CN101454020A

CN101454020A - Vaccination of young animals against lawsonia intracellularis infections

Info

Publication number: CN101454020A
Application number: CNA200780019257XA
Authority: CN
Inventors: 杰里米·克罗尔
Original assignee: Boehringer Ingelheim Vetmedica GmbH
Current assignee: Boehringer Ingelheim Vetmedica GmbH
Priority date: 2006-05-25
Filing date: 2007-05-24
Publication date: 2009-06-10
Also published as: TW200815027A; CL2007001499A1; AR061201A1

Abstract

The present invention provides a method of vaccinating a young animal against L. intracellularis infection comprising the step of administering to said animal 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

The young animal immunity inoculation is infected with the opposing lawsonia intracellularis

Related application

The application requires the priority of U.S.'s serial number 60/803,207 of submission on May 25th, 2006, and the instruction and the content of this patent all are incorporated herein by reference.

Technical field

The present invention is about being used for immunity inoculation to resist the hypertrophy enteritis that is called ileitis (ileitis) that is caused by obligate intracellular bacteria lawsonia intracellularis (Lawsonia Intracellularis), being preferably the improvement inoculation method of pig hypertrophy ileitis in general.Particularly, the invention provides a kind of by hugely beginning to inoculate young animal infects the enhancing protection that the antagonism lawsonia intracellularis is provided with the antagonism lawsonia intracellularis method from one (1).These young animal are preferably big to 21 days greatly at 1 day, and are more preferably big to 10 days greatly at 1 day, more preferably big to 9 days greatly at 1 day, more preferably big to 6 days greatly at 1 day, more preferably big at 1 or 2 day, and most preferably accept inoculation when big at 1 day.Particularly, this young animal is a year young piglet (young piglet), is preferably the preceding piglets (pre-weaned piglet) of wean.

Prior art

(Porcine proliferative enteropathy PPE) is the disease of natural generation to pig hypertrophy enteritis, and it can influence the pig of weaning to one's early years manhood (young adult).Determined that its pathogen is a lawsonia intracellularis.Lawsonia intracellularis is an obligate intracellular bacterial, can not cultivate with common bacteriological method on not celliferous conventional culture medium, it is generally acknowledged that its growth needs has cell.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) have discussed in the conventional organization culture bottle, have cultivated lawsonia intracellularis with IEC-18 rat monolayer enterocyte.United States Patent (USP) 5,714,375 and 5,885,823 (all introducing for referencial use in full) have been described in the suspension host cell and have been cultivated lawsonia intracellularis.

The pathogenic strain of lawsonia intracellularis and non-pathogenic attenuated strain are well known in the art.For example, WO96/39629 and WO 05/011731 have described the non-pathogenic attenuated strain of lawsonia intracellularis.The attenuated strain of lawsonia intracellularis can be referring to WO 02/26250 and WO 03/00665.

The feature of this disease at first is its naked eyes and microcosmic pathology, and is subsequently to confirm to have intracellular bacteria in the infected cell.The typical pathological characters of this disease is the epithelial hypertrophy of immaturity in ileum (end portion of small intestinal), large intestine or both crypts (crypt).The slice feature of infected tissue is that reddening of similar " rubber hose (garden hose) " thickens mucosa and intestinal infringement.Intestinal thickens final prevention normal bowel function, absorbability and trophic transfer.The clinical manifestation of this disease is chronic weight saving, not sturdy (unthriftiness), diarrhoea and dead.This disease is owing to infected animals death causes damage, medical treatment cost increases, weightening finish difficulty and efficiency of food conversion reduce and have the economics importance.The most noticeable in the 6-20 pig in age in week, observing the clinical case of ileitis.Yet, confirmed the existence of (by PCR) lawsonia intracellularis in the pig (3-4 age in week) soon of wean just, this shows that the situation that is exposed to lawsonia intracellularis occurs in plant and may come from positive female animal (Mauch and Bilkei (2004) the Vet Rec 155:532 of Lawson bacterium; People such as Marsteller (2003) .Swine Health Prod 11:127-130; People such as Stege (2004) .Vet Micro 104:197-206).These observed results emphasize to introduce the importance of preventative strategies can (such as inoculation early) in production system.

At present the vaccination strategies of the immunization of antagonism ileitis relates to and only the pig from three ages in week and the not contacted Lawson bacterium more than three ages in week is carried out the oral throwing of vaccine and give, and this is because be lower than the piglet of this age group because exposure of previous sow or inoculation and may have the maternal antibody that lawsonia intracellularis is positive.Before method of the present invention, it is generally acknowledged that the existence of maternal antibody or other galactagogue factors can disturb the effect of inoculating in these piglets potentially, because before the immune system of piglet can be discerned vaccine and begin to secrete himself antibody, during maternal antibody has and the ability of vaccine.Therefore, when facing maternal immunity, avoided inoculating young piglet.

Description of drawings

Fig. 1 is illustrated among the IFAT number of the colostrum sample that IgG, IgM and IgA are positive.

Fig. 2 is illustrated in the titer (twice titrating meansigma methods) of lawsonia intracellularis culture in the sow samples of latex with different I g content.

Summary of the invention

The present invention is based on two kinds of wonderful observed results.At first, during gastrointestinal tract, maternal antibody and sow emulsion do not manifest and are effective in the deactivation lawsonia intracellularis.Cultivate the titer that lawsonia intracellularis does not influence the lawsonia intracellularis culture in 4 hour nurturing period of room temperature with sow colostrum or emulsion.Has zero difference between the sample of high or low Ig content.Even under 1:20 dilution factor (5% lawsonia intracellularis culture/95% emulsion), do not observe influence to culture titer.In a word, in vitro do not detect of the direct influence of any tested person sow samples of latex to the titer of lawsonia intracellularis culture.Yet,, therefore maternal immunity (Holyoake, people such as P.K. (1994) J Clin Microbiol 32, the 1980-1985 pages or leaves of antagonism lawsonia intracellularis have been discussed because a year young piglet is not suffered from ileitis usually; Mauch, C.H.Y. and G.Bilkei (2004) Vet.Rec.155,532).So shown in the research, during gastrointestinal tract, maternal antibody and sow emulsion do not manifest and are effective in the deactivation lawsonia intracellularis.

Secondly, maternal antibody and sow emulsion are not disturbed lawsonia intracellularis antigen, and the foundation of the active protection infected of the antagonism lawsonia intracellularis that does not therefore prevent to be provided by inoculation.In fact, do not compare existing minimizing of the visible pathological manifestations of naked eyes relevant of the piglets of inoculation when big with this disease at 1 day with inoculating piglets.

Therefore, the present invention overcomes the not enough of prior art and provides new method to be used for animal is provided the enhancing protection of antagonism ileitis (lawsonia intracellularis infection).In detail, the invention provides a kind of method that young animal infects with the antagonism lawsonia intracellularis of inoculating, this method comprises throws the antigenic step of lawsonia intracellularis of giving this young animal effective dose.The age of this young animal be preferably one (1) huge or one day big more than.In addition, according on the other hand, the invention provides and a kind ofly be used for inoculation and have or be exposed to anti-lawsonia intracellularis antibody, especially be the method that the young animal of the anti-lawsonia intracellularis antibody of maternal source infects with the antagonism lawsonia intracellularis, this method comprises throws the antigenic step of lawsonia intracellularis of giving this young animal effective dose.The age of this young animal be preferably one (1) huge or one day big more than.

According on the other hand, the invention provides and a kind ofly be used for inoculation and have or be exposed to anti-lawsonia intracellularis antibody, especially be the method that the animal of the anti-lawsonia intracellularis antibody of maternal source infects with the antagonism lawsonia intracellularis, this method comprises throws the antigenic step of lawsonia intracellularis of giving this animal effective dose.

Term " inoculation " (" vaccination " or " vaccinating ") means and (but being not limited to) a kind of comprising animal is thrown the antigenic method of lawsonia intracellularis of giving as used in this article, and wherein this lawsonia intracellularis antigen causes the immunoreation that maybe can cause the antagonism lawsonia intracellularis in this animal body when this animal is given in throwing.

Term " animal " means (but being not limited to) bird as used in this article, fish reaches such as mammals such as cattle, pig, horse or primates.Yet according to a preferred embodiment of the invention, this animal is a pig, is preferably the preceding piglets of wean.

Therefore, according on the other hand, the invention relates to a kind of method that young animal infects with the antagonism lawsonia intracellularis of inoculating, this method comprises from one (1) and hugely begins to throw the antigenic step of the lawsonia intracellularis that gives this young animal effective dose, and wherein this animal is bird, fish or such as mammals such as cattle, pig, horse or primates.This animal is preferably mammal.This animal is pig more preferably.This animal most preferably is the preceding piglets of wean.

