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Wooden boards are commonly used for aging artisan cheeses. Although considered critical to the development of desired flavors and aromas, knowledge about the microbial communities associated with these boards is limited. To begin to address this need, we performed a 16S ribosomal RNA analysis of the bacterial communities present on the surface and within 5 wooden boards used for cheese ripening that were obtained from 3 cheese-processing facilities. The 5 boards were dominated by bacteria in the phyla Actinobacteria, Firmicutes, and Proteobacteria and displayed differences in both diversity and richness. Analysis of these boards also identified significant board-to-board variation. A total of 288 operational taxonomic units were identified across all samples, with 7 operational taxonomic units forming a core microbiota across all boards. Taken together, these data reflect the cheese-ripening environment, which appears to select for salt- and cold-tolerant bacteria.

Histophilus somni is a Gram-negative coccobacillus that causes diffuse vasculitis and intravascular thrombosis that can lead to multiple organ failure in cattle. Macrophages are important cellular mediators of fibrin deposition and removal at sites of inflammation. It has become evident that macrophages and other cells release microparticles (MPs) that have an array of biological activities, including pro-coagulant activity. We sought to determine whether monocyte-derived macrophages exposed to H. somni in vitro release MPs that activate the clotting cascade in a manner that could lead to thrombus formation. Bovine monocyte-derived macrophages were incubated with H. somni (at a 10:1 ratio) in RPMI with 10% heat inactivated fetal bovine serum for 6 hr at 37 °C with 5% CO2. Membrane-shed MPs were isolated from the conditioned media, washed twice with Ca2+ and Mg2+ free HBSS, and pro-coagulant activity assessed by a one-step plasma clotting assay. We observed greater pro-coagulant activity for MPs from H. somni stimulated macrophages than from unstimulated controls. Microparticle pro-coagulant activity was inhibited by addition of an anti-tissue factor antibody. We also observed co-localization of fluorescein-labeled H. somni cells and annexin V staining as evaluated by confocal microscopy. These results demonstrate that exposure to H. somni cells causes bovine monocyte-derived macrophages to release MPs that contain tissue factor, the first such report for bovine macrophages. We infer that if similar events occur in vivo they could amplify thrombus formation in bovine histophilosis.

Bacterial biofilms impair healing in 60% of chronic skin wounds. Various animal models (mice, rats, rabbits, and pigs) have been developed to replicate biofilm infected wounds in vivo. We developed a sustained wound infection model by applying preformed Pseudomonas aeruginosa biofilms on a wound dressing to full-thickness murine skin wounds. We bathed a commercially available wound dressing in P. aeruginosa for 48 h, allowing a biofilm to establish on the dressing prior to application to the wound. Dressings were removed from the wounds after 3 days at which time the wound beds contained ∼108 bacterial cells per gram tissue. Significant numbers of P. aeruginosa persisted within the skin wounds for up to 21 days. Un-inoculated wounds reached closure between 9 and 12 days. In contrast, biofilm-inoculated wounds achieved closure between 18 and 21 days. Histologic analysis confirmed decreased re-epithelialization and collagen deposition, coupled with increased inflammation, in the biofilm-inoculated wounds compared to un-inoculated controls. This novel model of delayed healing and persistent infection of full-thickness murine skin wounds may provide a robust in vivo system in which to test novel treatments to prevent wound infection by bacterial biofilms.

Mannheimia haemolytica and Pasteurella multocida are two bacterial species implicated in the bovine respiratory disease complex (BRDC) that is costly to the beef and dairy cattle industries. Both bacterial species are thought to occupy a similar niche as commensals in the upper respiratory tract. Many bacteria are thought to exist as biofilms in their hosts, perhaps in close proximity with other bacterial species. We previously showed that M. haemolytica forms biofilm on bovine respiratory epithelial cells in vitro. We are interested in the possibility that M. haemolytica and P. multocida co-exist as biofilms in the upper respiratory tract of cattle. In this study, we begin to explore this possibility by assessing the ability of M. haemolytica and P. multocida to form a biofilm on bovine respiratory epithelial cells in vitro. We found that M. haemolytica and P. multocida are separately able to form biofilms on bovine respiratory epithelial cells, but mutually inhibit one another when incubated together as a biofilm. Both the biofilm matrix (crystal violet stain) and bacterial numbers (CFU and PCR) were reduced when M. haemolytica and P. multocida were incubated together on fixed epithelial cells. This inhibition does not appear to result from a soluble factor, as neither conditioned medium nor separation of the two species by a transwell filter membrane reproduced the effect. We infer that when located in close proximity on the epithelial surface, M. haemolytica and P. multocida mutually regulate one another.

Although frozen dairy desserts have a strong record of safety, recent outbreaks of foodborne disease linked to ice creams have brought new attention to this industry. There is concern that small-scale frozen dessert equipment may not comply with or be reviewed against published comprehensive design and construction sanitation specifications (National Sanitation Foundation or 3-A sanitary standards). Equipment sanitary design issues may result in reduced efficacy of cleaning and sanitation, thus increasing the likelihood of postprocess contamination with pathogenic bacteria. In this context, and given that Listeria monocytogenes outbreaks are of great concern for the frozen dessert industry, a complementary study was conducted to evaluate the fate of L. monocytogenes in ice cream mix on a stainless steel surface. Our results showed that L. monocytogenes survived for up to 6 weeks at room temperature and 9 weeks at 4°C in contaminated ice cream on a stainless steel surface. Furthermore, chlorine- and acid-based surface sanitizers had no detrimental effect on the L. monocytogenes when used at a concentration and contact time (1 min) recommended by the manufacturer; significant reduction in CFU required 5 to 20 min of contact time.

