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Archived formalin fixed paraffin-embedded (FFPE) tissues are powerful tools in medicine, capable of harboring diagnostic and genetic answers to challenging clinical questions. Successful utilization of DNA derived from FFPE samples is dependent upon repairing DNA damage generated from the fixation process. Methods to repair FFPE DNA have been successful in human medicine for a variety of research and clinical applications, yet remain underutilized in veterinary medicine. Despite the available technology, our study is the first to evaluate the repair of FFPE derived DNA from veterinary species for single-nucleotide polymorphism (SNP) analysis using the Illumina OvineSNP50 BeadChip and Illumina FFPE QC and DNA Restore kit. To accomplish this, 48 ovine FFPE samples were run using the Illumina OvineSNP50 BeadChip with and without restoration. Compared to pre-restore data, we found increased sample call rates, SNP call frequency, and assay metrics for all samples post-restoration. Further, we utilized four sheep with available parallel fresh DNA and FFPE DNA to compare assay metrics and genotype calls between the two starting sample types. Although fresh samples generated increased call rates, we found 99% concordance in allele calls between restored FFPE and fresh DNA for all four samples. Our results indicate successful restoration and genotyping of ovine FFPE samples using this technology, with potential for utilization in other veterinary species.

This research was funded through grants from NIH to SMB (5R03AI083735-02) and from The Islamic Development Bank to MAI.

Paratuberculosis, or Johne’s Disease (JD) is a debilitating chronic enteritis mainly affecting ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). This organism causes worldwide economic losses to the livestock industry, and is of public health importance due to the potential zoonotic risk between MAP and Crohn’s disease (CD) in humans. Without economical treatments, or a vaccine capable of preventing infection without causing cross-reactions with bovine tuberculosis, test-and-cullmethods for disease control are imperative. Unfortunately, difficulties in diagnostics and long subclinical stage hinder adequate control and is further complicated by variation in MAP exposure outcome. Interestingly, the majority of infections result in asymptomatic presentation and never progress to clinical disease. One contributing factor is host genetics, where polymorphisms in innate immune genes have been found to influence resistance and susceptibility to disease. Candidate genes identified across studies overlap with those found in CD and tuberculosis including; Solute carrier family 11 member 1 gene (SLC11A1), Nucleotide-binding-oligomerization domain containing gene 2 (NOD2), Major histocompatibility complex type II (MHC-II), and Toll-like receptor (TLR) genes. This review will highlight evidence supporting the vital role of these genes in MAP infection outcome, associated challenges, and implications for the future of JD research. The authors would like to thank the Department of Biomedical Science and Pathobiology and the Virginia-Maryland College of Veterinary Medicine for funding and support. Published version

Feral swine cost around $1.5 billion each year in agricultural, environmental, and personal property damages. They are also the most widespread carriers of the zoonotic disease brucellosis, which threatens both livestock bio-security and public health. Currently, there is no approved vaccine against brucellosis in pigs. This is a preliminary report on the development of a live-attenuated B. suis vaccine that could be employed to deliver heterologous antigens to control swine populations. An attenuated vaccine strain provided significant protection against B. suis challenge in mice. Leucine auxotrophy in the vaccine strain allowed the over-expression of heterologous antigens without the use of antibiotic resistant markers. Vaccinated mice showed the development of antibodies against expressed antigen. Further evaluation is required to assess its ability to cause infertility using the mouse model prior to further testing for use as a tool for feral swine population and disease control.

Population and health management of wildlife is a key to environmental health, domestic herd health, and ultimately public health. Many different methods including: surgical sterilization, poison baits, and sponsored hunting programs have been used in the attempt to control populations of various nuisance animal species. Particular interest has been given to immunocontraception through wildlife vaccination protocols. This study specifically looked at the potential immunocontraceptive and protective properties of a Brucella abortus RB51 ΔleuB vaccine expressing Salmonella typhimurium FliC conjugated to porcine follicle stimulating hormone beta subunit (FSHβ) or gonadotropin releasing hormone (GnRH) DNA sequences. B. abortus RB51 ΔleuB pNS4-TrcD-FliC- FSHβ (RB51LFSHβ) and B. abortus RB51 ΔleuB pNS4-TrcD-FliC-GnRH (RB51LGnRH) were tested in a pilot breeding study with BALB/c mice, and a significant reduction in fertility characteristics was observed in both male and female mice. Ultimately, this study provides support to test these vaccine candidates in feral swine, a destructive invasive species in the United States of America., fertility control, feral swine, FSHβ/GnRH, Brucella, RB51, Salmonella, feral swine population control.

