TW200815027A - Vaccination of young animals against lawsonia intracellularis infections - Google Patents

Abstract

The present invention provides a method of vaccinating a young animal against L. intracellularis infection comprising the step of administering to said animals an effective dose of L. intracellularis antigen. It also provides a method of vaccinating an animal, preferably a young animal, having anti-L. intracellularis antibodies or is exposed to anti-L. intracellularis antibodies. In particular, those anti-L. intracellularis antibodies are maternally derived antibodies.

Description

200815027 IX. INSTRUCTIONS: [Technical field to which the invention pertains] The present invention relates to the use of vaccination against sputum caused by obligate intracellular bacterial cells (z mine (10) (10) " Proliferative enteritis of ileitis, preferably an improved method of porcine proliferative colitis. In particular, the present invention provides a method for vaccinating young animals against Phytophthora intracellularis by starting from one (1) day old Infection to provide a method of enhancing the protective protection against L. intracellularis. Such young animals> are preferably from 1 day to 21 days, more preferably from 1 day to 1 day, more preferably. It is from 5 days to 9 days, more preferably from 6 days to 6 days, more preferably in 2 days or 2 days, and it is best to receive vaccination at 1 day. In particular, the young animal is the year. Young pigs, preferably early weaned piglets. [Prior Art] Porcine proliferative enteritis (PPE) is a naturally occurring disease that affects pigs that are weaned to young adulthood. The pathogen has been determined to be intracellular Aspergillus I. Intracellular L. sonnei is a special , Intracellular bacterium which can not by normal bacteriological methods in conventional cell-free media and has been considered to be cultured cells need for their growth .s · McOrist et al., Infection and

Immunity, Vol. 61, No. 19, 4286-4292 (1993) and G. Lawson, 'J. 〇f Clinical Microbiology, Vol. 31, No. 5, 1136-1 142 (1993) discusses conventional tissue culture In the flask, ffiIEC_1 8 rat intestinal epithelial cells were monolayer cultured with L. intracellularis. The intracellular Lactobacillus strains are cultured in suspension host cells as described in U.S. Patent Nos. 5,714,375 and 5,885,823, the disclosures of each of which are incorporated herein by reference. The pathogenic and non-pathogenic strains of L. sinensis are well known in the art. For example, WO 96/39629 and wo 〇5/〇11731 describe non-pathogenic attenuated strains of L. intracellularis. W〇 〇2/26250 and W0 03/00665 describe the attenuated bacterial strains of Lactobacillus in the cell.

The disease is characterized primarily by its macroscopic and microscopic pathology, and subsequently by the evidence of intracellular bacteria in infected cells. The pathological characteristics of the disease are hyperplasia of immature epithelial cells in the ileum (the end portion of the small intestine), the large intestine, or both. The sliced features of the infected tissue are similar to the "garden hose" which turns reddened mucosa and intestinal damage. Intestinal thickening ultimately prevents normal bowel function, absorptive capacity and nutrient transfer. The clinical manifestations of the disease are chronic weight loss, imperfection, diarrhea and death. The disease is of economic importance due to loss of death in infected animals, increased cost of drug treatment, difficulty in gaining weight, and reduced food conversion. Most notable is the clinical case of ileitis observed in pigs aged 6-20 weeks. However, the presence of L. intracellularis (by pCR) has been demonstrated in recently weaned pigs (3-4 weeks old), indicating that exposure to L. intracellularis occurs on farms and may be sourced劳 劳 阳性 阳性 阳性 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( -206) These observations emphasize the importance of incorporating preventive strategies (such as early vaccination) into the production system. The current vaccination strategy against ileitis is only for the three weeks 120954.doc 200815027 coincidence - week劳 The above-mentioned Laxonia untreated pigs are vaccinated orally. The piglets below this age group may have maternal antibodies positive for L. intracellularis due to exposure or vaccination of the previous sow. Prior to the method of the invention, the presence of the parent antibody or other prolactin may potentially interfere with the efficacy of the vaccination in the piglets, since the mother's immune system recognizes the vaccine and begins to secrete its own antibodies, the mother The antibody has the ability to neutralize the vaccine. Therefore, it has been avoided to inoculate young piglets in the face of maternal immunization. [Invention] The present invention is based on two surprising observations. First, in the gastrointestinal , period mit and sow emulsion did not appear to be effective in passivating the intracellular L. sonnei. The sow colostrum or emulsion k month intracellular L. sonicia did not affect intracellular labor during 4 hours of incubation at room temperature. The titer of the Soniasis culture. There was no difference between the samples with high or low 4 content, even at 1:20 dilution (5% intracellular L. sinensis culture / 95% emulsion) To the effect on the titer of the culture. In summary, no direct effect of any test sow emulsion sample on the titer of the intracellular L. sonicate culture was detected in vitro. However, since young piglets usually do not suffer Ileitis, so maternal immunity against L. intracellularis has been discussed (Holyoake, Ρ·Κ. et al. (1994) J Clin Microbiol 32, pp. 198CU 1985; Mauch, C. Η. Υ··G · Bilkei (2004) Vet·R Ec· i55 5 3 2). As shown in this study, maternal antibodies and sow emulsions did not appear to be effective in inactivating intracellular Lactobacillus during the gastrointestinal tract. Secondly, the maternal antibody and sow emulsion did not interfere. Intracellular Lawsonia 120954.doc 200815027 Bacterial antigen 'and therefore does not prevent the establishment of an automatic protection against intracellular L. sonnei infection provided by vaccination. In fact, compared to unvaccinated piglets, 1 day The macroscopic inoculation of piglets has been reduced in the gross pathology associated with the disease. Accordingly, the present invention overcomes the deficiencies of the prior art and provides novel methods for providing enhanced protection against ileitis (L. intracellularis infection). In particular, the present invention provides a method for infusing a young animal against a, or, intracellular, S. cerevisiae infection, the method comprising administering an effective dose of the intracellular L. solani antigen to the young animal The steps. The age of the young animal is preferably one (1) day old or more than one day old. Further, according to another aspect, the present invention provides a method for inoculation of an anti-L. intracellularis antibody having or exposed to an anti-L. intracellularis, a maternal-derived anti-L. intracellularis antibody The animal comprises the step of administering an effective dose of the intracellular Laussum antigen of the 5th juvenile to the method of combating the intracellular L. solani infection. The age of the young animal is preferably one day or more. According to another aspect, the present invention provides an animal for vaccination against an intracellular L. lyrium antibody, in particular a parent-derived anti-L. intracellularis antibody, against an intracellular labour. A method of Soniasis infection, the method comprising the step of administering to the animal an effective amount of an intracellular Lawsonia antigen. The term "inoculation," (, or, aCCinating") used in this document is, but is not limited to, a method comprising administering to a animal an intracellular L. solani antigen, wherein the intracellular Loxaonia The bacterial antigen elicits or the immune response of the bacterium in the animal when the activity is administered 120954.doc 200815027.