Highlights • We devise a novel system that allows M. haemolytica biofilms to form on epithelial cells. • Mucin significantly decreased biofilm formation. • Prior infection with several respiratory viruses does not alter biofilm formation. • Biofilms on epithelial cells were more resistant to some antibiotics than biofilms on polystyrene., Mannheimia haemolytica is the most important bacterial agent associated with the bovine respiratory disease complex (BRDC), which causes worldwide economic losses to the cattle industry. M. haemolytica cells initially colonize the tonsillar crypts in the upper respiratory tract of cattle, from where they can subsequently descend into the lungs to cause disease. Many bacteria exist as biofilms inside their hosts. We hypothesize that M. haemolytica colonization of cattle during its commensal state may include biofilm formation. To begin to assess this possibility, we developed an in vitro system to study biofilm formation directly on bovine respiratory epithelial cells. Using fixed primary bovine bronchial epithelial cells, we observed M. haemolytica biofilm formation after a 48 h incubation period at 37 °C. Addition of mucin, the main component of mucus present in the upper respiratory tract, decreased M. haemolytica biofilm formation on bovine epithelial cells. We investigated the effects of prior viral infection of the epithelial cells on subsequent biofilm formation by M. haemolytica and found negligible effects. Utilization of this model system will provide new insights into the potential role of biofilm formation by M. haemolytica in the pathogenesis of BRDC.

Histophilus somni is a Gram negative coccobacillus that causes respiratory, reproductive and central nervous system disease in cattle. The hallmark of H. somni infection is diffuse vasculitis and intravascular thrombosis that can lead to an acute central nervous system disease known as thrombotic meningoencephalitis (TME). Because neutrophils are major players in the pathophysiology of septic meningitis, we sought to determine their role in H. somni-induced fibrin clot formation in vitro. Bovine brain endothelial cells (TBBE cells) were exposed to H. somni cells at a 1:25 ratio, respectively. Conditioned media (CM) were collected after a 6 h incubation at 37 °C with 5% CO2, and then incubated with bovine peripheral blood polymorphonuclear neutrophils (PMNs). Following incubation, fibrin clot formation and tissue factor activity were assessed by a re-calcified plasma clotting assay. We found greater tissue factor activity in cell lysates and CM from H. somni-stimulated TBBE cells than unstimulated control TBBE cells. In addition, PMNs exposed to CM or extracellular vesicles from H. somni-stimulated TBBE cells expressed von Willenbrand factor, exhibited increased fibrin clot formation, and displayed greater tissue factor activity than PMNs exposed to CM or extracellular vesicles from unstimulated control TBBE cells. These results suggest that bovine PMNs might acquire extracellular vesicles from endothelial cells that leads to thrombus formation in bovine brain microvasculature and contribute to the process that characterizes TME.

Chronic nonhealing skin wounds often contain bacterial biofilms that prevent normal wound healing and closure and present challenges to the use of conventional wound dressings. We investigated inhibition of Pseudomonas aeruginosa biofilm formation, a common pathogen of chronic skin wounds, on a commercially available biological wound dressing. Building on prior reports, we examined whether the amino acid tryptophan would inhibit P. aeruginosa biofilm formation on the three-dimensional surface of the biological dressing. Bacterial biomass and biofilm polysaccharides were quantified using crystal violet staining or an enzyme linked lectin, respectively. Bacterial cells and biofilm matrix adherent to the wound dressing were visualized through scanning electron microscopy. D-/L-tryptophan inhibited P. aeruginosa biofilm formation on the wound dressing in a dose dependent manner and was not directly cytotoxic to immortalized human keratinocytes although there was some reduction in cellular metabolism or enzymatic activity. More importantly, D-/L-tryptophan did not impair wound healing in a splinted skin wound murine model. Furthermore, wound closure was improved when D-/L-tryptophan treated wound dressing with P. aeruginosa biofilms were compared with untreated dressings. These findings indicate that tryptophan may prove useful for integration into wound dressings to inhibit biofilm formation and promote wound healing.

Additional interventions to reduce the risk of Salmonella in ground meat products are needed in the industry. Fungi in the genus Muscodor produce an array of volatile compounds with antimicrobial activity. A commercial mixture of these volatile compounds (all considered to be GRAS), in proportions similar to that produced by the fungus, was assessed for its inhibitory activity against Salmonella in vitro. The minimal inhibitory concentration of the volatiles mixture for growth of Salmonella enterica in Mueller-Hinton broth was 0.5% (v/v). Exposure to the vapor phase of the volatile compounds similarly inhibited visible growth of Salmonella on agar (up to 6 cm zone of inhibition). Addition of the volatiles mixture (0.25%–1.0% v/w) inhibited Salmonella by 1.5 and 2.8 log 10 CFU in ground turkey (85% or 93% lean, respectively) and 2.2 and 1.7 log 10 CFU in ground beef (73% or 93% lean, respectively) during a 5 day period at 8 °C. Addition of the volatiles also inhibited growth of normal microflora on ground turkey at 8 °C by approximately 5.3 log 10 CFU. These findings indicate this mixture of volatile compounds retards growth of spoilage organisms and Salmonella in ground meat.

