WO2011113801A1

WO2011113801A1 - Use of clostridium perfringens strain 23 to protect against necrotic enteritis

Info

Publication number: WO2011113801A1
Application number: PCT/EP2011/053825
Authority: WO
Inventors: Filip Van Immerseel; Richard Ducatelle; Anouk Lanckriet
Original assignee: Universiteit Gent
Current assignee: Universiteit Gent
Priority date: 2010-03-16
Filing date: 2011-03-15
Publication date: 2011-09-22
Anticipated expiration: 2012-09-16

Abstract

The present invention relates to the prevention of necrotic enteritis caused by an infection with the anaerobe bacterium Clostridium perfringens. More specifically, the invention relates to compounds produced by or derived from Clostridium perfringens strain 23 which are useful as vaccines to effectively protect against necrotic enteritis.

Description

Use of Clostridium perfringens strain 23 to protect against necrotic enteritis Technical field of the invention

The present invention relates to the prevention of necrotic enteritis caused by an infection with the anaerobic bacterium Clostridium perfringens . More specifically, the invention relates to compounds produced by or derived from Clostridium perfringens strain 23 which are useful as vaccines to effectively protect against necrotic enteritis.

Background art

Clostridium perfringens is the causative agent of necrotic enteritis, an intestinal disease that affects industrial poultry worldwide [1 , 2]. After the ban of growth promoting antibiotics in the European Union, necrotic enteritis has become much more widespread. It is mainly contained by using curative antibiotics or ionophore anticoccidials [13, 14]. However, the use of curative antibiotics holds the risk of inducing resistance among the C. perfringens population and the rest of the intestinal microflora. Anticoccidials are meant in the first place to control coccidiosis.

Vaccination would be a valuable approach for the prevention of necrotic enteritis. Indeed, C. perfringens strains are ubiquitous and notorious for the wide range of toxins and virulence factors they excrete in their environment [3]. The majority of the chicken strains are toxinotype A, meaning that they carry the pic gene encoding alpha toxin [4-7]. For a long time, it was believed that this alpha toxin, a metalloenzyme with lecithinase and sphingomyelinase activities, was the major virulence factor involved in necrotic enteritis [8]. Recently, a novel pore forming toxin, NetB, was discovered [9]. Whereas a pic deletion mutant from a virulent C. perfringens chicken strain is still capable of inducing necrotic lesions in the gut of experimentally infected broilers, a netB deletion mutant from the same strain is not [9, 10]. Besides alpha toxin and NetB, C. perfringens secretes other proteins like metalloproteinases, growth inhibiting factors, etc... that play a role in the pathogenesis of necrotic enteritis [1 1, 12].

Previous reports on vaccination experiments with supematants or toxoids mention a significant reduction in the number of animals with necrotic lesions or partial protection but not total protection [18]. Vaccination of parent birds with type A or C toxoid decreases the occurrence of enteritis or hepatitis lesions with 5 to 15% in the progeny [19, 20]. Fukata et al. (1988) already showed that immunization with alpha toxoid or active alpha toxin alone

only offers partial protection against necrotic enteritis [21 ]. These results were later confirmed by subcutaneous vaccination of broilers with recombinant alpha toxin after which only a moderate degree of protection was obtained [22]. Also vaccination with different recombinant proteins never resulted in total prevention of the development of necrotic lesions. Kulkarni et al. (2006) have found six antigenic secreted proteins in the supernatant that were unique to virulent C. perfringens strains [23]. Immunization of birds with the respective recombinant proteins alpha toxin, a hypothetical protein (presumably a metalloprotease) and pyruvate - ferredoxine oxidoredutase significantly reduced the number of birds with lesions after a severe 5-day challenge of unlimited access to infected feed. Immunization with two other immunogenic proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose 1 ,6 biphosphate aldolase, only partially protects against a mild 2 day challenge [24]. Vaccination with recombinant endo-beta-N-acetylglucosaminidase only reduces the number of positive animals after challenge with one of two virulent strains. The degree of protection by phosphoglyceromutase decreases when the severity of the challenge increases [25].

Hence, there is still an urgent need for a vaccine which significantly prevents the development of necrotic lesions due to an infection with C. perfringens in the majority of animals. Such a vaccine would be a valid alternative to the currently used antibiotics and anticoccidials.