The term "as used herein" as in the case of cattle, fish, and (but not limited to) birds, fish, and dried pigs or mammals, is more sinister; In the season of mussels, the animals are pigs, m, ,, 4. ^ are early weaned piglets. Therefore, according to another aspect, the hair & month is about inoculation of sputum to fight cells. The method of Intrasonia's ", nine scorpion scorpion M, and the genus Niagina sinensis infection, the method includes the initiation of the intracellular labor of the young animal from the first () Tianda The step of the Sonia 囷 antigen, wherein the animal is a mammal, a mammal, such as a cow, a pig, a horse or a long animal. The cockroach is a mammal. "pig. The animal is best for early weaned piglets. The term "young animal" as used herein refers to an animal that is as large as 20 days old, and the term ", young animal," preferably refers to an animal that is from 1 day to 10 days old. The term "young animal" preferably means 1 八 main y, more preferably 1 day to 8 days, K soil is 1 day to 7 Α λ, more preferably i day is as large as 6 days, more preferably 1 It is better to be 1 day or 4 days old, better than 1 day or 2 days old. It is better for animals of 1 or 2 days. The individual meaning of the term "year: animal" also refers to the age at which the animal is vaccinated with the intracellular Loxos® antigen. Thus, 'another aspect of the invention relates to a method for inoculating a young animal against an intracellular L. solani infection, the method comprising administering to the young animal an effective dose of the intracellular L. solani antigen The step wherein the cockroach animal is vaccinated from 1 day to 20 days old. According to another embodiment, the present invention relates to the method of inoculation, wherein the animal is inoculated with a fork from i Days to ι〇大大 120954.doc 200815027. According to another embodiment, the invention relates to the method of inoculation, wherein the animal is at! Inoculation is accepted when the day is 9 days old. According to another embodiment, the invention relates to the method of inoculation, wherein the animal is vaccinated at a height of 8 days from the day of the animal. According to another embodiment, the invention relates to the method of inoculation wherein the animal is vaccinated from 1 day up to 7 days old. According to another embodiment, the invention relates to the method of inoculation, wherein the animal is vaccinated from the day of the day to the age of 6 days. According to another embodiment, the invention relates to the method of inoculation, wherein the animal is at! Inoculation is given when the day is 5 days old. According to another embodiment, the invention relates to the method of inoculation, wherein the animal is at! Inoculation is accepted when the day is up to 4 days old. According to another embodiment, the invention relates to the method of inoculation, wherein the animal is vaccinated from day to day and up to 3 days old. According to another embodiment, the invention relates to the method of inoculation wherein the animal is vaccinated at 1 or 2 days of age. According to another embodiment, the invention relates to the method of inoculation, wherein the animal is vaccinated at the end of the day. The term "having or exposing to an antibody against L. intracellularis means, but is not limited to, having or exposing to preferably at least 1:4, more preferably more than 1 · 16 per ml, more preferably More than 1:64, more preferably 1:128 or more, more preferably 1 ·25 6 'more preferably 1:5 12 or more' and the best is 1:1 024 or more of the integrable anti-cell Lawsonia The antibody titer of the antibody. The anti-L. intracellularis antibody titer is preferably in the specific anti-L. intracellularis immunoassay, preferably in the assay as described in Example 4. Detected and quantified. These anti-L. intracellularis antibodies are preferably mother-derived antibodies. The term "exposure to anti-cellulosic antibodies" means (but not limited to 120954.doc -10) - 200815027 to obtain a nutrient such as colostrum or lotion, preferably at least 1:4 per ml or per gram of nutrient, more preferably 1:16 or more, more preferably [Μ above, more Preferably, the ratio is 1:128 or more, more preferably 1:256, more preferably the above, and the best is 1:1024 or more of the detectable anti-cell Lawsonia Facts antibody titer.

The term "having an anti-L. intracellularis antibody" shall mean, but is not limited to, preferably at least 1:4, more preferably more than 1 · 16 or more in the i ml serum of the animal. : 64 or more, more preferably 1:128 or more, more preferably 1.256, more preferably 1:512 or more, and most preferably 1:1 〇 24 or more detectable anti-L. intracellularis antibody Titration. Thus, according to another aspect, the present invention provides a method for vaccinating an animal against an intracellular L. solani infection, the method comprising the step of administering to the animal an effective amount of an intracellular Lawsonia antigen, Wherein the animal has or is exposed to at least 1:4, more preferably 1:16:, more preferably 1:64 or more, more preferably 1:128 or more, still more preferably Μ%, more preferably It is 1:512 or more, and the best is ten difficult to detect the anti-L. intracellularis antibody titer. Preferably, the antibodies are materally derived, and the body m titer is preferably present in the animal on the day of inoculation. Thus, according to another aspect, the present invention provides a method for vaccinating a young animal against an intracellular L. solani infection, the method comprising administering to the young animal an effective dose of L. intracellularis The step of the antigen wherein the young animal has or is exposed to preferably from 1:5 to 2, more preferably 1:16 or more, more preferably 丨: (10), more preferably on 1281, more preferably 1 : 256, more preferably 1:512 or more, the best bargain above 120954.doc 200815027 of the debt test anti-L. intracellularis antibody titer. These antibodies are preferably parent-derived antibodies. The age of the young animal is preferably between 1 day and 20 days old. The age of the young animal is preferably between 1 day and 10 days, more preferably between 1 day and 9 days, more preferably between 1 day and 8 days, more preferably 1 day. Between 7 days and a day, it is better between 〖big day and 6 days old, better between 1 day and 5 days, better between 〖days and 4 days, better. Between 1 day and 3 days, it is better for 丨 or 2 days, and the best is 1 day. ""an effective d〇se"" as used herein means, but is not limited to, elicited or capable of eliciting in an effective dose of an intracellular L. sonnei antigen. The amount of antigen of the animal's immune response. "Immunological or immunoreactive" to a composition or vaccine is a cellular and/or antibody-mediated immune response to a composition or vaccine of interest in a host. Thus, the term "primes or is capable of eliciting an immune response," It means, but is not limited to, an immunological process within the host, characterized in that the host develops a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. By Φ, the immune response ''includes, but is not limited to, one or more of the following effects: · Produces or activates anti-to B cells specific for the antigen included in the composition or vaccine of (4), auxiliary Ding cells, inhibitory tau cells and/or cytotoxin cells and/or yd τ fines. Preferably, the host will exhibit a therapeutic or protective immunological response to increase resistance to new infections and/or reduce the clinical severity of the disease. This protection will be demonstrated by a reduction or lack of symptoms associated with host infection as described above. 120954.doc -12- 200815027 The amount of antigen that is effective in eliciting an immune response or capable of eliciting an immune response in an animal depends on the composition of the vaccine and the course of the administration. Typically, when the killed bacterial antigen is used in a vaccine, the vaccine contains from about 〇3 to about 1 〇9 colony forming units (CFU) of bacteria per dose, preferably from about i 〇4 to about 10 per dose. (CFU) bacteria 'better about every dose containing about to about (CFU) 〇

Specifically, when the modified live cell L. sonnei bacteria is used for the vaccine (for example, a bacterial isolate designated as isolate B39〇3, ATcc accession number PTA-4926; and designated as isolate N34Np4) 〇wk bacteria 7 knives, ATCC accession number 55783, both described in w〇96/39629 and WO 05/01 1731), the recommended dose to be administered to susceptible animals is better, spoon 3.0 TCID5〇 ( Tissue culture half infection dose endpoint) / dose to about 6 〇 TCID50 / dose and optimally from about 4.0 TCID 5 〇 / dose to about 5 〇 TCID 5 〇 / dose. In a preferred embodiment, the titer of the vaccine is about 4.9 TCID50 per dose as determined by tissue culture half infection dose endpoint dilution assay (TCID5G). In general, the amount of immunogen will be between 5 and 5 μg and between 10 and 1〇9·0 tcid50, preferably between 1〇3.0 and 1〇6.0 TCIDM, more preferably 1 〇4.° with 1 〇5.° Between (10)- (when using purified bacteria). Preferably, the mother agent is at least 〇2 antigen, preferably from about 2 to about _ μg per dose, more preferably from about 4 至 to about 2 每 per dose, more preferably from about 0.35 to about from about 3 to about 1 〇〇, preferably about 0.4 to about 50 μ § per dose, more preferably about 45 to about 30 Å per dose, more preferably about 〇 6 to about 15 Å per dose, more preferably about about every dose. Preferably, from about 75 to about 8, more preferably from about 1 to about 6 per dose, and more preferably from about U to about 3.0 in each dose of the antigen. 120954.doc -13- 200815027 The term "enhanced protection, as used herein, means, but is not limited to, one or more of the former and one of the uninoculated control animals compared to the group of uninoculated control animals. A statistically significant decrease in clinical symptoms (cross-damage frequency, etc.) associated with A. faecalis infection. The term "statistically significant reduction in clinical symptoms n means, but is not limited to, in the infectious cell, L. solani After the bacteria were stimulated, the frequency of occurrence of at least one clinical symptom in the vaccinated animal group was at least 20%, preferably 3%, more preferably 50%, and most preferably 70% lower than in the uninoculated control group. The term "L. intracellularis" as used herein means by c.