Mannheimia haemolytica is the primary bacterial agent in the bovine respiratory disease complex. It is thought that M. haemolytica colonizes the tonsillar crypts of cattle as a commensal and subsequently descends into the lungs to cause disease. Many bacterial species persist in the host as biofilms. There is limited information about the ability of M. haemolytica to form biofilms. The aim of this study was to develop an in vitro model for M. haemolytica biofilm formation. We found that M. haemolytica required at least 36 h to form robust biofilms on plastic in vitro when incubated in RPMI-1640 tissue culture medium at 37 °C, with maximal biofilm formation being evident at 48 h. Biofilm formation was inhibited by adding the monosaccharides d(+) galactose and d(+) mannose to the growth medium. Addition of antibodies to the M. haemolytica surface protein OmpA also reduced biofilm formation. Upon evaluating the macromolecules within the biofilm extracellular polymeric substance we found it contained 9.7 μg/cm(2) of protein, 0.81 μg/cm(2) of total carbohydrate, and 0.47 μg/cm(2) of extracellular DNA. Furthermore, proteinase K treatment significantly decreased biofilms (P

Silver is a widely used antimicrobial agent, yet when impregnated in macroscopic dressings, it stains wounds, can lead to tissue toxicity and can inhibit healing. Recently, we reported that polymeric nanofilms containing silver nanoparticles exhibit antimicrobial activity at loadings and release rates of silver that are 100x lower than conventional dressings. Here we report fabrication of composite microfilm constructs that provide a facile way to transfer the silver-loaded polymeric nanofilms onto wounds in vivo. The construct is fabricated from a silver nanoparticle-loaded polymeric nanofilm that is laminated with a micrometer-thick soluble film of polyvinylalcohol (PVA). When placed on a moist wound, the PVA dissolves, leaving the silver-loaded nanofilm immobilized on the wound-bed. In vitro, the immobilized nanofilms release

The objectives of this study were to develop an animal model to study Listeria monocytogenes infection during the peri-parturient period and identify sources of maternal shedding of the pathogen. Peri-parturient mice were infected intragastrically with L. monocytogenes that expressed bacterial luciferase. Mice were then imaged in vivo over time. Secreted breast milk samples from mice infected after parturition were enriched and plated for culture and imaging. Bioluminescence imaging technology was able to detect luciferase emitting L. monocytogenes in vaginal secretions and maternal and fetal organs at 72 and 96 h post infection in mice infected prior to, or just after, parturition. The results from this study clearly show that L. monocytogenes is shed in vaginal secretions and disseminates to the mammary chain, from which it can be shed in the milk of peri-parturient mice.

We report a general and facile method that permits the transfer (stacking) of multiple independently fabricated and nanoscopically thin polymeric films, each containing a distinct bioactive agent, onto soft biomedically relevant surfaces (e.g., collagen-based wound dressings). By using polyelectrolyte multilayer films (PEMs) formed from poly(allyl amine hydrochloride) and poly(acrylic acid) as representative polymeric nanofilms and micrometer-thick water-soluble poly(vinyl alcohol) sacrificial films to stack the PEMs, we demonstrate that it is possible to create stacked polymeric constructs containing multiple bioactive agents (e.g., antimicrobial and antibiofilm agents) on soft and chemically complex surfaces onto which PEMs cannot be routinely transferred by stamping. We illustrate the characteristics and merits of the approach by fabricating stacks of Ga3+ (antibiofilm agent)- and Ag+ (antimicrobial agent)-loaded PEMs as prototypical examples of agent-containing PEMs and demonstrate that the stacked PE...

Bone marrow (BM) hematopoietic stem cells differentiate to common lymphoid progenitors (CLP) that emigrate to the thymus to form T cells or differentiate into immature B cells that then migrate to the spleen for maturation. Rapid in vivo suppression of BM progenitor cells by a single oral or intraperitoneal dose of 7,12‐dimethylbenz(a)anthracene (DMBA) subsequently decreased mature lymphoid populations in BM, spleen, and thymus. These suppressions depended on BM CYP1B1, but not on aryl hydrocarbon receptor (AhR) activity. Suppression of pre‐B colony formation at 6 h, correlated with subsequent decreases in mature BM, spleen, and thymus populations (48–168 h). Thymus T‐cell ratios were unaffected, suggesting low local toxicity. DMBA treatment suppressed progenitor cells 24‐h post treatment in wild type (WT), AhRb mice, but not in Cyp1b1‐ko mice. The stem cell populations were sustained. Benzo(a)pyrene (BP) mediated a similar progenitor suppression up to 6 h, but reversal rapidly ensued. This recovery was absent in mice with a polycyclic aromatic hydrocarbon (PAH)‐resistant, AhRd genotype. This AhR‐dependent progenitor recovery with BP induction accounts for the absence of suppression of B220+ BM and spleen populations at 48–168 h. However, DMBA and BP produced similar profiles for thymus cell suppression, independent of AhR genotype. Thus, lymphoid progenitors may exit the BM to the thymus prior to the BP reversal. This progenitor recovery is associated with elevated chemokines and cytokines that depend on AhR‐mediated induction of CYP1A1. This response increased constitutively in Cyp1b1‐ko BM, demonstrating that CYP1B1 metabolizes local stimulants that impact a basal progenitor protection process.

Histophilus somni resides as part of the normal microflora in the upper respiratory tract of healthy cattle. From this site, the organism can make its way into the lower respiratory tract, where it is one of the important bacterial agents of the respiratory disease complex. If H. somni cells disseminate to the bloodstream, they frequently result in thrombus formation. A series of in vitro investigations have examined potential mechanisms that might contribute to such thrombus formation. Earlier work showed that H. somni can stimulate some bovine endothelial cells to undergo apoptosis. More recent studies indicate that H. somni stimulates endothelial cell tissue factor activity and disrupts intercellular junctions. The net effect is to enhance procoagulant activity on the endothelium surface and to make the endothelial monolayer more permeable to molecules, leukocytes, and perhaps H. somni cells. H. somni also activates bovine platelets, which also can enhance tissue factor activity on the endothelium surface. When exposed to H. somni, bovine neutrophils and mononuclear phagocytes form extracellular traps in vitro. Ongoing research is investigating how the interplay among endothelial cells, platelets, and leukocytes might contribute to the thrombus formation seen in infected cattle.

