Histophilus somni-induced infections in cattle from southern Brazil.

The sudden death of three calves, one diarrheic calf, and one aborted fetus from four farms in southern Brazil was investigated. Two Histophilus somni-associated syndromes were identified: systemic histophilosis (n = 4) and abortion (n = 1). The principal pathological findings included vasculitis, meningoencephalitis with thrombosis, necrotizing myocarditis, renal infarctions, hepatic abscesses, and bronchopneumonia. PCR assays were used to amplify specific amplicons of the ovine herpesvirus 2, bovine herpesvirus 1 and -5, Listeria monocytogenes, H. somni, and pestivirus; bovine group A rotavirus (BoRV-A) and bovine coronavirus (BCoV) were investigated in calves with diarrhea. H. somni DNA was amplified in tissues from all calves and the brain of the aborted fetus with pathological alterations consistent with histophilosis. All other PCR assays were negative; BoRV-A and BCoV were not identified. These findings confirm the participation of H. somni in the pathological alterations observed in this study and represent the first description of histophilosis in cattle from Brazil.

Decoration of Histophilus somni lipooligosaccharide with N-acetyl-5-neuraminic acid enhances bacterial binding of complement factor H and resistance to killing by serum and polymorphonuclear leukocytes.

The incorporation of N-acetyl-5-neuraminic acid (Neu5Ac), or sialic acid, onto surface components of some bacterial species may enhance their virulence. We have previously shown that Neu5Ac can be incorporated onto the lipooligosaccharide (LOS) of the bovine pathogen Histophilus somni, resulting in diminished antibody binding and enhanced serum resistance (Inzana et al., 2002. Infect. Immun. 70, 4870). In the present study, we assessed the effect of sialylation of H. somni LOS on the interaction with bovine innate host defenses. Incubation of non-sialylated H. somni with pre-colostral calf serum (PCS) resulted in dose-dependent, complement-mediated killing of the bacteria by the alternative pathway. However, sialylated H. somni was significantly more resistant to killing at any of the concentrations of PCS used. Sialylated H. somni LOS activated and consumed less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized red blood cells, and by Western blotting of C(3) activation products. Sialylated H. somni bound more factor H and iC(3)b and less C(3) than non-sialylated bacteria, as determined by enzyme-linked immunosorbent assay, supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni LOS inhibited both polymorphonuclear leukocyte phagocytosis of (3)H-thymidine-labeled bacteria and intracellular killing of the bacteria, compared to non-sialylated bacteria. Furthermore, sialylated H. somni bound less non-specific antibodies in normal bovine sera than non-sialylated bacteria. Therefore, sialylation of H. somni LOS had profound effects on resistance of the bacteria to innate bovine host defenses, which should be taken into consideration during in vitro studies of H. somni.

Genetics and molecular specificity of sialylation of Histophilus somni lipooligosaccharide (LOS) and the effect of LOS sialylation on Toll-like receptor-4 signaling.

Histophilus somni is an etiologic agent of bovine respiratory and systemic diseases. Most pathogenic strains of H. somni that have been tested (36 of 42) are able to utilize N-acetyl-5-neuraminic acid (Neu5Ac) to sialylate their lipooligosaccharide (LOS). Homologs of all the genes required for transport, metabolism, and regulation of Neu5Ac in Haemophilus influenzae were identified in the sequenced genomes of H. somni. Three open reading frames (ORFs) in H. somni strain 2336 were identified that contained homology to genes required for LOS sialylation in related bacteria. ORF-1 (hssT-I), ORF-2 (hssT-II), and ORF-3 (neuA(Hs)) were predicted to encode for putative proteins with 37% amino acid homology to an α-(2-3)-sialyltransferase in H. influenzae, 43% amino acid homology to an Haemophilus ducreyi sialyltransferase, and 72% amino acid homology to an H. influenzae CMP-Neu5Ac synthetase, respectively. The specific enzyme activity of each ORF was determined using synthetic acceptor substrates. The HssT-I sialyltransferase primarily sialylated N-acetyllactosamine (LacNAc, Gal-ß-[1-4]-GlcNAc-R), which is expressed on strain 2336, whereas HssT-II preferentially sialylated lacto-N-biose (LNB, Gal-ß-[1-3]-GlcNAc-R), which is expressed on a phase variant of strain 2336: strain 738. Phase variation of the terminal galactose linkage in strain 738 from ß-(1-3)-(LNB) to ß-(1-4)-(LacNAc) was confirmed using monoclonal antibody reactivity and nuclear magnetic resonance spectroscopy. Sialylated LOS induced significantly less chemokine response from macrophages derived from Toll-like receptor (TLR)-4 knockout mice than from de-sialylated LOS. Furthermore, sialylated LOS induced significantly less NF-κB activity from mouse-derived bone marrow macrophages than de-sialylated LOS. Therefore, sialylation inhibited LOS signaling through TLR-4. In conclusion, H. somni utilizes linkage-specific sialyltransferases to sialylate its LOS to avoid innate host defense mechanisms despite simultaneous epitope phase variation.