The present invention discloses compounds produced by or derived from a specific strain of C. perfringens (= C. perfringens strain 23) which are very effective in preventing the development of necrotic lesions in the majority of animals infected with C. perfringens. In other words, the present invention discloses an effective vaccine against necrotic enteritis. Description of invention

The present invention is based on the surprising finding that vaccination with antigen preparations derived from C. perfringens strain 23 significantly prevents the development of necrotic lesions in the majority of vaccinated animals when challenged with pathogenic C. perfringens strains. Therefore, a first embodiment of the present invention relates to a composition comprising at least an immunoprotective fraction of Clostridium perfringens strain 23 for use as a medicament. The terms 'a composition comprising at least an immunoprotective fraction of Clostridium perfringens strain 23' relate to a composition comprising at least an antigen preparation derived of Clostridium perfringens strain 23. Said antigen preparation has immunoproptective properties and includes whole-cell bacterial preparations or preparations of components produced by or derived from Clostridium perfringens strain 23 or a combination thereof. Such antigen preparations may thus comprise whole -killed (inactive) bacteria, live-attenuated (weakened) bacteria or processed and/or artificial bacterial preparations, or combinations thereof. Processed bacterial preparations include preparations of bacterial proteins, which are partially or completely purified. These can be used alone or in combination with artificial antigen preparations such as preparations which are either in part or entirely obtained by synthetic or recombinant methods. Also processed bacterial preparations which are in part or entirely obtained by synthetic or recombinant methods are part of the present invention. The antigen preparation or immunoprotective fraction of the present invention can also be a cell lysate of Clostridium perfringens strain 23, i.e. a mixture obtained upon lysis of bacterial cells. Said lysate can be detoxified or inactivated, and/or further purified, and/or further fractionated using methods known to a skilled person in order to obtain a composition of enriched or purified immunoprotective antigens. For example, said antigen preparation can be a soluble, immunoprotective fraction of a sonicated bacterial culture, e.g. obtained after filtration.

According to a further embodiment of the invention, the composition comprising an antigen preparation or immunoprotective fraction of the present invention is an antigen preparation or immunoprotective fraction, of the supernatant of a culture of said Clostridium strain. In a specific embodiment, the composition of the present invention comprises a C. perfringens strain 23 culture supernatant. Said supernatant can be detoxified or inactivated, and/or further purified, and/or further fractionated using methods known to a skilled person in order to obtain a composition of enriched or purified immunoprotective antigens. Said supernatant can, for example, be derived from an overnight culture of said Clostridium strain which is concentrated by dialysis (against for example a 20kDa polyethyleneglycol solution) followed by further concentration and desalting using desalting columns, and, which is subsequently diluted in PBS. The antigen preparation or immunoprotective fraction of the present invention as indicated above will thus comprise immunoprotective organic compounds, preferably immunoprotective proteins or antigens, produced by Clostridium perfringens strain 23. Preferably, said proteins will be secreted by said bacterial strain.

The term 'immunoprotective' relates to a significant prevention of the development of necrotic lesions in the majority of animals vaccinated with antigen preparations derived from C. perfringens strain 23 and challenged with pathogenic C perfringens strains. This can, for example, be tested as follows: 1) vaccinate a group of animals with a composition comprising at least an immunoprotective fraction of Clostridium perfringens strain 23 according to the present invention using methods known in the art. Preferably, said immunoprotective fraction is an antigen preparation of Clostridium perfringens strain 23, more preferably a supernatant of a culture of Clostridium perfringens strain 23. For example, vaccinate birds twice (on day 3 and 12 post-hatching) subcutaneously with a 200 μΐ dose of supernatant of said bacteria containing 7 to 70 μg total protein ( possibly in addition of 50 μg/animal/vaccination adjuvant), 2) challenge the animals with C. perfringens bacteria using methods known in the art. For example, orally challenge on days 17, 18, 19 and 20 three times a day with approximately 4. 10 exp 8 cfu C. perfringens bacteria, 3) euthanize and perform a necropsy on the animals and investigate the intestinal lesions. For example, euthanize and perform a necropsy on the animals on days 22, 23 and 24 and score the intestinal lesions as described by Keyburn et al. (10).

The term 'a significant prevention of the development of necrotic lesions' means that at least 50%, 60%, 70%, preferably 80%, more preferably 90% or most preferably 100% of the necrotic lesions that would develop without prior vaccination will not develop due to the vaccine of the present invention. With the term 'majority of animals' is meant that at least 50%, 60%, 70%, preferably 80%, more preferably 90% or, most preferably 100% of all animals which are vaccinated with the vaccine of the present invention will show a significant prevention of the development of necrotic lesions. Taken together, the composition of the present invention is capable to reduce the number of animals having necrotic lesions with 50%, 60%, 70%, preferably 80%, more preferably 90% or most preferably 100% when compared to a group of control animals which did not receive the composition of the present invention.