In this study, we explored the use of vinegar, or vinegar and canola oil as a salad dressing, to reduce bacterial levels on spinach leaves. We found that incubation of spinach leaves with various types of vinegar substantially reduced the predominantly gram-negative microflora. A similar response was observed when spinach leaves were incubated with white vinegar mixed in various proportions with canola oil, as used in salad dressing. We assessed the effects of vinegar, or vinegar and oil, on spinach leaves that had been experimentally inoculated with a cocktail of Salmonella enterica strains. Allowing the mixture to sit at room temperature for at least 20 min resulted in a substantial reduction (up to 2.0 log CFU) in numbers of S. enterica. Vinegar and oil caused a limited reduction in CFU (0.5 log) for spinach leaves experimentally inoculated with a cocktail of Listeria monocytogenes strains. These findings suggest that mixing spinach leaves with vinegar and oil as a salad dressing can reduce the bacterial load associated with the spinach leaves, including Salmonella if it is present.

We report the design of polyelectrolyte multilayers (PEMs) that can be prefabricated on an elastomeric stamp and mechanically transferred onto biomedically-relevant soft materials, including medical-grade silicone elastomers (E′~450–1500 kPa; E′-elastic modulus) and the dermis of cadaver-skin (E′~200–600 kPa). Whereas initial attempts to stamp PEMs formed from poly(allylamine hydrochloride) and poly(acrylic acid) resulted in minimal transfer onto soft materials, we report that integration of micrometer-sized beads into the PEMs (thicknesses of 6–160 nm) led to their quantitative transfer within 30 seconds of contact at a pressure of ~196 kPa. To demonstrate the utility of this approach, PEMs were impregnated with a range of loadings of silver-nanoparticles and stamped onto the dermis of human cadaver-skin (a wound-simulant) that was subsequently incubated with bacterial cultures. Skin-dermis stamped with PEMs that released 0.25±0.01 μg cm−2 of silver ions caused a 6 log10 reduction in colony forming units of Staphylococcus epidermidis and Pseudomonas aeruginosa within 12 h. Significantly, this level of silver release is below that which is cytotoxic to NIH 3T3 mouse fibroblast cells. Overall, this study describes a general and facile approach for the functionalization of biomaterial surfaces without subjecting them to potentially deleterious processing conditions.

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5′ and 3′ regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein ( P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera ( P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle ( P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis -infected cattle mount a humoral response to both MAP1152 and MAP1156 cross-reactive epitopes. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immunopathogenesis of JD.

Silver is widely used as a biocidal agent in ointments and wound dressings. However, it has also been associated with tissue toxicity and impaired healing. In vitro characterization has also revealed that typical loadings of silver employed in ointments and dressings (approximately 100 microg/cm(2)) lead to cytotoxicity. In this paper, we report the results of an initial study that sought to determine if localization of carefully controlled loadings of silver nanoparticles within molecularly thin films immobilized on surfaces can lead to antimicrobial activity without inducing cytotoxicity. Polymeric thin films of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were prepared by layer-by-layer deposition and loaded with approximately 0.4 microg/cm(2) to approximately 23.6 microg/cm(2) of silver nanoparticles. Bacterial killing efficiencies of the silver-loaded films were investigated against Staphylococcus epidermidis, a gram-positive bacterium, and it was determined that as little as approximately 0.4 microg/cm(2) of silver in the polymeric films caused a reduction of 6log(10)CFU/mL (99.9999%) bacteria in suspensions incubated in contact with the films (water-borne assays). Significantly, whereas the antibacterial films containing high loadings of silver were found to be toxic to a murine fibroblast cell line (NIH-3T3), the polymeric films containing approximately 0.4 microg/cm(2) of silver were not toxic and allowed attachment, and growth of the mammalian cells. Thus, the results of this study go beyond prior reports by identifying silver-impregnated, polymeric thin films that are compatible with in vitro mammalian cell culture yet exhibit antibacterial activity. These results support the hypothesis that localization of carefully controlled loadings of silver nanoparticles within molecularly thin polymeric films can lead to antimicrobial activity without cytotoxicity. More broadly, this strategy of modifying surfaces with minimal loadings of bioactive molecules indicates the basis of approaches that may permit management of microbial burden in wound beds without impairment of wound healing.

Mannheimia haemolytica leukotoxin (LktA) is a member of the RTX toxin family that specifically kills ruminant leukocytes. Previous studies have shown that LktA induces apoptosis in susceptible cells via a caspase-9-dependent pathway that involves binding of LktA to mitochondria. In this study, using the bioinformatics tool MitoProt II we identified an N-terminal amino acid sequence of LktA that represents a mitochondrial targeting signal (MTS). We show that expression of this sequence, as a GFP fusion protein within mammalian cells, directs GFP to mitochondria. By immunoprecipitation we demonstrate that LktA interacts with the Tom22 and Tom40 components of the translocase of the outer mitochondrial membrane (TOM), which suggests that import of this toxin into mitochondria involves a classical import pathway for endogenous proteins. We also analysed the amino acid sequences of other RTX toxins and found a MTS in the N-terminal region of Actinobacillus pleuropneumoniae ApxII and enterohaemorrhagic Escherichia coli EhxA, but not in A. pleuropneumoniae ApxI, ApxIII, Aggregatibacter actinomycetemcomitans LtxA or the haemolysin (HlyA) from uropathogenic strains of E. coli. These findings provide a new evidence for the importance of the N-terminal region in addressing certain RTX toxins to mitochondria.

Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-alpha mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1beta antibody or human soluble TNF-alpha receptor (sTNF-alphaR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1beta antibody, sTNF-alphaR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease, a chronic intestinal infection in ruminants. Adenosine 5′-Triphosphate (ATP) has been reported to induce killing of several Mycobacterium species in human and murine macrophages. We investigated whether ATP secreted from M. avium subsp. paratuberculosis -infected bovine monocytes affects intracellular survival of the bacilli. Bovine monocytes constitutively secreted ATP during an 8-day incubation period in vitro ; however, M. avium subsp. paratuberculosis infection did not enhance ATP release. Removal of extracellular ATP by the addition of apyrase increased the viability of infected monocytes, but surprisingly decreased the number of viable intracellular bacilli. In contrast to previous reports, addition of extracellular ATP (1 mM) increased intracellular survival of M. avium subsp. paratuberculosis in bovine monocytes. Neither apyrase nor ATP altered production of reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI) by bovine monocytes. These results suggest that ATP release from infected bovine monocytes improves, rather than decreases, the intracellular survival of M. avium subsp. paratuberculosis .