Term " young animal " is meant 1 day greatly to 20 the biggest animals as used in this article.Term " young animal " preferably is meant 1 day greatly to 10 the biggest animals.It is big to 9 days greatly that term " young animal " more preferably is meant 1 day, more preferably 1 day big to 8 days greatly, more preferably 1 day big to 7 days greatly, more preferably 1 day big to 6 days greatly, more preferably 1 day big to 5 days greatly, and more preferably 1 day big to 4 days greatly, and more preferably 1 day big to 3 days greatly, more preferably 1 or 2 the biggest animals most preferably are meant 1 the biggest animal.Indivedual implications of term " young animal " (respective meaning) also are meant its age when inoculating animal for the first time with lawsonia intracellularis antigen.

Therefore, another aspect of the present invention is a method of inoculating young animal with the infection of antagonism lawsonia intracellularis about a kind of, this method comprises throws the antigenic step of lawsonia intracellularis give this young animal effective dose, and wherein this animal was accepted inoculation greatly when big to 20 days at 1 day.According to another embodiment, the invention relates to this inoculation method, wherein this animal was accepted inoculation greatly when big to 10 days at 1 day.According to another embodiment, the invention relates to this inoculation method, wherein this animal was accepted inoculation greatly when big to 9 days at 1 day.According to another embodiment, the invention relates to this inoculation method, wherein this animal was accepted inoculation greatly when big to 8 days at 1 day.According to another embodiment, the invention relates to this inoculation method, wherein this animal was accepted inoculation greatly when big to 7 days at 1 day.According to another embodiment, the invention relates to this inoculation method, wherein this animal was accepted inoculation greatly when big to 6 days at 1 day.According to another embodiment, the invention relates to this inoculation method, wherein this animal was accepted inoculation greatly when big to 5 days at 1 day.According to another embodiment, the invention relates to this inoculation method, wherein this animal was accepted inoculation greatly when big to 4 days at 1 day.According to another embodiment, the invention relates to this inoculation method, wherein this animal was accepted inoculation greatly when big to 3 days at 1 day.According to another embodiment, the invention relates to this inoculation method, wherein this animal was accepted inoculation when big at 1 or 2 day.According to another embodiment, the invention relates to this inoculation method, wherein this animal was accepted inoculation when big at 1 day.

Term " have or be exposed to anti-lawsonia intracellularis antibody " means (but being not limited to) to have or is exposed to and be preferably every milliliter of 1:4 at least, more preferably more than the 1:16, more preferably more than the 1:64, more preferably more than the 1:128, more preferably more than the 1:256, more preferably more than the 1:512, and most preferably be the above animal that detects anti-lawsonia intracellularis antigen titration degree of 1:1024.Those anti-lawsonia intracellularis antigen titration degree preferably are can detect and quantized in test as described in example 4 above preferably in the anti-lawsonia intracellularis immunity test of specificity.The anti-lawsonia intracellularis antibody of they is the antibody of maternal source more preferably.

Term " is exposed to anti-lawsonia intracellularis antibody " and means (but being not limited to) and obtains to be preferably every milliliter or every gram nutrient 1:4 at least with for example nutrient such as colostrum or emulsion feeding animals, more preferably more than the 1:16, more preferably more than the 1:64, more preferably more than the 1:128, more preferably more than the 1:256, more preferably more than the 1:512, and most preferably be the above fact that detects anti-lawsonia intracellularis antigen titration degree of 1:1024.

Term " has anti-lawsonia intracellularis antibody " and should mean (but being not limited to) and has in the 1ml of this animal serum and be preferably 1:4 at least, more preferably more than the 1:16, more preferably more than the 1:64, more preferably more than the 1:128, more preferably more than the 1:256, more preferably more than the 1:512, and most preferably be 1:1024 above detect anti-lawsonia intracellularis antigen titration degree.

Therefore, according on the other hand, the invention provides a kind of method that animal infects with the antagonism lawsonia intracellularis that is used to inoculate, this method comprises throws the antigenic step of lawsonia intracellularis of giving this animal effective dose, wherein this animal has or is exposed to and is preferably every milliliter of 1:4 at least, more preferably more than the 1:16, more preferably more than the 1:64, more preferably more than the 1:128, more preferably more than the 1:256, more preferably more than the 1:512, and most preferably be 1:1024 above detect anti-lawsonia intracellularis antigen titration degree.They's antibody is preferably the antibody of maternal source.They's antigen titration degree more preferably is present in those animal bodies in inoculation day.

Therefore, according on the other hand, the invention provides a kind of method that young animal infects with the antagonism lawsonia intracellularis that is used to inoculate, this method comprises throws the antigenic step of lawsonia intracellularis of giving this young animal effective dose, wherein this young animal has or is exposed to and is preferably every milliliter of 1:4 at least, more preferably more than the 1:16, more preferably more than the 1:64, more preferably more than the 1:128, more preferably more than the 1:256, more preferably more than the 1:512, most preferably be the anti-lawsonia intracellularis antigen titration degree that detects more than the 1:1024.They's antibody is preferably the antibody of maternal source.The age of this young animal be preferably 1 day greatly to 20 days big between.The more preferably 1 day age of this young animal greatly to 10 days big between, more preferably 1 day greatly to 9 days big between, more preferably 1 day greatly to 8 days big between, more preferably 1 day greatly to 7 days big between, more preferably 1 day greatly to 6 days big between, more preferably 1 day greatly to 5 days big between, more preferably 1 day greatly to 4 days big between, more preferably 1 day greatly to 3 days big between, more preferably 1 or 2 day is big, most preferably be 1 day big.

Term " effective dose " (" an effective dose ") means (but being not limited to) and gives causing in the antigenic animal of lawsonia intracellularis of this effective dose in throwing and maybe can cause the immunoreactive antigenic amount of this animal as used in this article.

" immunological response or immunoreation " to compositions or vaccine is compositions or vaccine generation cell immune response and/or the antibody-mediated immunoreation to being paid close attention in the host.Therefore, term " initiation maybe can cause immunoreation " means the immunology process in (but being not limited to) host, it is characterized in that this compositions or the cell immune response of vaccine and/or the antibody-mediated immunoreation of host's generation to being paid close attention to.Usually, " immunoreation " include, but is not limited to following active one or many persons: produce or activate specificity and be directed to and be included in compositions or the antigenic antibody in the vaccine, B cell, helper T lymphocyte, suppressor T lymphocyte and/or cytotoxic T cell and/or the yd T cell of being paid close attention to.The host preferably will represent treatment or protective immunity reaction so that strengthen clinical severity to the resistance and/or the reduction disease of new infection.This protective effect will reduce or lack and confirm by the amount of the symptom relevant with host infection as indicated above or seriousness.

Be effective in animal body and cause immunoreation and maybe can cause immunoreactive antigen amount and decide on the composition and the throwing process of giving of vaccine.Usually, when the bacterial antigens that kill were used for vaccine, the every dosage of vaccine contains had an appointment 10 ³To about 10 ⁹The antibacterial of the amount of individual colony-forming units (CFU), preferred every dosage contains has an appointment 10 ⁴To about 10 ⁸The antibacterial of individual (CFU), more preferably every dosage contains has an appointment 10 ⁵To about 10 ⁶Individual (CFU).

Particularly, (for example, the antibacterial separated strain of called after separated strain B3903, ATCC deposit number PTA-4926 when modified lawsonia intracellularis antibacterial alive is used for vaccine; And the antibacterial separated strain of called after separated strain N34NP40wk, ATCC deposit number 55783, the two is described among WO 96/39629 and the WO 05/011731), wait to throw the recommended dose of giving susceptible animal and be preferably about 3.0 TCID ₅₀(tissue culture's 50 3nfective dose terminal point)/agent is to about 6.0 TCID ₅₀/ agent and most preferably be about 4.0 TCID ₅₀/ agent is to about 5.0 TCID ₅₀/ agent.In a preferred embodiment, as by the 50 3nfective dose endpoint dilution assay method (TCID of tissue culture ₅₀) measure, the titer of vaccine is about 4.9TCID ₅₀/ agent.Generally speaking, immunogenic amount will be between 5 and 5000 micrograms and 10 ^2.0With 10 ^9.0TCID ₅₀Between, preferably 10 ^3.0With 10 ^6.0TCID ₅₀Between, more preferably 10 ^4.0With 10 ^5.0TCID ₅₀Between (when using purification of bacterial).

Usually with every dose at least 0.2 μ g antigen, preferably with every dose about 0.2 to about 400 μ g, more preferably with every dose about 0.3 to about 200 μ g, more preferably with every dose about 0.35 to about 100 μ g, more preferably with every dose about 0.4 to about 50 μ g, more preferably with every dose about 0.45 to about 30 μ g, more preferably with every dose about 0.6 to about 15 μ g, more preferably with every dose about 0.75 to about 8 μ g, more preferably with every dose about 1.0 to about 6 μ g and more preferably throw to the antigen intensive amount of about 3.0 μ g and give subunit vaccine with every dose about 1.3.