The term 'Clostridium perfringens strain 23' is a type A strain (i.e. is a strain that contains alpha toxin, but not iota, beta and epsilon toxin) and corresponds to isolate number 23 as described in Table 2 on page 147 of ref n° 5. The latter strain has further been deposited on January 29, 2010 with BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie - Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium (http://bccm.belspo.be/) and has accession number LMG S-25552 and deposited on June 4, 2010 with BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie - Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium (http ://bccm.belspo .be/) and has accession number LMG P-25816 and deposited on June 4, 2010 with BCCM/LMG Bacteria Collection,

Laboratorium voor Microbiologie - Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent,

Belgium (http://bccm.belspo.be/) and has accession number 5 LMG P-25816.

The term a 'medicament' as used above, also referred to as pharmaceutical drug, medicine or medication, refers to a composition comprising at least an antigen preparation or an immunoprotective fraction of Clostridium perfringens strain 23 intended for use in the medical diagnosis, cure, treatment, or prevention of disease wherein said disease preferably is necrotic enteritis. Necrotic enteritis refers to an intestinal disease that affects industrial poultry worldwide. Said medicament thus comprises at least one biologically active ingredient (= an antigen preparation or an immunoprotective fraction of Clostridium perfringens strain 23) and possibly at least one excipient or carrier which is the substance of the dosage form of the medicament, or the liquid the active ingredient is suspended in, and possibly other materials that are pharmaceutically inert. Suitable carriers, excipients or other materials are known to the skilled man. The 'medicament' or composition may be administered by any suitable method within the knowledge of the skilled man. A specific route of administration is oral or parenteral administration. Vaccination in ovo is also possible. However, the dosage and mode of administration will depend on the individual/animal.

The present invention relates in particular to a composition of the present invention for use to protect against necrotic enteritis in birds. Said birds are preferably chickens, ducks or turkeys and said chickens are preferably broilers or breeders.

In a further embodiment the present invention refers to any method known to the skilled person for the preparation of a vaccine against necrotic enteritis. In particular, the invention provides the use of the antigen preparation or immunoprotective fraction Clostridium perfringens strain 23 in the preparation of a medicament or vaccine for protecting a subject against necrotic enteritis. The latter method specifically relates to a vaccine against necrotic enteritis in birds. The latter vaccine comprises a composition according to the present invention. Alternatively, the vaccine can comprise, in addition to the antigen preparation of the present invention, a suitable adjuvant. A large number of adjuvants are known to a person skilled in the art. Examples are Freund's Complete and Incomplete adjuvants, mycolate -based adjuvants, bacterial liposaccharide, peptidoglycans, proteoglycans, streptococcal preparations, vegetable oils, liposomes, Biostim™, aluminium hydroxide, saponin, DEAE-dextran, neutral oils, the RIBI adjuvant system etc. The type of adjuvant will vary, depending on the type of antigen preparation and route of administration used. A preferred adjuvant is Quil A. The vaccines of the present invention can be in solid or liquid form such as tablets, capsules, powders, solutions, suspensions or emulsions. The present invention further encompasses a method of administering said vaccine or composition according to the invention to a subject in need thereof. The subject is thereby vaccinated by said vaccine or composition. The vaccines of the present invention can be administered orally, or, in injectable dosages by solution or suspension of these materials in a physiologically acceptable diluent with or without a pharmaceutical carrier as known in the art as is for example described in PCT/EP2007/009855. Alternatively said vaccines can be administered as aerosols as is for example described in PCT EP2007/009855. The administration of vaccines of the present invention may take place in a single dose or in a dose repeated once or several times after a certain period. The appropriate dosage varies according to various parameters, for example the individual/animal treated (adult animal, young animal including one-day old animal, or, in the case of birds, animals which didn't hatch yet when in ovo administration as described by Williams C.J. and Zedek A.S. 2010 Poultry Science 89: 189-193 is envisioned), the antigen itself, the mode and frequency of administration, the presence or absence of adjuvant and the type of adjuvant. For example, birds can be vaccinated twice (on day 3 and 12 post-hatching) subcutaneously with a 200 μΐ dose of supernatant of said bacteria containing 7 to 70 μg total protein ( possibly in addition of 50 μg/animal/vaccination adjuvant). Alternatively, dosages can be expressed in CFUs. Crude antigen preparations (i.e. containing traces of culture medium) may require higher dosages to be effective than partly purified or purified preparations. The vaccine of the present invention can be administered through any suitable route, such as in ovo or by mucosal (intranasal), parenteral, intramuscular, oral, intradermal, intraperitoneal, intravenous, transdermal/transcutaneous or subcutaneous administration, or any combination thereof. The different aspects of the present invention are illustrated by, but not limited to the examples detailed hereafter.