Listeria monocytogenes remains an important foodborne pathogen, and strategies designed to decrease the susceptibility of selected patient populations to foodborne pathogens are therefore desirable. Our objective was to determine if the poloxamer P85 was protective against L. monocytogenes infection. Caco-2 cells were treated with 0.1% (w/v) P85 and challenged with 10(7) L. monocytogenes EGD for 1 hour. A standard gentamicin protection assay was performed to determine invasion differences between the experimental groups. Effects of P85 on the pathogen were studied by measuring bacterial growth and ATP concentrations. In a murine model of listeriosis, FVB mice were administered 150 mg/kg P85 or vehicle control 45 minutes prior to intragastric inoculation of 10(7) L. monocytogenes. Dissemination of the pathogen from the gastrointestinal tract to the liver and spleen was determined 24 hours after bacterial challenge. Pretreatment of Caco-2 cells with P85 significantly decreased L. monocytogenes invasion compared to controls. Repletion of ATP reversed the protective effects of P85. No changes in bacterial ATP or growth profile were detected in P85-treated bacteria. Administration of P85 to mice prior to infection led to decreased dissemination to the liver and spleen compared to vehicle-treated mice. P85 is protective against L. monocytogenes infection when administered prior to bacterial challenge. Modulation of host ATP levels appears to be crucial for the protective effects of P85.

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.

Ready-to-eat (RTE) deli meats have been categorized as high-risk foods for contraction of foodborne listeriosis. Several recent listeriosis outbreaks have been associated with the consumption of RTE deli turkey meat. In this study, we examined whether the growth of Listeria monocytogenes F2365 on commercially prepared RTE deli turkey meat causes listerial cells to become more resistant to inactivation by synthetic gastric fluid (SGF). Listerial cells grown on turkey meat to late logarithmic-early stationary phase were significantly more resistant to SGF at pH 7.0, 5.0, or 3.5 than listerial cells grown in brain heart infusion (BHI) broth. The pH was lower in the fluid in packages of turkey meat than in BHI broth (6.5 versus 7.5). However, listerial cells grown in BHI broth adjusted to a lower pH (6.0) did not exhibit enhanced resistance to SGF. The lesser resistance to SGF of listerial cells grown in BHI broth may be due, in part, to the presence of glucose (0.2%). This study indicates the environment presented by the growth of L. monocytogenes on deli turkey meat affects its ability to survive conditions it encounters in the gastrointestinal tract.

Mycobacterium aviumsubsp.paratuberculosisis the etiologic agent of Johne's disease, a chronic granulomatous enteritis in ruminants. ATP has been reported to induce cell death of macrophages and killing ofMycobacteriumspecies in human and murine macrophages. In this study we investigated the short-term effect of ATP on the viability ofM. aviumsubsp.paratuberculosis-infected bovine mononuclear phagocytes and the bacilli within them. Addition of 5 mM ATP toM. aviumsubsp.paratuberculosis-infected bovine monocytes resulted in 50% cytotoxicity of bovine monocytes at 24 h. Addition of 2′(3′)-O-(4-benzoylbenzoyl) ATP triethylammonium salt (Bz-ATP), which is a longer-lived ATP homologue and purinergic receptor agonist, significantly increased the uptake of YO-PRO, which is a marker for membrane pore activation by P2X receptors. Addition of Bz-ATP also stimulated lactate dehydrogenase release and caspase-3 activity in infected bovine monocytes. Neither ATP nor Bz-ATP reduced the survival ofM. aviumsubsp.paratuberculosisin bovine mononuclear phagocytes. Likewise, addition of ATP or Bz-ATP was cytotoxic to murine macrophage cell lines (RAW 264.7 and J774A.1 cells) but did not affect the intracellular survival ofM. aviumsubsp.paratuberculosis, nor were the numbers of viableMycobacterium aviumsubsp.aviumorMycobacterium bovisBCG cells altered in bovine mononuclear phagocytes or J774A.1 cells following ATP or Bz-ATP treatment. These data suggest that extracellular ATP does not induce the killing of intracellularM. aviumsubsp.paratuberculosisin bovine mononuclear phagocytes.

“Haemophilus somnus” causes thrombotic meningoencephalitis in cattle. Our laboratory has previously reported thatH. somnushas the ability to adhere to, but not invade, bovine brain endothelial cells (BBEC) in vitro. The goal of this study was to determine ifH. somnusalters brain endothelial cell monolayer integrity in vitro, in a manner that would be expected to contribute to inflammation of the central nervous system (CNS). Monolayer integrity was monitored by measuring transendothelial electrical resistance (TEER) and albumin flux. BBEC incubated withH. somnusunderwent rapid cytoskeletal rearrangement, significant increases in albumin flux, and reductions in TEER. Decreased monolayer TEER was preceded by phosphorylation of the myosin regulatory light chain and was partially dependent on tumor necrosis factor alpha and myosin light-chain kinase but not interleukin-1β. Neither heat-killedH. somnus, formalin-fixedH. somnus, nor purified lipooligosaccharide altered monolayer integrity within a 2-h incubation period, whereas conditioned medium fromH. somnus-treated BBEC caused a modest reduction in TEER. The data from this study support the hypothesis that viableH. somnusalters integrity of the blood-brain barrier by promoting contraction of BBEC and increasing paracellular permeability of the CNS vasculature.

