Mixed species biofilms of Fusobacterium necrophorum and Porphyromonas levii impair the oxidative response of bovine neutrophils in vitro.

Biofilms composed of anaerobic bacteria can result in persistent infections and chronic inflammation. Host immune cells have difficulties clearing biofilm-related infections and this can result in tissue damage. Neutrophils are a vital component of the innate immune system and help clear biofilms. The comparative neutrophilic response to biofilms versus planktonic bacteria remains incompletely understood, particularly in the context of mixed infections. The objective of this study was to generate mixed species anaerobic bacterial biofilms composed of two opportunistic pathogens, Fusobacterium necrophorum and Porphyromonas levii, and evaluate neutrophil responses to extracellular fractions from both biofilms and planktonic cell co-cultures of the same bacteria. Purified bovine neutrophils exposed to culture supernatants from mixed species planktonic bacteria showed elevated oxidative activity compared to neutrophils exposed to biofilms composed of the same bacteria. Bacterial lipopolysaccharide plays a significant role in the stimulation of neutrophils; biofilms produced substantially more lipopolysaccharide than planktonic bacteria under these experimental conditions. Removal of lipopolysaccharide significantly reduced neutrophil oxidative response to culture supernatants of planktonic bacteria. Oxidative responses to LPS-removed biofilm supernatants and LPS-removed planktonic cell supernatants were similar. The limited neutrophil response to biofilm bacteria observed in this study supports the reduced ability of the innate immune system to eradicate biofilm-associated infections. Lipopolysaccharide is likely important in neutrophil response; however, the presence of other extracellular, immune modifying molecules in the bacterial media also appears to be important in altering neutrophil function.

Tulathromycin exerts proresolving effects in bovine neutrophils by inhibiting phospholipases and altering leukotriene B4, prostaglandin E2, and lipoxin A4 production.

The accumulation of neutrophils and proinflammatory mediators, such as leukotriene B4 (LTB4), is a classic marker of inflammatory disease. The clearance of apoptotic neutrophils, inhibition of proinflammatory signaling, and production of proresolving lipids (including lipoxins, such as lipoxin A4 [LXA4]) are imperative for resolving inflammation. Tulathromycin (TUL), a macrolide used to treat bovine respiratory disease, confers immunomodulatory benefits via mechanisms that remain unclear. We recently reported the anti-inflammatory properties of TUL in bovine phagocytes in vitro and in Mannheimia haemolytica-challenged calves. The findings demonstrated that this system offers a powerful model for investigating novel mechanisms of pharmacological immunomodulation. In the present study, we examined the effects of TUL in a nonbacterial model of pulmonary inflammation in vivo and characterized its effects on lipid signaling. In bronchoalveolar lavage (BAL) fluid samples from calves challenged with zymosan particles (50 mg), treatment with TUL (2.5 mg/kg of body weight) significantly reduced pulmonary levels of LTB4 and prostaglandin E2 (PGE2). In calcium ionophore (A23187)-stimulated bovine neutrophils, TUL inhibited phospholipase D (PLD), cytosolic phospholipase A2 (PLA2) activity, and the release of LTB4. In contrast, TUL promoted the secretion of LXA4 in resting and A23187-stimulated neutrophils, while levels of its precursor, 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], were significantly lower. These findings indicate that TUL directly modulates lipid signaling by inhibiting the production of proinflammatory eicosanoids and promoting the production of proresolving lipoxins.

Effects of growth hormone on the ontogenetic allometry of craniofacial bones.

Organism size is controlled by interactions between genetic and environmental factors mediated by hormones with systemic and local effects. As changes in size are usually not isometric, a considerable diversity in shape can be generated through modifications in the patterns of ontogenetic allometry. In this study we evaluated the role of timing and dose of growth hormone (GH) release on growth and correlated shape changes in craniofacial bones. Using a longitudinal study design, we analyzed GH deficient mice treated with GH supplementation commencing pre- and post-puberty. We obtained 3D in vivo micro-CT images of the skull between 21 and 60 days of age and used geometric morphometrics to analyze size and shape changes among control and GH deficient treated and non-treated mice. The variable levels of circulating GH altered the size and shape of the adult skull, and influenced the cranial base, vault, and face differently. While cranial base synchondroses and facial sutures were susceptible to either the direct or indirect effect of GH supplementation, its effect was negligible on the vault. Such different responses support the role of intrinsic growth trajectories of skeletal components in controlling the modifications induced by systemic factors. Contrary to the expected, the timing of GH treatment did not have an effect on catch-up growth. GH levels also altered the ontogenetic trajectories by inducing changes in their location and extension in the shape space, indicating that differences arose before 21 days and were further accentuated by a truncation of the ontogenetic trajectories in GHD groups.