Examples

Materials and methods

1.1 Strains

8 C. perfringens type A strains isolated from chickens and belonging to different genotypes, as analyzed by Pulsed Field Gel Electrophoresis (PFGE), were included [5]. All strains were used for supernatant production. All C. perfringens bacteria were grown anaerobically at 37°C in BHI broth (Oxoid, Basingstoke, UK). C. perfringens strains 56 and 61 were used as challenge strains in an in vivo necrotic enteritis model described below and have been shown to be highly virulent in the applied in vivo model before [15, 16]. A summary of the characteristics of the strains is given in Table 1.

1.2 Alpha toxin production and detection of the netB gene

The alpha toxin production levels of the strains were determined in earlier reports and is shown in table 1 [5].

The presence of the netB gene was determined by PCR using the primers AKP78 (5 '- GCTGGTGCTGGAATAAATGC-3', cf. SEQ ID NO.l) and AKP79 (5'- TCGCC ATTGAGT AGTTTCCC-3 ', cf. SEQ ID NO.2), as described by Keyburn et al. (2008) [9]. Lactate dehydrogenase cytotoxicity assays were performed as an indicator for NetB expression [9]. Overnight cultures were grown in BHI. The supematants were filter sterilized and dialyzed overnight against 10 mM Tris-HCl pH 8.5. To test for cytotoxicity, the culture supematants were added to the medium of chickens hepatoma (LMH) cells (ATCC CRL-21 17) in a 1 :4 dilution. The cells were incubated for 3 hours at 37°C and 5% C0₂. Lactate dehydrogenase release in the supernatant was used as an indicator of cytolysis and hence NetB production and was measured using the Cytotoxicity Detection Kit (Roche Applied Sciences, Penzberg, Germany). As a positive control, 10% Triton X-100 (Sigma Aldrich, St. Louis MO, USA) was added to the cells. The negative control consisted of 1 :4 diluted BHI. Percentage cytotoxicity was determined relative to the control groups. Experiments were repeated four times. For the LDH cytotoxicity assays, a one-way analysis of variance was used to detect significant differences in the relative cytotoxicity percentages of the supernatants (P<0.05) followed by a post-hoc Bonferroni test for multiple comparisons (P<0.0625). 1.3 Vaccines

In trials 1 and 2, supernatants derived from overnight cultures of the C. perfringens strains were concentrated by dialysis against a 20 kDa polyethyleneglycol (PEG, Sigma Aldrich, St. Louis MO, USA) solution, followed by further concentration and desalting using Centricon columns (Millipore, Billereca, MA, USA). The concentrated samples were diluted in PBS to a final concentration of 7 μg or 70 μg/200 μΐ respectively.

1.4 Experimental setup of the in vivo trials

The in vivo necrotic enteritis challenge model was applied as described previously [15]. Groups of 27 (trial 2 and 3) or 30 (trial 1) broilers were fed a wheat/rye -based (43%/7.5%) diet, with soybean meal as protein source.

In trial 1 , vaccination took place in 8 groups of birds with supernatants from 8 different strains (7, 1 1, 23, 37, 43, 48, 56, 61). On day 3 and day 12 post-hatching, the birds were vaccinated subcutaneously in the neck with a 200 μΐ dose of supernatant containing 7 and 70 μg total protein respectively. QuilA (Brenntag Biosector, Frederikssund, Denmark) was used as an adjuvant (50 μg/bird/vaccination). At the same time, one control group got placebo vaccinated with PBS and 50μg of QuilA/bird/vaccination. Another control group was left unvaccinated. In trial 2, only the supernatant of strain 23 was used for vaccination in the same way as described for trial 1. The control group was vaccinated with PBS and QuilA. In trial 3, no prior vaccination took place.

