Recombinant attenuated
The present invention discloses attenuated Clostridium perfringens organisms that express a substantially nontoxic alpha-toxin. The expressed alpha-toxin is a deletion mutein that relative to the alpha-toxin of the mature alpha-toxin of Clostridium perfringens strain 13, is missing at least nine consecutive amino acid residues including His 68 . The present invention also discloses attenuated organisms that encode the muteins, as well as the use of such attenuated organisms as vaccines.
1. An attenuated Clostridium perfringens organism constructed by integrating a nucleic acid molecule that encodes a substantially nontoxic mutein of Clostridium perfringens alpha-toxin into a chromosome of a non-attenuated Clostridium perfringens organism;
wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 minus at least 9 consecutive amino acid residues, with no more than 24 consecutive amino acid residues deleted;
wherein one of the deleted amino acid residues is His 68 ; and
wherein the attenuated Clostridium perfringens organism is substantially nontoxic due to a lack of a functional wild-type plc gene.
2. The attenuated Clostridium perfringens organism of claim 1 wherein the nucleic acid molecule is located at a position on the chromosome that is homologous to the location of a nucleic acid molecule encoding a wild-type alpha-toxin present in the non-attenuated Clostridium perfringens.
3. The attenuated Clostridium perfringens organism of claim 1 that is a type A Clostridium perfringens.
4. The attenuated Clostridium perfringens organism of claim 1 wherein the non-attenuated Clostridium perfringens from which it was constructed was isolated from a host animal that is either a mammal or an avian.
5. The attenuated Clostridium perfringens organism of claim 4 , wherein said mammal is selected from the group consisting of bovine, ovine, and porcine.
6. The attenuated Clostridium perfringens organism of claim 4 wherein said avian is a chicken, a turkey, a goose, a duck, a swan, a dove, a pigeon, a grouse, or a partridge.
7. A vaccine comprising the attenuated Clostridium perfringens organism of claim 1 .
8. The vaccine of claim 7 further comprising one or more of the following: a pharmacologically acceptable buffer, excipient, or an adjuvant.
9. A method of reducing the severity of a disease in an animal caused by C. perfingens comprising administering to the animal an immunologically effective dose of the vaccine of claim 7 .
10. The method of claim 9 wherein the vaccine is administered by one or more of the following routes: oral, intramuscular, intravenous, intradermal, subcutaneous, or intranasal.
11. The method of claim 9 wherein the vaccine is top-dressed on the feed of the animal.
12. The method of claim 9 wherein the vaccine is sprayed onto the animals to provide for oral administration.
13. An animal feed that comprises the vaccine of claim 7 .
14. The attenuated Clostridium perfringens organism of claim 1 that comprises at least one gene encoding a non- Clostridium perfringens polypeptide.
15. The attenuated Clostridium perfringens organism of claim 14 wherein said non- Clostridium perfringens polypeptide is a non-bacterial polypeptide.
16. The attenuated Clostridium perfringens organism of claim 15 wherein the non-bacterial polypeptide is an avian or mammalian polypeptide.
17. The attenuated Clostridium perfringens organism of claim 16 wherein the non-bacterial polypeptide is an avian polypeptide.
18. The attenuated Clostridium perfringens organism of claim 16 wherein the non-bacterial polypeptide is a cytokine.
19. The attenuated Clostridium perfringens organism of claim 18 wherein the cytokine is chicken IL-18.
20. A Clostridium perfringens organism that is CPERF/ΔαToxin 365-054 having ATCC Deposit No. PTA7364.
21. A Clostridium perfringens organism that is CPERF/ΔαToxin 365-053 having ATCC Deposit No. PTA7365.
22. The attenuated Clostridium perfringens organism of claim 1 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 18 consecutive amino acid residues deleted.
23. An isolated attenuated Clostridium perfringens organism constructed by integrating a nucleic acid molecule that encodes a substantially nontoxic mutein of Clostridium perfringens alpha-toxin into a chromosome of a non-attenuated Clostridium perfringens organism;
wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 in which 9 consecutive amino acid residues have been deleted;
wherein one of the deleted amino acid residues is His 68 ; and
wherein the attenuated Clostridium perfringens organism is substantially nontoxic due to a lack of a functional wild-type plc gene.
24. The attenuated Clostridium perfringens organism of claim 23 wherein the deleted 9 consecutive amino acid residues are amino acids 62 to 70 of the amino acid sequence of SEQ ID NO:3.
25. The attenuated Clostridium perfringens organism of claim 24 wherein the deleted 9 consecutive amino acids are replaced by a single leucine residue.
26. A vaccine comprising the attenuated Clostridium perfringens organism of claim 25 .
27. A vaccine comprising the Clostridium perfringens organism of claim 20 .
28. A vaccine comprising the Clostridium perfringens organism of claim 21 .
29. An attenuated Clostridium perfringens organism constructed by integrating a nucleic acid molecule that encodes a substantially nontoxic mutein of Clostridium perfringens alpha-toxin into a chromosome of a non-attenuated Clostridium perfringens organism;
wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 in which His 68 is deleted together with an additional 4 to 60 flanking amino acid residues; and
wherein the attenuated Clostridium perfringens organism is substantially nontoxic due to a lack of a functional wild-type plc gene.