Effective Method Of Treatment Of Porcine Circovirus And Lawsonia Intracellularis Infections

Abstract

The present invention relates to the use of porcine circovirus type 2 (PCV2) antigen and Lawsonia intracellularis (L. intracellularis) antigen for the prevention, reduction in severity of, lessening of the clinical symptoms associated therewith, reduced incidence in a herd, and treatment of swine against Porcine Circovirus Associated Diseases (PCVAD) and L. intracellularis associated diseases. In particular, the present invention provides a method for the treatment or prophylaxis of an animal against PCV2 infection and ileitis caused by L. intracellularis, or a method for reduction of clinical symptoms caused by or associated with a PCV2 and L. intracellularis infection, comprising the step of administering an effective amount of PCV2 antigen and L. intracellularis antigen to an animal in need of such treatment.

Description

RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application Ser. No. 60/869,512. The teachings and content of that application are hereby incorporated by reference herein.

SEQUENCE LISTING

[0002] A sequence listing in electronic format is being provided herewith. Applicants note that the sequence listing provided herewith is identical to the sequence listing of WO06/072065, which is entirely incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to the use of porcine circovirus type 2 (PCV2) antigen and Lawsonia intracellularis (L. intracellularis) antigen for the prevention, reduction of clinical symptoms associated with, and treatment of swine against Porcine Circovirus Associated Diseases (PCVAD) and L. intracellularis associated diseases.

[0005] 2. Background of the Invention

[0006] Porcine circovirus type 2 (PCV2) is a small (17-22 nm in diameter), icoshedral, non-enveloped DNA virus, which contains a single-stranded circular genome. PCV2 shares approximately 80% sequence identity with porcine circovirus type 1 (PCV1). However, in contrast with PCV1, which is generally non-virulent, infection of swine with PCV2 has recently associated with a number of disease syndromes which have been collectively named Porcine Circovirus Associated Diseases (PCVAD) (Allan et al, 2006, IPVS Congress). Postweaning Multisystemic Wasting Syndrome (PMWS) is generally regarded to be the major clinical manifestation of PCVAD. (Harding et al., 1997, Swine Health Prod; 5: 201-203; Kennedy et al., 2000, J Comp Pathol; 122: 9-24). PMWS affects pigs between 5-18 weeks of age. PMWS is clinically characterized by wasting, paleness of the skin, unthriftiness, respiratory distress, diarrhea, icterus, and jaundice. In some affected swine, a combination of all symptoms will be apparent while other affected swine will only have one or two of these symptoms. (Muirhead, 2002, Vet. Rec.; 150: 456) During necropsy, microscopic and macroscopic lesions also appear on multiple tissues and organs, with lymphoid organs being the most common site for lesions. Allan and Ellis, 2000; J Vet. Diagn. Invest., 12: 3-14). A strong correlation has been observed between the amount of PCV2 nucleic acid or antigen and the severity of microscopic lymphoid lesions. Mortality rates for swine infected with PCV2 can approach 80%. In addition to PMWS, PCV2 has been associated with several other infections including pseudorabies, porcine reproductive and respiratory syndrome (PRRS), Glasser's disease, streptococcal meningitis, salmonellosis, postweaning colibacillosis, dietetic hepatosis, and suppurative bronchopneumonia. However, research thus far has not confirmed whether any of these clinical symptoms are in fact, the direct result of a PCV2 infection. Moreover, it is not yet known whether any of these clinical symptoms can be effectively reduced or cured by an active agent directed against PCV2.

[0007] Approaches to treat PCV2 infections based on a DNA vaccine are described in U.S. Pat. No. 6,703,023. In WO 03/049703, production of a live chimeric vaccine is described. This vaccine comprises a PCV1 backbone in which an immunogenic gene of a pathogenic PCV2 strains replaces a gene of the PCV1 backbone. WO 99/18214 has provided several PCV2 strains and procedures for the preparation of a killed PVC2 vaccine. An effective ORF-2 based subunit vaccine has also been reported in WO 06/072065.

[0008] With growing prevalence of porcine circovirus type 2 associated disease (PCVAD) in nursery and finishing pigs in swine herds, accurate diagnosis of the disease is critical for effective health management within the swine industry. That is because some diseases, such as ileitis caused by Lawsonia intracellularis can cause clinical symptoms--e.g. diarrhea and weight loss--in nursery or finishing stage of pigs that can be confused with porcine circovirus type 2 associated disease (PCVAD).

[0009] Ileitis or Proliferative enteritis (PE) caused by L. intracellularis is a disease of high economic impact in swine worldwide. L. intracellularis is an obligate, intracellular bacterium, described for example in S. McOrist et al., Infection and Immunity, Vol. 61, No. 19, 4286-4292 (1993) and G. Lawson et al., J. of Clinical Microbiology, Vol. 31, No. 5, 1136-1142 (1993). The disease was first identified in swine and characterized by its gross and microscopic pathology, and later by the demonstration of the intracellular bacteria within affected cells. The characterizing pathological feature of the disease is the proliferation of immature epithelial cells in the crypts of the ileum (terminal part of the small intestine), the large intestine or both. Sections of infected tissue are characterized by a reddened thickening mucosa resembling a "garden hose," and enteric lesions. The gut thickening ultimately prevents normal gut function, absorption capabilities, and nutrient transfer. Clinical effects of the disease are chronic weight loss, unthriftiness, diarrhea, and death. The disease is of economic importance owing to death loss, increased medication costs, poor weight gain and decreased food conversion in affected animals. Clinical cases of ileitis are observed most notably in pigs 6-20 weeks of age. However, the presence of L. intracellularis has been confirmed by polymerase chain reaction (PCR) in recently weaned pigs (3-4 weeks of age), suggesting subclinical L. intracellularis exposure occur in the nursery and perhaps, originates from Lawsonia-positive dams (Mauch and Bilkei (2004) Vet Rec 155: 532; Marsteller et al. (2003). Swine Health Prod 11:127-130; Stege et al. (2004) Vet Micro 104: 197-206). Current vaccination strategies for the prevention treatment of proliferative enteritis are limited to swine. For example, U.S. Pat. Nos. 5,714,375 and 5,885,823 as well as WO 05/011731, all of which are herein incorporated by reference in their entireties, provide vaccines for the immunization of swine. Those vaccines are highly effective and known in the market as Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903 (Boehringer Ingelheim Vetmedica Inc., St Joseph, Mo., USA).

DISCLOSURE OF THE INVENTION

[0010] Even if PCVAD, caused by PCV2, may cause similar clinical symptoms as ileitis in swine and also reduce productivity of swine industry, its effects were yet not considered to serve or prolong ileitis. However, due to the confusion in clinical signs caused by both pathogens, PCV2 and L. intracellularis, a risk exists in respect to the vaccination strategy for swine herds and swine farms. Yet, no kind of co-vaccination strategy exists for the prevention or treatment of pigs against PCVAD and ileitis caused by L. intracellularis, in particular due to the absence of the specific awareness of that problem.

[0011] The present invention has identified that problem, and overcomes it by providing a distinct advance in the state of the art. According to a general aspect, the present invention provides a method for the treatment or prophylaxis of an animal against a PCV2 infection and ileitis caused by L. intracellularis, or a method for reduction of clinical symptoms caused by or associated with a PCV2 and L. intracellularis infection, comprising the step of administering an effective amount of PCV2 antigen and L. intracellularis antigen to an animal in need of such treatment. Preferably, said animal is swine. The treatment of swine with an appropriate PCV2 antigen and L. intracellularis antigen results in a better performance of the vaccinated pigs, in particular it results in a higher resistance against PCVAD and ileitis. Moreover, the immunogenic potential of the PCV2 antigen, preferably of Ingelvac.RTM. CircoFLEX.TM. (Boehringer Ingelheim Vetmedica, Inc, St Joseph, Mo., USA) can be enhanced by the administration of L. intracellularis antigen, preferably by Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903 (Boehringer Ingelheim Vetmedica Inc., St Joseph, Mo., USA) and vice versa. PCV2 clinical signs and disease manifestations are greatly magnified when L. intracellularis infection is present and vice versa.

[0012] The immunogenic compositions and vaccination strategies as provided herewith can reduce and lessen these effects greatly, and more than expected. In other words, an unexpected synergistic effect can be achieved when animals, preferably pigs, are treated with PCV2 antigen and L. intracellularis antigen. Preferably the treatment with PCV2 and L. intracellularis antigen occurs prior to any PCV2 and/or L. intracellularis infection of swine.