Endothelial cells were once viewed as relatively inert cells lining the vasculature. They are now recognized as active and responsive regulators of coagulation, platelet adhesion, fluid homeostasis, wound healing, leukocyte extravasation and vascular tone. Endothelial cells play a key role in the host response to infectious agents by regulating leukocyte trafficking, producing inflammatory cytokines and presenting antigen in association with major histocompatibility class II (MHC II) molecules. A number of infectious agents have a tropism for endothelial cells. Infection of endothelial cells can promote thrombosis, vascular leakage, and increased adherence and emigration of leukocytes. Furthermore, activation of a systemic inflammatory response, in the absence of direct endothelial cell infection, can also lead to endothelial cell dysfunction. The purpose of this review is to highlight the interactions between endothelial cells and infectious or inflammatory agents that contribute to coagulation disturbances, vasculitis and edema. A select group of viral and bacterial pathogens will be used as examples to demonstrate how endothelial cell dysfunction contributes to the pathogenesis of infectious and inflammatory disorders.

Histophilus somni is a gram-negative pleomorphic bacillus that causes respiratory disease primarily in cattle. Clinical signs of H. somni infection can include pneumonia, abortion, arthritis, septicemia, myocarditis, vasculitis, and an acute condition known as thrombotic meningoencephalitis (1, 3, 8, 10, 13). The ability of H. somni to modify its lipooligosaccharide (LOS) composition and structure is thought to play an important role in its virulence. H. somni can incorporate sialic acid into the outer core of the LOS molecule, and this event has been shown to be critical for resistance to serum-mediated killing (14). It has been reported that some strains of H. somni that do not incorporate sialic acid are unable to produce disease (5). Another modification is the addition of phosphorylcholine (ChoP) to the outer core of the LOS (12). H. somni exhibits extensive intrastrain variability in ChoP incorporation into the LOS molecule (12). The possible contribution of ChoP to H. somni pathogenesis is unknown. For other species of bacteria, ChoP can be incorporated into bacterial structures such as fibrillar proteins and cell wall components that are important for bacterial adherence to host cells. For example, the expression of ChoP by Streptococcus pneumoniae has been reported to contribute to pneumococcal adherence and invasion in the lung (6, 30) and the brain (20). Similarly, the expression of ChoP on the LOS of Haemophilus influenzae contributes to its binding and internalization by human epithelial cells (27, 28). This adherence was demonstrated to be due to an interaction between ChoP expressed on LOS and the platelet-activating factor (PAF) receptor on epithelial cells. ChoP has also been found in Actinobacillus actinomycetemcomitans, which facilitates binding to the PAF receptor on human vascular endothelial cells, followed by internalization of the bacteria (7, 23, 24). ChoP has also been detected in Bacillus spp. and Clostridium spp., and the lipopolysaccharide of Escherichia coli O26:B6 was found to activate human platelets through a PAF receptor-dependent pathway (9, 19). Our laboratory has previously reported that H. somni and its LOS can activate bovine platelets (16). We also found that H. somni, but not its LOS, could induce platelet aggregation. The mechanism by which H. somni induces platelet aggregation is unknown. It has previously been demonstrated that endotoxin and bacteria can adhere to and activate platelets from several different mammalian species (2, 11, 18, 21, 22, 31-33). For this study, we sought to investigate the interaction between H. somni and bovine platelets and determine if bacterial expression of ChoP affects platelet activation. We first wanted to ascertain whether platelet aggregation was induced by ChoP-expressing H. somni cells. Using colony immunoblotting with an anti-phosphorylcholine antibody (15), two H. somni variants of strain 7735 were selected for either high or low expression of ChoP. These populations were enriched through selective passage in culture. Bovine platelets (2.5 × 108 platelets) (isolation procedures were described previously [16]) were incubated with one of the two H. somni variants (multiplicity of infection [MOI] of 5:1) for 10 min in a Chronolog aggregometer. As a positive control, platelets were treated with PAF (10−6 M; Calbiochem) to induce irreversible aggregation within 5 min. It was found that ChoP+ H. somni induced platelet aggregation, while ChoP− H. somni did not (Fig. 1A and B). ChoP+ H. somni consistently induced approximately 15% aggregation, which was not reversible within a 30-min incubation period (data not shown). Upon microscopic examination, platelet aggregates could be observed following incubation with ChoP+ but not with ChoP− H. somni (Fig. 1C and D). In addition, we observed ChoP+ H. somni within bovine platelet aggregates. Pretreatment of ChoP+ H. somni with polymyxin B (10 μg/ml; Sigma) for 10 min resulted in a modest decrease in the ability of H. somni to induce aggregation (Fig. ​(Fig.1E1E). FIG. 1. ChoP+ but not ChoP− H. somni induces bovine platelet aggregation. Platelets (500 μl, 2.5 × 108 platelets/ml) were placed in siliconized glass cuvettes and incubated with ChoP+ or ChoP− H. somni (MOI of 5:1) ... We next