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.

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.

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.

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.

Broad-spectrum inhibitors against 3C-like proteases of feline coronaviruses and feline caliciviruses.

UNLABELLED: Feline infectious peritonitis and virulent, systemic calicivirus infection are caused by certain types of feline coronaviruses (FCoVs) and feline caliciviruses (FCVs), respectively, and are important infectious diseases with high fatality rates in members of the Felidae family. While FCoV and FCV belong to two distinct virus families, the Coronaviridae and the Caliciviridae, respectively, they share a dependence on viral 3C-like protease (3CLpro) for their replication. Since 3CLpro is functionally and structurally conserved among these viruses and essential for viral replication, 3CLpro is considered a potential target for the design of antiviral drugs with broad-spectrum activities against these distinct and highly important viral infections. However, small-molecule inhibitors against the 3CLpro enzymes of FCoV and FCV have not been previously identified. In this study, derivatives of peptidyl compounds targeting 3CLpro were synthesized and evaluated for their activities against FCoV and FCV. The structures of compounds that showed potent dual antiviral activities with a wide margin of safety were identified and are discussed. Furthermore, the in vivo efficacy of 3CLpro inhibitors was evaluated using a mouse model of coronavirus infection. Intraperitoneal administration of two 3CLpro inhibitors in mice infected with murine hepatitis virus A59, a hepatotropic coronavirus, resulted in significant reductions in virus titers and pathological lesions in the liver compared to the findings for the controls. These results suggest that the series of 3CLpro inhibitors described here may have the potential to be further developed as therapeutic agents against these important viruses in domestic and wild cats. This study provides important insights into the structure and function relationships of 3CLpro for the design of antiviral drugs with broader antiviral activities. IMPORTANCE: Feline infectious peritonitis virus (FIPV) is the leading cause of death in young cats, and virulent, systemic feline calicivirus (vs-FCV) causes a highly fatal disease in cats for which no preventive or therapeutic measure is available. The genomes of these distinct viruses, which belong to different virus families, encode a structurally and functionally conserved 3C-like protease (3CLpro) which is a potential target for broad-spectrum antiviral drug development. However, no studies have previously reported a structural platform for the design of antiviral drugs with activities against these viruses or on the efficacy of 3CLpro inhibitors against coronavirus infection in experimental animals. In this study, we explored the structure-activity relationships of the derivatives of 3CLpro inhibitors and identified inhibitors with potent dual activities against these viruses. In addition, the efficacy of the 3CLpro inhibitors was demonstrated in mice infected with a murine coronavirus. Overall, our study provides the first insight into a structural platform for anti-FIPV and anti-FCV drug development.

Outer membrane proteins of Fusobacterium necrophorum subsp. necrophorum and subsp. funduliforme.

Fusobacterium necrophorum, classified into subsp. necrophorum (Fnn) and subsp. funduliforme (Fnf), is frequently associated with necrotic infections of animals and humans. The outer membrane proteins (OMP) of many Gram negative bacteria play an important role in bacterial adhesion and establishment of infection. The OMP profile of F. necrophorum has not been well characterized. We analyzed OMP of bovine strains of Fnn and Fnf and human strains of F. necrophorum. Electrophoretic separations of extracted OMP of Fnn and Fnf strains of cattle showed a total of 19 and 20 protein bands, respectively. The most prominent protein band was 40 kDa in Fnn and 37.5 kDa in Fnf. The four human clinical strains examined had more heterogeneous banding patterns and had different profiles than those of bovine Fnf strains. A total of 11 protein bands in Fnn and 13 protein bands in Fnf were recognized by sera from cattle with liver abscesses. The intensities of many of the bands in Fnn were higher than that of Fnf. We conclude that the two subspecies of F. necrophorum differ in their OMP profiles and the difference may account for differences in their virulence and involvement in the pathogenesis of necrotic 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.

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.

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.

Microarray assessment of virulence, antibiotic, and heavy metal resistance in an agricultural watershed creek.

Potential risks associated with impaired surface water quality have commonly been evaluated by indirect description of potential sources using various fecal microbial indicators and derived source-tracking methods. These approaches are valuable for assessing and monitoring the impacts of land-use changes and changes in management practices at the source of contamination. A more detailed evaluation of putative etiologically significant genetic determinants can add value to these assessments. We evaluated the utility of using a microarray that integrates virulence genes with antibiotic and heavy metal resistance genes to describe and discriminate among spatially and seasonally distinct water samples from an agricultural watershed creek in Eastern Ontario. Because microarray signals may be analyzed as binomial distributions, the significance of ambiguous signals can be easily evaluated by using available off-the-shelf software. The FAMD software was used to evaluate uncertainties in the signal data. Analysis of multilocus fingerprinting data sets containing missing data has shown that, for the tested system, any variability in microarray signals had a marginal effect on data interpretation. For the tested watershed, results suggest that in general the wet fall season increased the downstream detection of virulence and resistance genes. Thus, the tested microarray technique has the potential to rapidly describe the quality of surface waters and thus to provide a qualitative tool to augment quantitative microbial risk assessments.

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.

Catecholamines increase conjugative gene transfer between enteric bacteria.