Term " strengthen property protection " means (but being not limited to) and does not compare with inoculating the control animal group through the inoculation animal groups as used in this article, and one or more clinical symptoms relevant with the lawsonia intracellularis infection (intersect and damage frequency etc.) is in statistically evident reduction in the former.Term " clinical symptoms is in statistically evident reduction " means (but being not limited to) after attacking with the infectiousness lawsonia intracellularis, occurrence frequency at least a clinical symptoms in the inoculation animal groups does not hang down at least 20% than inoculating in the matched group, preferred 30%, more preferably 50%, most preferably 70%.

Term " lawsonia intracellularis " means by people such as C.Gebhart as used in this article, Int ' 1.J.ofSystemic Bacteriology, the 43rd volume, the 3rd phase, people such as 533-538 (1993) and S.McOrist, Int ' 1.J.of Systemic Bacteriology, the 45th volume, the 4th phase, in the born of the same parents that 820-825 (1995) (each document by reference in full be incorporated herein) describes in detail crooked gram negative bacteria and include, but is not limited to WO 96/39629 and WO 05/011731 described in separated strain.In detail, term " lawsonia intracellularis " also means (but being not limited to) and is preserved in American type culture collection (ATCC) according to budapest treaty (Budapest Treaty), 10801 University Boulevard, Manassas, Virginia20110-2209 and ATCC deposit number are that PTA 4926 or ATCC deposit number are 55783 separated strain.Two kinds of separated strains are described in WO 96/39629 and WO 05/011731 respectively.Term " lawsonia intracellularis " also means (but being not limited to) any other lawsonia intracellularis bacterial strain or separated strain, these bacterial strains or separated strain preferably have at least one the immunogenic properties in the lawsonia intracellularis bacterial strain described in WO 96/39629 and the WO 05/011731, especially have according to budapest treaty and be preserved in ATCC, 10801 University Boulevard, Manassas, Virginia 20110-2209 and ATCC deposit number are that PTA 4926 or ATCC deposit number are at least one the immunogenic properties in 55783 the separated strain.

When a kind of bacterial strain or separated strain can be at least be detected in a kind of detection test that also is described in WO 06/01294 with one of anti-lawsonia intracellularis specific antibody described in the WO 06/01294, this bacterial strain or separated strain have the lawsonia intracellularis bacterial strain described in WO 96/39629 and the WO 05/011731, especially with at least one " immunogenic properties " in the separated strain of ATCC deposit number PTA 4926 or 55783 preservations of ATCC deposit number.These antibody preferably are selected from the antibody that is numbered 301:39,287:6,268:29,110:9,113:2 and 268:18.This detection test is preferably the sandwich ELISA described in the

embodiment

2 and 3 of WO06/12949, and antibody 110:9 is used as coupling antibody as capture antibodies and antibody 268:29.All antibody that disclosed among the WO 06/12949 are by the hybridoma manufacturing, these hybridomies are preserved in applied microbiology research center (Centre for Applied Microbiology and Research according to budapest treaty as patent preservation thing, CAMR) and European cell culture preservation center (ECACC), Salisbury, Wiltshire SP4 0JG, UK.Preservation date is on May 11st, 2004.With ECACC deposit number 04092204 preservation hybridoma cell line 110:9 successfully.With ECACC deposit number 04092201 preservation hybridoma cell line 113:2 successfully.With ECACC deposit number 04092202 preservation hybridoma cell line 268:18 successfully.With ECACC deposit number 04092206 preservation hybridoma cell line 269:29 successfully.With ECACC deposit number 04092203 preservation hybridoma cell line 287:6 successfully.With ECACC deposit number 04092205 preservation hybridoma cell line 301:39 successfully.

Term " lawsonia intracellularis antigen " means any compositions that (but being not limited to) comprises at least a antigenic material as used in this article, and this antigen can bring out, stimulates or strengthen the immunoreation of the infection that causes of antagonism lawsonia intracellularis when animal is given in throwing.This lawsonia intracellularis antigen is preferably complete lawsonia intracellularis antibacterial (especially being the deactivation form) (antibacterial that what is called is killed), modified lawsonia intracellularis antibacterial work or attenuation (so-called MLB), any subunit, polypeptide or component or any chimeric vector (at least one immunogenicity aminoacid sequence of each self-contained lawsonia intracellularis) of lawsonia intracellularis.Term " immunogenic protein ", " immunogenic polypeptide " or " immunogenicity aminoacid sequence " are meant and cause immunoreactive any aminoacid sequence that antagonism comprises the pathogen of this immunogenic protein, immunogenic polypeptide or immunogenicity aminoacid sequence in the hosts as used in this article.Particularly, " immunogenic protein " of lawsonia intracellularis, " immunogenic polypeptide " or " immunogenicity aminoacid sequence " mean and are coded in antigenic any aminoacid sequence of throwing the immunological response that causes the antagonism lawsonia intracellularis among the host who gives this " immunogenic protein ", " immunogenic polypeptide " or " immunogenicity aminoacid sequence ".

" immunogenic protein ", " immunogenic polypeptide " or " immunogenicity aminoacid sequence " include, but is not limited to any proteic full length sequence, its analog or its immunogenic fragments as used in this article.Term " immunogenic fragments " means and comprises one or more epi-position, and therefore causes the proteic fragment of the immunological response of the relevant pathogen of antagonism.Can use multiple epitope mapping technology well known in the art to discern these fragments.For example, referring to Epitope Mapping Protocols in Methods inMolecular Biology, the 66th volume (Glenn E.Morris compiles, 1996) Humana Press, Totowa, New Jersey.(its teaching and content are incorporated herein with way of reference.) for example, when these peptides still are attached to these holders, make these peptides and antibody response can measure linear epitope by reaching at synthetic simultaneously a large amount of peptides (these peptides are corresponding to the part of protein molecular) on the solid support.These technology are known in the art and for example be described in United States Patent (USP) the 4th, 708, No. 871; People such as Geysen (1984) Proc.Natl.Acad.Sci.USA 81:3998-4002; Among people such as Geysen (1986) Molec.Immunol.23:709-715.(its teaching and content are incorporated herein with way of reference.) similarly, come easily to determine comformational epitope (conformational epitope) by measuring amino acid whose space conformation (such as by for example X-ray crystal analysis art and two dimensional NMR).For example referring to above-mentioned Epitope MappingProtocol.In this definition, also comprise synthetic antigen, for example multi-epitope (polyepitope), flank epi-position and other reorganization or the synthetic antigen that obtains.For example, referring to people such as Bergmann (1993) Eur.J.Immunol.23:2777-2781; People such as Bergmann (1996), J.Immunol.157:3242-3249; Suhrbier, A. (1997), Immunol.and Cell Biol.75:402-408; People such as Gardner, (1998) 12th World AIDS Conference, Geneva, Switzerland, 28-July 3 June, 1998.(its teaching and content are incorporated herein with way of reference.)

Suitable lawsonia intracellularis antigen includes, but is not limited to they at EP 1219711, US 6,605,696, the antigen described in WO 96/39629, WO 97/20050, WO 00/69903, WO 00/69904, WO 00/69905, WO 00/69906, WO 02/38594, WO 02/26250, WO 03/006665, WO 04/033631, WO 05/026200 and the WO 05/011731.

Therefore, comprise aforesaid any lawsonia intracellularis antigen according to the present invention for the vaccine that uses, it causes the immunoreation that maybe can cause the antagonism lawsonia intracellularis.This vaccine preferably provides the enhancing protection of antagonism lawsonia intracellularis at least.

Therefore, according on the other hand, the invention relates to a kind of method that young animal infects with the antagonism lawsonia intracellularis of inoculating, this method comprises from one (1) and hugely begins to throw the antigenic step of the lawsonia intracellularis that gives this young animal effective dose, and wherein this lawsonia intracellularis antigen is to be selected from the group of being made up of one or more subunit of the modified lawsonia intracellularis antibacterial, the lawsonia intracellularis antibacterial that kills or the lawsonia intracellularis antibacterial that live.This vaccine preferably comprises modified lawsonia intracellularis antibacterial alive.Vaccine is Enterisol more preferably Ileitis B3903 (Boehringer Ingelheim Vetmedica, Inc.).As mentioned above, inoculation preferably at 1 day greatly to 20 days when big, more preferably at 1 day greatly to 10 days when big, more preferably at 1 day greatly to 9 days when big, more preferably at 1 day greatly to 8 days when big, more preferably at 1 day greatly to 7 days when big, more preferably at 1 day greatly to 6 days when big, more preferably at 1 day greatly to 5 days when big, more preferably at 1 day greatly to 4 days when big, more preferably at 1 day greatly to 3 days when big, more preferably at 1 or 2 day when big, and most preferably took place when big at 1 day.

