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Cytolysin LktA is one of the major pathogenicity factors of Mannheimia haemolytica (formerly Pasteurella haemolytica) that is the cause of pasteurellosis, also known as shipping fever pneumonia, causing substantial loss of sheep and cattle during transport. LktA belongs to the family of RTX-toxins (Repeats in ToXins) that are produced as pathogenicity factors by a variety of Gram-negative bacteria. Sublytic concentrations of LktA cause inflammatory responses of ovine leukocytes. Higher concentrations result in formation of transmembrane channels in target cells that may cause cell lysis and apoptosis. In this study we investigated channel formation by LktA in artificial lipid bilayer membranes made of different lipids. LktA purified from culture supernatants by polyethylene glycol 4000 precipitation and lyophilization had to be activated to frequently form channels by solution in 6 M urea. The LktA channels had a single-channel conductance of about 60 pS in 0.1 M KCl, which is about one tenth of the conductance of most RTX-toxins with the exception of adenylate cyclase toxin of Bordetella pertussis. The LktA channels are highly cation-selective caused by negative net charges. The theoretical treatment of the conductance of LktA as a function of the bulk aqueous concentration allowed a rough estimate of the channel diameter, which is around 1.5 nm. The size of the LktA channel is discussed with respect to channels formed by other RTX-toxins. We present here the first investigation of LktA in a reconstituted system.

Bovine viral diarrhea virus (BVDV) infection is a risk factor for development of shipping fever pneumonia in feedlot cattle, but the mechanisms are not well defined. The hypothesis of this study was that BVDV predisposes to bacterial pneumonia by downregulation of innate immune response in airway epithelial cells. The effects of noncytopathic (ncp)-BVDV (type 1 and type 2) infection on the constitutive and LPS-stimulated expression of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) in bovine tracheal epithelial cells (BTEC) were investigated. BTEC were isolated from healthy calves and cultured on collagen-coated wells. The effect of each BVDV isolate on expression of TAP and LAP in BTEC was sequentially investigated 3, 6, 12, 24 and 48 hours post infection with 6 x 105.5 of 50% tissue culture-infective dose (TCID50). The effect of each BVDV isolate on LPS-stimulated expression of TAP and LAP in BTEC was determined by treating BTEC in triplicate as follows: virus-infected and untreated, virus-infected and untreated, uninfected and LPS-treated, and virus-infected and LPS-treated. After 48 hours of viral infection, BTEC were treated with LPS or medium for 16 hours and total RNA was extracted from the cells. TAP and LAP mRNA expression was determined by real time RT-PCR relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The effect of the two ncp-BVDV isolates on LPS-stimulated secretion of lactoferrin in BTEC was investigated, 24 and 48 hours post infection. The concentration of lactoferrin in the supernatant medium was measured by enzyme linked immunosorbant assay (ELISA). The results showed that constitutive expression of TAP and LAP mRNA was not significantly (P>0.05) changed by BVDV infection. Treatment of BTEC with LPS resulted in a significant (P