Gebhan et al., Int’l. J. 〇f Systemic Bacteriology, Vol. 43, 笫 3 ’ 533-538 (1993) and S. McOrist et al., j· 0f

Systemic Bacteriology, Vol. 45, No. 4, 820-825 (1995) (incorporated herein by reference in its entirety herein in its entirety in its entirety herein in its entirety herein in the entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire all The isolates described in WO 96/39629 and WO 0 5/0 1 1 73 1. In particular, the term "Intracellular Lawsonia" is also (but not limited to) under the Budapest Treaty (Budapest)

Treaty) is deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 201 10-2209 and ATCC registration number PTA 4926 or ATCC registration number 55783. Two isolates are described in WO 96/39629 and WO 05/01 1731, respectively. The term "L. solani" also means, but is not limited to, any other strain or isolate of L. intracellularis, preferably having WO 96/39629 and WO 05 The immunogenic nature of at least one of the strains of L. intracellularis described in /01 173 1, in particular 120954.doc -14- 200815027 which is deposited under the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard , Manassas, Virginia 201 1 0-2209 and the immunogenic properties of at least one of the isolates ATCC Registry Number PTA 4926 or ATCC Accession Number 55783. When a strain or isolate can be detected in at least one of the anti-L. intracellularis-specific antibodies described in WO 06/01294 in a detection assay also described in WO 06/0 1294, The strain or isolate has the strain of L. intracellularis described in WO 96/39629 and WO 05/011731, in particular at least one of the isolates deposited under ATCC Accession No. PTA 4926 or ATCC Accession No. 5 5783. "Immunogenic properties". These antibodies are preferably selected from the reference numbers 301:39, 287:6, 268:29,

1 10:9, 113:2 and 268:18 antibodies. Preferably, the detection assay is a sandwich ELISA as described in Examples 2 and 3 of WO 06/12949, while antibody 110:9 is used as a capture antibody and antibody 268:29 is used as a conjugate antibody. All of the anti-systems disclosed in WO 06/12949 are manufactured by fusion tumor cells deposited under the Budapest Treaty as a patent deposit in Centre for Applied Microbiology and Research (CAMR) and European Collection of Cell Cultures (ECACC). , Salisbury, Wiltshire SP4 0JG, UK. The deposit period is May 11, 2004. HYBRIDOMA CELL LINE 110:9 was successfully registered with ECACC registration number 04092204. HYBRIDOMA CELL LINE 113 :2 was successfully registered with ECACC registration number 04092201. HYBRIDOMA CELL LINE 268:18 was successfully registered with EC ACC registration number 04092202. HYBRIDOMA CELL 120954.doc -15- 200815027 LINE 268:29 was successfully registered with ECACC registration number 04092206. HYBRIDOMA CELL LINE 287:6 was successfully registered with ECACC registration number 04092203. HYBRIDOMA CELL LINE 301:39 was successfully registered with ECACC Deposit Editor 04092205. The term "L. intracellularis antigen, as used herein, means, but is not limited to, any composition comprising a substance of at least one antigen that induces, stimulates or enhances an antagonist cell when administered to an animal. An immune response to an infection caused by L. solani. The intracellular L. sonnei antigen is preferably a complete intracellular L. sonnei bacterium (especially in a passivated form) (so-called killed bacteria), Modified live or attenuated intracellular L. sonnei bacteria (so-called MLB), any subunit of P. intracellularis, polypeptide or component or any chimeric vector (each containing intracellular Loxos At least one immunogenic amino acid sequence of a bacterium, as used herein, the term 'immunogenic protein,' an 'immunogenic polypeptide' or an immunogenic amino acid sequence" Any amino acid sequence in the host that elicits an immune response against a pathogen comprising the immunogenic protein, immunogenic polypeptide or immunogenic amino acid sequence. Specifically, a π-immunogenic protein, an "immunogenic polypeptide" or an "immunogenic amino acid sequence" of L. intracellularis means that the immunogenic protein is encoded in the administration. , , , an immunogenic polypeptide, or any amino acid sequence of an antigen that elicits an immunological response against Lawsonia intracellularis in a host of "immunogenic amino acid sequence". π immunogenic polypeptide" Or "free

And thus eliciting an antagonistic phase as used herein, immunogenic protein η, plague - its class - includes a 120954.doc -16-200815027 immunological response to pathogens. Many of these fragments can be identified using a number of antigen determinant targeting techniques well known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn Mor. Morris, ed., 1996) Humana Press, Totowa, New Jersey. (The teachings and contents are incorporated herein by reference.) For example, by simultaneously synthesizing a large number of peptides in a solid support (the peptides correspond to portions of the protein molecule) and when the peptides are still attached to the vectors Linear epitopes can be determined by reacting the peptides with an antibody. Such techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec Immunol. 23:709-715. (The teachings and contents are incorporated herein by reference.) Similarly, conformational epitopes are readily determined by determining the spatial conformation of an amino acid, such as by, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. . See, for example, the above Epitope Mapping Protocol. Synthetic antigens are also included in the definition, such as multiple anti-determinants, flanking epitopes, and other recombinant or synthetically derived antigens. See, for example, Bergmann et al. (1993) Eur J. Immunol. 23:2777-2781; Bergmann^ A (1996), J. Immunol. 157:3242-3249; Suhrbier, A. (1997), Immunol· and Cell Biol. 75: 402-408; Gardner et al., (1998) 12th World AIDS Conference, Geneva, Switzerland, June 28-July 3, 1998. (The teachings and contents are incorporated herein by reference.) Suitable L. intracellularis antigens include, but are not limited to, those in EP 1219711 - US 6,605,696, WO 96/39629, WO 97/20050, 120954. Doc -17- 200815027

WO 00/69903, WO 00/69904, wo 00/69905, WO 00/69906, W〇02/38594, WO 02/26250 > WO 03/006665, W〇04/033631, WO 05/026200 and WO 05 The antigen described in /01 173 1. The vaccine thus used in accordance with the present invention includes any of the Lactobacillus intracellularis antigens as described above which cause or are capable of eliciting an immune response against the intracellular Lactobacillus. Preferably, the vaccine provides at least enhanced protection against L. intracellularis. Thus, according to another aspect, the present invention relates to a method of inoculating a young animal against an intracellular L. solani infection, the method comprising administering an effective dose of the young animal from one (1) day old a step of a L. intracellularis antigen, wherein the intracellular L. sonnei antigen is selected from the group consisting of live modified S. cerevisiae bacteria, killed intracellular L. sonnei bacteria Or a group consisting of one or more subunits of L. intracellularis bacteria. Preferably, the vaccine comprises a modified live intracellular R. solani bacterium. The vaccine is preferably Enterisol® Ileitis B3903 (BoehHnger Ingelheim Vetmedica, Inc.). As described above, the inoculation is preferably from 1 day to 20 days, more preferably from 1 day to 1 day, more preferably from 1 day to 9 days, more preferably from 8 days to 8 days. When it is big, it is better when it is 1 day to 7 days, and it is better when it is 6 days old. It is better when it is 5 days old, more preferably 1 day to 4 days. When it is better, it is better when it is 3 days old, and it is better when it is 1 or 2 days old, and it is best when it is 1 day old. According to another aspect, the present invention provides a method for vaccinating an animal against an intracellular L. solani infection, the method comprising administering to the animal an intracellular Lausonia having a dose of 120954.doc -18-200815027 The step of the antigen, wherein the animal has or is exposed to preferably at least 1:4, more preferably 1:16 or more, more preferably 1:64 or more, still more preferably 1:128 or more, more preferably 1:256, more preferably 1 · 5 12 or more, and most preferably u 〇 24 or more detectable anti-L. intracellularis antibody titer' and wherein the intracellular L. sonnei antigen selection A group of free-lived, modified, intracellular L. sonnei bacteria, killed intracellular L. sonnei bacteria, or one of the intracellular Lactobacillus bacteria or sub-units. Preferably, the vaccine comprises a modified live L. intracellularis bacterium. More preferably, the vaccine is Ente:risol (D neitis B3903 (B〇ehringer Ingelheim Vetmedica, Inc.). Further, these antibodies are preferably maternal-derived antibodies. The titer of these antibodies is better in the inoculated sputum present in the animal body. According to another aspect, the present invention provides a method for vaccinating a young animal against intracellular Lawsonia's infection, the method comprising administering to the young animal an effective amount of the intracellular L. solani a step of antigen, wherein the young animal has or is exposed to preferably at least 丨:4 per ml, more preferably 1:1 6 or more, more preferably 1:64 or more, more preferably 1:128 or more, more preferably Is a 1:256, more preferably 1:512 or more, optimally 1:1 〇 24 or more detectable anti-L. intracellularis antibody titer, and wherein the intracellular L. serovar antigenic system A group consisting of one or more subunits of a free-lived modified L. intracellularis bacterium, a killed L. intracellularis bacterium or an intracellular L. sonnei bacterium. Preferably, the modified live intracellular L. solani bacteria are included. More preferably Enteris〇1⑧

