Title: CONSTRUCTION OF IN-FRAME, NON-POLAR, LKTA MUTANTS OF MANNHEIMIA HAEMOLYTICA USING A NEW TEMPERATURE CONDITIONAL PLASMID AND EFFICACY AS MODIFIED-LIVE MUCOSAL VACCINE CANDIDATES (ORAL PRESENT., INTL. PASTEURELLACEAE CONF.)
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 1, 2002
Publication Date: April 20, 2002
Citation: BRIGGS, R.E., TATUM, F.M. CONSTRUCTION OF IN-FRAME, NON-POLAR, LKTA MUTANTS OF MANNHEIMIA HAEMOLYTICA USING A NEW TEMPERATURE CONDITIONAL PLASMID AND EFFICACY AS MODIFIED-LIVE MUCOSAL VACCINE CANDIDATES. INTERNATIONAL PASTEURELLACEAE SOCIETY CONFERENCE. 2002. ABSTRACT p. 35, #34.
A 1.2 kb temperature-conditional plasmid origin of replication based on that of the 4.2 kb ampicillin-resistance plasmid of Mannheimia haemolytica was produced using a hydroxylamine mutagenesis technique. The origin was coupled to a kanamycin-resistance cassette based on that of Tn903 and a 1.0 kb fragment containing the ColE1 origin of replication. The hybrid plasmid supported replication in M. haemolytica at 30 C but not at 37 C. A 4kb fragment containing lktC and most of lktA was cloned into the new vector and an approximately 1kb deletion was introduced, corresponding with aa 34 to aa 378 of the encoded protein. Mannheimia transformed with this plasmid was expanded at 30 C, then passed to 37 C with kanamycin to select for products of single-crossover events. Passage at 30 C without selection allowed these products to resolve to either wild-type or products of a double-crossover event. Using the same replacement plasmid, it was possible to introduce clean mutations into serotypes 1, 5, 6, 7, 8, 9, and 12. Serotype 2 was insufficiently homologous 5' from the deletion and required a separate replacement plasmid. Each mutant expressed and transported a leukotoxin product of approximately 66 kDa MW. Calf trials were conducted by top-dressing the serotype 1 mutants onto feed for delivery and by conventional injection. Dramatic protection against experimental challenge was afforded by both delivery methods. All such exposed calves remained alert and feeding while unexposed controls became ill or died. Percentage lung involvement of controls averaged 32%, oral vaccinates 4%, and parenteral vaccinates 7%. Lung loads of M. haemolytica were about 10,000-fold lower in vaccinates than controls.