[0013] The term "prevention" or "treatment" as used herein means, but is not limited to a process which includes the administration of an L. intracellularis and PCV2 antigen to an animal, wherein said L. intracellularis antigen, when administered to said animal elicits or is able to elicit an immune response in said animal against L. intracellularis and wherein said PCV2 antigen, when administered to said animal elicits or is able to elicit an immune response in said animal against PCV2. Altogether, such treatment results in reduction of the clinical symptoms of PCVAD and/or ileitis.

[0014] The term reduction of clinical symptoms shall mean, but not limited to the reduction of any of the clinical symptoms associated with a L. intracellularis and/or PCV2 infection. For example, it should refer to any reduction of pathogen load, pathogen shedding, reduction in transmission of and reduction loss of weight gain and the extent diarrhea.

PCV2 Antigens

[0015] The term "PCV2 antigen", as used herein, refers to any immunogenic composition which can be used to prevent or treat a PCV2 infection-associated disease or condition in a subject. The term "antigen", as used herein, refers in general to an amino acid sequence, or nucleic acid, which elicits an immune response in a subject, when administered to that subject. Thus, an antigen can be of proteineous or nucleic acid origin. A proteineous antigen, as used herein, includes the full-length sequence of any immunogenic protein, as well as analogs or fragments thereof. In contrast, a nucleic acid antigen, as used herein, refers to a nucleic acid molecule that codes for a full-length sequence of any immunogenic protein, as well as analogs or fragments thereof. The term "immunogenic protein, analog or fragment" refers to a fragment of a protein which includes one or more epitopes and thus elicits the immune response in a host.

[0016] An "immune response" means but is not limited to the development in a subset of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, an "immune response" includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or a protective immunological (memory) response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction in number or severity of, or lack of one or more of the symptoms associated with the infection of the pathogen, in the delay of onset of viremia, in a reduced viral persistence, in a reduction of the overall viral load and/or in a reduction of viral excretion.

[0017] Thus a preferred PCV2 antigen, as used herein, can induce, stimulate or enhance the immune response against PCV2. In this context, the term PCV2 antigen encompasses subunit immunogenic compositions, as well as compositions containing whole killed, or attenuated and/or inactivated PCV2.

[0018] By the term "subunit immunogenic composition", a composition is meant that contains at least one immunogenic polypeptide or antigen, but not all antigens, derived from or homologous to an antigen of interest. Such a composition is substantially free of intact pathogen, e.g. PCV2. Thus, a "subunit immunogenic composition" is prepared from at least partially purified or fractionated (preferably substantially purified) immunogenic polypeptides from PCV2, or recombinant analogs thereof. A preferred subunit immunogenic composition comprises the PCV2 ORF-2 protein.

[0019] PCV2 ORF-2 protein is a highly conserved domain within PCV2 isolates and thereby, any PCV2 ORF-2 protein or DNA would be effective as the source of the PCV ORF-2 antigen. Preferred PCV2 ORF-2 proteins are those described in WO06/072065, in particular the PCV2 ORF-2 protein that is encoded by SEQ ID NO: 11. A further preferred PCV ORF-2 polypeptide is provided as SEQ ID NO: 5 in WO06/072065. However, it is understood by those of skill in the art that any of these sequences could vary by as much as 6-30% in sequence homology provided that they still retain the antigenic characteristics that render it useful in immunogenic compositions. The antigenic characteristics of the PCV2 ORF-2 antigen can be, for example, estimated by the challenge experiment as provided by Example 4 of WO06/072065. The antigenic characteristic of a modified PCV ORF-2 antigen is, for example, still retained, when the modified antigen confers at least 70%, preferably at least 80%, even more preferably more preferably 90% of the protective immunity as compared to the PCV2 ORF-2 protein, encoded by the polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 as provided in WO06/072065.

[0020] Beside a full-length PCV2 ORF-2 protein, immunogenic portions of PCV2 ORF-2 protein can also be used as PCV2 antigen. The term "immunogenic portion", as used herein, refers to truncated and/or substituted forms, or fragments of PCV2 ORF-2 protein and/or polynucleotide, respectively. Preferably, such truncated and/or substituted forms, or fragments will comprise at least 6 contiguous amino acids from the full-length PCV2 ORF-2 protein. More preferably, the truncated or substituted forms, or fragments will have at least 10, more preferably at least 15, and still more preferably at least 19 contiguous amino acids from the full-length PCV2 ORF-2 protein. Two preferred sequences in this respect are provided as SEQ ID NO: 9 and SEQ ID NO: 10 of WO06/072065. It is further understood that such sequences may be a part of larger fragments or truncated forms.

[0021] "Sequence Identity" as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are "identical" at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 85%, preferably 90%, even more preferably 95% "sequence identity" to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 85%, preferably 90%, even more preferably 95% identity relative to the reference nucleotide sequence, up to 15%, preferably 10%, even more preferably 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 15%, preferably 10%, even more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 15, preferably up to 10, even more preferably up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, even more preferably up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 15%, preferably up to 10%, even more preferably up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.

[0022] "Sequence homology", as used herein, refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and gaps are introduced if necessary. However, in contrast to "sequence identity", conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 15%, preferably up to 10%, even more preferably up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence. Preferably the homolog sequence comprises a stretch of at least 50, even more preferably at least 100, even more preferably at least 250, and even more preferably at least 500 nucleotides.

[0023] A "conservative substitution" refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, hydrophobicity, etc., such that the overall functionality does not change significantly.

[0024] "Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.

[0025] Thus, the PCV2 antigen as used herein, can be a PCV2 antigen that comprises or consists of: [0026] i) a PCV2 ORF-2 protein that comprises the sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 of WO06/07065; [0027] ii) a PCV2 ORF-2 protein that is at least 80% homologous to the polypeptide of i), [0028] iii) any immunogenic portion of the polypeptides of i) and/or ii) [0029] iv) the immunogenic portion of iii), comprising at least 10 contiguous amino acids included in the sequences of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 of WO06/072065, [0030] v) a polypeptide that is encoded by a DNA comprising the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 of WO06/072065. [0031] vi) any polypeptide that is encoded by a polynucleotide that is at least 80% homologous to the polynucleotide of v), [0032] vii) any immunogenic portion of the polypeptides encoded by the polynucleotide of v) and/or vi) [0033] viii) the immunogenic portion of vii), wherein polynucleotide coding for said immunogenic portion comprises at least 30 contiguous nucleotides included in the sequences of SEQ ID NO: 3, or SEQ ID NO: 4 of WO06/072065.

[0034] Preferably any of the immunogenic portions of PCV2 ORF-2 as described above have the immunogenic characteristics of PCV2 ORF-2 protein that is encoded by the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 of WO 06/07065.

[0035] The PCV2 antigen, preferably any of the PCV2 ORF-2 proteins described above and as used in the immunogenic composition in accordance with the present invention can be derived in any fashion including isolation and purification of PCV2 antigen, standard protein synthesis, and recombinant methodology. Preferred methods for obtaining PCV2 ORF-2 proteins are provided in WO06/072065, the teachings and content of which are hereby incorporated by reference in their entirety. Briefly, susceptible cells are infected with a recombinant viral vector containing PCV2 ORF-2 DNA coding sequences, PCV2 ORF-2 polypeptide is expressed by the recombinant virus, and the expressed PCV2 ORF-2 polypeptide is recovered from the supernatant by filtration and inactivated by any conventional method, preferably using binary ethylenimine, which is then neutralized to stop the inactivation process.

[0036] The PCV2 antigen, as used herein, can be a part of a PCV2 immunogenic composition that comprises i) any of the PCV2 ORF-2 proteins described above and ii) at least a portion of the viral vector expressing said PCV2 ORF-2 protein, preferably of a recombinant baculovirus. Moreover, said PCV2 immunogenic composition can comprise i) any of the PCV2 ORF-2 proteins described above, ii) at least a portion of the viral vector expressing said PCV2 ORF-2 protein, preferably of a recombinant baculovirus, and iii) a portion of the cell culture supernatant.

[0037] The PCV2 immunogenic composition, as used herein, also refers to a composition that comprises i) any of the PCV2 ORF-2 proteins described above, ii) at least a portion of the viral vector expressing said PCV2 ORF-2 protein, iii) a portion of the cell culture, iv) and inactivating agent, preferably BEI, to inactivate the recombinant viral vector, wherein about 90% of the components i) to iii) have a size smaller than 1 .mu.m. Preferably, BEI is present in concentrations effective to inactivate the baculovirus.