Bovine brucellosis is caused byBrucella abortus.The bacterial pathogen causes economic losses because it induces abortion in cattle. Vaccination of calves with liveB. abortusstrain 19 induces a certain level of protection but induces persistent antibodies against cell envelope lipopolysaccharide that make it difficult to Distinguish Infected from Vaccinated Animals (DIVA). Live vaccineB. abortusstrain RB51 was developed to elimínate such interfering antibodies and therefore, facilitate the differentiation of infected from vaccinated animals and help in the eradication of the disease. Vaccination with strain RB51 induces levels of protection similar to strain 19 but neither of the two vaccines give complete protection. We have been working to enhance protection induced by strain RB51 vaccine. ProtectiveBrucellaantigens can be over-expressed in strain RB51 by introducing a plasmid containing theleuBgene and the genes encoding such antigens. To avoid the expression of antibiotic resistance genes, we produced aleuBdeficient strain RB51 and introduced a plasmid containing theleuBgene and the genes to be over-expressed. This new strain maintains the plasmid and has induced significantly high protection levels in mice. In addition, it allowed the construction of an RB51 vaccine strain able to expressMycobacterium bovisprotective antigens so that the vaccine could protect against brucellosis and tuberculosis simultaneously.

Salmonella serotypes linked to tomato-associated outbreaks were evaluated for survival in soil and water over a 40-day period. Salmonella enterica serotypes Anatum, Baildon, Braenderup, Montevideo, Newport, and Javiana were inoculated separately into sterile soil and water, followed by plating onto TSAYE and XLT4 at 10-day intervals. Biofilm production by Salmonella serotypes was measured on both quartz particles (soil surrogate) and glass coverslips, and was evaluated using a crystal violet dye assay. Salmonella populations in soil and water over 40 days indicated no significant differences between Salmonella serotypes tested (p > 0.05). Over a 40-day period, there was a 1.84 ± 0.22 log CFU/g and 1.56 ± 0.54 CFU/mL decrease in populations of Salmonella in soil and water, respectively. Enumeration indicated that Salmonella population fluctuated in water but decreased linearly in soil. All serotypes tested produced the "red dry and rough" morphotype on Congo Red agar. Biofilm produced by all the Salmonella serotypes tested was significantly different on quartz particles than on glass coverslips (p

Background Multidrug resistant Acinetobacter baumannii (MDR-Ab) is a Gram-negative bacterium known for causing severe nosocomial infections, attributed in part to its formation of biofilm. Siderophore is a virulence factor known to support biofilm formation by regulating iron availability. In this study, we screened 44 isolates of MDR-Ab from our Gram-negative repository to determine the strains that phenotypically form biofilm and produce siderophore. The results were compared to Pseudomonas aeruginosa PAO1, which produces both biofilm and siderophore. Methods Isolates were grown overnight in minimal M9 medium supplemented with casamino acids and hydroxyquinones at 37°C. Bacterial cells were normalized (to OD 600=0.01) and a standard diluted 10-3 tube was used in the study. A 96-well plate was inoculated with 100 microliters of each isolate in quadruplicates. This process was repeated in Tygon tubes with 50 microliters of each isolate in triplicates. The plate and Tygon tubes were incubated statically for 48 hours at 30°C and then stained with crystal violet. The contents were dissolved in 33% glacial acetic acid and analyzed by spectrophotometry to measure biofilm formation. Siderophore secretion was measured in supernatants with Chrome Azurol S (CAS) reagent and production was observed on CAS agar plates. Results High levels of biofilm formation were observed in 8 strains of MDR-Ab in the 96-well plate (3, 4, 9, 22, 61, 1010, 1012, 1022) and 6 strains in Tygon tubes (3, 4, 16, 66, 1002, 1010) (Fig. 1). There was minimal siderophore production in MDR-Ab isolates compared to PAO1 in both the 96-well plate and Tygon tubes (Fig. 2). Only 4 strains lacked siderophore production on CAS agar and were inversely negative for the secretion in medium. Figure 1 Biofilm formation in a 96-well plate and Tygon tubes (A) High levels of biofilm formation were observed in MDR-Ab strain numbers 3, 4, 9, 22, 61, 1010, 1012, 1022 in the 96-well plate. (B) High levels of biofilm formation were observed in MDR-Ab strain numbers 3, 4, 16, 66, 1002, 1010 in Tygon tubes. Figure 2 Degree of siderophore production in a 96-well plate and Tygon tubes Siderophore production of MDR-Ab was limited compared to PAO1 after inoculation in a 96-well plate (A) and in Tygon tubes (B). Conclusion Many strains of MDR-Ab readily form biofilm. Overall siderophore production is lower in MDR-Ab compared to consistent production by PAO1, but this does not appear to affect MDR-Ab’s ability to form biofilm. Unlike in PAO1, biofilm formation in MDR-Ab may occur independently of siderophore production. This research serves as a basis for understanding future MDR-Ab biofilm elimination in patient catheters and indwelling devices. Disclosures All Authors: No reported disclosures