Direct and indirect anti-inflammatory effects of tulathromycin in bovine macrophages: inhibition of CXCL-8 secretion, induction of apoptosis, and promotion of efferocytosis.

Recent evidence indicates that immunomodulation by antibiotics may enhance their clinical efficacy. Specifically, drug-induced leukocyte apoptosis and macrophage efferocytosis have been shown to promote the resolution of inflammation in a variety of disease settings. Tulathromycin is a new macrolide antibiotic for the treatment of bovine respiratory disease. The direct antimicrobial effects of the drug alone do not fully justify its superior clinical efficacy, and we hypothesize that tulathromycin may have immunomodulating properties. We recently reported that tulathromycin promotes apoptosis and inhibits proinflammatory NF-κB signaling in bovine neutrophils. In this study, we investigated the direct and indirect anti-inflammatory effects of tulathromycin in bovine macrophages. The findings indicate that bovine monocyte-derived macrophages and alveolar macrophages readily phagocytose tulathromycin-induced apoptotic neutrophils both in vitro and in the airways of Mannheimia haemolytica-infected calves. Moreover, tulathromycin promotes delayed, concentration-dependent apoptosis, but not necrosis, in bovine macrophages in vitro. Activation of caspase-3 and detection of mono- and oligonucleosomes in bovine monocyte-derived macrophages treated with tulathromycin was observed 12 h posttreatment; pretreatment with a pan-caspase inhibitor (ZVAD) blocked the proapoptotic effects of the drug. Lastly, tulathromycin inhibited the secretion of proinflammatory CXCL-8 in lipopolysaccharide (LPS)-stimulated bovine macrophages; this effect was independent of caspase activation or programmed cell death. Taken together, these immunomodulating effects observed in bovine macrophages help further elucidate the mechanisms through which tulathromycin confers anti-inflammatory and proresolution benefits. Furthermore, these findings offer novel insights on how antibiotics may offer anti-inflammatory benefits by modulating macrophage-mediated events that play a key role in inflammation.

The impact of freezing and multiple freeze-thaw cycles on Brix refractometry estimates of immunoglobulin concentration in beef cattle colostrum.

Evaluation of immunoglobulin G (IgG) concentration in colostrum is important to guide on-farm management. Studies have shown that digital Brix refractometry accurately estimates colostrum IgG concentration in both dairy and beef cattle colostrum. Colostrum is often frozen in both clinical and research settings. The implications of this freezing on the accuracy of Brix refractometry measurements are largely unknown. The first objective of this study was to evaluate the agreement between digital Brix percentage measurements of IgG in beef cattle colostrum taken before and after different durations of freezing. The second objective was to evaluate the effects of multiple freeze-thaw (FT) cycles on Brix percentage measurements of IgG in beef cattle colostrum. There was good agreement between Brix percentages in fresh colostrum and after short (2 to 8 d), medium (4 to 7 mo), and long (3 y) periods of freezing (concordance correlation coefficient: 0.95, 0.96, and 0.96, respectively). Although there was no significant change in mean Brix percentages over 2 FT cycles (P > 0.05), mean Brix percentages decreased with 3 FT cycles (P = 0.017). Samples from the fourth and fifth FT cycles were observably coagulated, and these measurements were therefore deemed inaccurate. Data from this study indicate that freezing had minimal impact on digital Brix refractometer estimates of IgG concentration in beef cattle colostrum, but that samples stored for future testing should not undergo more than 2 FT cycles.