[0038] The PCV2 immunogenic composition, as used herein, also refers to a composition that comprises i) any of the PCV2 ORF-2 proteins described above, ii) at least a portion of the viral vector expressing said PCV2 ORF-2 protein, iii) a portion of the cell culture, iv) an inactivating agent, preferably BEI, to inactivate the recombinant viral vector, and v) a neutralization agent to stop the inactivation mediated by the inactivating agent, wherein about 90% of the components i) to iii) have a size smaller than 1 .mu.m. Preferably, if the inactivating agent is BEI, said composition comprises sodium thiosulfate in equivalent amounts to BEI.

[0039] Additionally, the PCV2 immunogenic composition, as used herein, can include one or more veterinary-acceptable carriers. As used herein, "a veterinary-acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Preferably, the PCV2 immunogenic composition comprises PCV2 ORF-2 protein, an adjuvant, preferably Carbopol, and physiological saline. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others. Adjuvants include aluminium hydroxide and aluminium phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsions can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane or squalene oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.). John Wiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997). A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative. Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Pat. No. 2,909,462 which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among them, there may be mentioned Carbopol 974P, 934P and 971P. Most preferred is the use of Carbopol, in particular the use of Carbopol 971P, preferably in amounts of about 500 .mu.g to about 5 mg per dose, even more preferred in an amount of about 750 .mu.g to about 2.5 mg per dose and most preferred in an amount of about 1 mg per dose. Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314, or muramyl dipeptide among many others.

[0040] The PCV2 immunogenic composition, as used herein, also refers to an immunogenic composition that comprises i) any of the PCV2 ORF-2 proteins described above, ii) at least a portion of the viral vector expressing said PCV2 ORF-2 protein, iii) a portion of the cell culture, iv) an inactivating agent to inactivate the recombinant viral vector preferably BEI, and v) an neutralization agent to stop the inactivation mediated by the inactivating agent, preferably sodium thiosulfate in equivalent amounts to BEI; and vi) a suitable adjuvant, preferably Carbopol 971; wherein about 90% of the components i) to iii) have a size smaller than 1 .mu.m. According to a further aspect, the PCV2 immunogenic composition, as used herein, further comprises a pharmaceutical acceptable salt, preferably a phosphate salt in physiologically acceptable concentrations. Preferably, the pH of said immunogenic composition is adjusted to a physiological pH, meaning between about 6.5 and 7.5.

[0041] The PCV2 immunogenic composition, as used herein, also refers to Ingelvac CircoFLEX.RTM., (Boehringer Ingelheim Vetmedica Inc, St Joseph, Mo., USA), CircoVac.RTM. (Merial SAS, Lyon, France), CircoVent (Intervet Inc., Millsboro, Del., USA), or Suvaxyn PCV2 One Dose.RTM. (Fort Dodge Animal Health, Kansas City, Kans., USA). The most preferred PCV2 antigen, as used herein, is Ingelvac CircoFLEX.RTM., (Boehringer Ingelheim Vetmedica Inc, St Joseph, Mo., USA)

Lawsonia intracellularis Antigens

[0042] The term "L. intracellularis" as used herein means the intracellular, curved gram-negative bacteria described in detail by C. Gebhart et al., Int'l. J. of Systemic Bacteriology, Vol. 43, No. 3, 533-538 (1993) and S. McOrist et al., Int'l. J. of Systemic Bacteriology, Vol. 45, No. 4, 820-825 (1995), each of which is incorporated herein by reference in their entireties, and includes but is not limited to the isolates described in WO 96/39629 and WO 05/011731. In particular, the term "L. intracellularis" also means, but is not limited to the isolates deposited under the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 and assigned ATCC accession number PTA 4926 or ATCC accession number 55783. Both isolates are described in WO 96/39629 and WO 05/011731, respectively. The term "L. intracellularis" also means, but is not limited to any other L. intracellularis bacteria strain, or isolate, preferably having the immunogenic properties of at least one of the L. intracellularis strains described in WO 96/39629 and WO 05/011731, in particular having the immunogenic properties of at least one of the isolates deposited under the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 and assigned ATCC accession numbers PTA 4926 or ATCC accession number 55783.

[0043] A strain or isolate has the "immunogenic properties" of at least one of the L. intracellularis strains described in WO 96/39629 and WO 05/011731, in particular, of the isolates deposited as ATCC accession numbers PTA 4926 or ATCC accession number 55783, when it is detectable at least with one of the anti-L. intracellularis specific antibodies, described in WO06/01294, in an detection assay that is also described in WO06/01294. Preferably those antibodies are selected from the antibodies having the reference numbers 301:39, 287:6, 268:29, 110:9, 113:2 and 268:18. Preferably, the detection assay is a sandwich ELISA as described in Examples 2 and 3 of WO06/12949, whereas antibody 110:9 is used as a capture antibody and antibody 268:29 is used as a conjugated antibody. All antibodies disclosed in WO 06/12949 are produced by hybridoma cells, which are deposited at the Centre for Applied Microbiology and Research (CAMR) and European Collection of Cell Cultures ("ECACC"), Salisbury, Wiltshire SP4 OJG, UK, as patent deposits according to the Budapest Treaty. The date of deposit was May 11, 2004. HYBRIDOMA CELL LINE 110:9 is successfully deposited under ECACC Acc. No. 04092204. HYBRIDOMA CELL LINE 113:2 is successfully deposited under ECACC Acc. No. 04092201. HYBRIDOMA CELL LINE 268:18 is successfully deposited under ECACC Acc. No. 04092202. HYBRIDOMA CELL LINE 268:29 is successfully deposited under ECACC Acc. No. 04092206. HYBRIDOMA CELL LINE 287:6 is successfully deposited under ECACC Acc. No. 04092203. HYBRIDOMA CELL LINE 301:39 is successfully deposited under ECACC Acc. No. 04092205.

[0044] The term "L. intracellularis antigen" as used herein means, but is not limited to any composition of matter, that comprises at least one antigen that can induce, stimulate or enhance the immune response against a L. intracellularis-caused infection, when administered to an animal. Preferably, said L. intracellularis antigen is a complete L. intracellularis bacterium, in particular in an inactivated form (a so called killed bacterium), a modified live or attenuated L. intracellularis bacterium (a so called MLB), any sub-unit, polypeptide or component of L. intracellularis, or any chimeric vector each comprises at least an immunogenic amino acid sequence of L. intracellularis. The terms "immunogenic protein", "immunogenic polypeptide" or "immunogenic amino acid sequence" as used herein refer to any amino acid sequence which elicits an immune response in a host against a pathogen comprising said immunogenic protein, immunogenic polypeptide or immunogenic amino acid sequence. In particular, an "immunogenic protein", "immunogenic polypeptide" or "immunogenic amino acid sequence" of L. intracellularis means any amino acid sequence that codes for an antigen which elicits an immunological response against L. intracellularis in a host to which said "immunogenic protein", "immunogenic polypeptide" or "immunogenic amino acid sequence" is administered.

[0045] Suitable L. intracellularis antigens include, but are not limited to those described in EP 1219711; U.S. Pat. No. 6,605,696; WO 96/39629; WO97/20050; WO 00/69903; WO 00/69904; WO 00/69905; WO 00/69906; WO 02/38594; WO 02/26250; WO 03/06665; WO 04/033631; WO 05/026200; WO 05/011731; WO 06/116763; and/or WO 06/113782, which are all incorporated entirely herein by reference.

[0046] Preferably, the L. intracellularis antigen is a modified live L. intracellularis bacteria. Most preferably, said L. intracellularis antigen is Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903 (Boehringer Ingelheim Vetmedica, Inc.).

Effective Amount of Porcine Circovirus and L. intracellularis Antigen

[0047] The amount of antigen that is effective to elicit an immune response or is able to elicit an immune response in an animal depends on the ingredients of the vaccine and the schedule of administration.