During all three trials, Nobilis Gumboro D78 Gumboro vaccine (Schering-Plough Animal

Health, Brussels, Belgium) was given in the drinking water on day 16 in all groups. From day 17 onwards, the same diet was used with the exception that fishmeal (30%) replaced soy bean as protein source. On days 17, 18, 19 and 20, oral challenge was performed three times a day with approximately 4.10⁸ cfu C. perfringens bacteria. On day 18, all birds were orally inoculated with a ten-fold dose of Paracox-5 (Schering-Plough Animal Health). In trial 1 , oral challenge was performed with virulent strain 56 whereas in trial 2, the chickens were challenged with one of two virulent strains 56 and 61. In trial 3, the virulence of strain 23 was compared to that of strain 56 in the necrotic enteritis challenge model. In trial 3, a negative control group was included consisting of birds to which only a 10-fold dose of Paracox-5 was administered.

On days 22, 23 and 24, each time one third of the birds in each group were euthanized and necropsied. Intestinal lesions in the small intestine (duodenum to ileum) were scored as described by Keyburn et /.[10]. Birds with lesion scores of 2 (1 to 5 necrotic lesions) or more were classified as necrotic enteritis positive. The data were analyzed with SPSS Statistics 17.0 software (SPSS Inc., Chicago) using the binary logistic regression method to compare the number of necrotic enteritis positive animals within the test groups. Bonferroni's correction for multiple comparisons was applied (P<0.05/n) for the first vaccination trial and significance was determined at P < 0.005. For all other trials, significance was determined at P<0.05.

2. Results

3.1 Detection of the netB gene and NetB cytotoxic effect towards LMH cells

PCR showed that strains 7, 1 1, 43 an 48 were netB negative, whereas strains 23, 37, 56 and 61 carry the netB gene. The in vitro cytopathic effect of all supernatants towards LMH cells was determined as an indicator for NetB activity [9, 17]. The relative cytotoxicity percentages of the supernatants from the different strains are shown in table 2. Statistical analysis indicated that there were significant differences between the in vitro cytotoxicity percentages of the 8 supernatants (ANOVA, P<0.05). The post-hoc Bonferroni method for multiple comparison confirmed that the supernatants of all (strains 7, 43 and 48) but one (strain 1 1) netB negative strains were significantly less cytotoxic towards LMH cells than the supernatants of netB positive isolates 23, 37, 56 and 61 (PO.00625).

3.2 Vaccination experiments

The vaccination experiment clearly showed that vaccination with the supernatant of netB negative strains 7, 11 and 43 did not result in immunoprotection (table 3). Vaccination with supernatant of netB positive strains 37 and 56 did not result in immunoprotection either. Vaccination with supernatant from strains 23 and 48 resulted in a significant decrease in number of birds with necrotic lesions (P<0.005). Only vaccination with supernatant of strain 23, a netB positive strain and low alpha toxin producer, totally prevented the development of necrotic lesions after severe challenge. The supernatant of strain 48 {netB negative) provided partial protection.

Since the toxins in the supernatants were not neutralized, a variable number of chicks died shortly after vaccination. High percentages of dead chicks were observed after vaccination with supernatant of strains 11 and 48 (high alpha toxin producers) and strain 61 (low alpha toxin producer).

In a second vaccination trial, none of the chickens that received subcutaneous vaccination with supernatant of strain 23 showed lesions, while 19% and 20% of the placebo vaccinated chickens were positive for necrotic lesions after challenge with virulent strain 56 and 61 respectively (table 3).

3.3 In vivo comparison of the necrotic enteritis inducing potential of strain 56 and 23

Table 4 shows that strain 23 is only capable of inducing lesions in half as many birds as strain 56 is.

TABLES

Table 1. Characteristics of the strains.

Strain number Origin¹ Alpha toxin production²

7 healthy Low

11 healthy High

23 healthy Low

37 diseased Moderate

43 diseased Moderate

48 diseased High

56 diseased Moderate

61 diseased Low

¹ Health status of the flock the strain was isolated from. Diseased means that there was a necrotic enteritis outbreak in the flock.

As determined in [5], comparison between chicken strains. Table 2. Lactate dehydrogenase cytotoxicity assay of LMH cells treated with 1 :4 diluted supematants of different strains.

