              10  VP1 + Oil                                               
                          64  256  256   512                              
Caique        14  VP1 +  Oil                                              
                          2048                                            
                              4096 512   1024                             
Ringneck Parakeet                                                         
              2   Acemannan                                               
                          &lt;2  16   32    16                               
Blue and Gold Macaw                                                       
              5   Acemannan                                               
                          128 2048 1024  2048                             
Ducorp&#39;s Cockatoo                                                         
              6   Acemannan                                               
                          &lt;2  4    16    32                               
Caique        7   Acemannan                                               
                          1024                                            
                              4096 8192  2048                             
Blue-crowned Conure                                                       
              13  Acemannan                                               
                          &lt;2  4    16    16                               
Umbrella Cockatoo                                                         
              15  Acemannan                                               
                          512 4096 8192  4096                             
African Grey Parrot                                                       
              17  Acemannan                                               
                          4   16   32    16                               
Ducorp&#39;s Cockatoo                                                         
              3   Equimune                                                
                          &lt;2  8    8     8                                
Caique        12  Equimune                                                
                          2048                                            
                              32,768                                      
                                   131,072                                
                                         65,536                           
Caique        26  Equimune                                                
                          1024                                            
                              4096 16,384                                 
                                         16,384                           
__________________________________________________________________________
 *Bird died                                                               
 
    
     
                       TABLE 4                                                     
______________________________________                                    
Summary of the reciprocal VN antibody titers that occurred                
following intramuscular vaccination with antigen mixed                    
with either Acemannan or Equimune adjuvant                                
                        Days Post Initial                                 
         Bird           Vaccination                                       
Species    #      Adjuvant  42     49    63                               
______________________________________                                    
Ringneck   2      Acemannan 16     32    16                               
Parakeet                                                                  
Blue and Gold                                                             
           5      Acemannan 2048   1024  1024                             
Macaw                                                                     
Ducorp&#39;s   6      Acemannan 32     32    4                                
Cockatoo                                                                  
Caique     7      Acemannan 2048   8192  4096                             
Umbrella   8      Acemannan 1024   1024  1024                             
Cockatoo                                                                  
Sulphur-crested                                                           
           9      Acemannan 256    1024  512                              
Cockatoo                                                                  
Ducorp&#39;s   11     Acemannan 16     32    64                               
Cockatoo                                                                  
Blue-crowned                                                              
           13     Acemannan 16     32    32                               
Conure                                                                    
Umbrella   15     Acemannan 4096   8192  4096                             
Cockatoo                                                                  
African Grey                                                              
           17     Acemannan 16     1024  512                              
Parrot                                                                    
Ducorp&#39;s   20     Acemannan 2      4     16                               
Cockatoo                                                                  
Goffin&#39;s Cockatoo                                                         
           24     Acemannan 64     128   256                              
Ducorp&#39;s   3      Equimune  8      8     32                               
Cockatoo                                                                  
Sun Conure 4      Equimune  16,384 16,384                                 
                                         8192                             
Red-fronted                                                               
           10     Equimune  512    2048  4096                             
Conure                                                                    
Caique     12     Equimune  65,536 16,384                                 
                                         65,536                           
Caique     14     Equimune  1024   2048  32,768                           
Green Conure                                                              
           16     Equimune  131,072                                       
                                   32,768                                 
                                         16,384                           
Amazon Parrot                                                             
           18     Equimune  32     512   512                              
African Grey                                                              
           19     Equimune  64     256   256                              
Parrot                                                                    
African Grey                                                              
           21     Equimune  8      32    64                               
Parrot                                                                    
Pionus Parrot                                                             
           25     Equimune  4      8     16                               
Caique     26     Equimune  16,384 16,384                                 
                                         4096                             
______________________________________                                    
 
    
     
                                           TABLE 5                                 
__________________________________________________________________________
Summary of reciprocal VN antibody titers in birds vaccinated by the       
subcutaneous route when divided into birds that were seronegative or      
seropositive prior to vaccination                                         
                Bird        Days Post Initial Vaccination                 
Species         #   Adjuvant                                              
                            0   14   28    42                             
__________________________________________________________________________
SERONEGATIVE GROUP                                                        
Ringneck Parakeet                                                         
                2   Acemannan                                             
                            &lt;2  16   32    16                             
Ducorp&#39;s Cockatoo                                                         
                3   Equimune                                              
                            &lt;2  8    8     8                              
Ducorp&#39;s Cockatoo                                                         
                6   Acemannan                                             
                            &lt;2  4    16    32                             
Umbrella Cockatoo                                                         
                8   Oil     &lt;2  128  256   1024                           
Sulphur-crested Cockatoo                                                  
                9   Antigen Only                                          
                            &lt;2  1024 512   256                            
Ducorp&#39;s Cockatoo                                                         
                11  Oil     &lt;2  4    4     16                             
Blue-crowned Conure                                                       
                13  Acemannan                                             
                            &lt;2  4    16    16                             
African Grey Parrot                                                       
                17  Acemannan                                             
                            4   16   32    16                             
Amazon Parrot   18  E3      2   4    16    32                             
African Grey Parrot                                                       
                19  Oil     &lt;2  128  128   64                             
Ducorp&#39;s Cockatoo                                                         
                20  E3      2   4    4     2                              
African Grey Parrot                                                       
                21  Antigen Only                                          
                            &lt;2  16   16    8                              
African Grey Parrot                                                       
                22  E3      &lt;2  2    *     *                              
Golden Conure   23  E3      &lt;2  64   *     *                              
Goffin&#39;s Cockatoo                                                         
                24  E3      &lt;2  64   32    64                             
Pionus Parrot   25  E3      &lt;2  2    4     4                              
SEROPOSITIVE GROUP                                                        
Caique          1   E3      512 1024 *     *                              
Sun Conure      4   E3      512 16,384                                    
                                     16,384                               
                                           16,384                         
Blue and Gold Macaw                                                       
                5   Acemannan                                             
                            128 2048 1024  2048                           
Caique          7   Acemannan                                             
                            1024                                          
                                4096 8192  2048                           
Caique          12  Equimune                                              
                            2048                                          
                                32,768                                    
                                     131,072                              
                                           65,536                         
Umbrella Cockatoo                                                         
                15  Acemannan                                             
                            512 4096 8192  4096                           
Green Conure    16  Oil     --  2048 32,768                               
                                           131,072                        
Caique          26  Equimune                                              
                            1024                                          
                                4096 16,384                               
                                           16,384                         
__________________________________________________________________________
 *Bird died                                                               
 