According on the other hand, the invention provides a kind of method that animal infects with the antagonism lawsonia intracellularis that is used to inoculate, this method comprises throws the antigenic step of lawsonia intracellularis of giving this animal effective dose, wherein this animal has or is exposed to and is preferably every milliliter of 1:4 at least, more preferably more than the 1:16, more preferably more than the 1:64, more preferably more than the 1:128, more preferably more than the 1:256, more preferably more than the 1:512, and most preferably be the anti-lawsonia intracellularis antigen titration degree that detects more than the 1:1024, and wherein this lawsonia intracellularis antigen is the modified lawsonia intracellularis antibacterial that is selected from by living, the group that one or more subunit of lawsonia intracellularis antibacterial that kills or lawsonia intracellularis antibacterial is formed.This vaccine preferably comprises modified lawsonia intracellularis antibacterial alive.Vaccine more preferably

Ileitis B3903 (BoehringerIngelheim Vetmedica, Inc.).In addition, they's antibody is preferably the antibody of maternal source.They's antigen titration degree more preferably is present in those animal bodies in inoculation day.

According on the other hand, the invention provides a kind of method that young animal infects with the antagonism lawsonia intracellularis that is used to inoculate, this method comprises throws the antigenic step of lawsonia intracellularis of giving this young animal effective dose, wherein this young animal has or is exposed to and is preferably every milliliter of 1:4 at least, more preferably more than the 1:16, more preferably more than the 1:64, more preferably more than the 1:128,1:256 more preferably, more preferably more than the 1:512, most preferably be the anti-lawsonia intracellularis antigen titration degree that detects more than the 1:1024, and wherein this lawsonia intracellularis antigen is the modified lawsonia intracellularis antibacterial that is selected from by living, the group that one or more subunit of lawsonia intracellularis antibacterial that kills or lawsonia intracellularis antibacterial is formed.This vaccine preferably comprises modified lawsonia intracellularis antibacterial alive.Vaccine more preferably

IleitisB3903 (Boehringer Ingelheim Vetmedica, Inc.).In addition, they's antibody is preferably the antibody of maternal source.They's antigen titration degree more preferably is present in those animal bodies in inoculation day.The age of this young animal be preferably 1 day greatly to 20 days big between.The more preferably 1 day age of this young animal greatly to 10 days big between, more preferably 1 day greatly to 9 days big between, more preferably 1 day greatly to 8 days big between, more preferably 1 day greatly to 7 days big between, more preferably 1 day greatly to 6 days big between, more preferably 1 day greatly to 5 days big between, more preferably 1 day greatly to 4 days big between, more preferably 1 day greatly to 3 days big between, more preferably 1 or 2 day is big, most preferably be 1 day big.

According on the other hand, the invention relates to a kind of method that young animal infects with the antagonism lawsonia intracellularis of inoculating, the method includes the steps of: from one (1) huge begin to throw give the about 3.0TCID of this young animal ₅₀To about 6.0 TCID ₅₀The modified lawsonia intracellularis antibacterial of work of dosage.Preferably, this antibacterial is a vaccine

Ileitis B3903 (Boehringer Ingelheim Vetmedica, Inc.) in included antibacterial.As mentioned above, inoculation preferably at 1 day greatly to 20 days when big, more preferably at 1 day greatly to 10 days when big, more preferably at 1 day greatly to 9 days when big, more preferably at 1 day greatly to 8 days when big, more preferably at 1 day greatly to 7 days when big, more preferably at 1 day greatly to 6 days when big, more preferably at 1 day greatly to 5 days when big, more preferably at 1 day greatly to 4 days when big, more preferably at 1 day greatly to 3 days when big, more preferably at 1 or 2 day when big, and most preferably took place when big at 1 day.

Therefore, according on the other hand, the invention relates to a kind of method that animal infects with the antagonism lawsonia intracellularis of inoculating, the method includes the steps of: throw and give about 3.0 TCID of this animal ₅₀To about 6.0TCID ₅₀The modified lawsonia intracellularis antibacterial of work of dosage, wherein this animal has or is exposed to and is preferably every milliliter of 1:4 at least, more preferably more than the 1:16, more preferably more than the 1:64, more preferably more than the 1:128,1:256 more preferably, more preferably more than the 1:512, and most preferably be 1:1024 above detect anti-lawsonia intracellularis antigen titration degree.This antibacterial is preferably vaccine IleitisB3903 (Boehringer Ingelheim Vetmedica, Inc.) in included antibacterial.

Therefore, according on the other hand, the invention relates to a kind of method that young animal infects with the antagonism lawsonia intracellularis of inoculating, the method includes the steps of: throw and give the about 3.0TCID of this young animal ₅₀To about 6.0TCID ₅₀The modified lawsonia intracellularis antibacterial of work of dosage, wherein this young animal has or is exposed to and is preferably every milliliter of 1:4 at least, more preferably more than the 1:16, more preferably more than the 1:64, more preferably more than the 1:128,1:256 more preferably, more preferably more than the 1:512, and most preferably be 1:1024 above detect anti-lawsonia intracellularis antigen titration degree.This antibacterial is preferably vaccine

According on the other hand, the invention relates to a kind of method that young animal infects with the antagonism lawsonia intracellularis of inoculating, this method comprises from one (1) and hugely begins to throw the antigenic step of the lawsonia intracellularis that gives this young animal effective dose, and wherein this young animal is negative to lawsonia intracellularis and anti-lawsonia intracellularis maternal antibody.As mentioned above, inoculation preferably at 1 day greatly to 20 days when big, more preferably at 1 day greatly to 10 days when big, more preferably at 1 day greatly to 9 days when big, more preferably at 1 day greatly to 8 days when big, more preferably at 1 day greatly to 7 days when big, more preferably at 1 day greatly to 6 days when big, more preferably at 1 day greatly to 5 days when big, more preferably at 1 day greatly to 4 days when big, more preferably at 1 day greatly to 3 days when big, more preferably at 1 or 2 day when big, and most preferably took place when big at 1 day.

According on the other hand, the present invention also is about the antigenic novel medical application of a kind of lawsonia intracellularis, it is used for preparation and begins medicament (being preferably vaccine combination) that the young animal inoculation is infected with the antagonism lawsonia intracellularis when huge one (1), wherein this young animal be one (1) huge or one day big when above through this lawsonia intracellularis antigen inoculation of effective dose.This inoculation preferably at 1 day greatly to 20 days when big, more preferably at 1 day greatly to 10 days when big, more preferably at 1 day greatly to 9 days when big, more preferably at 1 day greatly to 8 days when big, more preferably at 1 day greatly to 7 days when big, more preferably at 1 day greatly to 6 days when big, more preferably at 1 day greatly to 5 days when big, more preferably at 1 day greatly to 4 days when big, more preferably at 1 day greatly to 3 days when big, more preferably at 1 or 2 day when big, and most preferably took place when big at 1 day.

According on the other hand, the present invention also is about the antigenic new medical application of a kind of lawsonia intracellularis, it is used to prepare the medicament (being preferably vaccine combination) that the inoculation animal infects with the antagonism lawsonia intracellularis, wherein this animal has or is exposed to and is preferably every milliliter of 1:4 at least, more preferably more than the 1:16, more preferably more than the 1:64, more preferably more than the 1:128,1:256 more preferably, more preferably more than the 1:512, and most preferably be 1:1024 above detect anti-lawsonia intracellularis antigen titration degree.

According on the other hand, the present invention also is about the antigenic novel medical application of a kind of lawsonia intracellularis, it is used to prepare the medicament (being preferably vaccine combination) that the inoculation young animal infects with the antagonism lawsonia intracellularis, wherein this young animal has or is exposed to and is preferably every milliliter of 1:4 at least, more preferably more than the 1:16, more preferably more than the 1:64, more preferably more than the 1:128,1:256 more preferably, more preferably more than the 1:512, and most preferably be 1:1024 above detect anti-lawsonia intracellularis antigen titration degree.This inoculation preferably at 1 day greatly to 20 days when big, more preferably at 1 day greatly to 10 days when big, more preferably at 1 day greatly to 9 days when big, more preferably at 1 day greatly to 8 days when big, more preferably at 1 day greatly to 7 days when big, more preferably at 1 day greatly to 6 days when big, more preferably at 1 day greatly to 5 days when big, more preferably at 1 day greatly to 4 days when big, more preferably at 1 day greatly to 3 days when big, more preferably at 1 or 2 day when big, and most preferably took place when big at 1 day.

