Characterization of Three New Outer Membrane Adhesion Proteins in Fusobacterium necrophorum.

Fusobacterium necrophorum, an anaerobic Gram-negative pathogen, causes necrotic cattle infections, impacting livestock health and the US feedlot industry. Antibiotic administration is the mainstay for treating F. necrophorum infections, although resistance hampers their effectiveness. Vaccination, especially targeting outer membrane proteins (OMPs) due to their antigenic properties and host specificity, offers an alternative to antibiotics. This study identified high-binding-affinity adhesion proteins from F. necrophorum using binding and pull-down assays with bovine adrenal gland endothelial cells (EJG). Four OMP candidates (17.5 kDa/OmpH, 22.7 kDa/OmpA, 66.3 kDa/cell surface protein (CSP), and a previously characterized 43 kDa OMP) were expressed as recombinant proteins and purified. Rabbit polyclonal antibodies to recombinant OMPs were generated, and their ability to inhibit bacterial binding in vitro was assessed. The results show that treatment with individual polyclonal antibodies against 43 kDa significantly inhibited bacterial adhesion, while other antibodies were less potent. However, combinations of two or more antibodies showed a more prominent inhibitory effect on host-cell adhesion. Thus, our findings suggest that the identified OMPs are involved in fusobacterial attachment to host cells and may have the potential to be leveraged in combination for vaccine development. Future in vivo studies are needed to validate their roles and test the feasibility of an OMP-based subunit vaccine against fusobacterial infections.

Outer-Membrane Vesicles of Fusobacterium necrophorum: A Proteomic, Lipidomic, and Functional Characterization.

Outer-membrane vesicles (OMVs) are extruded nanostructures shed by Gram-negative bacteria, containing periplasmic contents, and often including virulence factors with immunogenic properties. To assess their potential for use in vaccine development, we purified OMVs from the Fusobacterium necrophorum subspecies necrophorum, an opportunistic necrotic infection-causing pathogen, and characterized these structures using proteomics, lipid-profiling analyses, and cytotoxicity assays. A proteomic analysis of density-gradient-purified F. necrophorum OMVs identified 342 proteins, a large proportion of which were outer-membrane proteins (OMPs), followed by cytoplasmic proteins, based on a subcellular-localization-prediction analysis. The OMPs and toxins were among the proteins with the highest intensity identified, including the 43-kDa-OMP-, OmpA-, and OmpH-family proteins, the cell-surface protein, the FadA adhesin protein, the leukotoxin-LktA-family filamentous adhesin, the N-terminal domain of hemagglutinin, and the OMP transport protein and assembly factor. A Western blot analysis confirmed the presence of several OMPs and toxins in the F. necrophorum OMVs. The lipid-profiling analysis revealed phospholipids, sphingolipids, and acetylcarnitine as the main lipid contents of OMVs. The lactate-dehydrogenase-cytotoxicity assays showed that the OMVs had a high degree of cytotoxicity against a bovine B-lymphocyte cell line (BL-3 cells). Thus, our data suggest the need for further studies to evaluate the ability of OMVs to induce immune responses and assess their vaccine potential in vivo.

Albumin Deficiency Reduces Hepatic Steatosis and Improves Glucose Metabolism in a Mouse Model of Diet-Induced Obesity.

Serum albumin facilitates the transport of free fatty acids (FFAs) from adipose tissue to other organs. It was not known if impeding this process could protect from hepatic steatosis and metabolic dysfunction in obesity. We tested whether albumin knockout (Alb-/-) mice would exhibit a reduction in plasma FFA concentration, reduced hepatic lipid accumulation, and improved glucoregulation as compared to wild-type (WT) mice. Male homozygous albumin knockout mice (Alb-/-) and WT controls were fed a low-fat diet (LFD) or high-fat diet (HFD). Alb-/- mice exhibited a similar body weight gain and body composition as WT on both diets. Despite HFD-induced obesity, Alb-/- mice were protected from various comorbidities. Compared to WT mice on the HFD, Alb-/- exhibited lower plasma FFA levels, lower blood glucose levels during glucose tolerance and insulin tolerance tests, and lower hepatic steatosis and inflammation. Alb-/- mice on HFD also exhibited elevated expression of multiple genes in the liver and adipose tissues, such as peroxisome proliferator-activated receptor α in both tissues, as well as glucose transporter-4 and adiponectin in adipose tissues. The results indicate that albumin's FFA transport function may be involved in the development of hepatic lipid accumulation and dysregulated glucose metabolism in obesity.