PCV2 Antigen:

[0048] Typically, when an inactivated virus or a modified live virus preparation is used in the combination vaccine, an amount of the vaccine containing about 10.sup.2.0 to about 10.sup.9.0 TCID.sub.50 per dose, preferably about 10.sup.3.0 to about 10.sup.8.0 TCID.sub.50 per dose, more preferably, about 10.sup.4.0 to about 10.sup.8.0 TCID.sub.50 per dose. In particular, when modified live PCV2 is used in the vaccines, the recommended dose to be administered to the susceptible animal is preferably about 10.sup.3.0 TCID.sub.50 (tissue culture infective dose 50% end point)/dose to about 10.sup.6.0 TCID.sub.50/dose and more preferably about 10.sup.4.0 TCID.sub.50/dose to about 10.sup.5.0 TCID.sub.50/dose. In general, the quantity of antigen will be between 0.2 and 5000 micrograms, and between 10.sup.2.0 and 10.sup.9.0 TCID.sub.50, preferably between 10.sup.3.0 and 10.sup.6.0 TCID.sub.50, and more preferably between 10.sup.4.0 and 10.sup.5.0 TCID.sub.50, when purified antigen is used.

[0049] Sub-unit viral vaccines are normally administered with an antigen inclusion level of at least 0.2 .mu.g antigen per dose, preferably with about 0.2 to about 400 .mu.g/dose, still more preferably with about 0.3 to about 200 .mu.g/dose, even more preferably with about 0.35 to about 100 .mu.g/dose, still more preferably with about 0.4 to about 50 .mu.g/dose, still more preferably with about 0.45 to about 30 .mu.g/dose, still more preferably with about 0.6 to about 15 .mu.g/dose, even more preferably with about 0.75 to about 8 .mu.g/dose, even more preferably with about 1.0 to about 6 .mu.g/dose, and still more preferably with about 1.3 to about 3.0 .mu.g/dose.

[0050] If PCV ORF-2 antigen is used, the PCV ORF-2 antigen inclusion level is at least 0.2 .mu.g/PCV2 ORF-2 protein as described above per dose of the final antigenic composition (.mu.g/dose), more preferably from about 0.2 to about 400 .mu.g/dose, still more preferably from about 0.3 to about 200 .mu.g/dose, even more preferably from about 0.35 to about 100 .mu.g/dose, still more preferably from about 0.4 to about 50 .mu.g/dose, still more preferably from about 0.45 to about 30 .mu.g/dose, still more preferably from about 0.6 to about 15 .mu.g/dose, even more preferably from about 0.75 to about 8 .mu.g/dose, even more preferably from about 1.0 to about 6 .mu.g/dose, still more preferably from about 1.3 to about 3.0 .mu.g/dose, even more preferably from about 1.4 to about 2.5 .mu.g/dose, even more preferably from about 1.5 to about 2.0 .mu.g/dose, and most preferably about 1.6 .mu.g/dose.

L. intracellularis:

[0051] Typically, when killed L. intracellularis antigen is used in the vaccine, the vaccine contains an amount of about 10.sup.5 to about 10.sup.9 colony forming units (CFU) of the L. intracellularis bacterium per dose, preferably, about 10.sup.6 to about 10.sup.8 (CFU) of the bacterium per dose.

[0052] In particular, when modified live L. intracellularis bacteria are used in the vaccines, e.g. the bacteria isolates designated isolate B3903, ATCC accession No. PTA-4926 and designated isolate N34NP40wk, ATCC accession No. 55783 (both described in WO 96/39629 and WO 05/011731), the recommended dose to be administered to the susceptible animal is preferably about 4.5 log 10 TCID.sub.50 (tissue culture infective dose 50% end point)/dose to about 9.0 log 10 TCID.sub.50/dose and more preferably about 4.9 log 10 TCID.sub.50/dose to about 6.9 log 10 TCID.sub.50/dose. In a preferred embodiment, the titer of the vaccine is about 5.9 log 10 TCID.sub.50/dose as determined by Tissue Culture Infective Dose 50% endpoint dilution assay (TCID.sub.50). In general, the quantity of immunogen will be between 50 and 5000 micrograms, and between 4.5 log 10 and 9.0 log 10 TCID.sub.50, more preferably between 4.9 log 10 and 6.9 log 10 TCID.sub.50, when purified bacteria are used.

[0053] Sub-unit vaccines, for example those described in WO 06/116763 or WO 06/113782, are normally administered with an antigen inclusion level of at least 2 .mu.g antigen per dose, preferably with about 2 to about 500 .mu.g/dose, still more preferably with about 5 to about 400 .mu.g/dose, even more preferably with about 8 to about 300 .mu.g/dose, still more preferably with about 10 to about 200 .mu.g/dose, still more preferably with about 10 to about 150 .mu.g/dose, still more preferably with about 10 to about 100 .mu.g/dose, still more preferably with about 10 to about 75 .mu.g/dose, still more preferably with about 10 to about 50 .mu.g/dose, and still more preferably with about 10 to about 20 .mu.g/dose. Administration preferably occurs via parenteral route such as intra muscularly or subcutaneously for example.

Administration of PCV2 and L. intracellularis Antigen

Method of Treatment

[0054] As already mentioned above, according to a general aspect, the present invention provides a method for the treatment or prophylaxis of swine against a PCV2 infection and ileitis caused by L. intracellularis, or a method for reduction of clinical symptoms caused by or associated with a PCV2 and L. intracellularis infection, comprising the step of administering an effective amount of PCV2 antigen and L. intracellularis antigen to an animal in need of such treatment. Preferably, the PCV antigen is Ingelvac.RTM. CircoFLEX.TM., and the L. intracellularis antigen is Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903. Vaccination with both, PCV2 and L. intracellularis antigen should be done prior to infection of pigs with PCV2 and/or L. intracellularis and can occur simultaneously or consecutively. For example, the positive clinical effects seen by the prophylactic use of PCV2 antigen, preferably Ingelvac.RTM. CircoFLEX.TM. can be enhanced by the administration of L. intracellularis antigen, preferably by Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903. Administration of PCV2 antigen, preferably Ingelvac.RTM. CircoFLEX.TM. prior to administration of L. intracellularis antigen, e.g. Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903 is preferred. Reduction of clinical symptoms associated with a PCV2 infection, enhance the effect of the L. intracellularis antigen when administered to pigs. However, administration of L. intracellularis antigen, preferably Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903 prior to PCV2 antigen, preferably Ingelvac.RTM. CircoFLEX.TM. is also advantageous for the overall performance of the pigs and within the meaning of the present invention.

[0055] It has been found that L intracellularis antigen, in particular Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903 can be effectively administered to pigs at day one (1) of age or later. Preferably, administration is done before week 12 of age. Thus, this finding allows vaccination of pigs prior to their exposure to L. intracellularis. As already mentioned, treatment of pigs with L. intracellularis antigen enhances the positive clinical effects mediated by the PCV2 antigen. PCV2 antigen, in particular Ingelvac.RTM. CircoFLEX.TM. is administered to pigs at week three (3) of age or later. Preferably, PCV2 antigen, in particular, Ingelvac.RTM. CircoFLEX.TM. is administered not later than week 12 of age, preferably not later than week eight (8) of age, and more preferably not later than week six (6) of age, because clinical symptoms of PCVAD often strikes when pigs are 8 to 16 weeks of age. Thus, according to one aspect, the L. intracellularis antigen, preferably Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903, is administered at day one (1) of age or later, preferably at days one (1) to 21 of age, whereas the PCV2 antigen, preferably Ingelvac.RTM. CircoFLEX.TM. is administered at week three (3) of age to week 12 of age, preferably to week six (6) of age. The antigens are preferably administered in amounts as described above. In cases where Ingelvac.RTM. CircoFLEX.TM. and Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903 are used, administration of one (1) dose of each antigen is preferred.

[0056] According to a further aspect, the interval between the administration of L. intracellularis antigen and PCV2 antigen should be at least 1 to 20 days. However, administration on the same day is also possible. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 2 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 3 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 4 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 5 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 6 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 7 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 8 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 9 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 10 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 11 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 12 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 13 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 14 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 15 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 16 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be at least 17 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 18 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 19 to 20 days. According to a further aspect of the invention the interval between the administration of L. intracellularis antigen and PCV2 antigen should be about 20 days.