According to above-mentioned these medical applications on the other hand, this lawsonia intracellularis antigen is to be selected from the group of being made up of one or more subunit of the modified lawsonia intracellularis antibacterial, the lawsonia intracellularis antibacterial that kills or the lawsonia intracellularis antibacterial that live.This lawsonia intracellularis antigen is preferably modified lawsonia intracellularis antibacterial alive.More preferably, these animals are through about 3.0 TCID ₅₀To about 6.0 TCID ₅₀The modified lawsonia intracellularis antibacterial of work of dosage throw and give.

The preparation that comprises the antigenic vaccine combination of lawsonia intracellularis is a prior art and known to those skilled in the art.For example, those who familiarize themselves with the technology additional component that can determine can to comprise in the said composition (also referring to Remington ' s Pharmaceutical Sciences. (1990). the 18th edition, Mack Publ., Easton).The professional can use known injectable, the last acceptable sterile solution of physiology.Be suitable for non-ly through enteral administration or dabbling promptly using for (ready-to-use) solution for preparation, isotonic aqueous solution can easily obtain such as saline or corresponding plasma protein solution.These vaccine combinations can the lyophilizing attitude or the drying agent form exist, for example, as assembly test kit (kits of parts), its can be before using under aseptic condition directly with known Injectable solution reconstruction.

In addition, immunogenicity of the present invention and vaccine combination can comprise the acceptable supporting agent of one or more veterinary." the acceptable supporting agent of veterinary " comprises any solvent, disperse medium, coating materials, adjuvant, stabilizing agent, diluent, antiseptic, antibacterial agent and antifungal, isotonic agent, absorption delayed-action activator and analog thereof as used in this article.

" diluent " can comprise water, saline (saline), glucose, ethanol, glycerol and analog thereof.Isotonic agent can comprise sodium chloride, glucose, mannitol, Sorbitol and lactose etc.Stabilizing agent comprises the alkali salt of albumin and ethylenediaminetetraacetic acid etc.

" adjuvant " can comprise aluminium hydroxide and aluminum sulfate as used herein, Saponin is Quil A, QS-21 (Cambridge Biotech Inc. for example, Cambridge MA), GPI-0100 (GalenicaPharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsion, O/w emulsion, water-in-oil-in-water compositions.Emulsion can be especially based on following material: light liquid attitude paraffin oil (European Pharmacopoeia type); Isoprenoid oil such as squalane or Squalene; By alkene, especially be isobutene. or the oligomeric oil that obtains of decene; Contain the acids of straight chained alkyl or the esters of alcohols, more in particular be vegetable oil, ethyl oleate, propylene glycol two (caprylic/capric) ester, three (caprylic/capric) glyceride or Rikemal PO 200s; The esters of branched chain fatty acid or alcohol especially is the isostearic acid esters.Oil and emulsifier combination are used to form emulsion.These emulsifying agents are preferably the agent of nonionic show activity, especially be according to circumstances through the ester of sorbitan ester, mannide ester (for example anhydrous mannitol oleate), glycol ester, polyglycerin ester, propylene glycol ester and oleic acid, isostearic acid, ricinoleic acid or the hydroxy stearic acid of ethoxylation, and polyoxypropylene-polyoxyethylene block copolymer (be the Pluronic product especially, be in particular L121).Referring to people such as Hunter, The Theory and Practical Application of Adjuvants (Stewart-Tull, D.E.S. compiles).John Wiley and Sons, NY, people such as 51-94 page or leaf (1995) and Todd, Vaccine 15:564-570 (1997).(its teaching and content are incorporated this paper into way of reference in view of the above.)

For example, might use " VaccineDesign; The Subunit and Adjuvant Approach " that compile by M.Powell and M.Newman, Plenum Press, the 147th page of 1995 the above SPT emulsion and the 183rd page of same book the above emulsion MF59.(its teaching and content are incorporated this paper into way of reference in view of the above.)

Another example of adjuvant is the chemical compound that is selected from the copolymer of the polymer of acrylic or methacrylic acid and maleic anhydride and thiazolinyl derivant.Favourable adjuvant compound is the polymer of acrylic or methacrylic acid of connection, especially with the polymer of the crosslinked acrylic or methacrylic acid of the polyalkenyl ether of saccharide or polyalcohols.These chemical compounds known by term carbomer (carbomer) (Phameuropa the 8th volume, the 2nd phase, in June, 1996).Being familiar with this operator also can be with reference to United States Patent (USP) the 2nd, 909, No. 462, it is described this type of and has at least 3 hydroxyls, be preferably the acrylate copolymer of the polyhydroxylated compound crosslink that is no more than 8 hydroxyls, the hydrogen atom of at least 3 hydroxyls is had the unsaturated aliphatic group displacement of at least 2 carbon atoms.Preferred group contains the group of 2 to 4 carbon atoms for they, and for example vinyl, pi-allyl and other alkene are unsaturated group.Itself can contain these unsaturated groups such as other substituent groups such as methyl.With carbopol (Carbopol) is that (BF Goodrich, Ohio USA) is particularly suitable for the product sold of title.Itself and allyl sucrose or crosslinked with pi-allyl isoamyl tetrol.Wherein, can mention Carbopol 974P, 934P and 971P.Most preferably use Carbopol 971P.In the copolymer of maleic anhydride and thiazolinyl derivant, copolymer EMA (Monsanto) is arranged, it is the copolymer of maleic anhydride and ethylene.The dissolving of these polymer in water produces acid solution, and it will be neutralized, and preferably is neutralized to the physiology pH value to produce assist agent solution, incorporates immunogenic composition, immune composition or vaccine combination itself again in this assist agent solution into.

Suitable in addition adjuvant includes, but is not limited to RIBI adjuvant system (Ribi Inc.), block copolymer (CytRx; Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, avridine (Avridine) lipid-amine adjuvant, from heat labile enterotoxin (reorganization or other modes), cholera toxin, IMS 1314 or the Romurtide etc. of escherichia coli (E.coli).

Preferably with the amount interpolation adjuvant of the about 100 μ g of every dosage to about 10mg.More preferably with the amount interpolation adjuvant of the about 100 μ g of every dosage to about 10mg.More preferably with the amount interpolation adjuvant of the about 500 μ g of every dosage to about 5mg.More preferably with the amount interpolation adjuvant of the about 750 μ g of every dosage to about 2.5mg.Most preferably add adjuvant with the amount of the about 1mg of every dosage.

Vaccine combination can further comprise one or more other immunomodulators, such as interleukin, interferon or other cytokines.Vaccine combination also can comprise Geneticin (Gentamicin) and thimerosal (Merthiolate).Can easily determine to be applicable to the adjuvant in the context of the present invention and the amount and the concentration of additive though have the knack of the skilled worker, the present invention has been contained and has been comprised about 50 μ g to about 2000 μ g adjuvants and be preferably the compositions of about 250 μ g adjuvants in every milliliter of vaccine combination dosage.In another preferred embodiment, the present invention is contained and is comprised about 1 μ g/ml to the antibiotic of about 60 μ g/ml and more preferably less than the antibiotic vaccine combination of about 30 μ g/ml.

With any conventional approaches, most preferably gavage (oral drench) and vaccine is thrown given animal via per os, be preferably mammal and pig more preferably.Waiting to throw the dosage that gives will decide on concrete case, but under any circumstance, it is to be enough to bring out the protection antibody of antagonism ileitis or cell-mediated immunoreactive amount.

Usually vaccine of the present invention is thrown with one or more dosage and given susceptible animal, the preferred throwing given year young piglet and/or had anti-lawsonia intracellularis antibody or be exposed to the piglets of anti-lawsonia intracellularis antibody.Can after initial inoculation, give vaccine alive or that kill with 2 to 4

weekly intervals

1 or 2 throwings.For attenuated live vaccine, potion is preferred.As mentioned above, for the first time or single throwing give preferably at 1 day greatly to 20 days when big, more preferably at 1 day greatly to 10 days when big, more preferably at 1 day greatly to 9 days when big, more preferably at 1 day greatly to 8 days when big, more preferably at 1 day greatly to 7 days when big, more preferably at 1 day greatly to 6 days when big, more preferably at 1 day greatly to 5 days when big, more preferably at 1 day greatly to 4 days when big, more preferably at 1 day greatly to 3 days when big, more preferably at 1 or 2 day when big, and most preferably carried out when big at 1 day.

As if needing or must throwing for the second time and give, then after vaccine is given in throwing for the first time, carry out in about 1 to about 4 weeks throwing the second time giving.According on the other hand, throw in any previous inoculation and to inoculate with 3 to 12 months interval after giving.Preferably carrying out the throwing of vaccine dose subsequently with 6 months to 1 year interval gives.In aspect another is preferred, the animal that inoculated before about 2 to 3 ages in week should inoculate.Preferably carrying out the throwing of vaccine dose subsequently with the interval in 1 year gives.