L'évaluation de la concentration d'immunoglobuline G (IgG) dans le colostrum est importante pour guider la gestion à la ferme. Des études ont montré que la réfractométrie Brix numérique estime avec précision la concentration d'IgG du colostrum dans le colostrum des bovins laitiers et de boucherie. Le colostrum est souvent congelé dans les milieux cliniques et de recherche. Les implications de cette congélation sur la précision des mesures de réfractométrie Brix sont largement inconnues. Le premier objectif de cette étude était d'évaluer la concordance entre les mesures numériques du pourcentage de Brix d'IgG dans le colostrum de bovins de boucherie prises avant et après différentes durées de congélation. Le deuxième objectif était d'évaluer les effets de plusieurs cycles de congélation-décongélation (FT) sur les mesures du pourcentage Brix d'IgG dans le colostrum de bovins de boucherie. Il y avait un bon accord entre les pourcentages de Brix dans le colostrum frais et après des périodes de congélation courtes (2 à 8 jours), moyennes (4 à 7 mois) et longues (3 ans) (coefficient de corrélation de concordance : 0,95, 0,96 et 0,96, respectivement). Bien qu'il n'y ait pas eu de changement significatif dans les pourcentages moyens de Brix sur deux cycles FT (P > 0,05), les pourcentages moyens de Brix ont diminué avec trois cycles FT (P = 0,017). Les échantillons des quatrième et cinquième cycles FT étaient coagulés de manière observable, et ces mesures ont donc été jugées inexactes. Les données de cette étude indiquent que la congélation a eu un impact minimal sur les estimations du réfractomètre numérique Brix de la concentration d'IgG dans le colostrum de les bovins de boucherie, mais que les échantillons stockés pour les tests futurs ne doivent pas subir plus de deux cycles FT.(Traduit par Docteur Serge Messier).

Protoporphyrin IX derived from dual-species anaerobic biofilms of Fusobacterium necrophorum and Porphyromonas levii attenuates bovine neutrophil function.

Host immune cells and clinical interventions often fail to eradicate biofilm-mediated infections, resulting in chronic inflammation. The role of the biofilm three-dimensional structure in this tolerant phenotype has been studied extensively; however, the impact of small molecules released from biofilm-bacteria in modulating host immune function is less well understood. A model of mixed-species biofilms composed of Fusobacterium necrophorum and Porphyromonas levii was developed to evaluate bovine neutrophil responses to bioactive molecules released from either biofilm or planktonic bacteria. We hypothesized that different soluble extracellular factors (ECFs) would be released from planktonic and biofilm bacteria, resulting in altered neutrophil function. Neutrophils exposed to ECFs from planktonic bacteria showed significantly elevated levels of reactive oxygen species (ROS). In contrast, biofilm components from these same species of bacteria failed to induce such a response. Size-exclusion filtration of ECFs revealed that the bioactive molecule causing neutrophil ROS responses was below 3 kDa. Intensive heat, nuclease, lipase, or protease treatments of the <3 kDa fractions did not alter neutrophil functional responses. Protoporphyrin IX (PPIX) is an important heme precursor and growth requirement for many anaerobes. Porphyromonas species can accumulate environmental PPIX at the cell surface as a strategy to protect the bacteria from oxidative stress and we investigated the direct interaction of bovine neutrophils with PPIX. In the present study, evidence suggests that the accumulation of protoporphyrin in these dual-species biofilm ECFs attenuates neutrophil ROS production and chemotaxis. The diminished neutrophil response to biofilm ECFs via the action of PPIX may represent a biofilm immune-evasion strategy that could assist in explaining the ineffectual host clearance of biofilm-mediated infections involving these bacteria.

Anti-Inflammatory benefits of antibiotic-induced neutrophil apoptosis: tulathromycin induces caspase-3-dependent neutrophil programmed cell death and inhibits NF-kappaB signaling and CXCL8 transcription.

Clearance of apoptotic neutrophils is a central feature of the resolution of inflammation. Findings indicate that immuno-modulation and induction of neutrophil apoptosis by macrolide antibiotics generate anti-inflammatory benefits via mechanisms that remain obscure. Tulathromycin (TUL), a new antimicrobial agent for bovine respiratory disease, offers superior clinical efficacy for reasons not fully understood. The aim of this study was to identify the immuno-modulating effects of tulathromycin and, in this process, to establish tulathromycin as a new model for characterizing the novel anti-inflammatory properties of antibiotics. Bronchoalveolar lavage specimens were collected from Holstein calves 3 and 24 h postinfection, challenged intratracheally with live Mannheimia haemolytica (2 × 10(7) CFU), and treated with vehicle or tulathromycin (2.5 mg/kg body weight). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and enzyme-linked immunosorbent assay (ELISA) revealed that tulathromycin treatment significantly increased leukocyte apoptosis and reduced levels of proinflammatory leukotriene B(4) in M. haemolytica-challenged calves. In vitro, tulathromycin concentration dependently induced apoptosis in freshly isolated bovine neutrophils from healthy steers in a capase-3-dependent manner but failed to induce apoptosis in bovine fibroblasts, epithelial cells, and endothelial cells, as well as freshly isolated bovine blood monocytes and monocyte-derived macrophages. The proapoptotic effects of TUL were also, in part, drug specific; equimolar concentrations of penicillin G, oxytetracycline, and ceftiofur failed to cause apoptosis in bovine neutrophils. In addition, tulathromycin significantly reduced levels of phosphorylated IκBα, nuclear translocation of NF-κB p65, and mRNA levels of proinflammatory interleukin-8 in lipopolysaccharide (LPS)-stimulated bovine neutrophils. The findings illustrate novel mechanisms through which tulathromycin confers anti-inflammatory benefits.