Cdc14 phosphatase contributes to cell wall integrity and pathogenesis in Candida albicans.

The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. Here, we identified an invariant motif in the disordered C-terminal tail of fungal Cdc14 enzymes that is required for full enzyme activity. Mutation of this motif reduced Cdc14 catalytic rate and provided a tool for studying the biological significance of high Cdc14 activity. A S. cerevisiae strain expressing the reduced-activity hypomorphic mutant allele (cdc14hm ) as the sole source of Cdc14 proliferated like the wild-type parent strain but exhibited an unexpected sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. Sensitivity to echinocandins was also observed in Schizosaccharomyces pombe and Candida albicans strains lacking CDC14, suggesting this phenotype reflects a novel and conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. In C. albicans, the orthologous cdc14hm allele was sufficient to elicit echinocandin hypersensitivity and perturb cell wall integrity signaling. It also caused striking abnormalities in septum structure and the same cell separation and hyphal differentiation defects previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reduced Cdc14 activity on virulence in Galleria mellonella and mouse models of invasive candidiasis. Partial reduction in Cdc14 activity via cdc14hm mutation severely impaired C. albicans virulence in both assays. Our results reveal that high Cdc14 activity is important for C. albicans cell wall integrity and pathogenesis and suggest that Cdc14 may be worth future exploration as an antifungal drug target.

Comparison between Symptomatic and Asymptomatic Mice after Clostridioides difficile Infection Reveals Novel Inflammatory Pathways and Contributing Microbiota.

Clostridioides difficile causes the highest number of nosocomial infections. Currently, treatment options for C. difficile infection (CDI) are very limited, resulting in poor treatment outcomes and high recurrence rates. Although the disease caused by CDI is inflammatory in nature, the role of inflammation in the development of CDI symptoms is contradictory and not completely understood. Hence, the use of anti-inflammatory medication is debatable in CDI. In the current study, we evaluated the genetic and microbiome profiles of mice after infection with C. difficile. These mice were categorized based on the severity of CDI and the results were viewed accordingly. Our results indicate that certain genes are upregulated in severe CDI more than in the moderate case. These include oncostatin-M (OSM), matrix metalloprotease 8 (MMP8), triggering receptor expressed on myeloid cells 1 (Trem-1), and dual oxidase 2 (Duox2). We also investigated the microbiome composition of CDI mice before and after infecting with C. difficile. The results show that C. difficile abundance is not indicative of diseases severity. Certain bacterial species (e.g., Citrobacter) were enriched while others (e.g., Turicibacter) were absent in severe CDI. This study identifies novel inflammatory pathways and bacterial species with a potential role in determining the severity of CDI.

Comparative Genomic Analysis of Fusobacterium necrophorum Provides Insights into Conserved Virulence Genes.

Fusobacterium necrophorum is a Gram-negative, filamentous anaerobe prevalent in the mucosal flora of animals and humans. It causes necrotic infections in cattle, resulting in a substantial economic impact on the cattle industry. Although infection severity and management differ within F. necrophorum species, little is known about F. necrophorum speciation and the genetic virulence determinants between strains. To characterize the clinical isolates, we performed whole-genome sequencing of four bovine isolates (8L1, 212, B17, and SM1216) and one human isolate (MK12). To determine the phylogenetic relationship and evolution pattern and investigate the presence of antimicrobial resistance genes (ARGs) and potential virulence genes of F. necrophorum, we also performed comparative genomics with publicly available Fusobacterium genomes. Using up-to-date bacterial core gene (UBCG) set analysis, we uncovered distinct Fusobacterium species and F. necrophorum subspecies clades. Pangenome analyses revealed a high level of diversity among Fusobacterium strains down to species levels. The output also identified 14 and 26 genes specific to F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, respectively, which could be essential for bacterial survival under different environmental conditions. ClonalFrameML-based recombination analysis suggested that extensive recombination among accessory genes led to species divergence. Furthermore, the only strain of F. necrophorum with ARGs was F. necrophorum subsp. funduliforme B35, with acquired macrolide and tetracycline resistance genes. Our custom search revealed common virulence genes, including toxins, adhesion proteins, outer membrane proteins, cell envelope, type IV secretion system, ABC (ATP-binding cassette) transporters, and transporter proteins. A focused study on these genes could help identify major virulence genes and inform effective vaccination strategies against fusobacterial infections. IMPORTANCE Fusobacterium necrophorum is an anaerobic bacterium that causes liver abscesses in cattle with an annual incidence rate of 10% to 20%, resulting in a substantial economic impact on the cattle industry. The lack of definite biochemical tests makes it difficult to distinguish F. necrophorum subspecies phenotypically, where genomic characterization plays a significant role. However, due to the lack of a good reference genome for comparison, F. necrophorum subspecies-level identification represents a significant challenge. To overcome this challenge, we used comparative genomics to validate clinical test strains for subspecies-level identification. The findings of our study help predict specific clades of previously uncharacterized strains of F. necrophorum. Our study identifies both general and subspecies-specific virulence genes through a custom search-based analysis. The virulence genes identified in this study can be the focus of future studies aimed at evaluating their potential as vaccine targets to prevent fusobacterial infections in cattle.