In following examples, further describe the present invention, provide following examples only for illustrative purposes, should not be construed as have restricted.In fact, other variants of the present invention should be that those who familiarize themselves with the technology is apparent.

Open case of all that quoted and patent are to incorporate in full by reference herein.

Embodiment

Embodiment 1

In from the colostrum of sow (giving birth to more than 1 nest) and first farrowing sow (first nest) and samples of latex, detect the specific antibody of lawsonia intracellularis tool

Materials and methods

After farrowing, obtain the colostrum sample from 25 sows and 25 first farrowing sows (gilt) in the 24h.In week age of sucking first and second week obtained samples of latex from identical pig.4 ℃ twice with sample with the fats portion of the centrifugal 10min of 2000g with separating emulsions.The water sample of sample partly is stored under-20 ℃ until analysis.In IFAT, after the initial dilution factor of 1:20 begins continuous 2 times of dilutions, detect the specific antibody of anti-lawsonia intracellularis.Remove for each sample, the anti-pig IgG of FITC labelling, IgM and IgA antibody are used to detect outside the inhomogeneity Ig, as described among the embodiment 5 and other places, carry out IFAT.Obtain parallel blood sample in back 24 hours in farrowing from sow and first farrowing sow.According to the indication of manufacturer with

Ileitis ELISA checks these samples.

The result

Be summarized in the result of antibody test in the colostrum sample among Fig. 1.This figure is illustrated among the IFAT sample number that obtains positive findings for the dilution IgG of difference, IgM and IgA.The colostrum sample is not negative in all checks.Only a colostrum sample and the colostrum sample from first farrowing sow from sow is the IgG feminine gender, and one from the sample of first farrowing sow in and three samples from sow do not detect specific IgM.Eight negative in 25 samples that first farrowing sow and sow obtain respectively to the specific IgA score of lawsonia intracellularis tool.In the samples of latex that obtains in week age of sucking second, only two IgG from the sample of sow obtain the titer of 1:20, and an IgA score from the sample of first farrowing sow positive (1:20).In the 3rd week of age of sucking, obtain the IgG positive findings at the dilution factor of 1:20 from the sample of a sow and three first farrowing sows.Every other sample from age of sucking second week and the 3rd week is measured as IgG, IgM and IgA feminine gender.All blood samples of taking from sow after farrowing in the 24h exist

Positive among the Ileitis ELISA, and 2 samples obtain negative findings in 25 samples that first farrowing sow obtains.From no obvious dependency between the result of blood sample and the result from the colostrum sample.Can sum up,, exist the specific IgG of lawsonia intracellularis tool, IgM and IgA antibody in first Ruzhong from sow and first farrowing sow.Yet, in the samples of latex that a week is obtained after farrowing, only in 6% sample, can detect the lawsonia intracellularis specific antibody of low titer.

List of references

1.McOrist, people such as S. (2003) Pig J.51,26-35

2.Collins, people such as A.M. (2001) Allen D.Leman Swine, Conference

3.Holyoake, people such as P.K. (1994) J Clin Microbiol 32,1980-1985, Kruse, P.E. (1983) Ann.Rech.Vet.14,349-353

4.Bollwein，J.(2004)Doctoral?thesis，LMU，Munich

5.Knittel, people such as J.P. (1998) AJVR 59,722-726

Embodiment 2

In the direct influence of emulsion antibody during gastrointestinal tract to lawsonia intracellularis

Materials and methods

After birth, obtain the colostrum sample from sow and first farrowing sow in the 24h.In week age of sucking first and second week obtained the sow samples of latex.4 ℃ twice with sample with the fats portion of the centrifugal 10min of 2000g with separating emulsions.The water sample of sample partly is stored in-20 ℃ until analysis.In IFAT, after the initial dilution factor of 1:20 begins continuous 2 times of dilutions, detect the specific antibody of anti-lawsonia intracellularis.Remove for each sample, the anti-pig IgG of FITC labelling, IgM and IgA antibody are used to detect outside the inhomogeneity Ig, as described among the embodiment 5 and other places (5), carry out IFAT.Select to have the samples of latex of different I g content based on IFAT result.In colostrum or sow samples of latex, cultivate the lawsonia intracellularis culture with different dilution factors in room temperature.With twice of each sample test.(0 hour) reaches the 50 3nfective dose (TCID of tissue culture that measured lawsonia intracellularis after 4 hours in room temperature when cultivating beginning ₅₀).The undiluted sample of culture is served as the contrast of tested performance.Each TCID that surveys ₅₀During value, sample 20 times is homogenized by No. 20 pins.The McCoy cell is at 75cm ²The monolayer of using DMEM/HAM ' s F12 culture medium culturing to 100% to cover with in the tissue culture flasks with the cell trypsinization, is sub-packed on four 96 hole microdroplet plates.With 10 ^-1To 10 ^-7The sample of serial dilution respectively inoculates six inferior on the fresh McCoy cell.37 ℃ little have a liking under the oxygen condition cultivate 6 days after, by ice-cold acetone/methanol (50:50, v/v) fixed cell.By using the anti-Lawson's bacteria antibody of specific monoclonal and carrying out labelling with the anti-mouse antibodies of FITC labelling subsequently, detect the growth of lawsonia intracellularis.To contain one or more each hole and be judged to the positive with the McCoy cell of 5 or more fluorescence lawsonia intracellularis.Obtain TCID by using according to the 50% terminal point formula of Spearmann and Karber (6) ₅₀

The result

General introduction is from TCID among Fig. 1 ₅₀The result of test and IgG, IgA and the IgM titer of 8 colostrums (1-5 number) and emulsion (6-8 number) sample.In all experiments, the control sample of culture obtains desired TCID ₅₀Value.During room temperature was cultivated 4 hours, none took place the substantial variations of lawsonia intracellularis titer in described test.Therefore, so shown in the research, as if during gastrointestinal tract, maternal antibody and sow emulsion are not effective in the deactivation lawsonia intracellularis.

List of references

1.van Aken; N. wait people (2002) Proc.17 ^ThIPVS, Ames, Iowa, USA

2.Kruse，P.E.(1983)Ann.Rech.Vet.14，349-353

3.Collins, people such as A.M. (2001) Allen D.Leman Swine, Conference

4.Holyoake, people such as P.K. (1994) J Clin Microbiol 32, the 1980-1985 pages or leaves

5.Knittel, people such as J.P. (1998) AJVR 59,722-726.

6.Karber，G.(1931)Arch.exp.Path.Pharma.162，480

7.Mauch, C.H.Y. and G.Bilkei (2004) Vet.Rec.155,532

Embodiment 3

The anti-lawsonia intracellularis of inoculation one the biggest piglets infects effect

Materials and methods

This research relates to 1 the biggest lawsonia intracellularis experimental group negative and anti-Lawson's starter body negative antibody suckling piglets by three and forms.Research the 0th day, 20 pigletss of the 1st group (by inoculating) were separately accepted oral dose

Ileitis (vaccine separated strain B3903) (by specification (3) indication).20 placebo that pigletss acceptance is made up of growth medium of the 2nd group (contrast).The piglets that makes all groups is in research wean in the 20th day.At the 21st day, the piglets of the 1st group and the 2nd group was accepted via tube feed (gavage) to contain 3.5 * 10 ⁹The small intestinal homogenate of individual toxicity lawsonia intracellularis 2688/2685.10 pigs of the 3rd group (strict contrast) are not accepted vaccine, placebo or germ attack any period during studying.At the 42nd day, the people is genuine to be made the pig euthanasia of all groups and carries out obduction.Main efficacy parameter comprises special naked eyes and microcosmic infringement by the caused ileum of lawsonia intracellularis, caecum and colon.Minor parameter comprises clinical health state (behavior, body condition and feces hardness), average every day weight increase amount, feces discharge of bacteria (PCR) and seroconversion (IFAT) (1).

The result

When obduction (the 42nd day), compare with nonvaccinated contrast pig (ileum=1.16, caecum=0.42, colon=0.16), be subjected to inoculator's (the 1st group) to have significantly (p＜0.0003) less average gross lesion score (ileum (0.21), caecum (0.0) and colon (0.0)).With compare remarkable (p＜0.0001) higher (8.89cm) of small intestinal infringement average total length (ileum, caecum and colon) in matched group through inoculation pig (1.42cm).The average naked eyes intestinal of the 3rd group (strict matched group) damages to such an extent that be divided into 0.22, is considered to normal.