[0057] As already mentioned, combined vaccination of pigs with both antigens (L. intracellularis and PCV2) reduces the overall porcine circovirus load and virus persistence in the body, as well as the immunosuppressive effect of porcine circovirus and therefore enhances the overall performance of vaccinated pigs. Moreover, administration of PCV2 antigen, preferably Ingelvac.RTM. CircoFLEX.TM. surprisingly can enhance the resistance against other pathogens and also significantly enhance the potency of the L. intracellularis antigen, in particular with respect to reduction in loss of weight gain. Thus, according to a further aspect of the present invention, the administration of PCV2 antigen, preferably Ingelvac.RTM. CircoFLEX.TM. to pigs occurs at week three (3) to week 12 of age, preferably at week three (3) to week eight (8) of age week, more preferably at week three (3) to week (6) of age and prior to or simultaneous with the administration of the L. intracellularis antigen, preferably Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903. Preferably, vaccination with PCV2 antigen occurs prior to vaccination with L. intracelluaris antigen, e.g. about 1 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 2 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 3 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 4 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 5 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 6 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 7 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 8 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 9 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 10 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 11 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 12 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 13 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 14 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 15 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 16 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 17 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 18 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 19 to 20 days prior to vaccination with L. intracellularis antigen. Even more preferably, vaccination with PCV2 antigen occurs about 20 days prior to vaccination with L. intracellularis antigen.

[0058] According to a further aspect, at least one further dose of L. intracellularis and/or PCV2 antigen as described above is administered to the pigs, wherein said subsequent administration(s) are given at least 14 days beyond the initial or any former administration. The PCV2 antigen and/or L. intracellularis antigen can be administered with an immune stimulant. However, in cases of Ingelvac.RTM. CircoFLEX.TM. and Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903, an immune stimulant can be used, but its use is not necessary. Preferably, said immune stimulant is given at least twice. Preferably, at least 3 days, more preferably at least 5 days, and even more preferably at least 7 days are in between the first and the second or any further administration of the immune stimulant. Preferably, the immune stimulant is given at least 10 days, preferably 15 days, even more preferably 20, and even more preferably at least 22 days beyond the initial administration of the PCV2 and/or L. intracellularis antigens as described above. A preferred immune stimulant is, for example, keyhole limpet hemocyanin (KLH), preferably emulsified with incomplete Freund's adjuvant (KLH/ICFA). However, it is herewith understood, that any other immune stimulant known to a person skilled in the art can also be used. The term "immune stimulant" as used herein, means any agent or composition that can trigger the immune response, preferably without initiating or increasing a specific immune response, for example the immune response against a specific pathogen. It is further instructed to administer the immune stimulant in a suitable dose.

[0059] Preferably on the specific antigen administered, the L. intracellularis antigen and/or PCV2 antigen may be applied through intravenous, intravascular, intramuscular, intranasal, intraarterial, intraperitoneal, oral, subcutaneous, intradermal, intracutaneous, intralobal, intramedullar, or intrapulmonary routes. In case of Ingelvac.RTM. CircoFLEX.TM., intramuscular administrations are preferred. In cases of Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903, oral application is preferred.

[0060] Besides a general vaccination of herds with L. intracellularis antigen and PCV2 antigen in order to reduce clinical symptoms caused by or associated with PCV2 infection and/or L. intracellularis, an individual vaccination program can be developed for each farm or swine herd which is affected by swine pathogens, preferably by PCV2 and L. intracellularis. As already mentioned, incidence of L. intracellularis and PCV2 within a swine herd can affect the efficacy of the treatment with PCV2 antigen and L. intracellularis treatment. In particular, incidence of PCV2 can affect efficacy of the treatment with L. intracellularis and vice versa. It is therefore appropriate to estimate first the incidence of L. intracellularis and/or PCV2 in a herd of a farm prior to vaccination with L. intracellularis antigen and/or PCV2 antigen. Incidence of L. intracellularis for example can be estimated by any diagnostic assay, which allows the detection L. intracellularis antigen or specific anti-L. intracellularis antibodies. Specific embodiments of those assays are described for example in WO 06/0202730 and WO 06/012949. PCV2 incidence can be estimated by an assay described in WO06/072065. After incidence is estimated, an individual vaccination program can be developed, which reflects the PCV2 and L. intracellularis incidence of that farm or herd. Thus, according to a further aspect the present invention relates to a method for the treatment or prophylaxis of an animal, preferably swine against a PCV2 infection and ileitis caused by L. intracellularis, or a method for reduction of clinical symptoms caused by or associated with a PCV2 and/or L. intracellularis infection, comprising the steps of: [0061] i. detecting the incidence of L. intracellularis and/or PCV2 in a swine herd, [0062] ii. administering to pigs of L. intracellularis positive herds an effective amount of L. intracellularis antigen and/or PCV2 antigen; and/or [0063] iii. administering to pigs of PCV2 positive herds an effective amount of L. intracellularis antigen and/or PCV2 antigen.

[0064] Preferably, administration of L. intracellularis antigen and/or PCV2 antigen occurs prior to infection of pigs with L. intracellularis and/or PCV2, most preferably prior to exposure of pigs with L. intracellularis, and administration of PCV2 antigen should be done prior to infection of pigs with PCV2. Suitable administration regimes are described above.

Kit of Parts

[0065] A further aspect relates to a kit, comprising a container which comprises any of the PCV2 antigens as described herein, preferably Ingelvac.RTM. CircoFLEX.TM., a container which comprises any of the L. intracellularis antigens as described herein, preferably Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903, and an instruction manual, including the information for the administration of the PCV2 and L. intracellularis antigen. Preferably, the instruction manual includes the administration information as described supra for the co-vaccination of pigs with PCV2 and L. intracellularis antigen. For example said instruction manual comprises the information, that pigs should be treated with the PCV2 antigen and/or L. intracellularis antigen prior to infection of said pigs with PCV2 and/or L. intracellularis. Moreover, said instruction manual also can comprise the information, that pigs should be vaccinated with the L. intracellularis antigen prior to the vaccination with the PCV2 antigen. According to a further aspect, the instruction manual comprises the information, that the PCV2 antigen should be administered prior to the administration of L. intracellularis. The sequence in treatment may be influenced by the incidence of both pathogens within a herd or farm and the effects of one antigen to the other. For example, L. intracellularis antigen enhances the immunological potency of the PCV2 antigen, whereas the reduction of clinical symptoms caused by or associated with PCV2 can enhance the effectiveness of the L. intracellularis antigen, in particular in respect to reduction in loss of weight gain. Thus, in cases where incidence of PCV is predominant, vaccination against PCV2 should be performed first and vice versa. Moreover the instruction manual can also comprise the information, that the pigs can be treated with the L. intracellularis antigen at day 1 of age or later, but preferably not later than week 12 of age. Moreover, the instruction manual can also comprise the information, that the vaccination with the PCV2 antigen should occur at week three (3) of age or later, but preferably not later than at week 12 of age, preferably not later than at week (eight) 8, most preferably not later than at week six (6) of age. Moreover, the instruction manual also comprises the information that it is more effective to vaccinate the pigs consecutively with an interval in between the administration of the two antigens of about one (1) to 20 days, and more specifically as described above. In the case of Ingelvac.RTM. CircoFLEX.TM., the instruction manual also includes the information that each pig is preferably treated with one (1) dose intramuscularly. In the case of Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903, the instruction manual also comprises the information, that pigs are preferably treated with one (1) dose administered orally. Moreover, according to a further aspect, said instruction manual can comprise the information of a second or further administration(s) of at least one further dose of a PCV2 and/or L. intracellularis antigen, wherein any subsequent administration is at least 14 days beyond the initial or any former administration. Preferably, said instruction manual also includes the information, to administer an immune stimulant. Preferably, said immune stimulant shall be given at least twice. Preferably, at least 3, more preferably at least 5, even more preferably at least 7 days are between the first and the second or any further administration of the immune stimulant. Preferably, the immune stimulant is given at least 10 days, preferably 15, even more preferably 20, even more preferably at least 22 days beyond the initial administration of the PCV2 and/or L. intracellularis antigen. A preferred immune stimulant is for example is keyhole limpet hemocyanin (KLH), still preferably emulsified with incomplete Freund's adjuvant (KLH/ICFA). However, it is herewith understood, that any other immune stimulant known to a person skilled in the art can also be used. "Immune stimulant" as used herein, means any agent or composition that can trigger the immune response, preferably without initiating or increasing a specific immune response, for example the immune response against a specific pathogen. It is further instructed to administer the immune stimulant in a suitable dose. Moreover, the kit may also comprise a container, including at least one dose of the immune stimulant, preferably one dose of KLH, or KLH/ICFA.