Physical Activity Shapes the Intestinal Microbiome and Immunity of Healthy Mice but Has No Protective Effects against Colitis in MUC2-/- Mice.

The interactions among humans, their environment, and the trillions of microbes residing within the human intestinal tract form a tripartite relationship that is fundamental to the overall health of the host. Disruptions in the delicate balance between the intestinal microbiota and host immunity are implicated in various chronic diseases, including inflammatory bowel disease (IBD). There is no known cure for IBD; therefore, novel therapeutics targeting prevention and symptom management are of great interest. Recently, physical activity in healthy mice was shown to be protective against chemically induced colitis; however, the benefits of physical activity during or following disease onset are not known. In this study, we examine whether voluntary wheel running is protective against primary disease symptoms in a mucin 2-deficient (Muc2-/- ) lifelong model of murine colitis. We show that 6 weeks of wheel running in healthy C57BL/6 mice leads to distinct changes in fecal bacteriome, increased butyrate production, and modulation in colonic gene expression of various cytokines, suggesting an overall primed anti-inflammatory state. However, these physical activity-derived benefits are not present in Muc2-/- mice harboring a dysfunctional mucosal layer from birth, ultimately showing no improvements in clinical signs. We extrapolate from our findings that while physical activity in healthy individuals may be an important preventative measure against IBD, for those with a compromised intestinal mucosa, a commonality in IBD patients, these benefits are lost.IMPORTANCE Perturbation in the gut microbial ecosystem has been associated with various diseases, including inflammatory bowel disease. Habitual physical activity, through its ability to modulate the gut microbiome, has recently been shown to prophylactically protect against chemically induced models of murine colitis. Here, we (i) confirm previous reports that physical activity has limited but significant effects on the gut microbiome of mice and (ii) show that such changes are associated with anti-inflammatory states in the gut, such as increased production of beneficial short-chain fatty acids and lower levels of proinflammatory immune markers implicated in human colitis; however, we also show that (iii) these physical activity-derived benefits are completely lost in the absence of a healthy intestinal mucus layer, a hallmark phenotype of human colitis.

Effects of Azithromycin on Behavior, Pathologic Signs, and Changes in Cytokines, Chemokines, and Neutrophil Migration in C57BL/6 Mice Exposed to Dextran Sulfate Sodium.

Here we characterized the murine dextran sulfate sodium (DSS) model of acute colitis. Specifically, we evaluated azithromycin and metronidazole treatment regimens to assess their effects on animal wellbeing, pathologic changes, barrier function, cytokine and chemokine profiles, and neutrophil migration in colon tissue. Azithromycin treatment significantly reduced the severity of colitis, as assessed through body weight change, water consumption, macroscopic lesions, and animal behaviors (activity level, climbing, and grooming), but did not alter food consumption or feeding behavior. Mucosal barrier function (evaluated by using FITC-labeled dextran) was decreased after DSS exposure; azithromycin did not significantly alter barrier function in mice with colitis, whereas metronidazole exacerbated the colitis-related deficit in barrier function. In addition, metronidazole appeared to exacerbate disease as assessed through water consumption and animal behaviors (overall activity, climbing, grooming, and drinking) but had no effect on weight loss, macroscopic lesions, or eating behavior. Pathologic changes were typical for DSS treatment. Antibiotic treatment resulted in reduced levels of proinflammatory cytokines and chemokines and decreased neutrophil adhesion and emigration in DSS-exposed mice. The results highlight the importance of clinical and behavioral assessments in addition to laboratory evaluation as tools to evaluate animal welfare and therapeutic efficacy in disease models. Data from this study suggest that azithromycin may convey some benefits in the mouse DSS colitis model through modulation of the immune response, including neutrophil migration into tissues, whereas metronidazole may exacerbate colitis.

Influence of timing (pre-puberty or skeletal maturity) of ovariohysterectomy on mRNA levels in corneal tissues of female rabbits.