Ileitis B3903 (Boehdnger Ingelheim Vetmedica, Inc). This 120954.doc -19- 200815027 Tubo antibody is preferably a W-derived antibody. These antibody titers are more present in the animal. The age of the young animal is preferably between one day and a large one. The age of the young animal is better! Between the day and the age of 10 days, 'better than 1 day to W is better between i days and 8 days, more preferably between 1 day and 7 days, better! Between 6 days old, 'better is better! Between day and day to 5 days, better ^ between day and day to 4 days' is better between i days and 3 days, better _ Tianda The best is 1 day old.

According to another aspect, the present invention relates to a method for inoculating a young animal against an intracellular L. solani infection, the method comprising the steps of: administering the young animal about one (1) day old 3 0 TCID5. Live modified L. intracellularis bacteria up to a dose of about 6 〇 Tcm5G. Preferably, the bacterium is a bacterium included in the vaccine Enteris 〇 18 Ilehis B39 〇 3 (Ingelheim Vetmedica, Inc. on B〇e). As described above, the inoculation is preferably from 1 day to 2 days, more preferably from 1 day to 1 day, more preferably from 1 day to 9 days, more preferably from 1 day to 1 day. When it is 8 days old, it is better when it is 1 day to 7 days, and it is better when it is 6 days old. It is better when it is 5 days old, and better when it is bigger than 4 days. When it is big, it is better to be better when it is 1 or 2 days, and it is best when it is 1 day old. Thus, according to another aspect, the present invention relates to a method of vaccinating an animal against an intracellular L. solani infection, the method comprising the steps of: administering a dose of about 3.0 TCIDm to about 6.00 TCIDw of the animal. A live modified, intracellular L. solani bacterium, wherein the animal has or violent vines preferably at least 1:4 per ml, more preferably 1:1 6 or more, more preferably 1:64 120954 .doc -20- 200815027 Above, more preferably 1:128 or more, more preferably 1:256, more preferably 1:512 or more, and the best is 1:1024 or more detectable anti-cell Lawsonia Bacterial antibody titer. Preferably, the bacterium is a vaccine included in the vaccine Enteris〇l@丨B3903 (Boehringer Ingelheim Vetmedica, Inc.). Thus, according to another aspect, the present invention relates to a method of inoculating a young animal against an intracellular L. solani infection, the method comprising the steps of administering the young animal about 3 TCjDw to about 6 〇. A live modified, intracellular L. sonicella bacterium having a dose of TCID5, wherein the young animal has or is exposed to preferably at least 1:4, more preferably 1:16 or more per ml, more preferably 1 : 64 or more, more preferably 1:128 or more, more preferably 1:256, more preferably 1:512 or more, and most preferably 1:1 〇 24 or more detectable anti-L. intracellularis antibody Titration. The bacterium is preferably a vaccine Enteris〇1@

Bacteria included in Ileitis B3903 (Boehringer Ingelheim Vetmedica, Inc.). As described above, the inoculation is preferably from 1 day to 2 days, more preferably from 1 day to 10 days, more preferably from 1 day to 9 days, more preferably from 1 day to 8 days. When it is big, it is better when it is 7 days old, better when it is 1 day to 6 days. It is better when it is 5 days old, more preferably 1 day to 4 days. The day is better, preferably from 1 day to 3 days, more preferably 1 or 2 days old, and the best occurs at 1 day. According to another aspect, the present invention relates to a method of inoculating a young animal against an intracellular L. solani infection, the method comprising administering an effective dose of the young animal to the intracellular dose from one (1) day old The step of the Lawsonia antigen is 'the young animal is negative for the intracellular L. sonnei and the anti-cell 120954.doc -21 - 200815027. As described above, the inoculation is preferably from 1 day to 20 days, more preferably from 1 day to 10 days, more preferably 1 day, more preferably from 1 day to 8 days, more preferably. When the time is from 1 day to 7 = large, it is better when it is 1 day to 6 days, more preferably it is 5 days old, and more preferably 1 day to 4 days, more preferably 1 day. When it is as large as 3 days, it is better when it is 1 or 2 days old, and it is best when it is big. According to another sad form, the present invention also relates to a new medical use of intracellular Lawsonia® antigen for preparing vaccination against young animals at a (1) day old to counteract intracellular labor. An agent (preferably a vaccine composition) infected with a Soniabacterium, wherein the young animal is vaccinated with an effective dose of the intracellular L. solani antigen at one (1) day old or more than one day: the vaccination is more When the weather is from 1 day to 汕天大, it is better when it is as big as 10 days, preferably from 1 day to 9 days, and better when it is 8 days old, better. When the time is from 1 day to 7 days, it is better when it is from 1 day to 6 days, more preferably from 1 day to 5 days, more preferably from 1 day to 4 days, more preferably in the day. When the time is _ to 3 days old, it is better when it is 1 or 2 days old, and it is best when it is big. According to another aspect, the present invention is also directed to a new drug of the intracellular Loxosian antigen, which is used for the preparation of a medicament for vaccinating an animal against an intracellular L. sonnei infection (preferably In the case of a vaccine composition), the cockroach has or is preferably exposed to at least 〗 〖4, more preferably 1.1 Å or more, more preferably 1:64 or more, still more preferably 1:128 or more, more preferably ^. 56, more preferably 1:512 or more, and most preferably a detectable anti-L. intracellularis antibody titer of l: i 〇 24 or more. According to another sorrow, the present invention also relates to a novel medical use of an intracellular Loxos 120954.doc -22-200815027 囷 antigen for the preparation of a vaccination against young animals against intracellular Lawsonia An agent for the infection of bacteria (preferably a vaccine composition), wherein the young animal has or is preferably exposed to at least, preferably more than 1:16, more preferably 1:64 or more, more preferably more, more preferably Preferably, the ratio is 1:256, more preferably 1:S12 or higher, and the best is ι:ι〇24 to detect the anti-L. intracellularis antibody titer. The inoculation is preferably from 1 day to 20 days, more preferably from 1 day to 1 day, more preferably from 1 day to 9 days, more preferably from day to day to 8 days. More preferably, when the sky is as large as 7 days, it is better when it is 1 day to 6 days, and more preferably when it is as large as 5 days, even better when it is 1 day to 4 days. When it is 1 day to 3 days old, it is more preferably 1 or 2 days old and most preferably occurs at 1 day. According to another aspect of the medical use described above, the L. intracellularis k-derived line is selected from a live modified, intracellular L. lysostus bacterium, killed intracellular L. A group of bacteria or one or more subunits of L. intracellularis bacteria. The intracellular L. solani antigen is preferably a live modified S. cerevisiae bacterium. More preferably, the animals are administered by live modified, S. cerevisiae bacteria at a dose of from about 3.0 TCIDw to about 6.0 TCID^t. The manufacture of vaccine compositions comprising L. intracellularis antigens is at the state of the art and is known to those skilled in the art. For example, those skilled in the art will be able to determine additional components that may be included in the composition (see also Remington's Pharmaceutical Sciences. (1990). 18th ed. Mack Publ., Easton). A known injectable, physiologically acceptable sterile solution can be used by a person skilled in the art. For the preparation of a liquid suitable for parenteral injection or 120954.doc -23-200815027, a liquid m such as saline or a corresponding plasma protein solution is also obtained. Such vaccine compositions may be in the form of an as-dry or drywood formulation which may be reconstituted directly under sterile conditions with an injectable solution prior to (d), e.g., as a kit of parts. The immunogenic and vaccine compositions of the invention may comprise - or a plurality of pharmaceutically acceptable carriers. As used herein, "veterinary acceptable carrier" includes any solvent, dispersion medium, coating agent, adjuvant, stabilizer, dilution

', preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents and the like. "Diluent" may include water, physiological saline, dextrose, ethanol, glycerol, and the like. The isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, lactose, etc. Stable. The agent includes an alkaline salt of albumin and ethylenediaminetetraacetic acid, etc. As used herein, the adjuvant "may include aluminum hydroxide and aluminum sulfate, and the example ^ Quil A > QS. 21 (Cambridge Biotech Inc. 5 Cambridge MA) > GPI.〇l〇〇 (Galenica Pharmaceuticals, Inc. 5 Birmingham, AL) saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion may in particular be based on the following substances: light liquid paraffin oil (European Pharmacopoeia type); isoprene oils such as squalane or squalene; oligomeric oils derived from olefins, especially isobutylene or decene; a g of a linear alkyl group or an alcohol, more particularly a vegetable oil, ethyl oleate, propylene glycol di(octanoic acid/capric acid) ester, tris(caprylic/capric acid) glyceride or propylene glycol dioleate Acid S曰, a branched fatty acid or a transesterified ester, especially an isostearate. The oil is used in combination with an emulsifier to form an emulsion. Preferably, the emulsifiers are nonionic 120954.doc -24-200815027 surfactants - especially ethoxylated sorbitan esters, mannitol esters (eg anhydrous mannitol oleate) ), glycol esters, polyglycerols, propylene glycol i and oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid esters' and polyoxypropylene-polyoxyethylene block copolymers (especially Pluromc products, Especially for L121). See H leg as others, 7}^

Theory and Practical Application of Adjuvants (Stewart-

Tull, D. E. S., ed.). John Wiley and Sons, NY, pp. 51-94 (1995) and Todd et al., Vaccine 15: 564_57 (1997). (The teachings and contents are hereby incorporated by reference.) The case of 牛 ' σ ' has been used by M. Poweii and μ· Newman, Vaccine Design, The Subunit and Adjuvant Approach 11, Plenum Press, 1995 The spT lotion described on page 147 and the lotion described on page 183 of the same book] ^^^9. (The teachings and contents thereof are hereby incorporated by reference.) Another example of an adjuvant is a compound selected from the group consisting of a polymer of acrylic acid or methacrylic acid and a copolymer of maleic anhydride and an alkenyl derivative. Advantageous adjuvant compounds are polymers of acrylic acid or methacrylic acid which are especially crosslinked with polyalkenyl ethers of saccharides or polyols. These compounds are known by the term carbomer (1>]: 1&11161^〇1^Volume 8, No. 2, Η% June). A person skilled in the art can also refer to U.S. Patent No. 2,9,9,462, the disclosure of which is incorporated herein by reference to the entire disclosure of the entire disclosure of the disclosure of the entire disclosure of At least 3 of the hydroxyl groups are replaced by an unsaturated aliphatic group having at least 2 carbon atoms. Preferred groups are those having 2 to 4 carbon atoms, such as vinyl, ene 120954.doc -25-200815027 propyl and other ethylenically unsaturated groups. The unsaturated groups themselves may contain other substituents such as a methyl group. The product sold under the name Carb〇p〇1 (BF Goodrich, Ohio, USA) is particularly suitable. It is crosslinked with allyl sucrose or with propyl isoprene. Among them, mention may be made of 974P, 934P and 971P. Best use of Cabopol 97lp. Among the copolymers of maleic anhydride and an alkenyl derivative, there is a copolymer ema (Monsanto) which is a copolymer of maleic anhydride and ethylene. Dissolution of such polymers in water produces an acidic solution which will be neutralized, preferably neutralized to physiological pH to produce an adjuvant solution into which the immunogenic, immunological or vaccine composition will be incorporated. itself.

Further suitable adjuvants include, but are not limited to, RIBI adjuvant systems (RiM he·), block copolymers (CytRx, Atlanta GA), SAF-M (Chiron A, Avridine lipids • coli Heat-labile enterotoxin

Emeryville CA), monophosphoryl lipid a, plasto-amine adjuvant, from E. coli (E. col or other means), cholera toxin, IMS 13 14 or cell wall dipeptide. Preferably, the adjuvant is added in an amount of from about 100 to about 10 mg per dose. More preferably, the adjuvant is added in an amount of from about 100 to about 1 mg per dose. More preferably, the adjuvant is added in an amount of from about 500 Å to about 5 mg per dose. More preferably, the adjuvant is added in an amount of from about 75 〇pg to about 2.5 mg per dose. Best to add about 1 mg per dose

I20954.doc -26- 200815027 i and / / □ ' but the present disclosure encompasses a composition comprising from about 50 to about 2000 μ guanidine and preferably about 25 yang adjuvant per milliliter of vaccine composition dose. In another preferred embodiment, the invention encompasses a vaccine composition comprising an antibiotic of from about i pg/ml to about 6 〇 pg/ml and more preferably less than about 3 〇 pg/mi. The vaccine is administered to the animal, preferably a mammal and more preferably a pig, preferably by oral administration, in any conventional manner. The dosage to be administered will depend on the particular case, but in any event, it is an amount sufficient to elicit a protective antibody or cell-mediated immune response against ileitis. The vaccine of the present invention is usually administered to a susceptible animal in one or more doses, preferably to young piglets and/or to antibodies against L. intracellularis or to antibodies against L. intracellularis. The little pig. The live or killed vaccine can be administered 1 or 2 times at intervals of 2 to 4 weeks after the initial vaccination. For a live attenuated vaccine, one dose is preferred. As described above, the first or single administration is preferably from 1 day to 20 days, more preferably from 1 day to 1 day, more preferably from 1 day to 9 days, more preferably. When the sky is as big as 8 days, it is better when it is 1 day to 7 days. It is better when it is 〗 〖Day to 6 days, better in 〗 〖Day to 5 days, better in 丨When it is 4 days old, it is better when it is from 2 days to 3 days, more preferably 1 or 2 days old, and it is best when it is big. If a second administration is required or necessary, a second administration is performed about 1 to about 4 weeks after the first administration of the vaccine. According to another aspect, re-inoculation is performed at intervals of 3 to 12 months after any previous inoculation. The subsequent dose of the vaccine is administered on a purely 6 months to one year basis. In another preferred case, animals vaccinated before about 2 to 3 weeks of age should be vaccinated. The subsequent dose of the vaccine is preferably administered on a one-year basis from 120954.doc -27- 200815027. The purpose of the present invention is further described in the following examples, and the following examples are merely for the purpose of saying that the term 'for months' should not be construed as limiting. The actual variant should be obvious to those skilled in the art. All publications and patents cited herein are incorporated by reference. ^八王乂 [Embodiment]