Sequence CWU 1

1

1118DNAArtificial SequenceThis is a modified Kozak's sequence 1ccgccatg 826DNAArtificial SequenceThis is a recombinant Eco R1 sequence. 2gaattc 63713DNAPorcine circovirus 3cagctatgac gtatccaagg aggcgttacc gcagaagaag acaccgcccc cgcagccatc 60ttggccagat cctccgccgc cgcccctggc tcgtccaccc ccgccaccgc taccgttgga 120gaaggaaaaa tggcatcttc aacacccgcc tctcccgcac cttcggatat actgtggaga 180aggaaaaatg gcatcttcaa cacccgcctc tcccgcacct tcggatatac tgtgacgact 240ttgttccccc gggagggggg accaacaaaa tctctatacc ctttgaatac tacagaataa 300gaaaggttaa ggttgaattc tggccctgct cccccatcac ccagggtgat aggggagtgg 360gctccactgc tgttattcta gatgataact ttgtaacaaa ggccacagcc ctaacctatg 420acccatatgt aaactactcc tcccgccata caatccccca acccttctcc taccactccc 480gttacttcac acccaaacct gttcttgact ccactattga ttacttccaa ccaaataaca 540aaaggaatca gctttggctg aggctacaaa cctctagaaa tgtggaccac gtaggcctcg 600gcactgcgtt cgaaaacagt aaatacgacc aggactacaa tatccgtgta accatgtatg 660tacaattcag agaatttaat cttaaagacc ccccacttaa accctaaatg aat 7134713DNAPorcine circovirus 4ccgccatgac gtatccaagg aggcgttacc gcagaagaag acaccgcccc cgcagccatc 60ttggccagat cctccgccgc cgcccctggc tcgtccaccc ccgccaccgc taccgttgga 120gaaggaaaaa tggcatcttc aacacccgcc tctcccgcac cttcggatat actgtcaagg 180ctaccacagt cacaacgccc tcctgggcgg tggacatgat gagatttaat attgacgact 240ttgttccccc gggagggggg accaacaaaa tctctatacc ctttgaatac tacagaataa 300gaaaggttaa ggttgaattc tggccctgct cccccatcac ccagggtgat aggggagtgg 360gctccactgc tgttattcta gatgataact ttgtaacaaa ggccacagcc ctaacctatg 420acccatatgt aaactactcc tcccgccata caatccccca acccttctcc taccactccc 480gttacttcac acccaaacct gttcttgact ccactattga ttacttccaa ccaaataaca 540aaaggaatca gctttggctg aggctacaaa cctctagaaa tgtggaccac gtaggcctcg 600gcactgcgtt cgaaaacagt aaatacgacc aggactacaa tatccgtgta accatgtatg 660tacaattcag agaatttaat cttaaagacc ccccacttga accctaagaa ttc 7135233PRTPorcine circovirus 5Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg1 5 10 15Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45Leu Ser Arg Thr Phe Gly Tyr Thr Val Lys Ala Thr Thr Val Thr Thr 50 55 60Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asp Asp Phe Val65 70 75 80Pro Pro Gly Gly Gly Thr Asn Lys Ile Ser Ile Pro Phe Glu Tyr Tyr 85 90 95Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn 115 120 125Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr 130 135 140Ser Ser Arg His Thr Ile Pro Gln Pro Phe Ser Tyr His Ser Arg Tyr145 150 155 160Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro 165 170 175Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ser Arg Asn 180 185 190Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Lys Tyr Asp 195 200 205Gln Asp Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe 210 215 220Asn Leu Lys Asp Pro Pro Leu Lys Pro225 2306233PRTPorcine circovirus 6Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg1 5 10 15Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45Leu Ser Arg Thr Phe Gly Tyr Thr Val Lys Ala Thr Thr Val Thr Thr 50 55 60Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asp Asp Phe Val65 70 75 80Pro Pro Gly Gly Gly Thr Asn Lys Ile Ser Ile Pro Phe Glu Tyr Tyr 85 90 95Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn 115 120 125Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr 130 135 140Ser Ser Arg His Thr Ile Pro Gln Pro Phe Ser Tyr His Ser Arg Tyr145 150 155 160Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro 165 170 175Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ser Arg Asn 180 185 190Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Lys Tyr Asp 195 200 205Gln Asp Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe 210 215 220Asn Leu Lys Asp Pro Pro Leu Glu Pro225 2307756DNAArtificial SequenceThis sequence is from porcine circovirus type 2, open reading frame 2, together with a portion from the pGEM T-easy vector. 7gcggccgcgg gaattcgatc cgccatgacg tatccaagga ggcgttaccg cagaagaaga 60caccgccccc gcagccatct tggccagatc ctccgccgcc gcccctggct cgtccacccc 120cgccaccgct accgttggag aaggaaaaat ggcatcttca acacccgcct ctcccgcacc 180ttcggatata ctgtcaaggc taccacagtc acaacgccct cctgggcggt ggacatgatg 240agatttaata ttgacgactt tgttcccccg ggagggggga ccaacaaaat ctctataccc 300tttgaatact acagaataag aaaggttaag gttgaattct ggccctgctc ccccatcacc 360cagggtgata ggggagtggg ctccactgct gttattctag atgataactt tgtaacaaag 420gccacagccc taacctatga cccatatgta aactactcct cccgccatac aatcccccaa 480cccttctcct accactcccg ttacttcaca cccaaacctg ttcttgactc cactattgat 540tacttccaac caaataacaa aaggaatcag ctttggctga ggctacaaac ctctagaaat 600gtggaccacg taggcctcgg cactgcgttc gaaaacagta aatacgacca ggactacaat 660atccgtgtaa ccatgtatgt acaattcaga gaatttaatc ttaaagaccc cccacttgaa 720ccctaagaat tctatcacta gtgaattcgc ggccgc 756810387DNAArtificial SequenceThis is the porcine circovirus type 2, ORF2 construct, which includes baculovirus and pGEM T-easy coding sequences. 8aagctttact cgtaaagcga gttgaaggat catatttagt tgcgtttatg agataagatt 60gaaagcacgt gtaaaatgtt tcccgcgcgt tggcacaact atttacaatg cggccaagtt 120ataaaagatt ctaatctgat atgttttaaa acacctttgc ggcccgagtt gtttgcgtac 180gtgactagcg aagaagatgt gtggaccgca gaacagatag taaaacaaaa ccctagtatt 240ggagcaataa tcgatttaac caacacgtct aaatattatg atggtgtgca ttttttgcgg 300gcgggcctgt tatacaaaaa aattcaagta cctggccaga ctttgccgcc tgaaagcata 360gttcaagaat ttattgacac ggtaaaagaa tttacagaaa agtgtcccgg catgttggtg 420ggcgtgcact gcacacacgg tattaatcgc accggttaca tggtgtgcag atatttaatg 480cacaccctgg gtattgcgcc gcaggaagcc atagatagat tcgaaaaagc cagaggtcac 540aaaattgaaa gacaaaatta cgttcaagat ttattaattt aattaatatt atttgcattc 600tttaacaaat actttatcct attttcaaat tgttgcgctt cttccagcga accaaaacta 660tgcttcgctt gctccgttta gcttgtagcc gatcagtggc gttgttccaa tcgacggtag 720gattaggccg gatattctcc accacaatgt tggcaacgtt gatgttacgt ttatgctttt 780ggttttccac gtacgtcttt tggccggtaa tagccgtaaa cgtagtgccg tcgcgcgtca 840cgcacaacac cggatgtttg cgcttgtccg cggggtattg aaccgcgcga tccgacaaat 900ccaccacttt ggcaactaaa tcggtgacct gcgcgtcttt tttctgcatt atttcgtctt 960tcttttgcat ggtttcctgg aagccggtgt acatgcggtt tagatcagtc atgacgcgcg 1020tgacctgcaa atctttggcc tcgatctgct tgtccttgat ggcaacgatg cgttcaataa 1080actcttgttt tttaacaagt tcctcggttt tttgcgccac caccgcttgc agcgcgtttg 1140tgtgctcggt gaatgtcgca atcagcttag tcaccaactg tttgctctcc tcctcccgtt 1200gtttgatcgc gggatcgtac ttgccggtgc agagcacttg aggaattact tcttctaaaa 1260gccattcttg taattctatg gcgtaaggca atttggactt cataatcagc tgaatcacgc 1320cggatttagt aatgagcact gtatgcggct gcaaatacag cgggtcgccc cttttcacga 1380cgctgttaga ggtagggccc ccattttgga tggtctgctc aaataacgat ttgtatttat 1440tgtctacatg aacacgtata gctttatcac aaactgtata ttttaaactg ttagcgacgt 1500ccttggccac gaaccggacc tgttggtcgc gctctagcac gtaccgcagg ttgaacgtat 1560cttctccaaa tttaaattct ccaattttaa cgcgagccat tttgatacac gtgtgtcgat 1620tttgcaacaa ctattgtttt ttaacgcaaa ctaaacttat tgtggtaagc aataattaaa 1680tatgggggaa catgcgccgc tacaacactc gtcgttatga acgcagacgg cgccggtctc 1740ggcgcaagcg gctaaaacgt gttgcgcgtt caacgcggca aacatcgcaa aagccaatag 1800tacagttttg atttgcatat taacggcgat tttttaaatt atcttattta ataaatagtt 1860atgacgccta caactccccg cccgcgttga ctcgctgcac ctcgagcagt tcgttgacgc 1920cttcctccgt gtggccgaac acgtcgagcg ggtggtcgat gaccagcggc gtgccgcacg 1980cgacgcacaa gtatctgtac accgaatgat cgtcgggcga aggcacgtcg gcctccaagt 2040ggcaatattg gcaaattcga aaatatatac agttgggttg tttgcgcata tctatcgtgg 2100cgttgggcat gtacgtccga acgttgattt gcatgcaagc cgaaattaaa tcattgcgat 2160tagtgcgatt aaaacgttgt acatcctcgc ttttaatcat gccgtcgatt aaatcgcgca 2220atcgagtcaa gtgatcaaag tgtggaataa tgttttcttt gtattcccga gtcaagcgca 2280gcgcgtattt taacaaacta gccatcttgt aagttagttt catttaatgc aactttatcc 2340aataatatat tatgtatcgc acgtcaagaa ttaacaatgc gcccgttgtc gcatctcaac 2400acgactatga tagagatcaa ataaagcgcg aattaaatag cttgcgacgc aacgtgcacg 2460atctgtgcac gcgttccggc acgagctttg attgtaataa gtttttacga agcgatgaca 2520tgacccccgt