PURPOSE: Corneal thickness and curvature are reported to be influenced by hormonal changes associated with menstrual cycle, pregnancy, or menopause. However, the molecular mechanisms leading to these alterations are not clearly understood. The present study focuses on gene expression patterns (mRNA levels) in corneal tissues following surgically induced menopause in an animal model. The impact of lower hormone levels on mRNA levels in corneal tissues after pre-puberty ovariohysterectomy (OVX) was compared to that in skeletally mature adult animals. METHODS: Skeletally mature adult female rabbits were either left unoperated (control) or were subjected to OVX at 54 weeks of age using an approved protocol. The central (approximately 6 mm) and the peripheral corneal tissues were harvested from normal and OVX rabbits eight weeks after surgery. In a second study, young sexually immature rabbits at eight weeks of age were subjected to OVX and corneal tissues were collected when the animals were 22 and 32 weeks of age. In both experiments, RNA was isolated from corneal tissues and RT-PCR was used to assess mRNA levels for several relevant molecules. RESULTS: When mature animals were examined eight weeks after OVX, mRNA levels for molecules such as the estrogen receptor, decorin, collagen I, collagen V, and several growth factors were found to be significantly decreased in central corneal tissues. Interestingly, no corresponding changes in mRNA levels were observed for these same molecules in peripheral corneal tissues. When young, pre-pubertal animals were subjected to OVX, mRNA levels were found to be mainly unchanged for the OVX animals at 22 weeks of age i.e., after 14 weeks of low hormone conditions. However, significant decreases in mRNA levels for a similar subset of molecules were observed when the animals were at least 32 weeks of age, i.e., after 24 weeks of a low hormone environment. Examination of peripheral corneal tissues did not show significant changes in mRNA levels due to OVX at either 22 or 32 weeks of age except for collagens I and V at 32 weeks of age. CONCLUSIONS: These results indicate significant alterations in mRNA levels in the central corneal tissues of rabbits following OVX. Interestingly, peripheral corneal tissues show very little alteration in mRNA levels for the same molecules. Furthermore, OVX had a more rapid impact on mRNA levels in mature animals than in skeletally immature animals. Thus, loss of hormone producing tissues during growth and maturation apparently delayed the impact of hormone removal compared to loss after maturity had been attained and growth stimuli are likely absent. Therefore, specific areas of the cornea are more responsive to hormone levels than others. The impact of the loss of hormones is influenced by the maturation state of the rabbit, but mRNA levels for a similar subset of genes are affected by OVX in both age groups.

Evaluation of the enteric microflora of captive whooping cranes (Grus americana) and sandhill cranes (Grus canadensis).

The enteric flora of captive whooping cranes (Grus americana) and sandhill cranes (Grus canadensis) has not been well described, despite its potential importance in the understanding of both the normal condition of the intestinal physiology of these animals and the altered colonization within disease states in these birds. Nineteen whooping cranes and 23 sandhill cranes housed currently at the Calgary Zoo or its affiliated Devonian Wildlife Conservation Centre (DWCC) in Calgary, Alberta were sampled from October 2004-February 2005 by collecting aerobic and anaerobic cloacal swabs from each bird. There were seven major groupings of bacteria isolated from both species of crane. Gram-positive cocci, coliforms, and gram-negative bacilli were the most prevalent types of bacteria isolated for both crane species, with Escherichia coli, Enterococcus faecalis, and Streptococcus Group D, not Enterococcus the bacterial species isolated most commonly. There was a significant difference in the average number of isolates per individual between the two crane species but no differences between age or gender categories within crane species. Campylobacter sp. were isolated from five whooping cranes. The potential zoonotic pathogen Campylobacter jejuni was isolated from one whooping crane and C. upsaliensis was isolated from a second. Three other isolates were unspeciated members of the Campylobacter genus and likely belong to a species undescribed previously. The evaluation of the enteric cloacal flora of whooping cranes and sandhill cranes illustrates that differences exist between these two closely related crane species, and highlights the potential implications these differences may have for current practices involving captive wildlife. Zoo Biol 0:1-13, 2007. (c) 2007 Wiley-Liss, Inc.

Evaluation of the effects of short-chain fatty acids and extracellular pH on bovine neutrophil function in vitro.