Example 1: colostrum and lotion from sows (more than (4) and gilts (first). Detection of antibody materials and methods specific to L. intracellularis in the mouth & The colostrum samples were obtained from 25 mothers and 25 gilts within 24 hours after autment. The emulsion samples were obtained from the same pigs during the first week and the second week of lactation. The samples were centrifuged twice at 2 g. I took it to isolate the fat portion of the emulsion. Store the water sample of the sample down until analysis. Detecting anti-L. intracellularis after 1-2 times of initial dilution from 1:20 initial dilution in IFAT Specific antibodies. In addition to FITC-labeled anti-porcine igG, IgM and IgA antibodies were used to detect different classes for each sample, rIFAT was introduced as described in Example 5 and elsewhere. Parallel blood samples from sows and gilts were obtained and tested by the Enterisol® Ileitis ELISA according to the manufacturer's instructions. Results The results of antibody detection in colostrum samples are summarized in Figure 1. This figure is shown in IF AT. Get igG, IgM and IgA samples with different dilutions Sexuality 120954.doc -28· 200815027 Number of fruit. No colostrum samples were negative in all tests. Only one from the beginning of the sow; fL-like Όσ and a colostrum sample from the gilt were yin H In a sample from a gilt and three samples from a sow, there is no specificity! ± IgM is detectable. It is obtained from the mother and the sow, respectively. The IgA-specific knife of the lausonia is negative. In the emulsion sample obtained during the second week of lactation, only two IgGs from the sow sample get a titer of 1:2, and one comes from The IgA score of the sample of the mother and the pig was positive (1:2〇). In the third week of lactation, the sample from one sow and two gilts gave a positive result at a dilution of 1:2〇. All other samples from the second and third weeks of lactation were negative for IgG, IgM, and IgA. All blood samples taken from sows within 24 h after birth were positive in the Enterisol® Ileitis ELISA. Two of the twenty-five samples obtained by the gilts received negative results. Results from blood samples There was no significant correlation between the results from the colostrum samples. It can be inferred that in the colostrum from sows and gilts, there are Ig-like G, Μ and A specific for the intracellular L. Antibodies. However, in the emulsion samples obtained one week after calving, low titers of L. intracellularis-specific antibodies were detected in only 6% of the samples. References • 1· McOdst, S Et al. (2003) Pig J· 51, 26-35 2· Collins, A. Μ· et al. (2001) Allen D· Leman Swine

Conference 3. Holyoake, Ρ·Κ· et al. (1994) J Clin Microbiol 32, 1980, 1985, 120954.doc -29- 200815027

Knise, pE (1983) Ann· Rech· Vet, 14,349-353 4· B〇11Wem, J· (20〇4) Doctoral thesis, LMU, Munich 5. KmUe1, Jp• et al. (1998) AJVR 59, 722-726. Example 2 Direct effects of emulsion antibodies on L. intracellularis during gastrointestinal tract Materials and methods colostrum samples were obtained from sows and gilts within 24 hours of birth. Sow emulsion samples were taken during the first and second weeks of lactation. In; 4. The sample was centrifuged twice at 2 〇〇〇 g for 1 〇 min & to separate the fat portion of the emulsion. Store the water sample of the sample at -2 °C until analysis. Specific antibodies against L. intracellularis were detected in IFAT after a 2-fold dilution from the initial dilution of 1:20. In addition to FITC-labeled anti-porcine β IgM and IgA antibodies for each sample, in addition to the different classes of ig, such as the example $ and

Enter rIFAT as described elsewhere (5). Based on the IFAT results, different "Summer-containing emulsion samples were selected. The intracellular Lactobacillus cultures were incubated in different concentrations in colostrum or sow emulsion samples at room temperature. Each sample was tested for two-person. The tissue culture half-infection dose (TCID5〇) of the intracellular L. sonnei was measured at the beginning of the incubation (〇 hours) and at room temperature after 4 hours. The undiluted sample of the culture served as a control for the test efficacy. For TCIDw For each determination of the value, the sample was homogenized by 20 passes through a 20 gauge needle. A 75 cm2 tissue culture flask from a 1% by weight monolayer of McCoy cells grown in F12 medium grown in DMEM/HAM was used. The cells were trypsinized and divided into four 96-well microtiter plates. 1 (^ to 1〇-7 serial dilutions of I 120954.doc -30-200815027 were each applied to fresh McCoy cells six times. At 37t in micro After 6 days of aerobic conditions, the cells were fixed by ice-cold acetone/methanol (50:50, v/v) by using a specific monoclonal strain of Lactobacillus antibody and then by using Mouse antibody labeling to detect the growth of L. solani in cells It was judged that 3 or more of the wells of McCoy cells having 5 or more fluorescent L. intracellularis were positive. TCID5〇 was obtained by using the 501⁄4 endpoint formula according to Sparmann and Karber (6). Results The results from the TCIDm test and the IgG, IgA and IgM titers of the colostrum (No.) and the ritual (Sections 6-8) were summarized. In all experiments, the control samples of the culture were obtained. The expected value of Ding was depreciated. During the period of 4 months in the mouth of the month, there was no change in the titer of the intracellular L. sonnei in the test, and therefore, as shown in this study, During parenteral tract, maternal antibodies and sow emulsions do not appear to be effective in inactivating intracellular Lactobacillus.

L.van Aken; N. et al. (2〇〇2) Pr〇c 17th lpvs, Ames, i〇wa, USA 2· Kruse, Ρ·Ε· (1983) Ann·Rech· Vet· 14, 349-353 3· C〇llins, A. M et al. (2〇〇1) Alleil D· Leman Swine,