With compare remarkable (p＜0.02) higher (2.47) of the average microcosmic of the Lawson bacterium specificity of ileum infringement score in matched group through inoculation pig (0.53).In addition, and compare through inoculation group, total microcosmic infringement score (p＜0.006) and the positive pig percentage ratio of IHC (p＜0.0001) are significantly higher in matched group.Aspect caecum and colon, matched group has than through slightly high any average microcosmic infringement score and the positive animal percentage ratio of IHC (p〉0.05) of inoculation group.(p＜0.05) significantly and is compared through inoculation group, and the more contrast pigs in germ attack (the 35th day) 2 week back are the PCR positive.In research last day (the 42nd day), to compare through the pig (3/19) of inoculation group with they, the pig of matched group (8/19) is discharged slightly more lawsonia intracellularis (p〉0.05).During studying between the 1st group and the 2nd group average every day the weight increase amount or average clinical score do not have significantly (p〉0.05) difference.Generally speaking, main efficacy parameter (naked eyes and microcosmic small intestinal infringement progress) shows, when to 1 day big, maternal antibody is negative, when the piglets of not contacted Lawson bacterium is thrown and gives, single oral dose Ileitis is effectively to the mithridatism germ attack.In this research, the effectiveness that potential non-Lawson bacterium specificity lactogenic characteristic does not disturb or hinder vaccine that the mithridatism lawsonia intracellularis is attacked in colostrum and the emulsion between age of sucking.

Table 1. lawsonia intracellularis feces excreter's ratio.

^A.bSame letter represents not have significant difference (p＜0.05).

^ΦThe analysis that do not take statistics of strict matched group.

^*Each group has a pig because the adverse health state that has nothing to do with lawsonia intracellularis is removed.

List of references

1.McOrist Deng people (1993) Infection and Immunity 61:4286-4292

2.Knittel Deng people (1998) Am J Vet Res 59:722-726.

3.Kroll Deng people (2004) Am J Vet Res 65:559-565

4.Mauch and Bilkei (2004) Vet Rec 155:532

5.Marsteller Deng people (2003) .Swine Health Prod 11:127-130

6.Stege Deng people (2004) .Vet Micro 104:197-206

Embodiment 4

Be used to detect and quantize the IFAT of anti-lawsonia intracellularis antibody

Materials and methods:

Before research beginning, the negative sow of 16 health, pregnancy and lawsonia intracellularis is divided into 2 groups at random: 8 are hyperimmune group (A group), and 8 are given matched group (B group) for the placebo throwing.Inverse is the 55th, 35 and 14 day before farrowing, and each sow of A group is with commercially available

Ileitis gavages by direct per os and carries out hyperimmune.Throw with the method and to give vaccine dose and relate to and use 10ml aseptic plastic syringe that vaccine is applied to the rear portion, oral cavity.Matched group (B group) is accepted isodose by the placebo that the McCoy cell is formed that do not infect that is suspended in the growth medium.Make A group farrowing sow hyperimmune in the hope of before farrowing, inducing high-caliber maternal immunity power.Make each farrowing sow group stay in a plurality of chambers that separate (to avoid cross-contamination) but be in the same terms (temperature, ventilation and swinery (pen) size).The sow of each chamber is closed in identical swinery.

Become pregnant and the date unanimity of farrowing although the research person makes great efforts to make, sow is also not all farrowing on the same day.Truth is, during farrowing occurred in 10 days.For prevent change between the swinery in a plurality of inoculations day and germ attack day so that this research excessive, the average date of farrowing between the farro wing period is defined as studying the 0th day.Therefore, when inoculation (the 21st day) pig be that 3 week ± 5 are day big.

At the 21st day, choose 100 health, small weaning pigs and be divided into six processed group at random by nest (litter).Residence restriction and condition are similar to above-mentioned sow group.Divide the to the 1st to 3 group at random with the piglets that hyperimmune sow (A group) is given birth to, and after this be called " hyperimmune source " piglets in the research.To contrast the piglets that sow (B group) given birth to and divide the to the 4th to 6 group at random, and after this be called " placebo source " piglets in the research.The 1st group and the 4th group (being respectively 20 pig/groups) gavage by direct per os and give single 2ml dosage

Ileitis.The 2nd group and the 5th group (being respectively 20 pig/groups) give isodose placebo (the not McCoy cell+culture medium of Gan Raning).The 3rd group and the 6th group (10 pig/groups) are " strict negative control group ", and it does not accept vaccine or placebo treatment during this research, and is not subjected to germ attack.Behind weaning period the 22nd day, the people is genuine makes sow euthanasia and carry out obduction to be used to assess because the small intestinal infringement progress that PE causes.

At the 42nd day of research, accept every dosage 1 * 10 via the gastric tube feed for the 1st, 2,4 and 5 group ^7.3TCID ₅₀Allos toxicity pure culture lawsonia intracellularis separated strain N101494.After 21 days of germ attack, check clinical symptoms, diarrhoea, behavior and the body condition relevant with PE of all pigletss every day, (1=is clinical normal to provide 1 to 4 grade scoring according to the order of severity; The 4=serious disease).Research the 21st, 42 and 63 day pig is weighed to calculate average every day of the weight increase amount of every group of pig.Calculate average weight increase amount every day (ADWG) to analyze and the relevant treatment effect of pig normal growth performance.Pig is being weighed at first with the group average weight radix before obtaining to accept vaccine or placebo treatment.Find all group sizes evenly (difference＜0.63 kilogram/pig).The first crucial ADWG assessment betides the 21st day (inoculation) to the 42nd day (germ attack) period between group, to measure the immediate effect of vaccination or placebo inoculation.Be the 42nd day to 63 days (obduction) second assessment period, to measure the effect of attacking with toxicity pure culture lawsonia intracellularis.

Research the 63rd day, the people is genuine to be made all pig euthanasia and carries out obduction to be used to assess the small intestinal infringement progress that causes owing to PE.

The result

Maternal antibody detects

A group (hyperimmune) sow detects lawsonia intracellularis specific IgG, IgA and IgM antibody at serum and first Ruzhong between the farro wing period.During farrowing, 63% A group sow (5/8 sows) antagonism Lawson bacterium IgG antibody is the serum antibody positive, and 0% sow (0/8) is positive in the B group (contrast).In addition, the living piglets of A group sow institute only detects serum IgG antibody when farrowing day (the 0th day) to 5 ages in week (the 28th day), see Fig. 1.A group sow has 50%, 75% and 12.5% to detect anti-Lawson bacterium IgG, IgA and IgM in first Ruzhong respectively.The first Ruzhong average antibody concentration of A group sow is 1:14 (IgG, scope=1 is to 1:64), 1:10 (IgA, scope=1 is to 1:32) and 1:4 (IgM, scope=1 is to 1:4).The sow of B group does not just have any detectable anti-Lawson bacterium IgM or IgG antibody in the Ruzhong at it during this period.A pig in the B group sow is the IgA positive in the titer of 1:16.

The parent protection

Nonvaccinated contrast pig in relatively the 2nd group (A group sow institute live pig) and the 5th group (B group sow institute live pig) is to assess the potential parent protective effect that the mithridatism lawsonia intracellularis is exposed through the hyperimmune sow of vaccine (A organizes sow).

The average naked eyes of all groups of general introduction and microcosmic infringement score in the table 1.Compare with the 2nd group of pig (27.5%), Lawson bacterium special damage percentage ratio is higher in ileum of the 5th group of pig (77%) and the colon.At the 63rd day of research, to compare with the 5th group, average gross lesion score of the ileum of the 2nd group of pig and ileum and the average microcosmic of colon (IHC) infringement score all remarkable (p＜0.05) are lower.

, in arbitrary group feces, do not detect lawsonia intracellularis and discharge to the 42nd day (germ attack) in the 21st day (inoculation) studying, see Fig. 3 by PCR.The the 2nd and the 5th group feces was the PCR positive since the 49th day, and had the pig of discharging lawsonia intracellularis during this period of 5-25% and 15-72% to keep positive in the 63rd day (research stops) respectively.At the 63rd day of research, compare with the 5th group (72%), detect significantly (p＜0.05) less feces discharge (25%) by PCR in the 2nd group.

Research the 63rd day, compare with the 2nd group (25%), find the higher positive percentage ratio (45%) of PCR of organizing in the ileum of the 5th group of pig.The 2nd group of pig is lawsonia intracellularis PCR feminine gender in tonsil, mesenteric lymph node and colon.The 3.4th joint is described as mentioned, observes the different PCR positives in the colon of the 5th group of pig and mesentery lymphoid tissue.

The comparison of average weight recruitment between all test groups of general introduction in the table 2.The 21st day (inoculation), the 2nd group with the 5th group between average initial body weight consistent, pig heavily is respectively 6.35 and 6.10 kilograms/pig.The 21st day (inoculation) to the 42nd day (germ attack), ADWG did not have significant difference between the 2nd group (0.40 kilogram/pig) and the 5th group (0.41 kilogram/pig).Yet,, comparing during 21 days assessment phases of the 63rd day (research stop) in the 42nd day (germ attack) of research with the 5th group of pig (0.40 kilogram/pig), the ADWG of the 2nd group of pig (0.46 kilogram/pig) significantly (p＜0.05) is higher.

