Attenuated
The present invention provides attenuated M. haemolitica strains that elicit an immune response in animal against M. haemolitica , compositions comprising said strains, methods of vaccination against M. haemolitica , and kits for use with such methods and compositions. The invention further provides multi-valent vaccines, which provide protective immunity when administered in an effective amount to animals susceptible to “shipping fever” or bovine respiratory disease.
1. A gene-deleted M. haemolytica bacterium, useful for the prevention of bovine respiratory disease, in a bovine animal, caused by M. haemolytica , comprising a deletion within the LktCA gene locus encoding acylase (LktC) and leukotoxin A (LktA); and wherein the gene-deleted M. haemolytica bacterium secretes a truncated LktCA having an amino acid sequence set forth in SEQ ID NO: 18.
2. The gene-deleted M. haemolytica bacterium of claim 1 , which is attenuated relative to virulent wildtype parental bacterium, and wherein the parental bacterium causes bronchopneumonia in the lungs of affected cattle, and has the designation serotype A1 D153.
3. The gene-deleted M. haemolytica bacterium of claim 1 , which is attenuated relative to virulent wildtype parental bacterium, and wherein the parental bacterium causes bronchopneumonia in the lungs of affected cattle, and has the designation serotype A6 D174.
4. A method of producing a gene-deleted M. haemolytica bacterium that is useful for the prevention of bovine respiratory disease, in a bovine animal, caused by M. haemolytica , comprising the step of making a deletion within the LktCA gene locus encoding acylase (LktC) and leukotoxin A (LktA); wherein, as a result of the deletion, the gene-deleted M. haemolytica bacterium secretes a truncated LktCA having an amino acid sequence set forth in SEQ ID NO: 18, thereby producing the gene-deleted M. haemolytica bacterium.
5. The method of claim 4 , wherein the gene-deleted M. haemolytica bacterium is attenuated relative to virulent wildtype parental bacterium, and wherein the parental bacterium causes bronchopneumonia in the lungs of affected cattle, and has the designation serotype A1 D153.
6. The method of claim 4 , wherein the gene-deleted M. haemolytica bacterium is attenuated relative to virulent wildtype parental bacterium, and wherein the parental bacterium causes bronchopneumonia in the lungs of affected cattle, and has the designation serotype A6 D174.
7. A method of confirming the presence of the deletion in the gene-deleted M. haemolytica bacterium of claim 1 , comprising the following steps:
a. providing a sample of the gene-deleted M. haemolytica bacterium;
b. providing a sample of the wildtype M. haemolytica bacterium that was used to produce the gene-deleted M. haemolytica bacterium;
c. performing PCR amplification reactions separately on each the gene-deleted and wildtype samples, using suitable LktCA forward and LktCA reverse primers; and
d. determining that the PCR amplicon is ˜2.3 kb for the gene-deleted M. haemolytica bacterium and ˜5.0 kb for the wildtype M. haemolytica bacterium; thereby confirming the presence of the deletion in the gene-deleted M. haemolytica bacterium.
8. The method of claim 7 , wherein the PCR reactions are performed with primers having the sequence as set forth in SEQ ID NO: 1 and 2.
9. The gene-deleted M. haemolytica bacterium of claim 1 further comprising a polynucleotide having a sequence of SEQ ID NO: 16 or SEQ ID NO: 17.
10. The gene-deleted M. haemolytica bacterium of claim 1 wherein DNA sequences encoding a temperature sensitive origin and a kanamycin resistance gene have been deleted from said gene-deleted M. haemolytica bacterium.
11. A kit comprising a first container comprising said gene-deleted M. haemolytica bacteria of claim 1 , a second container comprising an adjuvant, and instructions on administering said gene-deleted M. haemolytica bacteria and said adjuvant to an animal.
12. A kit comprising (i) a first container comprising an attenuated M. haemolytica serotype A1 D153 comprising a mutated LktCA gene locus that encodes a truncated LktCA having an amino acid sequence set forth in SEQ ID NO: 18, (ii) a second container comprising an attenuated M. haemolytica serotype A6 D174 comprising a mutated LktCA gene locus that encodes a truncated LktCA having an amino acid sequence set forth in SEQ ID NO: 18, (iii) a third container comprising an adjuvant, and (iv) instructions on administering said attenuated M. haemolytica and said adjuvant to an animal.