Abstract
Mannheimia haemolytica serotype A2 is the principal cause of pneumonic mannheimiosis in ovine and caprine livestock; this disease is a consequence of immune suppression caused by stress and associated viruses and is responsible for significant economic losses in farm production worldwide. Gram-negative bacteria such as M. haemolytica produce outer membrane (OM)-derived spherical structures named outer membrane vesicles (OMVs) that contain leukotoxin and other biologically active virulence factors. In the present study, the relationship between M. haemolytica A2 and bovine lactoferrin (BLf) was studied. BLf is an 80 kDa glycoprotein that possesses bacteriostatic and bactericidal properties and is part of the mammalian innate immune system. Apo-BLf (iron-free) showed a bactericidal effect against M. haemolytica A2, with an observed minimal inhibitory concentration (MIC) of 16 µM. Sublethal doses (2–8 µM) of apo-BLf increased the release of OMVs, which were quantified by flow cytometry. Apo-BLf modified the normal structure of the OM and OMVs, as observed through transmission electron microscopy. Apo-BLf also induced lipopolysaccharide (LPS) release from bacteria, disrupting OM permeability and functionality, as measured by silver staining and SDS and polymyxin B cell permeability assays. Western blot results showed that apo-BLf increased the secretion of leukotoxin in M. haemolytica A2 culture supernatants, possibly through its iron-chelating activity. In contrast, holo-BLf (with iron) did not have this effect, possibly due to differences in the tertiary structure between these proteins. In summary, apo-BLf affected the levels of several M. haemolytica virulence factors and could be evaluated for use in animals as an adjuvant in the treatment of ovine mannheimiosis.
Highlights
Mannheimia haemolytica is a normal inhabitant of the nasal cavity and tonsil crypts of healthy ruminants
Lf is a multifunctional glycoprotein that has several activities, the most studied of which is as an antimicrobial compound
Apo-bovine lactoferrin (BLf) showed a bactericidal effect toward M. haemolytica A2, a result that is in agreement with those of other reports, such as for Streptococcus pneumoniae [35], Vibrio cholerae [36], antibiotic resistant strains of Helicobacter pylori [37], Staphylococcus aureus and Escherichia coli [38], Pseudomonas fluorescens in ground beef [39], and Actinobacillus pleuropneumoniae [40]
Summary
Mannheimia haemolytica is a normal inhabitant of the nasal cavity and tonsil crypts of healthy ruminants. When animals suffer from shipping stress and/ or an acute infection by Mycoplasma bovis (in cattle) or viruses (e.g., parainfluenza-3, adenovirus, and respiratory syncytial virus), they become immunocompromised. This condition leads to the rapid proliferation of M. Mannheimia haemolytica A2 produces several virulence factors that together lead to acute fibrinous pleuropneumonia in sheep. The most important of these virulence factors is the leukotoxin (Lkt), a member of the RTX family of toxins from Gram-negative bacteria that is primarily secreted during the bacterial logarithmic growth phase. Lkt is a 104-kDa, thermolabile soluble protein that is toxic to ruminant macrophages, leukocytes and erythrocytes. The N-terminal region of Lkt has been shown to interact through nonspecific (electrostatic) contacts and through a specific protein receptor (β-integrin) of the target cells to mediate pore formation and lysis [5,6,7,8,9,10]
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