Conference 4· Holyoake, Ρ·Κ·荨人(1994) j Clin Microbiol 32, pp. 1980. 1985 Page 5. Kmtte1, LP• et al. (1998) AJVR 59, 722-726· 120954.doc 200815027 6. Karber, G. (1931) Arch. exp. Path. Pharma· 162,480 7· Mauch, C_ Η. Y. and G. Bilkei (2004) Vet· Rec· 155, 532 Example 3 Inoculation of a large pig to fight cells Efficacy Materials and Methods for L. nausea Infections This study consisted of three experimental groups involving 1 day old L. intracellularis and anti-Lausonia's maternal antibody negative lactating piglets. On the third day of the study, 20 piglets from Group 1 were each received (inoculated) an oral dose of Enterisol® Ileitis (vaccine isolate B39〇3) (according to the labeling instructions (3)). Twenty piglets from Group 2 (control) received a placebo consisting of growth medium. All groups of piglets were weaned on the second day of study. On day 2, the piglets of Groups 1 and 2 received a gut slurry containing 35 virulence of L. oxysporum 2688/2685 via tube feeding. The third group of ι〇 pigs (rigorous controls) did not receive vaccine, placebo ^ at any time during the study period. On day 42, all groups of pigs were humanely euthanized and necropsy was performed. The main efficacy parameters include the gross and microscopic damage of the ileum, cecum and colon caused by the intracellular L. sonnei. Secondary parameters included clinical health status (behavior, body condition, and stool hardness), mean increase per venous, fecal excretion (PCR), and A, Taigu * V, and seroconversion (IFAT) (1). Results At autopsy (day 42), gamma soil was significantly less inoculated (Ρ<0._)3) compared to uninoculated control pigs (ileum = 1.16, cecum = 0.42 and colon 0.16). Zhiping (different, 丄 丄 有 相 相 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( · 42 cm) The mean total intestinal damage length (ileum, cecum, and colon) was significantly higher (ρ < 0·0001) (8·89 cm) in the control group. The average of the third group (strict control group) The score of gross eye damage was 0.22 and was considered normal. Compared with the inoculated pigs (0.53), the average microscopic damage score of the ileum of the ileum was significantly higher in the control group (ρ < 〇 · 〇 2) (2·47). In addition, the total microscopic damage score (ρ<0.006) and the percentage of IHC positive pigs (ρ<〇·〇〇〇ι) were significantly higher in the control group than in the vaccinated group. In the intestine and colon, the control group had a higher average microscopic damage score and a percentage of me-positive animals than the inoculated group (ρ > 0·〇5). Significantly (ρ < 0.05 ), compared with the vaccinated group, pigs in the vaccinated group (3/19) phase & 2 weeks after challenge, and pigs in the control group (8/19) ranked more intracellular lausonia There was no significant (p刈〇5) difference in mean maternal weight gain or mean clinical score between the sputum group and the second group during the study period. In summary, the main efficacy parameters (development of the naked eye and microscopic intestinal damage) indicate that In the case of a 1-day-old mother, the mother-in-law negative antibody, and the untreated piglets of Lawsonia, a single oral dose of Entens〇l Ileitis was effective for toxicity. In this study, we were feeding Xiang ^ ^ ^Does not interfere with or hinder the efficacy of the vaccine against toxic intracellular L. sinensis to stimulate the rain, the non-Lousoniabacterium specific prolactin properties are potentially present%> 120954.doc -33- 200815027 Table I. The ratio of the stool excretion of L. sonnei in the cell. Group * Group ID 0 to 21 day PCR positive 28 曰 PCR positive 35 曰 PCR positive 42nd 曰PCR positive 1 inoculation 0/19a 0/19a 0/19a 3/19a 2 challenge control l/19a 0/19a 5/19b 8/19 a 3 Φ strictly controlled 1/9 0/9 0/9 0/9 a'b The same letter indicates no significant difference (ρ < 0.05). g Φ is not statistically analyzed for the strict control group. *Because there is no relevant intracellular labor The poor health status of the Sonia strain, removing one pig from each group. References 1 · McOrist et al. (1 993) Infection and Immunity 61: 42 86-4292. 2. Knittel et al. (1998) Am J Vet Res 59:722-726. 3 Kroll et al. (2004) Am J Vet Res 65: 559-565 • 4. Mauch and Bilkei (2004) Vet Rec 1 55: 532 5. Marsteller et al. (2003)· Swine Health Prod 11:127-130, 6· Stege et al. (2004)· Vet Micro 104: 197-206 Example 4 IF AT Materials and Methods for Detecting and Quantifying Antibodies Against L. intracellularis: Before the start of the study, 16 healthy, pregnant and intracellular Lactobacillus negative Sows were randomly divided into 2 groups: 8 were in the hyperimmune group (group A) and 8 were 120954.doc -34-200815027 placebo was administered to the control group (group B). Each of the sows in Group A was hyperimmunized with the commercially available Enteris〇i® Hems by direct level oral administration on the 55th, 35th and 14th days before the birth. Dosing a vaccine dose by this method involves applying the vaccine to the back of the mouth using a sterile plastic such as a syringe. The control group (Group B) received a placebo consisting of uninfected McCoy cells suspended in growth medium in equal doses. Group A pregnant sows were hyperimmunized to induce high levels of maternal immunity prior to birth. Each pregnant sow group is housed in a separate room (to avoid contamination and contamination) and under the same conditions (temperature, ventilation, and shed size). The sows in each room were kept in the same shed. Although the investigators have tried to have consistent pregnancy and litter size, sows are not all born in the same day. The truth is that the birthing occurs in 10 days. To prevent the presence of multiple vaccination and challenge dates that would result in excessive variability between pigs in the study, the average date of birth during the litter period was determined as day 0 of the study. Therefore, at the time of inoculation (day 21), the pig was 3 weeks ± 5 days old. On the 2nd day, a hundred healthy, weaned piglets were selected by sex and randomly divided into six treatment groups. The residence restrictions and conditions are similar to the sow group mentioned above. Piglets from hyperimmune sows (Group A) were randomly assigned to 3 groups and were identified as "Superimmuno-derived M, pigs for use in the remainder of the study. Piglets from control sows (Group B) were randomly assigned to groups of 4 to 6 and identified as "placebo-derived" "pigs for use in the remainder of the study. By direct oral administration] Groups and Group 4 (2 pigs/group, respectively) with a single -2 ml dose of Enterisol8 lie Hill s. Groups 2 and 5 (both pigs/group, respectively) were given an equal dose of placebo (not Infected with McCoy cells + medium. Groups 3 and 6 (1〇/group) were designated as, strictly negative control group, and during the study period they did not receive the 120954.doc-35-200815027 vaccine or Placebo treatments were not exposed to challenge. On the 22nd day after the weaning period, the sows were humanely euthanized and autopsied for assessing the development of intestinal damage due to rush. On the 42nd day of the study, paragraphs 1, 2 Groups 4 and 5 received a heterologous toxic pure culture of the intracellular L. serovar strain NHH494 per mg5G via intragastric tuberculosis. The PE-related bed symptoms of all baboons were tested daily 21 days after challenge. Behavior and physical condition and given a score of 1 to 4 depending on the severity (Bu clinically normal; 4 = serious illness) Pigs were weighed on Days 2 1 42 and 63 to calculate the average daily weight gain for each group. The average daily weight gain (adwg) was calculated to analyze the therapeutic effect on the normal growth performance of ticks. Pigs were weighed to obtain a baseline mean group weight before receiving vaccine or placebo treatment. All groups were found to be uniform in size (difference < 0.63 kg/pig). The first critical ADWG assessment period between groups occurred on day 21 (vaccination) Immediately by day 42 (excitation exposure) to measure the immediate effect of vaccination or placebo vaccination. The second evaluation period was from day 42 to 63 (autopsy) to measure toxic pure cultures of intracellular Lactobacillus The effect of the exposure was stimulated. On the 63rd day of the study, all pigs were humanely euthanized and autopsied for evaluation of intestinal damage due to PE. Results Maternal antibody detection during group A in the litter period (hyperimmunization) In the serum and colostrum of sows, 4 antibodies were tested for specific IgG, IgA and IgM antibodies in the cells. Group A of 63% (5/8 sows) at birth Sows against Lawsonia 1 20954.doc -36- 200815027 The illgG antibody is positive for serum antibodies, while the females in the b group (control) are positive for 母% (〇π隹). In addition, only from the day of birth (days) to 5 At the age of weeks (Day 28), serum IgG antibodies were detected in piglets from group A sows, as shown in Figure 1. Anti-Laomycin IgG was detected in the colostrum of group A sows. , lgA • and 1gM were 50%, 75%, and 12.5%, respectively. The average colostrum in group A sows. The antibody concentration was kMQgG, ranging from i:64), 1:10 (IgA, range to 1: 32) and i.4 (lgM 'range 1 to 1:4). Group B sows did not have any detectable IgM or IgG anti-L. serovar antibodies in their colostrum during this period. One of the B pigs was positive for IgA at a titer of 1:16. Maternal protection was performed on a comparison of unvaccinated, control pigs from Group 2 (sows from sow group A) and group 5 (from pigs in group B) to assess the sows that were derived from vaccine hyperimmunization ( The potential of maternal protection of the toxicity of L. intracellularis in the toxicity of group A sows. The average gross and microscopic damage scores for all groups are summarized in Table 1. Compared with Group 2 pigs (27.5%), Group 5 pigs (77%) had a higher percentage of L. sinensis-specific lesions in the ileum and colon. On day 63 of the study, the second group of pigs had a significant (p<〇〇5) lower mean ocular damage score in the ileum and an average microscopic (mc) lesion in the ileum and colon compared to the fifth group. * Minute. On the 21st day (inoculation) to 42nd (excitation) days of the study, there was apparently no detectable excretion of L. intracellularis in the feces of either group (as known), see Figure 3. On the 49th day, the pigs in the 2nd and 5th groups began to show _ positive and the pigs that discharged the intracellular L. sonicia during this period had 120954.doc -37- 200815027 5% to 25% and 15% to 72 respectively. % remained positive until day 63 (study terminated). On the 63rd day of the study, compared with the fifth group (72%), in the second group, significant fecal excretion (25%) was detected by PCR#, which was significant (ρ < 0.05). On day 63 of the study, a higher percentage of positive PCR (45%) was found in the ileum of the pigs in Group 5 compared to Group 2 (25%). Group 2 pigs were PCr negative for L. intracellularis in the tonsils, mesenteric lymph nodes and colon. As described in Section 3.4 above, different PCR positives were observed in the colon and mesenteric lymphocytes of Group 5 pigs. A comparison of the average weight gain between all test groups is summarized in Table 2. On Day 21 (vaccination), the average initial body weight between Group 2 and Group 5 was consistent, with pig weights of 6.3 and 6.10 kg/pig. From day 21 (inoculation) to day 42 (excitation), there was no significant difference in ADWG between group 2 (〇·40 kg/pig) and group 5 (〇·41 kg/pig). However, during the 21-day evaluation period from the 42nd day (excitation) to the 63rd day (end of study), the second group of pigs (0.46 kg) compared with the fifth group of pigs (〇·4〇kg/pig) The ADWG of the / pig) is clearly significant (ρ < 0·05) higher. Vaccine efficacy in hyperimmune pigs A comparison of Group 1 Enterixol® Ileitis vaccinated pigs with Group 2 unvaccinated and control pigs to confirm protective immunity against PE after vaccination in 3 week old pigs To achieve vaccination in the face of maternal immunization. Both pigs are derived from sows that are hyperimmunized with vaccine (Group A sows). In addition, primary and secondary efficacy parameters between Group 1 and Group 4 vaccine-treated pigs were analyzed to determine if the vaccine was similar in efficacy against L. oxysporum challenge in virulent cells. The average gross and microscopic damage scores for all groups are summarized in Table 1. Compared with the 1 120954.doc -38- 200815027 group of pigs (12.5%), the second group of pigs (27.5%) had a percentage of sputum-specific damage in the ileum and colon. On day 63 of the study, the group of pigs had a significant (p<〇〇5) lower mean gross damage score (ileum) and a lower numerical average (IHC) damage compared to group 2. Score (ileal and colon). In addition, there was no significant difference between the average gross and microscopic damage scores or the severity of the lesions in the vaccinated pigs (Groups 1 and 4). On the 21st day of the study (vaccination) to the 35th day, in any group (1 or 2 groups), there was apparently no detectable discharge of L. intracellularis in the feces (as known by pCR). 3. On day 42 (excitation), pigs in the first group began to be PCR positive and 11% to 16% of the pigs that discharged the intracellular Lactobacillus during this period remained positive until day 63 (end of study). On day 49, the feces of the second group of pigs began to be PCR positive and 5% to 25% of the pigs that discharged the intracellular Lactobacillus during this period remained positive until the 63rd day (end of study). Group 4 pigs were not PCR positive for L. sonnei DNA until day 42 (excitation) and remained positive until the efflux rate was reduced from 25% to 5% until day 63 (end of study). There were no significant differences in the ratio of intracellular L. serrata fecal excretion between Group 1 and Group 2 and Group 1 and Group 4 during the study period. On study day 63 (study termination), a slightly higher percentage of tissue PCR positives (25%) was found in the ileum of Group 2 pigs compared to Group 1 (20%). At the end of the study, the PCR positive frequency (5%) of Group 4 tissue was clearly lower than Group 1 and Group 2. It was noted that there was no significant difference in PCR positive between Group 1 and Group 2 and Group 1 and Group 4, respectively. All three groups of pigs were PCR negative for L. intracellularis in the tonsils, mesenteric lymph nodes and colon. I20954.doc -39- 200815027 Table 2 summarizes the comparison of mean weight gain across all test groups. On day 21 (vaccination), the average initial body weight between Group 1 and Group 2 was consistent, with pig weights of 6.53 and 6.35 kg/pig. On the 21st day, between Group 1 and Group 4, the average body weight was also observed (6.53 kg/pig and 6.44 kg/pig, respectively). Day 21 (inoculation) to Day 42 (excitation), 1 & (〇·4〇kg/pig) and Group 2 (〇·4 1kg/pig) or between Group and Group 4 (〇 There was no significant difference in adwg between 44 kg/pig). During the 21-day evaluation period from day 42 (excitation) to day 63 (end of study), the first group of pigs (0.45 kg/pig) compared to the fourth group of pigs (〇51 kg/pig) The significant difference (p < 〇〇 5) is obvious. Note ~ ~ In this study, nine brothers 2 days to 63 days, the first group. There is no significant difference between the two groups of ADWG. Example 5