Strain the supernatant Relative percentage Standard deviation

was taken from cytotoxicity

7 1.20 2.05

1 1 61.04 10.41

23 75.41 14.74

37 85.81 16.65

43 13.07 4.05

48 42.92 18.10

56 87.03 11.87

61 89.31 15.33

^Percentage cytotoxicity of the supematants were determined relative to control wells treated with 10% Triton X-100 (positive control) or 1 :4 diluted BHI (negative control). The values are averages of 4 independent trials.

Table 3. Total number of birds with macroscopic necrotic enteritis lesions (lesion score >2) in the vaccination experiments

Number of Number of dead

Strain used Percentage of

Vaccination animals with chicks due to

Trial for animal with

Group lesions/total vaccination/total challenge lesions

number number⁸

1 SN of strain 7 56 20/29 69 % 0/30

SN of strain 56 8/13 61 % 16/30

11

SN of strain 56 0/29^a 0 % 0/30

23

SN of strain 56 13/24 54 % 2/30

37

SN of strain 56 23/30 76 % 0/30

43

SN of strain 56 3/17^a 17 % 13/30

48

SN of strain 56 12/24 50 % 2/30

56

SN of strain 56 4/12 33 % 18/30

61

PBS (placebo) 56 12/25 47 % 0/30

no vaccination 56 15/26 56 % 0/30

2 PBS 56 5/25 20 %

SN of strain 56 0/25^b 0 %

23

PBS 61 5/26 19 %

SN of strain 61 0/25^c 0 %

23

SN=supernatant §only chicks that died shortly after vaccination are mentioned

a values with superscripts differ significantly (P<0.005 for trial 1) from both the PBS- vaccinated as the non-vaccinated group (trial 1)

b'^c Values with superscripts differ significantly (P<0.05) from the control group vaccinated with PBS group and infected with the respective strain (trial 2)

Table 4. Number of birds with macroscopic necrotic enteritis lesions (lesion score >2).

Number of positive birds/ Percentage of birds with

Strain used for challenge

total number lesions

^"23 9/25 36 %

56 18/26 69 %

Negative control 0/29 0 %

Taken together, vaccination with two supematants out of eight, i.e. those from strains 23 and 48, significantly protected the birds against necrotic enteritis (trial 1). Vaccination with supernatant of strain 23 resulted in full protection against necrotic enteritis after challenge with two different virulent strains (trial 2). This is the first time that vaccination with supernatant has such a profound effect.

When the immunoprotective potential of the 8 supematants is compared with the level of in vitro alpha toxin expression, the presence of netB gene or level of presumed Netb-associated hepatocyte toxicity of the respective strains, it shows that the supematants of 3 out of 4 netB negative strains confer no immunoprotective potential at all. A netb gene in combination with a cytopathic effect of the supematants towards LMH cells was not associated with immunoprotective potential for the supematants of strains 37, 56 and 61. The immunoprotective potential of the supematants doesn't solely depend on the strain's alpha toxin expression level either: strain 23 produces low levels of alpha toxin in vitro whereas strain 48 produces high levels. This indicates that the immunity against necrotic enteritis cannot be attributed to alpha toxin or NetB on its own. References

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Claims

1. A composition comprising an antigen preparation of a culture of Clostridium perfringens strain 23 as deposited with the BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie - Universiteit Gent, with accession number LMG P-25816, for use as a medicament.

2. A composition according to claim 1 wherein said antigen preparation is the supernatant of a culture of said Clostridium strain.

3. A composition according to claims 1 and 2 for use to protect against necrotic enteritis in birds

4. A composition according to claim 3 wherein said birds are chickens or turkeys.

5. A composition according to claim 4 wherein said chickens are broilers or breeders.

6. A composition according to claim 2 wherein said supernatant is derived from an overnight culture of said Clostridium strain which is concentrated by dialysis followed by further concentration and desalting using desalting columns, and, which is subsequently diluted in PBS.

7. A composition according to anyone of claims 1 to 6 wherein said antigen preparation comprises immunoprotective organic compounds secreted by said Clostridium strain.

8. A composition according to claim 7 wherein said organic compounds are proteins.

9. Use of a composition according to claims 1 -8 for the preparation of a vaccine against necrotic enteritis in birds.

10. A vaccine comprising a composition according to claims 1 -8.

1 1. A method for preparing a vaccine according to claim 10, comprising the steps of:

a. providing an overnight culture of Clostridium strain 23 as deposited with the BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie - Universiteit Gent, with accession number LMG P-25816,

b. concentrating said culture by dialysis,

c. further concentrating and desalting said culture using desalting columns, and,

d. subsequently diluting said culture fraction in PBS.