Effect of Paclitaxel Stereochemistry on X-ray-Triggered Release of Paclitaxel from CaWO4/Paclitaxel-Coloaded PEG-PLA Nanoparticles.

For many locally advanced tumors, the chemotherapy-radiotherapy (CT-RT) combination ("chemoradiation") is currently the standard of care. Intratumoral (IT) CT-based chemoradiation has the potential to overcome the limitations of conventional systemic CT-RT (side effects). For maximizing the benefits of IT CT-RT, our laboratory has previously developed a radiation-controlled drug release formulation, in which anticancer drug paclitaxel (PTX) and radioluminescent CaWO4 (CWO) nanoparticles (NPs) are co-encapsulated with poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) block copolymers ("PEG-PLA/CWO/PTX NPs"). These PEG-PLA/CWO/PTX NPs enable radiation-controlled release of PTX and are capable of producing sustained therapeutic effects lasting for at least one month following a single IT injection. The present article focuses on discussing our recent finding about the effect of the stereochemical structure of PTX on the efficacy of this PEG-PLA/CWO/PTX NP formulation. Stereochemical differences in two different PTX compounds ("PTX-S" from Samyang Biopharmaceuticals and "PTX-B" from Biotang) were characterized by 2D heteronuclear/homonuclear NMR, Raman spectroscopy, and circular dichroism measurements. The difference in PTX stereochemistry was found to significantly influence their water solubility (WS); PTX-S (WS ≈ 4.69 µg/mL) is about 19 times more water soluble than PTX-B (WS ≈ 0.25 µg/mL). The two PTX compounds showed similar cancer cell-killing performances in vitro when used as free drugs. However, the subtle stereochemical difference significantly influenced their X-ray-triggered release kinetics from the PEG-PLA/CWO/PTX NPs; the more water-soluble PTX-S was released faster than the less water-soluble PTX-B. This difference was manifested in the IT pharmacokinetics and eventually in the survival percentages of test animals (mice) treated with PEG-PLA/CWO/PTX NPs + X-rays in an in vivo human tumor xenograft study; at short times (<1 month), concurrent PEG-PLA/CWO/PTX-S NPs produced a greater tumor-suppression effect, whereas PEG-PLA/CWO/PTX-B NPs had a longer-lasting radio-sensitizing effect. This study demonstrates the importance of the stereochemistry of a drug in a therapy based on a controlled release formulation.

Sheep as a Potential Model of Intradiscal Infection by the Bacterium Cutibacterium acnes.

The anaerobic bacterium Cutibacterium acnes has been increasingly linked to the development of degenerative disc disease (DDD), although causality is yet to be conclusively proven. To better study how this organism could contribute to the aetiology of DDD, improved animal models that are more reflective of human disc anatomy, biology and mechanical properties are required. Against this background, our proof-of concept study aimed to be the first demonstration that C. acnes could be safely administered percutaneously into sheep intervertebral discs (IVDs) for in vivo study. Following our protocol, two sheep were successfully injected with a strain of C. acnes (8.3 × 106 CFU/disc) previously recovered from a human degenerative disc. No adverse reactions were noted, and at one-month post inoculation all triplicate infected discs in our first animal grew C. acnes, albeit at a reduced load (5.12 × 104 to 6.67 × 104 CFU/disc). At six months, no growth was detected in discs from our second animal indicating bacterial clearance. This pilot study has demonstrated the feasibility of safe percutaneous injection of C. acnes into sheep IVDs under fluoroscopic guidance. The design of follow-up sheep studies to investigate the potential of C. acnes to drive pathological changes within infected discs should now be pursued.