considered the possibility that H. somni may interact with the PAF receptor on platelets. To exclude the contribution of platelet-derived PAF, we incubated platelets with previously described selective inhibitors of phospholipase A2, AACOCF3 (10 μM; Calbiochem) or cPLA (10 μM; Calbiochem), for 10 min prior to the addition of ChoP+ H. somni (17, 26). Some platelets were preincubated with the PAF receptor antagonist WEB 2170 (10 μg/ml) at a concentration that was previously demonstrated to inhibit platelet activation by PAF (29). Platelets preincubated with WEB 2170 but not inhibitors of PAF synthesis demonstrated a significant diminution in platelet aggregation following incubation with ChoP+ H. somni (Fig. ​(Fig.22). FIG. 2. Platelet aggregation is inhibited by the PAF receptor antagonist WEB 2170. Platelets (500 μl, 2.5 × 108 platelets/ml) were pretreated for 10 min with the PAF receptor antagonist WEB 2170 (10 μg/ml) or the PAF synthesis inhibitor ... To study PAF receptor signaling, we used an inhibitor of downstream signaling through phospholipase Cβ (D609, 200 μM; Calbiochem) and the PAF receptor antagonist WEB 2170 (10 μg/ml). Using an antibody against P-selectin as a marker of platelet activation (BD Bioscience), we observed low baseline P-selectin levels in unstimulated platelets. Following incubation with PAF (10−7 M) or ChoP+ H. somni (MOI of 5:1), we observed a significant increase in platelet activation (Fig. 3A and B). However, neither PAF antagonist resulted in a significant decrease in platelet P-selectin expression induced by ChoP+ H. somni, suggesting that PAF receptor activation is not required for platelet activation by ChoP+ H. somni. In addition, D609 inhibited PAF-induced aggregation but not aggregation induced by ChoP+ H. somni (data not shown). We next compared platelet activation in response to ChoP+ or ChoP− H. somni as determined by flow cytometry evaluation of P-selectin expression and fibrinogen binding. The latter event, which is required for platelet aggregation, was detected using an antibody against platelet-bound fibrinogen (American Diagnostica, Stamford, CT). We found that both ChoP+ and ChoP− H. somni cells induced significant platelet P-selectin expression (Fig. ​(Fig.3C).3C). Surprisingly, ChoP+ H. somni did not induce a significant level of platelet fibrinogen binding compared to platelets stimulated with PAF. The lack of fibrinogen binding suggests that H. somni may induce platelet aggregate formation using a mechanism distinct from activation induced by PAF. FIG. 3. H. somni-induced platelet activation is independent of PAF receptor signaling. Bovine platelets (2.5 × 108) were incubated for 10 min at 37°C with ChoP+ or ChoP− H. somni (MOI of 5:1) or PAF (10−7 M). The platelets ... Transmission electron microscopy (TEM) was used to visualize whether the expression of ChoP by H. somni affected the adherence of the bacteria to platelets or platelet morphology. Bovine platelets were incubated with ChoP+ or ChoP− H. somni for 10 min before being fixed and embedded for TEM. Unactivated platelets demonstrated their characteristic discoid shape, with no significant changes in morphology (Fig. ​(Fig.4A).4A). Following incubation with ChoP+ or ChoP− H. somni, bovine platelets exhibited spreading, pseudopod formation, and changes in morphology indicative of platelet activation (Fig. ​(Fig.4B).4B). Although platelet spreading and pseudopod formation were observed following exposure to either ChoP+ or ChoP− H. somni, only ChoP+ H. somni cells were capable of inducing platelet aggregation (Fig. 4C and D). In addition, obvious binding of platelets to ChoP+ H. somni only was observed. Closer examination of this interaction revealed surface structures on ChoP+ H. somni that appeared to facilitate the adherence of the bacteria to the platelets (Fig. 4E and F). FIG. 4. Transmission electron microscopy of platelets activated by H. somni. Platelets (500 μl, 2.5 × 108 platelets/ml) were placed in siliconized glass cuvettes and incubated with ChoP+ or ChoP− H. somni (MOI of 5:1) in a Chrono-Log ... In this study, we demonstrated that ChoP expression by H. somni plays a role in its interactions with bovine platelets. Preincubation of platelets with the PAF receptor antagonist WEB 2170 prevented platelet aggregation. However, the ineffectiveness of downstream PAF receptor signaling inhibitors suggests that PAF receptor signaling was not required for platelet activation or aggregation. One possible explanation may reflect the location of the ChoP molecule on H. somni LOS, where it is coupled to the primary glucose residue that is bound to heptose I (4). Steric interference from sugars in the outer core may make ChoP less accessible to interactions with the extracellular environment. In contrast, the ChoP group on H. influenza LOS is located on a terminal glycose on either heptose I or heptose III, where it may be more accessible for PAF receptor binding (25). Our findings suggest that H. somni may interact with the PAF receptor, but the resulting events are dissimilar to those by which PAF activates platelets. Upon careful examination, we conclude that aggregation reflected cross-linking between platelets and bacteria, a response that was not observed in bacteria that lacked ChoP (Fig. 1C and D and 4B to D). Future work will be directed at determining if H. somni ChoP interacts directly with the PAF receptor or through another platelet receptor and how these interactions may contribute to platelet activation.

