Mucosal and parenteral vaccination against pneumonic pasteurellosis in cattle with a modified-live in-frame lktA deletion mutant of Mannheimia haemolytica

abstract A new temperature-conditional shuttle vector, pBB80C, was constructed and utilized to generate an in-frame deletion in the leukotoxin structural gene of Mannheimia haemolytica serotype 1. Culture super-natants from the mutant contained no detectable cytotoxicity to BL-3 lymphocyte targets, and containeda new protein with an approximate molecular weight of 66 kDa which was reactive to anti-leukotoxinmonoclonal antibody. No protein reactive to anti-LktA monoclonal antibody was detected at themolecular weight 100e105 kDa of native LktA. Calves vaccinated mucosally by top-dressing the livemutant onto feed, or parenterally by subcutaneous injection, were resistant to virulent challenge withthe parent strain. Serologic antibody response, reduction in lung lesion, and reduction in pulmonaryinfectious load were greater among calves mucosally vaccinated than those which were vaccinated byinjection.Published by Elsevier Ltd. 1. IntroductionMannheimia haemolytica is the cause of signiﬁcant economiclosses to the beef, dairy, sheep, goat industries in the United Statesdue tobovine respiratorydisease (BRD) [1e4]. Theorganism is partof the normal nasopharyngeal ﬂora in cattle, sheep, and goats[1e3]. Under conditions of stress or concurrent respiratory viral-infection, M. haemolytica selectively proliferates in the naso-pharynxandcanbecomeestablishedinthelungstocauseﬁbrinouspneumonia [1]. Unfortunately, the current marketing system in theUnited States for feeder cattle inherently results in stress [1,2].Coupled with commonplace commingling of calves from multiplesources, and their various associated viral and bacterial pathogens,it should come as little surprise that Mannheimia is a predominantcause of stocker and feedlot losses [4,5].Management and control of BRD is variably achieved withacombinationofappropriatehusbandrytoreducestress,theuseoftherapeutic and metaphylactic antimicrobials, and immunizationfor common viral and bacterial agents [2,4,5]. While the value ofmanagement and husbandry for control of respiratory diseasecannot be overemphasized, there are concerns regarding excessiveagricultural use of antimicrobials and the development ofantibiotic-resistance, antibiotic residues in food products, andanimal welfare [3]. Effective alternate preventative measures aretherefore needed. Immunization, particularly against viral BRDpathogens, is widespread in North America. A major obstacle totheir effective use, however, is timely administration before antic-ipated periods of stress to allow an adequate immune responseprior to peak disease susceptibility [1,4,5]. In an effort to improvevaccine efﬁcacy, reduce the time required for induction of immu-nity, and to reduce the stress associated with handling of cattle,mucosal delivery of bacterial BRD vaccines has been a subject ofrecent interest [3,6].Previous work has shown that mutant M. haemolytica whichexpress no leukotoxin elicit dramatically reduced injury afterexperimental pulmonary instillation compared to their wild-typeparent strains [7]. The strains were therefore effectively attenu-ated. It is believed, however, that an immune response to leuko-toxin is important to disease resistance [1,3]. This experiment wasdesigned to produce attenuated strains of M. haemolytica whichexpress and transport inactive but immunogenic leukotoxinprotein for potential use as a mucosal or parenteral vaccine. Theproducts were constructed to be non-revertible and to contain noforeign DNA or antibiotic-resistance genes.