OBJECTIVE: To investigate the effects of short-chain fatty acids (SCFAs) and pH on neutrophil oxidative burst, phagocytosis, and morphology after exposure to acetate, propionate, butyrate, or succinate at pH 5.5 and 6.7. SAMPLE POPULATION: Neutrophils isolated from bovine blood samples and Porphyromonas levii, Prevotella spp, and Bacteroides fragilis isolated from lesions of cattle with acute interdigital phlegmon (foot rot). PROCEDURES: Bacteria were cultured in strictly anaerobic conditions. Bacterial SCFA production was measured with high-performance liquid chromatography. Neutrophils were isolated, stimulated with phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OZ), and incubated with dihydroethidium or dichlorofluorescein diacetate to measure production of O(2)and H(2)O(2), respectively. Phagocytosis was assessed after exposure to serum-opsonized bacteria. Cellular morphology was assessed with differential staining. RESULTS: All bacteria produced at least 3 of the 4 SCFAs. Production of both O(2) and H(2)O(2) was markedly curtailed in PMA-stimulated neutrophils exposed to SCFA at pH 5.5, compared with production at pH 6.7. Succinate caused a significant dose-dependent decrease in O(2) production at pH 6.7 in OZ-stimulated neutrophils. Monoprotic SCFAs elicited a significant increase in H(2)O(2) production in OZ-stimulated neutrophils at pH 6.7 but a significant decrease at pH 5.5. Monoprotic SCFAs significantly increased phagocytosis at pH 6.7 but decreased phagocytic activity at pH 5.5. Cellular necrosis was observed in cells exposed to SCFAs at pH 5.5. CONCLUSIONS AND CLINICAL RELEVANCE: Establishment and persistence of anaerobic bacteria in cattle with foot rot infection may result in part from neutrophil dysfunction secondary to the effects of bacterially secreted SCFA in acidotic microenvironments.

Use of polymyxin as an endotoxin blocker in the prevention of diffuse lamellar keratitis in an animal model.

PURPOSE: To determine whether bacterial endotoxin, lipopolysaccharide (LPS), could induce diffuse lamellar keratitis (DLK) in an animal model and whether DLK could be prevented by endotoxin blockers such as polymyxin. METHODS: Laser in situ keratomileusis (LASIK) flaps were created in rabbit eyes. The stromal bed was treated with 20 microg of Burkholderia cepacia LPS or balanced salt solution (BSS). Development of DLK, histological degree of inflammation, and presence of LPS detected by anti-LPS antibody were evaluated after 48 hours. In a second experiment, all eyes received LPS and were randomly assigned to receive either polymyxin in the form of two drops of Polytrim (Allergan, Irvine, Calif) on the stromal bed or two drops of BSS. RESULTS: In the animal model study, LPS was significantly associated with the development of DLK (P<.05, n=30). Infiltration with polymorphonuclear cells and presence of DLK were found in LPS treated eyes but not in controls. In the second experiment, 4 (27%) of 15 eyes that received polymyxin in addition to LPS developed DLK compared to 18 (95%) of 19 eyes that received only LPS (P<.05, n=34). There was a trend towards higher flap displacement in polymyxin treated eyes but this was not significant (P=.07). CONCLUSIONS: Diffuse lamellar keratitis in a rabbit model can be caused by bacterial endotoxin (LPS). Endotoxin blockers, such as polymyxin, are effective in decreasing the incidence of endotoxin-induced DLK in a rabbit model.

Diffuse lamellar keratitis complicating laser in situ keratomileusis: post-marketing surveillance of an emerging disease in British Columbia, Canada, 2000-2002.

PURPOSE: To describe a surveillance system and summarize data between January 2000 and December 2002 regarding diffuse lamellar keratitis (DLK), a complication of laser in situ keratomileusis (LASIK) surgery. SETTING: Community-based clinics in British Columbia, Canada, in which LASIK surgery is performed. METHODS: Monthly, all clinics in which LASIK is performed reported the number of LASIK procedures and nonnominal cases of DLK (by grade and onset date) to the British Columbia Centre for Disease Control. Diffuse lamellar keratitis outbreaks were investigated, and prevention and control measures were recommended. RESULTS: From 2000 to 2002, approximately 72,000 LASIK procedures were performed, with a mean DLK incidence rate of 0.67% (95% confidence interval, 0.61-0.73). The overall proportion of DLK cases attributed to outbreaks was 64%, decreasing from 72% in 2000 to 40% in 2003. CONCLUSIONS: An effective DLK surveillance program was implemented at all laser refractive clinics in British Columbia. Reported DLK incidence was 0.67 cases per 100 procedures, with 64% occurring in outbreaks.

Effects of oral administration of tilmicosin on pulmonary inflammation in piglets experimentally infected with Actinobacillus pleuropneumoniae.