Platelet activation by Histophilus somni and its lipooligosaccharide induces endothelial cell proinflammatory responses and platelet internalization.

Histophilus somni is a gram-negative coccobacillus that causes respiratory and reproductive disease in cattle. The hallmark of systemic H. somni infection is diffuse vascular inflammation that can lead to an acute central nervous system disease known as thrombotic meningoencephalitis. Previously, we demonstrated that H. somni and its lipooligosaccharide (LOS) activate bovine platelets, leading to expression of P selectin, CD40L, and FasL. Because activated platelets have been reported to induce endothelial cell cytokine production and adhesion molecule expression, we sought to determine if bovine platelets induce proinflammatory and procoagulative changes in bovine pulmonary artery endothelial cells. Endothelial cells were incubated with platelets activated with adenosine diphosphate, H. somni, or H. somni LOS. Incubation with activated bovine platelets significantly increased expression of in adhesion molecules (intercellular adhesion molecule 1, E selectin) and tissue factor, as measured by flow cytometry, real-time polymerase chain reaction, and Western blot analysis. Activated platelets also up-regulated expression of endothelial cell IL-1beta, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1alpha as determined by real-time polymerase chain reaction and an IL-1beta enzyme-linked immunosorbent assay. An interesting and surprising finding was that bovine platelets activated by H. somni or its LOS were internalized by bovine endothelial cells as visualized by transmission electron microscopy. This internalization seemed to correlate with endothelial cell activation and morphological changes indicative of cell stress. These findings suggest that activated platelets might play a role in promoting vascular inflammation during H. somni infection.

Haemophilus somnus activation of brain endothelial cells: potential role for local cytokine production and thrombosis in central nervous system (CNS) infection.

Thrombotic meningoencephalitis (TME) is a neurological condition in cattle characterized by fibrinopurulent meningitis with hemorrhage, abscess formation and thrombotic vasculitis throughout the central nervous system. The etiologic agent of TME is Haemophilus somnus, a gram-negative pleomorphic coccobacillus. Although the pathogenesis of TME is not well understood, the propensity of H. somnus to cause vasculitis and intravascular thrombosis suggests a critical role for the interactions between the bacteria and endothelial cells in inciting the disease. The goal of this study was to determine if H. somnus elicits an inflammatory and procoagulative response in bovine brain microvascular endothelial cells (BBEC) in vitro. We demonstrate that BBEC exposed to H. somnus secrete significant levels of the proinflammatory and procoagulative cytokines TNF-alpha and IL-1beta. BBEC treated with H. somnus also display increased levels of IL-6 mRNA, another cytokine associated with coagulopathy in vivo. H. somnus-treated BBEC exhibited increased procoagulant activity and tissue factor expression and activity, along with a decreased ability to activate protein C and decreased expression of thrombomodulin mRNA. These changes would be expected to promote thrombus formation in vessels of the CNS, and potentially contribute to the pathogenesis of TME.

Complete genome sequence of Haemophilus somnus (Histophilus somni) strain 129Pt and comparison to Haemophilus ducreyi 35000HP and Haemophilus influenzae Rd.