SummaryThrombotic 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 micro- vascular endothelial cells (BBEC) in vitro. We demonstrate that BBEC exposed to H. somnus secrete significant levels of the proinflammatory and procoagulative cytokines TNF-α and IL-1β. 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.

Antimicrobial surfaces with covalently attached biocidal functionalities only kill microbes that come into direct contact with the surfaces (contact-killing surfaces). Herein, the activity of contact-killing surfaces is shown to be enhanced by using gradients in the concentration of soluble chemoattractants (CAs) to attract bacteria to the surfaces. Two natural and nonbiocidal CAs (aspartate and glucose) were used to attract bacteria to model surfaces decorated with quaternary ammonium groups (known to kill bacteria that come into contact with them). These results demonstrate the killing of Escherichia coli and Salmonella typhimurium, two common pathogens, at levels 10- to 20-times greater than that of the native surfaces alone. This approach is general and provides new strategies for the design of active or dynamic contact-killing surfaces with enhanced antimicrobial activities.

A 2014 multistate listeriosis outbreak was linked to consumption of caramel-coated apples, an unexpected and previously unreported vehicle for Listeria monocytogenes. This outbreak was unanticipated because both the pH of apples (3 log10 in apples with sticks, whereas only a 1-log10 increase was observed even after 1 week for caramel-coated apples without sticks. When stored at 7°C, apples with sticks exhibited an approximately 1.5-log10 increase in L. monocytogenes levels at 28 days, whereas no growth was observed in apples without sticks. We infer that insertion of a stick into the apple accelerates the transfer of juice from the interior of the apple to its surface, creating a microenvironment at the apple-caramel interface where L. monocytogenes can rapidly grow to levels sufficient to cause disease when stored at room temperature., IMPORTANCE Neither caramel nor apples are a food where the pathogenic bacterium Listeria monocytogenes should grow, as caramel does not contain enough free water and apples are too acidic. Caramel-coated apples, however, were recently linked to a deadly outbreak of listeriosis. We hypothesized that inserting a stick into the apple releases juice to the interface between the apple and caramel, providing a more hospitable environment than either component alone. To test this hypothesis, apples were inoculated with L. monocytogenes prior to caramel dipping. Some apples had sticks inserted into them before dipping, while others did not. No growth of L. monocytogenes occurred on refrigerated caramel apples without sticks, whereas slow growth was observed on refrigerated caramel apples with sticks. In contrast, significant pathogen growth was observed within 3 days at room temperature on caramel apples with sticks inserted. Food producers should consider interfaces between components within foods as potential niches for pathogen growth.

The persistence of bacterial biofilms in chronic wounds delays wound healing. Although Ga(3+) can inhibit or kill biofilms, precipitation as Ga(OH)3 has prevented its use as a topical wound treatment. The design of a microfilm construct comprising a polyelectrolyte film that releases noncytotoxic concentrations of Ga(3+) over 20 d and a dissolvable micrometer-thick film of polyvinylalcohol that enables facile transfer onto biomedically important surfaces is reported. By using infrared spectroscopy, it is shown that the density of free carboxylate/carboxylic acid and amine groups within the polyelectrolyte film regulates the capacity of the construct to be loaded with Ga(3+) and that the density of covalent cross-links introduced into the polyelectrolyte film (amide-bonds) controls the release rate of Ga(3+) . Following transfer onto the wound-contact surface of a biologic wound dressing, an optimized construct is demonstrated to release ≈0.7 μg cm(-2) d(-1) of Ga(3+) over 3 weeks, thus continuously replacing Ga(3+) lost to precipitation. The optimized construct inhibits formation of P. aeruginosa (two strains; ATCC 27853 and PA01) biofilms for up to 4 d and causes pre-existing biofilms to disperse. Overall, this study provides designs of polymeric constructs that permit facile modification of the wound-contacting surfaces of dressings and biomaterials to manage biofilms.

Chronic nonhealing skin wounds often contain bacterial biofilms that prevent normal wound healing and closure and present challenges to the use of conventional wound dressings. We investigated inhibition of Pseudomonas aeruginosa biofilm formation, a common pathogen of chronic skin wounds, on a commercially available biological wound dressing. Building on prior reports, we examined whether the amino acid tryptophan would inhibit P. aeruginosa biofilm formation on the three-dimensional surface of the biological dressing. Bacterial biomass and biofilm polysaccharides were quantified using crystal violet staining or an enzyme linked lectin, respectively. Bacterial cells and biofilm matrix adherent to the wound dressing were visualized through scanning electron microscopy. D-/L-tryptophan inhibited P. aeruginosa biofilm formation on the wound dressing in a dose dependent manner and was not directly cytotoxic to immortalized human keratinocytes although there was some reduction in cellular metabolism or enzymatic activity. More importantly, D-/L-tryptophan did not impair wound healing in a splinted skin wound murine model. Furthermore, wound closure was improved when D-/L-tryptophan treated wound dressing with P. aeruginosa biofilms were compared with untreated dressings. These findings indicate that tryptophan may prove useful for integration into wound dressings to inhibit biofilm formation and promote wound healing.

Histophilus somni resides as part of the normal microflora in the upper respiratory tract of healthy cattle. From this site, the organism can make its way into the lower respiratory tract, where it is one of the important bacterial agents of the respiratory disease complex. If H. somni cells disseminate to the bloodstream, they frequently result in thrombus formation. A series of in vitro investigations have examined potential mechanisms that might contribute to such thrombus formation. Earlier work showed that H. somni can stimulate some bovine endothelial cells to undergo apoptosis. More recent studies indicate that H. somni stimulates endothelial cell tissue factor activity and disrupts intercellular junctions. The net effect is to enhance procoagulant activity on the endothelium surface and to make the endothelial monolayer more permeable to molecules, leukocytes, and perhaps H. somni cells. H. somni also activates bovine platelets, which also can enhance tissue factor activity on the endothelium surface. When exposed to H. somni, bovine neutrophils and mononuclear phagocytes form extracellular traps in vitro. Ongoing research is investigating how the interplay among endothelial cells, platelets, and leukocytes might contribute to the thrombus formation seen in infected cattle.

Haemophilus somnuscan cause a devastating fibrinopurulent meningitis with thrombotic vasculitis and encephalitis in cattle. The mechanisms used byH. somnusto migrate from the bloodstream into the central nervous system (CNS) are unknown. In this study, we demonstrate thatH. somnusadheres to, but does not invade, bovine brain endothelial cells (BBEC) in vitro. The number of adherentH. somnuswas significantly increased by prior activation of the BBEC with tumor necrosis factor alpha (TNF-α). Addition of exogenous glycosaminoglycans significantly reducedH. somnusadherence to resting and TNF-α-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 adherentH. 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 ofH. somnusto BBEC. Using heparin-labeled agarose beads, we demonstrated a high-molecular-weight heparin-binding protein expressed byH. somnus. Heparin was also shown to bindH. somnusin a 4°C binding assay. These data suggest that heparin-binding proteins onH. somnuscould serve as initial adhesins to sulfated proteoglycans on the endothelial cell surface, thus contributing to the ability ofH. somnusto infect the bovine CNS.