OBJECTIVES: To determine the effects of oral administration of tilmicosin in piglets experimentally infected with Actinobacillus pleuropneumoniae. ANIMALS: Forty 3-week-old specific-pathogen free piglets. PROCEDURES: Piglets were assigned to 1 of 4 groups as follows: 1) uninfected sham-treated control piglets; 2) infected untreated piglets that were intratracheally inoculated with 10(7) CFUs of A pleuropneumoniae; 3) infected treated piglets that were intratracheally inoculated with A pleuropneumoniae and received tilmicosin in feed (400 ppm [microg/g]) for 7 days prior to inoculation; or 4) infected treated piglets that were intratracheally inoculated with A pleuropneumoniae and received chlortetracycline (CTC) in feed (1100 ppm [microg/gl) for 7 days prior to inoculation. Bronchoalveolar lavage (BAL) fluid and lung tissue specimens of piglets for each group were evaluated at 3 or 24 hours after inoculation. For each time point, 4 to 6 piglets/group were studied. RESULTS: Feeding of CTC and tilmicosin decreased bacterial load in lungs of infected piglets. Tilmicosin delivered in feed, but not CTC, enhanced apoptosis in porcine BAL fluid leukocytes. This was associated with a decrease in LTB4 concentrations in BAL fluid of tilmicosin-treated piglets, compared with untreated and CTC-treated piglets, and also with a significant decrease in the number of pulmonary lesions. Tilmicosin inhibited infection-induced increases in rectal temperatures, as measured in untreated and CTC-treated piglets. Pulmonary neutrophil infiltration and prostaglandin E2 concentrations in the BAL fluid were not significantly different among groups at any time. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of tilmicosin to infected piglets induces apoptosis in BAL fluid leukocytes and decreases BAL fluid LTB4 concentrations and inflammatory lung lesions.

Immunomodulatory effects of tulathromycin on apoptosis, efferocytosis, and proinflammatory leukotriene B4 production in leukocytes from Actinobacillus pleuropneumoniae-or zymosan-challenged pigs.

OBJECTIVE: To investigate the anti-inflammatory and immunomodulatory properties of tulathromycin in vitro and in experimental models of Actinobacillus pleuropneumoniae-induced pleuropneumonia and zymosan-induced pulmonary inflammation in pigs. ANIMALS: Blood samples from six 8- to 30-week-old healthy male pigs for the in vitro experiment and sixty-five 3-week-old specific pathogen-free pigs. PROCEDURES: Neutrophils and monocyte-derived macrophages were isolated from blood samples. Isolated cells were exposed to tulathromycin (0.02 to 2.0 mg/mL) for various durations and assessed for markers of apoptosis and efferocytosis. For in vivo experiments, pigs were inoculated intratracheally with A pleuropneumoniae, zymosan, or PBS solution (control group) with or without tulathromycin pretreatment (2.5 mg/kg, IM). Bronchoalveolar lavage fluid was collected 3 and 24 hours after inoculation and analyzed for proinflammatory mediators, leukocyte apoptosis, and efferocytosis. RESULTS: In vitro, tulathromycin induced time- and concentration-dependent apoptosis in neutrophils, which enhanced their subsequent clearance by macrophages. In the lungs of both A pleuropneumoniae- and zymosan-challenged pigs, tulathromycin promoted leukocyte apoptosis and efferocytosis and inhibited proinflammatory leukotriene B4 production, with a concurrent reduction in leukocyte necrosis relative to that of control pigs. Tulathromycin also attenuated the degree of lung damage and lesion progression in A pleuropneumoniae-inoculated pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Tulathromycin had immunomodulatory effects in leukocytes in vitro and anti-inflammatory effects in pigs in experimental models of A pleuropneumoniae infection and nonmicrobial-induced pulmonary inflammation. These data suggested that in addition to its antimicrobial properties, tulathromycin may dampen severe proinflammatory responses and drive resolution of inflammation in pigs with microbial pulmonary infections.

In vitro expression of tumor necrosis factor-alpha, interleukin 1beta, and interleukin 8 mRNA by bovine macrophages following exposure to Porphyromonas levii.

The objective was to evaluate the pro-inflammatory response of bovine macrophages towards Porphyromonas levii, an etiologic agent of acute interdigital phlegmon, by evaluating the mRNA expression of tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and interleukin 8 (IL-8). Bovine macrophages detect the presence of bacteria, such as P. levii, and respond by upregulating transcription of the genes for pro-inflammatory cytokines TNF-alpha and IL-1beta in addition to the neutrophil chemoattractant IL-8. Monocytes were isolated from blood obtained from Holstein steers. These cells were cultured and differentiated into macrophages over 7 d, followed by exposure to P. levii, Escherichia coli lipopolysaccharide (LPS), or tissue culture medium for 1, 1.5, 2, 4, 6, 8,12, or 24 h. Total RNA was extracted, and reverse transcriptase polymerase chain reaction was conducted to examine the presence of TNF-alpha, IL-1beta, or IL-8 mRNA. Products were visualized on agarose gels to determine the presence or absence of cytokine mRNA amplified DNA. Bovine macrophages, when exposed to P. levii or E. coli LPS, produced mRNA for TNF-alpha, IL-1beta, and IL-8. Expression of all 3 cytokines was higher in the P. levii and LPS-exposed macrophages at all time points examined, compared with tissue culture medium-treated cells. Expression of these cytokines by macrophages is likely directly involved in orchestration of the immune response, and particularly in neutrophil recruitment to affected tissues in acute interdigital phlegmon.