High dose of vesicular stomatitis virus-vectored Ebola virus vaccine causes vesicular disease in swine without horizontal transmission.

ABSTRACTThe recent impact of Ebola virus disease (EVD) on public health in Africa clearly demonstrates the need for a safe and efficacious vaccine to control outbreaks and mitigate its threat to global health. ERVEBO® is an effective recombinant Vesicular Stomatitis Virus (VSV)-vectored Ebola virus vaccine (VSV-EBOV) that was approved by the FDA and EMA in late 2019 for use in prevention of EVD. Since the parental virus VSV, which was used to construct VSV-EBOV, is pathogenic for livestock and the vaccine virus may be shed at low levels by vaccinated humans, widespread deployment of the vaccine requires investigation into its infectivity and transmissibility in VSV-susceptible livestock species. We therefore performed a comprehensive clinical analysis of the VSV-EBOV vaccine virus in swine to determine its infectivity and potential for transmission. A high dose of VSV-EBOV resulted in VSV-like clinical signs in swine, with a proportion of pigs developing ulcerative vesicular lesions at the nasal injection site and feet. Uninoculated contact control pigs co-mingled with VSV-EBOV-inoculated pigs did not become infected or display any clinical signs of disease, indicating the vaccine is not readily transmissible to naïve pigs during prolonged close contact. In contrast, virulent wild-type VSV Indiana had a shorter incubation period and was transmitted to contact control pigs. These results indicate that the VSV-EBOV vaccine causes vesicular illness in swine when administered at a high dose. Moreover, the study demonstrates the VSV-EBOV vaccine is not readily transmitted to uninfected pigs, encouraging its safe use as an effective human vaccine.

Leukotoxin production by Fusobacterium necrophorum strains in relation to severity of liver abscesses in cattle.

Fusobacterium necrophorum, a Gram-negative anaerobe, is the primary etiologic agent of liver abscesses of beef cattle. The bacterium, a member of the microbial community of the rumen, travels to the liver via portal circulation to cause abscesses. The severity of liver abscesses vary from mild with one or two small abscesses to severe with medium to large multiple abscesses. Leukotoxin, a secreted protein, is the critical virulence factor involved in the infection. Our objective was to compare leukotoxin production between strains of F. necrophorum isolated from mild and severe liver abscesses collected from slaughtered cattle. The quantification of leukotoxin was based on assays to measure cytotoxicity and protein antigen concentration. One-hundred strains, 50 from mild and 50 from severe abscesses, were utilized in the study. Cell-free supernatants were prepared from cultures grown in anaerobic broth at 9 and 24 h incubations. The leukotoxic activity was quantified by measuring cytotoxicity based on the release of lactic dehydrogenase from bovine lymphocyte cells, BL3, treated with the culture supernatant. Leukotoxin protein concentration was quantified by a sandwich ELISA assay with a leukotoxin-specific monoclonal antibody as the capture antibody. The leukotoxin activity and concentration were highly variable among the strains within each severity of liver abscesses. Although the leukotoxic activity was unaffected by incubation time, leukotoxin protein concentration was consistently higher at 24 h compared to 9 h incubation. Strains from severe liver abscesses had significantly higher leukotoxic activity and higher protein concentration compared to strains from mild liver abscesses (P < 0.0001) at both 9 and 24 h culture supernatants. Across all strains, the correlation coefficients between leukotoxic activity and leukotoxin concentration at 9 and 24 h were 0.14 (P = 0.17) and 0.47 (P < 0.0001), respectively. In conclusion, strains isolated from severe liver abscesses had significantly higher leukotoxic activities and leukotoxin protein concentrations compared to strains isolated from mild liver abscesses.

The Serotonin Neurotransmitter Modulates Virulence of Enteric Pathogens.