    
     
                                           TABLE 6                                 
__________________________________________________________________________
Summary of post-vaccinational reactions in a                              
group of mixed species psittaciformes                                     
              Bird        Days Post Initial Vaccination                   
Species       #   Adjuvant                                                
                          0  14   28    42    49    63                    
__________________________________________________________________________
Umbrella Cockatoo                                                         
              8   Oil     0  4    4     3     3     3                     
                             1st site                                     
                                  2nd site                                
                                        bilateral                         
                                              bilateral                   
                                                    bilateral             
Ducorp&#39;s Cockatoo                                                         
              11  Oil     0  0    0     2     2     0                     
                                        2nd site                          
                                              2nd site                    
Green Conure  16  Oil     0  2    3     2     2     2                     
                             1st site                                     
                                  2nd site                                
                                        2nd site                          
                                              2nd site                    
                                                    2nd site              
African Grey Parrot                                                       
              19  Oil     0  2    4     3     3     2                     
                             1st site                                     
                                  2nd site                                
                                        bilateral                         
                                              bilateral                   
                                                    bilateral             
Red-fronted Conure                                                        
              10  VP1 + Oil                                               
                          0  3    4     4     3     3                     
                             1st site                                     
                                  2nd site                                
                                        bilateral                         
                                              bilateral                   
                                                    bilateral             
Caique        14  VP1 + Oil                                               
                          0  3    3     3     2     3                     
                             1st site                                     
                                  2nd site                                
                                        2nd site                          
                                              3rd site                    
                                                    3rd site              
Sulfur-crested Cockatoo                                                   
              9   Antigen Only                                            
                          0  0    0     0     0     0                     
African Grey Parrot                                                       
              21  Antigen Only                                            
                          0  0    1     0     2     0                     
                                  1st site    3rd site                    
Caique        1   E3      0  2    *     *     *     *                     
                             1st site                                     
Sun Conure    4   E3      0  0    0     2     2     0                     
                                        2nd site                          
                                              3rd site                    
Amazon Parrot 18  E3      0  0    1     2     2     0                     
                                  2nd site                                
                                        2nd site                          
                                              2nd site                    
Ducorp&#39;s Cockatoo                                                         
              20  E3      0  0    2     2     2     0                     
                                  1st site                                
                                        1st site                          
                                              1st site                    
African Grey Parrot                                                       
              22  E3      0  2    *     *     *     *                     
                             1st site                                     
Golden Conure 23  E3      0  2    *     *     *     *                     
                             1st site                                     
Goffin&#39;s Cockatoo                                                         
              24  E3      0  0    0     2     2     0                     
                                        2nd site                          
                                              2nd site                    
Pionus Parrot 25  E3      0  0    2     0     0     0                     
                                  2nd site                                
Ringneck Parakeet                                                         
              2   Acemannan                                               
                          0  0    0     0     0     0                     
Blue and Gold Macaw                                                       
              5   Acemannan                                               
                          0  0    0     0     0     0                     
Ducorp&#39;s Cockatoo                                                         
              6   Acemannan                                               
                          0  0    0     0     0     0                     
Caique        7   Acemannan                                               
                          0  0    0     0     0     0                     
Blue-crowned Conure                                                       
              13  Acemannan                                               
                          0  0    0     0     0     0                     
Umbrella Cockatoo                                                         
              15  Acemannan                                               
                          0  0    0     0     0     0                     
African Grey Parrot                                                       
              17  Acemannan                                               
                          0  1    0     0     0     0                     
                             1st site                                     
Ducorp&#39;s Cockatoo                                                         
              3   Equimune                                                
                          0  0    0     0     1     2                     
                                              3rd site                    
                                                    3rd site              
Caique        12  Equimune                                                
                          0  0    0     0     0     0                     
Caique        26  Equimune                                                
                          0  0    1     2     2     2                     
                                  2nd site                                
                                        2nd site                          
                                              3rd site                    
                                                    2nd                   
__________________________________________________________________________
                                                    site                  
 *Bird died. 0 = no reaction; 1 = slight reaction (hyperemia, skin        
 discoloration); 2 = mild reaction (small scab formation, thickening of th
 skin); 3 = moderate reaction (subcutaneous mass &lt;0.5 cm in diameter or   
 &lt;0.5 cm area of necrosis); 4 = severe reaction (&gt;0.5 cm subcutaneous mass
 or &gt;0.5 cm area of necrosis).