Biofilm bacteria: formation and comparative susceptibility to antibiotics.

The Calgary Biofilm Device (CBD) was used to form bacterial biofilms of selected veterinary gram-negative and gram-positive pathogenic bacteria from cattle, sheep, pigs, chicken, and turkeys. The minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) of ampicillin, ceftiofur, cloxacillin, oxytetracycline, penicillin G, streptomycin, tetracycline, enrofloxacin, erythromycin, gentamicin, tilmicosin, and trimethoprim-sulfadoxine for gram-positive and -negative bacteria were determined. Bacterial biofilms were readily formed on the CBD under selected conditions. The biofilms consisted of micro-colonies encased in extracellular polysaccharide material. Biofilms composed of Arcanobacterium (Actinomyces) pyogenes, Staphylococcus aureus, Staphylococcus hyicus, Streptococcus agalactiae, Corynebacterium renale, or Corynebacterium pseudotuberculosis were not killed by the antibiotics tested but as planktonic bacteria they were sensitive at low concentrations. Biofilm and planktonic Streptococcus dysgalactiae and Streptococcus suis were sensitive to penicillin, ceftiofur, cloxacillin, ampicillin, and oxytetracycline. Planktonic Escherichia coli were sensitive to enrofloxacin, gentamicin, oxytetracycline and trimethoprim/ sulfadoxine. Enrofloxacin and gentamicin were the most effective antibiotics against E. coli growing as a biofilm. Salmonella spp. and Pseudomonas aeruginosa isolates growing as planktonic populations were sensitive to enrofloxacin, gentamicin, ampicillin, oxytetracycline, and trimethoprim/sulfadoxine, but as a biofilm, these bacteria were only sensitive to enrofloxacin. Planktonic and biofilm Pasteurella multocida and Mannheimia haemolytica had similar antibiotic sensitivity profiles and were sensitive to most of the antibiotics tested. The CBD provides a valuable new technology that can be used to select antibiotics that are able to kill bacteria growing as biofilms.

Chemotaxis, phagocytosis, and oxidative metabolism in bovine macrophages exposed to a novel interdigital phlegmon (foot rot) lesion isolate, Porphyromonas levii.

OBJECTIVE: To examine the host response toward Porphyromonas levii, by evaluating chemotaxis, phagocytosis, and oxidative burst of bovine macrophages in vitro. SAMPLE POPULATION: Cultured bovine macrophages obtained from monocytes harvested from blood samples of 15 Holstein steers. Porphyromonas levii was isolated from the foot rot lesion of an acutely affected feedlot steer. PROCEDURE: Monocytes were cultured for macrophage differentiation over 7 days. Porphyromonas levii was cultured in strict anaerobic conditions for experimentation. Chemotaxis was evaluated by quantifying macrophage migration toward P. levii in Boyden chambers. Phagocytosis was assessed by quantification of macrophages engulfing P. levii following incubation with or without anti-P. levii serum or purified IgG. Oxidative burst was measured by use of the nitroblue tetrazolium reduction assay. RESULTS: Chemotaxis toward P. levii was not significantly different from control values at any of the tested bacterial concentrations. Phagocytosis of P. levii was approximately 10% at a 10:1 bacterium to macrophage ratio and did not change significantly over time. When higher proportions of P. levii were tested for phagocytosis, the 1,000:1 bacterium to macrophage ratio had a significant increase, compared with the 10:1 test group. Opsonization of P. levii with high-titeranti-P. levii serum or anti-P. levii IgG produced a significant increase in macrophage phagocytosis. Oxidative production significantly increased compared with control in the 1,000:1 test group only. CONCLUSIONS AND CLINICAL RELEVANCE: Porphyromonas levii may evade host detection by decreased chemotaxis, phagocytosis, and oxidative burst by macrophages. Acquired immunity may be beneficial for clearance of P. levii in foot rot lesions in cattle.