The gut-brain axis is crucial to microbial-host interactions. The neurotransmitter serotonin is primarily synthesized in the gastrointestinal (GI) tract, where it is secreted into the lumen and subsequently removed by the serotonin transporter, SERT. Here, we show that serotonin decreases virulence gene expression by enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium, a murine model for EHEC. The membrane-bound histidine sensor kinase, CpxA, is a bacterial serotonin receptor. Serotonin induces dephosphorylation of CpxA, which inactivates the transcriptional factor CpxR controlling expression of virulence genes, notably those within the locus of enterocyte effacement (LEE). Increasing intestinal serotonin by genetically or pharmacologically inhibiting SERT decreases LEE expression and reduces C. rodentium loads. Conversely, inhibiting serotonin synthesis increases pathogenesis and decreases host survival. As other enteric bacteria contain CpxA, this signal exploitation may be engaged by other pathogens. Additionally, repurposing serotonin agonists to inhibit CpxA may represent a potential therapeutic intervention for enteric bacteria.

Leukotoxic activity of Fusobacterium necrophorum of cattle origin.

Fusobacterium necrophorum is a Gram negative, rod-shaped and aero tolerant anaerobe. In animals, it is an opportunistic pathogen frequently associated with necrotic infections, generally called necrobacillosis, such as calf diphtheria, foot rot and liver abscesses in cattle. Two subspecies exist: subsp. necrophorum and subsp. funduliforme. Among several virulence factors, leukotoxin (Lkt) is considered to be a major factor and a protective antigen. The objective of the study was to utilize BL3 cells and measure the release of lactic dehydrogenase to quantify Lkt activity of F. necrophorum. The assay was used to examine the effects of storage and handling conditions, growth media, polymyxin B addition on the cytotoxicity and evaluate Lkt activities of F. necrophorum strains isolated from bovine liver abscesses and foot rot. The Lkt activity peaked at 9 h of incubation. There was a significant decrease in the cytotoxicity measured in the samples after each freeze and thaw cycle. No difference was observed in the cytotoxicity for the samples handled aerobically versus anaerobically. Lkt activities of strains grown in anaerobic Brain-Heart Infusion broth were higher compared to Vegitone broth. A small reduction in the cytotoxicity activity was observed after the addition of polymyxin. The Lkt activity was consistently higher in strains of subsp. necrophorum than subsp. funduliforme of liver abscess origin. Among the strains isolated from cattle foot rot, Lkt activities of subsp. necrophorum strains appear to be much more variable. Use of BL3 cells in combination of lactic acid dehydrogenase assay appears to be a simple and valid assay to measure Lkt activity of F. necrophorum.

Effects of supplemental zinc sulfate on growth performance, carcass characteristics, and antimicrobial resistance in feedlot heifers.

Effects of supplemental Zn as Zn sulfate on feedlot performance, carcass traits, and antimicrobial resistance were evaluated using 480 crossbred heifers (BW = 385 kg ± 13.08) in a randomized complete block design. Heifers were blocked by BW and randomly assigned within block to diets with 0, 30, 60, or 90 mg supplemental Zn/kg DM. Heifers were housed in dirt-surfaced pens (20 animals per pen; 6 pens per treatment) equipped with fence-line feed bunks and automatic water fountains. Heifers were fed once daily to ensure ad libitum intake. Plasma was collected on day 0 from five randomly selected heifers per pen and repeated on days 63 and 115 to determine plasma Zn concentrations. Random samples of freshly voided feces were collected from the surface of each pen the day of harvest to determine antibiotic resistance. Heifers were transported on day 144 to a commercial abattoir where hot carcass weight (HCW) and incidence of liver abscesses were recorded at harvest and HCW, dressed yield, ribeye area, 12th rib fat, quality and yield grades were recorded after 36 h of refrigeration. Plasma Zn concentration increased (P = 0.02) linearly in response to increasing concentrations of dietary Zn. Final BW and ADG were unaffected by supplementation (P ≥ 0.29). Quantified levels of resistance to ceftriaxone and tetracycline among fecal Escherichia coli were not impacted (P > 0.05) by dietary zinc concentrations. Increasing Zn concentrations tended to decrease (linear effect, P = 0.07) DMI, resulting in a linear (P = 0.03) and tendency for quadratic (P = 0.12) improvement in feed efficiency with increasing Zn concentration. No differences were detected for HCW, dressed yield, ribeye area, 12th rib fat, percentages of carcasses grading Select or Choice, or yield grade (P > 0.53), but added Zn tended to affect percentage of carcasses that graded Prime, peaking at 60 mg/kg added Zn (quadratic effect, P = 0.07). In vitro fermentations were performed using ruminal fluid cultures containing 0, 30, 60, 90, 120, or 150 mg Zn/kg substrate DM to determine impact of Zn on gas production, VFA concentrations, and in vitro DM disappearance (IVDMD). There were no effects of Zn on in vitro gas production, IVDMD, or most VFA (P > 0.15), but isovalerate decreased linearly in response to added Zn (P = 0.05). Supplementing finishing heifers up to 60 mg Zn/kg diet DM improved feed efficiency compared to other treatments.