Haemophilus somnus can be either a commensal of bovine mucosal surfaces or an opportunistic pathogen. Pathogenic strains of H. somnus are a significant cause of systemic disease in cattle. We report the genome sequence of H. somnus 129Pt, a nonpathogenic commensal preputial isolate, and the results of a genome-wide comparative analysis of H. somnus 129Pt, Haemophilus influenzae Rd, and Haemophilus ducreyi 35000HP. We found unique genes in H. somnus 129Pt involved in lipooligosaccharide biosynthesis, carbohydrate uptake and metabolism, cation transport, amino acid metabolism, ubiquinone and menaquinone biosynthesis, cell surface adhesion, biosynthesis of cofactors, energy metabolism, and electron transport. There were also many genes in common among the three organisms. Our comparative analyses of H. somnus 129Pt, H. influenzae Rd, and H. ducreyi 35000HP revealed similarities and differences in the numbers and compositions of genes involved in metabolism, host colonization, and persistence. These results lay a foundation for research on the host specificities and niche preferences of these organisms. Future comparisons between H. somnus 129Pt and virulent strains will aid in the development of protective strategies and vaccines to protect cattle against H. somnus disease.

Bovine plasma proteins increase virulence of Haemophilus somnus in mice.

The role of bovine serum or plasma proteins in Haemophilus somnus virulence was investigated in a mouse model of septicemia. An increase in virulence was detected when the organism was pre-incubated for 5 min and inoculated with fetal calf serum. When purified bovine serum or plasma proteins were pre-incubated with H. somnus before inoculating into mice, transferrin was found to increase virulence. Bovine lactoferrin was also noted to increase virulence, but to a lesser extent and had a delayed time course when compared with transferrin. Using an ELISA assay, an increased amount of H. somnus whole cells and culture supernatant bound to bovine transferrin when the organism was grown in iron-restricted media. Lactoferrin also bound to H. somnus, but binding was not affected by growth in iron-restricted media and it was eliminated with 2M NaCl, which reversed charge mediated binding. Transferrin, but not lactoferrin, supported growth of H. somnus on iron-depleted agar based media using a disk assay. Therefore, lactoferrin increased virulence by an undetermined mechanism whereas transferrin increased virulence of H. somnus by binding to iron-regulated outer-membrane proteins (IROMPs) and providing iron to the pathogen.

Roles of cellular activation and sulfated glycans in Haemophilus somnus adherence to bovine brain microvascular endothelial cells.

Haemophilus somnus can cause a devastating fibrinopurulent meningitis with thrombotic vasculitis and encephalitis in cattle. The mechanisms used by H. somnus to migrate from the bloodstream into the central nervous system (CNS) are unknown. In this study, we demonstrate that H. somnus adheres to, but does not invade, bovine brain endothelial cells (BBEC) in vitro. The number of adherent H. somnus was significantly increased by prior activation of the BBEC with tumor necrosis factor alpha (TNF-alpha). Addition of exogenous glycosaminoglycans significantly reduced H. somnus adherence to resting and TNF-alpha-activated BBEC. Heparinase digestion of the endothelial cell's glycocalyx or sodium chlorate inhibition of endothelial cell sulfated glycan synthesis significantly reduced the number of adherent H. somnus. In contrast, addition of hyaluronic acid, a nonsulfated glycosaminoglycan, had no inhibitory effect. These findings suggest a critical role for both cellular activation and sulfated glycosaminoglycans in adherence of H. somnus to BBEC. Using heparin-labeled agarose beads, we demonstrated a high-molecular-weight heparin-binding protein expressed by H. somnus. Heparin was also shown to bind H. somnus in a 4 degrees C binding assay. These data suggest that heparin-binding proteins on H. somnus could serve as initial adhesins to sulfated proteoglycans on the endothelial cell surface, thus contributing to the ability of H. somnus to infect the bovine CNS.

Haemophilus somnus possesses two systems for acquisition of transferrin-bound iron.

Haemophilus somnus strain 649 was found to acquire iron from ovine, bovine, and goat transferrins (Tfs). Expression of Tf receptors, as evaluated by solid-phase binding assays, required the organisms to be grown under iron-restricted conditions in the presence of Tf. Competition binding assays revealed the presence of two distinct Tf-binding receptor systems, one specific for bovine Tf and the other capable of binding all three ruminant Tfs. Affinity isolation procedures using total membranes yielded three putative bovine Tf-binding polypeptides and one putative ovine and goat Tf-binding polypeptide. PCR amplification followed by DNA sequence analyses revealed that H. somnus strain 649 possesses genes that encode a bipartite TbpA-TbpB receptor along with a homolog of the Histophilus ovis single-component TbpA receptor. Expression of TbpB and the single-component TbpA would appear to be subject to a form of phase variation involving homopolymeric nucleotide tracts within the structural genes.