Efficacy of vaccines in the hyperimmune pig

With in the 1st group with

The pig of Ileitis inoculation with the 2nd group in nonvaccinated contrast pig compare, to confirm the inoculation in the time of can realizing facing maternal immunity by the protective immunity of resisting PE after the assessment pig inoculation in 3 ages in week.Pig in two groups hyperimmune sow of vaccine (A organizes sow) of all hanging oneself.In addition, analyze the main and secondary efficacy parameter between the pig that vaccine is handled of the 1st group and the 4th group, to judge, whether the effect that vaccine is attacked the mithridatism lawsonia intracellularis is similar.

The average naked eyes of all groups of general introduction and microcosmic infringement score in the table 1.Compare with the 1st group of pig (12.5%), Lawson bacterium special damage percentage ratio is higher in ileum of the 2nd group of pig (27.5%) and the colon.At the 63rd day of research, to compare with the 2nd group, the average gross lesion score (ileum) of the 1st group of pig significantly (p＜0.05) is lower, and average microcosmic (IHC) infringement score (ileum and colon) numerical value is lower.In addition, in the pig (the 1st group and the 4th group) of accepting inoculation, there is not significant difference between average naked eyes and microcosmic infringement score or the infringement order of severity.

During the 21st day (inoculation) studying was to the 35th day, in the feces of arbitrary group (1 or 2 group), do not detect lawsonia intracellularis and discharge by PCR, see Fig. 3.The feces of the 1st group of pig is the PCR positive since the 42nd day (germ attack), and the pig of discharging lawsonia intracellularis during this period has 11-16% to keep positive in the 63rd day (research stops).The feces of the 2nd group of pig was the PCR positive since the 49th day, and the pig of discharging lawsonia intracellularis during this period has 5-25% to keep positive in the 63rd day (research stops).The 4th group of pig just becomes the Lawson bacterium DNA PCR positive up to the 42nd day (germ attack), and the positive is retained to the 63rd day (research stop), during discharge person's ratio be reduced to 5% from 25%.Lawsonia intracellularis feces excreter ratio does not have the sign of significant difference between the 1st group and the 2nd group, the 1st group and the 4th group during studying.

In the 63rd day (research stop) of research, compare with the 1st group (20%), find the slightly high positive percentage ratio (25%) of PCR of organizing in the ileum of the 2nd group of pig.When research stops, find that the 4th group the positive percentage ratio of PCR (5%) of organizing compares the 1st group and the 2nd group low.The positive no significant difference of PCR between the 1st group and the 2nd group, the 1st group and the 4th group.All three groups of pigs are the PCR feminine gender to lawsonia intracellularis in tonsil, mesenteric lymph node and colon.

The comparison of average weight recruitment between all test groups of general introduction in the table 2.The 21st day (inoculation), the 1st group with the 2nd group between average initial body weight consistent, pig heavily is respectively 6.53 and 6.35 kilograms/pig.The 21st day, the 1st group with the 4th group between also observe consistent average weight (being respectively 6.53 kilograms/pig and 6.44 kilograms/pig).The 21st day (inoculation) to the 42nd day (germ attack), and ADWG does not have significant difference between the 1st group (0.40 kilogram/pig) and the 2nd group (0.41 kilogram/pig) or between the 1st group and the 4th group (0.44 kilogram/pig).The 42nd day (germ attack) during 21 days assessment phases of the 63rd day (research stop), there were significant differences (p＜0.05) for the ADWG of the 1st group of pig (0.45 kilogram/pig) and the 4th group of pig (0.51 kilogram/pig).At the 21st day to the 63rd day of this research, ADWG did not have significant difference between the 1st group and the 2nd group.

Embodiment 5

Detect and quantize anti-lawsonia intracellularis antibody with IFAT

By Immunofluorescent Antibody Test (IFAT), use be fixed on the 96 hole polystyrene microdroplet plates Lawson bacterium holoantigen and at the FITC traget antibody of pig IgG people 1998 such as () knittel, test is from the existence of the IgG antibody of antagonism lawsonia intracellularis in the blood serum sample of the blood of each sow and pig.By the FITC traget antibody slightly modified IFA test of using at pig IgM and IgA.This modified method is used at each sow colostrum detection these antibody except that IgG, to be used to measure the concentration of different Lawson bacterium specific immune globulin.Do 2 times of dilutions with colostrum is duplicate in PBS, and shift (100 μ l/ hole) and wrap as mentioned above by in 96 well culture plates of Lawson bacterium to two groups.These plates were cultivated 30 minutes at 37 ℃, then with PBS washing 3 times.With in advance in PBS the anti-pig IgM of 1:200 dilution or IgA FITC coupling antibody (Kirkegaard and Perry Laboratories Inc.) is added in the culture plate, then repeats to cultivate and washing step.Use the UV microscopy to detect the titer of the anti-Lawson's bacteria antibody of each species specificity.The positive percentage of IFAT that calculates various immunoglobulins when mean droplet degree of deciding value is used between group relatively, to obtain the frequency and the content of IgG between sow, IgM and IgA colostral antibody.

List of references

1.Knittel Deng people (1998) Am J Vet Res 59:722-726.

Claims

1. method, it is used to inoculate young animal and infects with antagonism lawsonia intracellularis (L.intracellularis) or be used to inoculate the animal that has anti-lawsonia intracellularis antibody or be exposed to anti-lawsonia intracellularis antibody, and this method comprises throws the antigenic step of lawsonia intracellularis of giving effective dose.

2. the process of claim 1 wherein inoculation when big to 9 days greatly of these animals at 1 day.

3. claim 1 or 2 method, wherein these animals inoculation when big to 6 days greatly at 1 day.

4. the method for one of claim 1-3, wherein these animals inoculation when big at 1 or 2 day.

5. the method for one of claim 1-4, wherein these animals have or are exposed to every milliliter of serum or the fluid detectable anti-lawsonia intracellularis antigen titration degree of 1:4 at least.

6. the method for one of claim 1-4, wherein these animals have or are exposed to every milliliter of serum or the fluid detectable anti-lawsonia intracellularis antigen titration degree of 1:64 at least.

7. the method for one of claim 1-6, wherein this lawsonia intracellularis antigen is to be selected from the group of being made up of one or more subunit of the modified lawsonia intracellularis antibacterial, the lawsonia intracellularis antibacterial that kills or the lawsonia intracellularis antibacterial that live.

8. the method for one of claim 1-6, the wherein modified lawsonia intracellularis antibacterial of this lawsonia intracellularis antigen for living.

9. the method for claim 8, wherein this young animal is to throw to give about 3.0TCID ₅₀To about 6.0TCID ₅₀This modified lawsonia intracellularis antibacterial that lives of dosage.

10. the method for one of claim 1-9, wherein these animals be bird, fish, and mammal such as cattle, pig, horse and primate.

11. the method for claim 10, wherein these animals are pig.

12. the antigenic purposes of lawsonia intracellularis, it is used to prepare the inoculation young animal with the medicament that the antagonism lawsonia intracellularis infects or inoculation has anti-lawsonia intracellularis antibody or is exposed to the animal of anti-lawsonia intracellularis antibody, and wherein these animals are inoculated this lawsonia intracellularis antigen of effective dose.

13. the purposes of claim 12, these animals be inoculation when big to 9 days greatly at 1 day.

14. the purposes of claim 12 or 13, these animals be inoculation when big to 6 days greatly at 1 day.

15. the purposes of one of claim 12-14, wherein these animals inoculation when big 1 day or 2 days.

16. the purposes of one of claim 12-15, wherein these animals have or are exposed to every milliliter of serum or the fluid detectable anti-lawsonia intracellularis antigen titration degree of 1:4 at least.

17. the purposes of one of claim 12-16, wherein these animals have or are exposed to every milliliter of serum or the fluid detectable anti-lawsonia intracellularis antigen titration degree of 1:64 at least.

18. the purposes of one of claim 12-17, wherein this lawsonia intracellularis antigen is to be selected from the group of being made up of one or more subunit of the modified lawsonia intracellularis antibacterial, the lawsonia intracellularis antibacterial that kills or the lawsonia intracellularis antibacterial that live.

19. the purposes of one of claim 12-18, the wherein modified lawsonia intracellularis antibacterial of this lawsonia intracellularis antigen for living.

20. the purposes of claim 19, wherein this young animal is to throw to give about 3.0TCID ₅₀To about 6.0TCID ₅₀This modified lawsonia intracellularis antibacterial that lives of dosage.

21. the purposes of one of claim 12-20, wherein these animals are bird, fish, and mammal such as cattle, pig, horse and primate.

22. the purposes of claim 21, wherein these animals are pig.