Antimicrobial resistance of Enterococcus faecium strains isolated from commercial probiotic products used in cattle and swine.

Probiotics, an antibiotic alternative, are widely used as feed additives for performance benefits in cattle and swine production systems. Among bacterial species contained in probiotics, Enterococcus faecium is common. Antimicrobial resistance (AMR), particularly multidrug resistance, is a common trait among enterococci because of their propensity to acquire resistance and horizontally transfer AMR genes. Also, E. faecium is an opportunistic pathogen, and in the United States, it is the second most common nosocomial pathogen. There has been no published study on AMR and virulence potential in E. faecium contained in probiotic products used in cattle and swine in the United States. Therefore, our objectives were to determine phenotypic susceptibilities or resistance to antimicrobials, virulence genes (asa1, gelE, cylA, esp, and hyl) and assess genetic diversity of E. faecium isolated from commercial products. Twenty-two commercially available E. faecium-based probiotic products used in cattle (n = 13) and swine (n = 9) were procured and E. faecium was isolated and species confirmed. Antimicrobial susceptibility testing to determine minimum inhibitory concentrations was done by micro-broth dilution method using National Antimicrobial Resistance Monitoring Systems Gram-positive Sensititre panel plate (CMV3AGPF), and categorization of strains as susceptible or resistant was as per Clinical Laboratory and Standards Institute's guidelines. E. faecium strains from 7 products (3 for swine and 4 for cattle) were pan-susceptible to the 16 antimicrobials tested. Strains from 15 products (6 for swine and 9 for cattle) exhibited resistance to at least one antimicrobial and a high proportion of strains was resistant to lincomycin (10/22), followed by tetracycline (4/22), daptomycin (4/22), ciprofloxacin (4/22), kanamycin (3/22), and penicillin (2/22). Four strains were multidrug resistant, with resistant phenotypes ranging from 3 to 6 antimicrobials or class. None of the E. faecium strains were positive for any of the virulence genes tested. The clonal relationships among the 22 E. faecium strains were determined by pulsed-field gel electrophoresis (PFGE) typing. A total of 10 PFGE patterns were observed with 22 strains and a few of the strains from different probiotic products had identical (100% Dice similarity) PFGE patterns. In conclusion, the E. faecium strains in a few commercial probiotics exhibited AMR to medically-important antimicrobials, but none contained virulence genes.

Developing a xenograft human tumor model in immunocompetent mice.

Animal models are essential to cancer research, but current xenograft models are limited in their utility especially due to the lack of an immune system. Here we demonstrate that a xenograft tumor model can be developed in immunocompetent mice by tolerizing murine fetuses to human tumor cells. A375 human melanoma cells were injected into day E14 fetuses and after birth mice were challenged with A375 cells to determine their ability to develop tumors. Intravenous injections of cells resulted in metastatic-like lung tumors, which were verified to be human in origin by immunohistochemistry and PCR. These results were replicated with several other human tumor types: BxPC3 (human pancreatic adenocarcinoma), MDA-MB-231 (human breast adenocarcinoma), M21 (human melanoma), and HeLa (human cervical adenocarcinoma). Development of an immunocompetent xenograft tumor model would allow the further elucidation of the interaction of the immune system with therapy in both preclinical research and patient derived xenografts.

Mucoepidermoid Carcinoma in the Skull of an Orange-winged Amazon Parrot (Amazona amazonica).