agtgacaacg atcacgccca aaagaactgc cgactacaaa attaccgagt 2580atgtcggtga cgttaaaact attaagccat ccaatcgacc gttagtcgaa tcaggaccgc 2640tggtgcgaga agccgcgaag tatggcgaat gcatcgtata acgtgtggag tccgctcatt 2700agagcgtcat gtttagacaa gaaagctaca tatttaattg atcccgatga ttttattgat 2760aaattgaccc taactccata cacggtattc tacaatggcg gggttttggt caaaatttcc 2820ggactgcgat tgtacatgct gttaacggct ccgcccacta ttaatgaaat taaaaattcc 2880aattttaaaa aacgcagcaa gagaaacatt tgtatgaaag aatgcgtaga aggaaagaaa 2940aatgtcgtcg acatgctgaa caacaagatt aatatgcctc cgtgtataaa aaaaatattg 3000aacgatttga aagaaaacaa tgtaccgcgc ggcggtatgt acaggaagag gtttatacta 3060aactgttaca ttgcaaacgt ggtttcgtgt gccaagtgtg aaaaccgatg tttaatcaag 3120gctctgacgc atttctacaa ccacgactcc aagtgtgtgg gtgaagtcat gcatctttta 3180atcaaatccc aagatgtgta taaaccacca aactgccaaa aaatgaaaac tgtcgacaag 3240ctctgtccgt ttgctggcaa ctgcaagggt ctcaatccta tttgtaatta ttgaataata 3300aaacaattat aaatgctaaa tttgtttttt attaacgata caaaccaaac gcaacaagaa 3360catttgtagt attatctata attgaaaacg cgtagttata atcgctgagg taatatttaa 3420aatcattttc aaatgattca cagttaattt gcgacaatat aattttattt tcacataaac 3480tagacgcctt gtcgtcttct tcttcgtatt ccttctcttt ttcatttttc tcctcataaa 3540aattaacata gttattatcg tatccatata tgtatctatc gtatagagta aattttttgt 3600tgtcataaat atatatgtct tttttaatgg ggtgtatagt accgctgcgc atagtttttc 3660tgtaatttac aacagtgcta ttttctggta gttcttcgga gtgtgttgct ttaattatta 3720aatttatata atcaatgaat ttgggatcgt cggttttgta caatatgttg ccggcatagt 3780acgcagcttc ttctagttca attacaccat tttttagcag caccggatta acataacttt 3840ccaaaatgtt gtacgaaccg ttaaacaaaa acagttcacc tcccttttct atactattgt 3900ctgcgagcag ttgtttgttg ttaaaaataa cagccattgt aatgagacgc acaaactaat 3960atcacaaact ggaaatgtct atcaatatat agttgctgat atcatggaga taattaaaat 4020gataaccatc tcgcaaataa ataagtattt tactgttttc gtaacagttt tgtaataaaa 4080aaacctataa atattccgga ttattcatac cgtcccacca tcgggcgcgg atcagatctg 4140cagcggccgc gggaattcga tccgccatga cgtatccaag gaggcgttac cgcagaagaa 4200gacaccgccc ccgcagccat cttggccaga tcctccgccg ccgcccctgg ctcgtccacc 4260cccgccaccg ctaccgttgg agaaggaaaa atggcatctt caacacccgc ctctcccgca 4320ccttcggata tactgtcaag gctaccacag tcacaacgcc ctcctgggcg gtggacatga 4380tgagatttaa tattgacgac tttgttcccc cgggaggggg gaccaacaaa atctctatac 4440cctttgaata ctacagaata agaaaggtta aggttgaatt ctggccctgc tcccccatca 4500cccagggtga taggggagtg ggctccactg ctgttattct agatgataac tttgtaacaa 4560aggccacagc cctaacctat gacccatatg taaactactc ctcccgccat acaatccccc 4620aacccttctc ctaccactcc cgttacttca cacccaaacc tgttcttgac tccactattg 4680attacttcca accaaataac aaaaggaatc agctttggct gaggctacaa acctctagaa 4740atgtggacca cgtaggcctc ggcactgcgt tcgaaaacag taaatacgac caggactaca 4800atatccgtgt aaccatgtat gtacaattca gagaatttaa tcttaaagac cccccacttg 4860aaccctaaga attctatcac tagtgaattc gcggccgccg gccgctccag aattctagaa 4920ggtacccggg atcctttcct gggacccggc aagaaccaaa aactcactct cttcaaggaa 4980atccgtaatg ttaaacccga cacgatgaag cttgtcgttg gatggaaagg aaaagagttc 5040tacagggaaa cttggacccg cttcatggaa gacagcttcc ccattgttaa cgaccaagaa 5100gtgatggatg ttttccttgt tgtcaacatg cgtcccacta gacccaaccg ttgttacaaa 5160ttcctggccc aacacgctct gcgttgcgac cccgactatg tacctcatga cgtgattagg 5220atcgtcgagc cttcatgggt gggcagcaac aacgagtacc gcatcagcct ggctaagaag 5280ggcggcggct gcccaataat gaaccttcac tctgagtaca ccaactcgtt cgaacagttc 5340atcgatcgtg tcatctggga gaacttctac aagcccatcg tttacatcgg taccgactct 5400gctgaagagg aggaaattct ccttgaagtt tccctggtgt tcaaagtaaa ggagtttgca 5460ccagacgcac ctctgttcac tggtccggcg tattaaaaca cgatacattg ttattagtac 5520atttattaag cgctagattc tgtgcgttgt tgatttacag acaattgttg tacgtatttt 5580aataattcat taaatttata atctttaggg tggtatgtta gagcgaaaat caaatgattt 5640tcagcgtctt tatatctgaa tttaaatatt aaatcctcaa tagatttgta aaataggttt 5700cgattagttt caaacaaggg ttgtttttcc gaaccgatgg ctggactatc taatggattt 5760tcgctcaacg ccacaaaact tgccaaatct tgtagcagca atctagcttt gtcgatattc 5820gtttgtgttt tgttttgtaa taaaggttcg acgtcgttca aaatattatg cgcttttgta 5880tttctttcat cactgtcgtt agtgtacaat tgactcgacg taaacacgtt aaataaagct 5940tggacatatt taacatcggg cgtgttagct ttattaggcc gattatcgtc gtcgtcccaa 6000ccctcgtcgt tagaagttgc ttccgaagac gattttgcca tagccacacg acgcctatta 6060attgtgtcgg ctaacacgtc cgcgatcaaa tttgtagttg agctttttgg aattatttct 6120gattgcgggc gtttttgggc gggtttcaat ctaactgtgc ccgattttaa ttcagacaac 6180acgttagaaa gcgatggtgc aggcggtggt aacatttcag acggcaaatc tactaatggc 6240ggcggtggtg gagctgatga taaatctacc atcggtggag gcgcaggcgg ggctggcggc 6300ggaggcggag gcggaggtgg tggcggtgat gcagacggcg gtttaggctc aaatgtctct 6360ttaggcaaca cagtcggcac ctcaactatt gtactggttt cgggcgccgt ttttggtttg 6420accggtctga gacgagtgcg atttttttcg tttctaatag cttccaacaa ttgttgtctg 6480tcgtctaaag gtgcagcggg ttgaggttcc gtcggcattg gtggagcggg cggcaattca 6540gacatcgatg gtggtggtgg tggtggaggc gctggaatgt taggcacggg agaaggtggt 6600ggcggcggtg ccgccggtat aatttgttct ggtttagttt gttcgcgcac gattgtgggc 6660accggcgcag gcgccgctgg ctgcacaacg gaaggtcgtc tgcttcgagg cagcgcttgg 6720ggtggtggca attcaatatt ataattggaa tacaaatcgt aaaaatctgc tataagcatt 6780gtaatttcgc tatcgtttac cgtgccgata tttaacaacc gctcaatgta agcaattgta 6840ttgtaaagag attgtctcaa gctcgccgca cgccgataac aagccttttc atttttacta 6900cagcattgta gtggcgagac acttcgctgt cgtcgacgta catgtatgct ttgttgtcaa 6960aaacgtcgtt ggcaagcttt aaaatattta aaagaacatc tctgttcagc accactgtgt 7020tgtcgtaaat gttgtttttg ataatttgcg cttccgcagt atcgacacgt tcaaaaaatt 7080gatgcgcatc aattttgttg ttcctattat tgaataaata agattgtaca gattcatatc 7140tacgattcgt catggccacc acaaatgcta cgctgcaaac gctggtacaa ttttacgaaa 7200actgcaaaaa cgtcaaaact cggtataaaa taatcaacgg gcgctttggc aaaatatcta 7260ttttatcgca caagcccact agcaaattgt atttgcagaa aacaatttcg gcgcacaatt 7320ttaacgctga cgaaataaaa gttcaccagt taatgagcga ccacccaaat tttataaaaa 7380tctattttaa tcacggttcc atcaacaacc aagtgatcgt gatggactac attgactgtc 7440ccgatttatt tgaaacacta caaattaaag gcgagctttc gtaccaactt gttagcaata 7500ttattagaca gctgtgtgaa gcgctcaacg atttgcacaa gcacaatttc atacacaacg 7560acataaaact cgaaaatgtc ttatatttcg aagcacttga tcgcgtgtat gtttgcgatt 7620acggattgtg caaacacgaa aactcactta gcgtgcacga cggcacgttg gagtatttta 7680gtccggaaaa aattcgacac acaactatgc acgtttcgtt tgactggtac gcggcgtgtt 7740aacatacaag ttgctaacgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 7800gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 7860atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 7920cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 7980tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 8040agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 8100aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 8160gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 8220tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 8280cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 8340ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 8400cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 8460atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 8520agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 8580gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 8640gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 8700tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 8760agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 8820gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 8880aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 8940aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 9000ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 9060gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 9120aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 9180ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 9240tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 9300ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 9360cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 9420agcactgcat aattctctta

ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 9480gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 9540gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 9600acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 9660acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 9720agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 9780aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 9840gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 9900tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa 9960aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct 10020ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag 10080acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc 10140ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg 10200cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga 10260agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc 10320aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc 10380cagtgcc 10387920PRTPorcine circovirus 9Ser Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His His Pro Pro Ser1 5 10 15His Leu Gly Gln 201019PRTPorcine circovirus 10Pro Arg His His Tyr Arg Pro Arg Arg Lys Asn Gly Ile Phe Asn Thr1 5 10 15Thr Leu Ser11233PRTArtificial SequenceThis is an amino acid sequence for porcine circovirus type 2, open reading frame 2. 11Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg1 5 10 15Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45Leu Ser Arg Thr Phe Gly Tyr Thr Val Lys Ala Thr Thr Val Arg Thr 50 55 60Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asp Asp Phe Val65 70 75 80Pro Pro Gly Gly Gly Thr Asn Lys Ile Ser Ile Pro Phe Glu Tyr Tyr 85 90 95Arg Ile Lys Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn 115 120 125Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr 130 135 140Ser Ser Arg His Thr Ile Pro Gln Pro Phe Ser Tyr His Ser Arg Tyr145 150 155 160Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro 165 170 175Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ser Arg Asn 180 185 190Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp 195 200 205Gln Asp Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe 210 215 220Asn Leu Lys Asp Pro Pro Leu Lys Pro225 230

Claims

1. A method for the treatment or prophylaxis of an animal against PCV2 infection and ileitis caused by L. intracellularis, or a method for reduction of clinical symptoms caused by or associated with a PCV2 and L. intracellularis infection, comprising the step of administering an effective amount of PCV2 antigen and L. intracellularis antigen to an animal in need of such treatment.

2. The method according to claim 1, wherein said animal is swine.

3. The method according to claim 1, wherein said PCV2 antigen is an inactivated killed PCV2, modified life PCV2, or a subunit of PVC2.

4. The method according to claim 1, wherein said PCV2 antigen is PCV2 ORF2.

5. The method according to claim 1, wherein said PCV2 antigen is selected from the group consisting of Ingelvac.RTM. CircoFLEX.TM., CircoVac.RTM. CircoVent or Suvaxyn PCV2 One Dose.RTM..

6. The method according to claim 5, wherein said PCV2 antigen is Ingelvac.RTM. CircoFLEX.TM..

7. The method according to claim 1, wherein said L. intracellularis antigen is a modified live L. intracellularis.

8. The method according to claim 1, wherein said L. intracellularis antigen is Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903.

9. The method according to claim 1, wherein said PCV2 antigen is Ingelvac.RTM. CircoFLEX.TM. and the L. intracellularis antigen is Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903.RTM..

10. The method according to claim 1, wherein said L. intracellularis antigen is administered prior to the administration of said PCV2 antigen.

11. The method according to claim 1, wherein said L. intracellularis antigen is administered at day one of age to week 12 of age.

12. The method according to claim 1, wherein said PCV2 antigen is administered prior to the administration of said L. intracellularis antigen.

13. The method according to claim 1, wherein said PCV2 antigen is administered at week 3 to week 12 of age.

14. The method according to claim 1, wherein 2 to 20 days are in between the administration of both antigens.

15. The method according to claim 1, further comprising the step of administering an immune stimulant.

16. The method of claim 1, wherein prior to the administration of one or both antigens, the incidence of L. intracellularis and/or PCV2 is determined within a herd or farm.

17. A kit of parts, comprising a container which comprises a PCV2 antigen, preferably Ingelvac.RTM. CircoFLEX.TM., a container which comprises L. intracellularis antigen, preferably Enterisol.RTM. Ileitis or Enterisol.RTM. Ileitis B3903, and an instruction manual, including the information for the administration of said PCV2 and L. intracellularis antigen.

18. The kit of parts according to claim 17, wherein said container which comprises the PCV2 antigen and said container which comprises the L. intracellularis antigen are different containers.

19. The kit of parts according to claim 17, wherein said instruction manual includes the information that the L. intracellularis antigen is administered prior to the administration of the PCV2 antigen.

20. The kit of parts according to claim 17, wherein said instruction manual includes the information that the L. intracellularis antigen is administered at day one of age to week 12 of age.

21. The kit of parts according to claim 17, wherein said instruction manual includes the information that the PCV2 antigen is administered prior to the administration of the L. intracellularis antigen.

22. The kit of parts according to claim 17, wherein said instruction manual includes the information that 2 to 20 days are in between the administration of both antigens.

23. The kit of parts according to claim 17, wherein said instruction letter includes the information that the PCV antigen is administered at week 3 of age to week 12 of age.

24. The kit of parts according to claim 17, further comprising an immune stimulant and wherein said instruction manual includes the information that an immune stimulant is also administered.

25. The kit of parts according to claim 17, wherein said instruction manual includes the information that the incidence of the L. intracellularis and/or PCV2 is determined within the farm or specific herd prior to administration of the antigens.