Isat for detecting and quantifying antibodies against L. intracellularis using the immunofluorescence antibody test (IFAT) using the whole antigen of L. serrata immobilized on a 96-well polystyrene microtiter plate and The FITC-labeled antibody of porcine IgG (knittel et al. 1998) tested the presence of IgG antibodies against L. intracellularis in serum samples from the blood of each sow and pig. The IFA test was slightly modified by using FITC-labeled antibodies against porcine IgM and IgA. This modified procedure was used to detect such antibodies other than IgG in each sow colostrum for determination of the concentration of different Lawsonia-specific immunoglobulins. The colostrum was diluted 2-fold in duplicate in PBS and transferred (丨〇〇μΐ/well) to two groups of Lausonia-coated 96-well plates as described above. The inoculated plates were incubated at 37 ° C for 30 minutes and then washed 3 times with pBS. Add anti-pig Ig]v^IgA FITC 120954.doc -40-200815027 〆, irk 抗体 antibody (Kirkegaard and Perry Laboratories, Inc.) diluted in PBS in PBS to the culture dish and then repeat the incubation and Washing step. The titer of each specific anti-Laomycin antibody was measured using UV microscopy. The IFAT positive percentage and the average titer value of each immunoglobulin were calculated for comparison between groups to determine the frequency and content of IgG, IgM and IgA colostrum antibodies in sows. References 1 Knittel et al. (1998) Am J Vet Res 59:722-726. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the number of colostrum samples positive for IgG, Igiy [and IgA in IFAT. Figure 2 shows the titer of the intracellular Lactobacillus culture in the sow emulsion samples with different Ig contents (average of two titrations). 120954.doc 41

Claims (1)

200815027 X. Patent application scope: 1. A pharmaceutical composition for inoculation of young animals against L. intracellularis (J: · infection or for inoculation with anti-cell nematode antibodies or exposure An animal that is resistant to an intracellular L. solani antibody. The pharmaceutical composition comprises an effective amount of an intracellular Lawsonian reading antigen. 2. The pharmaceutical composition of claim 1, wherein the animal is as large as 1 day. Inoculation at 9 days of age. 3. The pharmaceutical composition of claim 1 or 2 wherein the animals are vaccinated from 1 day to 6 days. 4. The pharmaceutical composition according to claim 1 or 2, wherein The animals are vaccinated at 1 or 2 days. 5. The pharmaceutical composition of claim 2 or 2, wherein the animals have or are exposed to at least 1:4 per milliliter of serum or fluid. a pharmaceutical composition of the method of claim 1 or 2, wherein the animal has or is exposed to at least 1:64 per milliliter of serum or fluid, and is detectable against intracellular Lausonia. Cell antibody titer 7. As requested in claim 1 or 2 a composition, wherein the intracellular L. sonnei antigenic line is selected from the group consisting of live modified S. cerevisiae bacteria, killed intracellular L. solani bacteria, or intracellular lausonia A pharmaceutical composition comprising one or more subunits of the bacterium. The pharmaceutical composition of claim 1 or 2, wherein the intracellular L. sonnei antigen is a live modified, intracellular, lausonia The medicinal composition of claim 8, wherein the young animal is administered a live modified Rev. intracellularis of about 3. TCIDsq to a dose of about 6.0 TCIDm. 10. The pharmaceutical composition of claim 1 or 2, wherein the animal is a bird, a fish, and a mammal such as a cow, a pig, a horse, and a primate. 1L. The pharmaceutical composition of claim 10, wherein The animals are pigs. 12. The use of the intracellular L. solani antigen for the preparation of a vaccination against young animals for combating intracellular L. sonnei infection or for vaccination with anti-cellular labor Sonia virus antibody or exposure to anti-fine An agent of an animal of the genus L. solani antibody, wherein the animal is vaccinated with an effective amount of the intracellular L. solani antigen. 13 · The use of the item 12 is as follows: 'The animals are from 1 day to 9 Inoculated at the time of the day. 14. For the purposes of claim 12 or 13, the animals are vaccinated from 1 day up to 6 days old. The use of claim 12 or 13 is the use of 1 or 2 of the animals. Vaccination at day 1 0. The use of claim 12 or 13 wherein the animals have or are exposed to at least 1:4 per milliliter of serum or fluid to detect an anti-L. intracellularis antibody titer. 7. The use of claim 1 or 13 wherein the animal has or is exposed to at least 1·64 of the detectable anti-L. intracellularis antibody titer per milliliter of serum or fluid. 18. The use of claim 12 or 13, wherein the intracellular L. sonnei antigen is selected from the group consisting of live modified S. cerevisiae bacteria, killing 120954.doc 200815027 dead cell A group consisting of one or more subunits of A. sinensis bacteria, or intracellular locus bacteria. 19. The use of claim 12 or 13, wherein the intracellular Loxos is a live modified Rev. intracellular bacteria. 20. The use of claim 19, wherein the young animal is named TCID5. The live modified S. solani bacteria to a dose of about 6.0 TCIDwt. 2! The use of claim 12 or π, wherein the animals are high, ritual animals such as cattle, pigs, horses and primates. 22. The use of claim 21, wherein the animals are pigs. N. faecalis anti-bacterial: about 3.0 intracellular fish, and feeding 120954.doc