A 33-year-old female intact orange-winged Amazon parrot (Amazona amazonica) presented for a slowly growing mass over the right eye. A computed tomography scan performed with and without intravenous contrast revealed a heterogeneous mixed soft tissue and mineral-dense mass with a small area of non-contrast-enhancing fluid density located between the orbits at the caudal aspect of the nasal passages, with associated lysis of the right caudal nasal passage and the right frontal bone. Following euthanasia, the mass was found to consist of soft tissue between the right eye and nostril over the right frontal bone. Lysis of the underlying bone resulted in a bony defect leading into the infraorbital sinus along the dorsorostral aspect of the right eye. Histopathology revealed an unencapsulated, poorly demarcated, highly cellular neoplasm composed of islands and trabeculae of neoplastic cells embedded in abundant loose fibrovascular stroma which completely obliterated the cortical bone and sinuses of the rostral skull and infiltrated the surrounding muscle and soft tissue. Histologically, the tumor was consistent with a high-grade mucoepidermoid carcinoma, characterized by the presence of epidermoid, intermediate, and mucous-producing cell types. No evidence of metastasis was identified. The tissue of origin was suspected to be salivary or nasal mucous glands, but was difficult to confirm due to distortion of normal tissue architecture as a result of the tumor. Although mucoepidermoid carcinomas are a common salivary gland tumor in human medicine, they are not well recognized in avian species, and no specific case reports exist describing this pathology in an Amazon parrot. Despite the lack of distinct salivary glands in most avian species, mucoepidermoid carcinomas can occur, can cause significant clinical disease, and should be included as a differential diagnosis for avian patients presenting with similar lesions.

Bacterial Adrenergic Sensors Regulate Virulence of Enteric Pathogens in the Gut.

UNLABELLED: Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, which is largely used as a surrogate EHEC model for murine infections, are exposed to several host neurotransmitters in the gut. An important chemical exchange within the gut involves the neurotransmitters epinephrine and/or norepinephrine, extensively reported to increase virulence gene expression in EHEC, acting through two bacterial adrenergic sensors: QseC and QseE. However, EHEC is unable to establish itself and cause its hallmark lesions, attaching and effacing (AE) lesions, on murine enterocytes. To address the role of these neurotransmitters during enteric infection, we employed C. rodentium Both EHEC and C. rodentium harbor the locus of enterocyte effacement (LEE) that is necessary for AE lesion formation. Here we show that expression of the LEE, as well as that of other virulence genes in C. rodentium, is also activated by epinephrine and/or norepinephrine. Both QseC and QseE are required for LEE gene activation in C. rodentium, and the qseC and qseE mutants are attenuated for murine infection. C. rodentium has a decreased ability to colonize dopamine ß-hydroxylase knockout (Dbh(-/-)) mice, which do not produce epinephrine and norepinephrine. Both adrenergic sensors are required for C. rodentium to sense these neurotransmitters and activate the LEE genes during infection. These data indicate that epinephrine and norepinephrine are sensed by bacterial adrenergic receptors during enteric infection to promote activation of their virulence repertoire. This is the first report of the role of these neurotransmitters during mammalian gastrointestinal (GI) infection by a noninvasive pathogen. IMPORTANCE: The epinephrine and norepinephrine neurotransmitters play important roles in gut physiology and motility. Of note, epinephrine and norepinephrine play a central role in stress responses in mammals, and stress has profound effects on GI function. Bacterial enteric pathogens exploit these neurotransmitters as signals to coordinate the regulation of their virulence genes. The bacterial QseC and QseE adrenergic sensors are at the center of this regulatory cascade. C. rodentium is a noninvasive murine pathogen with a colonization mechanism similar to that of EHEC, enabling the investigation of host signals in mice. The presence of these neurotransmitters in the gut is necessary for C. rodentium to fully activate its virulence program, in a QseC/QseE-dependent manner, to successfully colonize its murine host. Our study data provide the first example of epinephrine and norepinephrine signaling within the gut to stimulate infection by a bacterial pathogen in a natural animal infection.

Cell Based Drug Delivery: Micrococcus luteus Loaded Neutrophils as Chlorhexidine Delivery Vehicles in a Mouse Model of Liver Abscesses in Cattle.

The recent WHO report on antibiotic resistances shows a dramatic increase of microbial resistance against antibiotics. With only a few new antibiotics in the pipeline, a different drug delivery approach is urgently needed. We have obtained evidence demonstrating the effectiveness of a cell based drug delivery system that utilizes the innate immune system as targeting carrier for antibacterial drugs. In this study we show the efficient loading of neutrophil granulocytes with chlorhexidine and the complete killing of E. coli as well as Fusobacterium necrophorum in in-vitro studies. Fusobacterium necrophorum causes hepatic abscesses in cattle fed high grain diets. We also show in a mouse model that this delivery system targets infections of F. necrophorum in the liver and reduces the bacterial burden by an order of magnitude from approximately 2•106 to 1•105.