The ability of pathogenic bacteria to sense and respond to periods of host stress is critical to their lifestyle. Adrenaline and norepinephrine are catecholamines that mediate acute host stress in vertebrates and invertebrates. Catecholamines are also used as environmental cues to enhance growth, motility and virulence of bacterial pathogens via specific binding receptors. Incidence of multidrug resistant and highly virulent bacterial pathogens is on the rise, and majority of the genes for antimicrobial resistance (AMR) and virulence are carried on horizontally transferable genetic elements. Conjugation machinery offers an efficient method for acquisition of AMR and virulence genes, which may be responsible for propelling the evolution of pathogenic bacteria. Here we show that norepinephrine (NE) at physiological concentrations enhances horizontal gene transfer (HGT) efficiencies of a conjugative plasmid from a clinical strain of Salmonella Typhimurium to an Escherichia coli recipient in vitro. Expressions of plasmid encoded transfer (tra) genes necessary for conjugation were also significantly upregulated in the presence of NE. Phentolamine, an α-adrenergic receptor antagonist, negated the effects of NE on conjugation more strongly than propranolol, a ß-adrenergic receptor antagonist. This study for the first time provides evidence that innate mediators of acute host stress may influence evolution and adaptation of bacterial pathogens.

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.

Gelatinase contributes to the pathogenesis of endocarditis caused by Enterococcus faecalis.

The Gram-positive pathogen Enterococcus faecalis is a leading agent of nosocomial infections, including urinary tract infections, surgical site infections, and bacteremia. Among the infections caused by E. faecalis, endocarditis remains a serious clinical manifestation and unique in that it is commonly acquired in a community setting. Infective endocarditis is a complex disease, with many host and microbial components contributing to the formation of bacterial biofilm-like vegetations on the aortic valve and adjacent areas within the heart. In the current study, we compared the pathogenic potential of the vancomycin-resistant E. faecalis V583 and three isogenic protease mutants (ΔgelE, ΔsprE, and ΔgelE ΔsprE mutants) in a rabbit model of enterococcal endocarditis. The bacterial burdens displayed by GelE(-) mutants (ΔgelE and ΔgelE ΔsprE mutants) in the heart were significantly lower than those of V583 or the SprE(-) mutant. Vegetations on the aortic valve infected with GelE(-) mutants (ΔgelE and ΔgelE ΔsprE mutants) also showed a significant increase in deposition of fibrinous matrix layer and increased chemotaxis of inflammatory cells. In support of a role for proteolytic modulation of the immune response to E. faecalis, we also demonstrate that GelE can cleave the anaphylatoxin complement C5a and that this proteolysis leads to decreased neutrophil migration in vitro. In vivo, a decreased heterophil (neutrophil-like cell) migration was observed at tissue sites infected with GelE-producing strains but not at those infected with SprE-producing strains. Taken together, these observations suggest that of the two enterococcal proteases, gelatinase is the principal mediator of pathogenesis in endocarditis.

Microbial ecological response of the intestinal flora of Peromyscus maniculatus and P. leucopus to heavy metal contamination.

Heavy metal contamination negatively affects natural systems including plants, birds, fish and bacteria by reducing biodiversity at contaminated sites. At the Tri-State Mining District, efforts have been made to remediate sites to mitigate the detrimental effects that contamination has caused on human health. While the remediation effort has returned the site to within federal safety standards, it is unclear if this effort is sufficient to restore floral and faunal communities. Intrinsic to ecosystem and organism health is the biodiversity and composition of microbial communities. We have taken advantage of recent advances in sequencing technology and surveyed the bacterial community of remediated and reference soils as well as the intestinal microbial community of two ubiquitous rodent species to provide insight on the impacts of residual heavy metal contamination on the ecosystem. Rodents found on the remediated site had reduced body mass, smaller body size and lower body fat than animals on reference sites. Using bar-coded, massively parallel sequencing, we found that bacterial communities in both the soil and Peromyscus spp. gastrointestinal tracts had no difference in diversity between reference and remediated sites but assemblages differed in response to contamination. These results suggest that niche voids left by microbial taxa that were unable to deal with the remnant levels of heavy metals on remediated sites were replaced by taxa that could persist in this environment. Whether this replacement provided similar ecosystem services as ancestral bacterial communities is unknown.

Development of microarray and multiplex polymerase chain reaction assays for identification of serovars and virulence genes in Salmonella enterica of human or animal origin.

Salmonella enterica is an important enteric pathogen consisting of many serovars that can cause severe clinical diseases in animals and humans. Rapid identification of Salmonella isolates is especially important for epidemiologic monitoring and controlling outbreaks of disease. Although immunologic and DNA-based serovar identification methods are available for rapid identification of isolates, they are time consuming or costly or both. In the current study, 2 molecular methods for identification of Salmonella serovars were developed and validated. A 70-mer oligonucleotide spotted microarray was developed that consisted of probes that detected genes responsible for genetic variation among isolates of Salmonella that can be used for serotyping. A multiplex polymerase chain reaction (PCR) assay was also developed, which is capable of identifying 42 serovars, thus providing a valuable prediction of the pathogenicity of the isolates by detecting the presence of virulence genes sseL, invA, and spvC. The gene spvC was the best predictor of pathogenicity. In a blind study, traditional serologic methods were correlated at 93.3% with the microarray-based method and 100% with the multiplex PCR-based serovar determination.