SUMMARY Two experiments were conducted to determine the effects of Lactobacillus plantarum (DFM) on performance in broilers challenged with avian pathogenic Escherichia coli (APEC) and its effects on Salmonella colonization following S. Typhimurium challenge. In experiment 1, broilers were reared from d zero to 28 and orally challenged with 1 × 108 CFU of APEC on d 7. Treatments included a non-challenged positive control (PC), challenged negative control (NC), NC + 0.05, 0.10, or 0.20% DFM, and NC + 0.05% antibiotic (BMD). Body weight (BW) was not different among treatments. Mortality-corrected feed conversion ratio (FCRm) was increased with APEC challenge in comparison to the PC, while BMD returned FCRm to that of the PC (P ≤ 0.05). Treatment with 0.10% DFM resulted in the largest relative spleen weight (SW: % BW) at d 14, but also resulted in the lowest number of APEC associated mortalities, suggesting an enhanced immune response. In experiment 2, one-day-old broilers were orally challenged with 1 × 108 CFU of S. Typhimurium and fed a control diet (PC), or the same diet with 0.10 or 0.20% DFM. Intestinal samples were collected for determination of Salmonella colonization by the most probable number method at d 3 and d 7 post inoculation, and bioluminescence imaging using In-Vivo Imaging System at d 7 post inoculation. Treatment with DFM reduced total Salmonella content on d 7, but changes in bioluminescence were not significant. Overall, DFM treatment was concentration specific, but did result in positive responses to both APEC challenge and total Salmonella counts.

Studies indicate that persistent Salmonella colonization occurs in poultry that are infected early in life, leading to both food safety and public health concerns. Development of improved preharvest Salmonella management strategies is needed to reduce poultry product contamination. The objective of this study was to evaluate the efficacy of a product containing medium chain fatty acids (MCFA) for reducing early Salmonella colonization in turkey poults. Day-of-hatch turkeys were provided a standard starter diet supplemented with MCFA at 0 (negative and positive controls), 1.5, 3, 4.5, or 6 lbs/ton of feed. Positive control and MCFA treated birds were also crop-gavaged with 108 colony forming units (CFU) of bioluminescent Salmonella Typhimurium. Gastrointestinal tissue samples were collected at 3 days postinoculation for bioluminescence imaging (Meckel's diverticulum to the cloaca) and selective enumeration (cecal contents). Quantification of bioluminescence indicated that the 4.5 and 6 lbs/ton MCFA groups had significantly less colonization than the positive control group (p = 0.0412 and p

The presence of dust is ubiquitous in the produce growing environment and its deposition on edible crops could occur. The potential of wind-distributed soil particulate to serve as a vehicle for S. Newport transfer to tomato blossoms and consequently, to fruits, was explored. Blossoms were challenged with previously autoclaved soil containing S. Newport (9.39log CFU/g) by brushing and airborne transfer. One hundred percent of blossoms brushed with S. Newport-contaminated soil tested positive for presence of the pathogen one week after contact (P

Brucella abortus RB51 is an attenuated, stable, spontaneous rough mutant derived in the laboratory from the virulent strain B. abortus 2308. Previous studies discovered that the wboA gene, which encodes a glycosyltransferase required for synthesis of the O-polysaccharide, is disrupted in strain RB51 by an IS711 element. However, complementation of strain RB51 with a functional wboA gene (strain RB51WboA) does not confer it a smooth phenotype but results in low levels of cytoplasmic O-polysaccharide synthesis. In this study, we asked if increasing the potential availability of bactoprenol priming precursors in strain RB51WboA would increase the levels of O-polysaccharide synthesis and enhance the protective efficacy against virulent Brucella challenge. To achieve this, we overexpressed the wbkF gene, which encodes a putative undecaprenyl-glycosyltransferase involved in bactoprenol priming for O-polysaccharide polymerization, in strain RB51WboA to generate strain RB51WboAKF. In comparison to strain RB51WboA, strain RB51WboAKF expressed higher levels of O-polysaccharide, but was still attenuated and remained phenotypically rough. Mice immunized with strain RB51WboAKF developed increased levels of smooth LPS-specific serum antibodies, primarily of IgG2a and IgG3 isotype. Splenocytes from mice vaccinated with strain RB51WboAKF secreted higher levels of antigen-specific IFN-gamma and TNF-alpha and contained more numbers of antigen-specific IFN-gamma secreting CD4(+) and CD8(+) T lymphocytes when compared to those of the RB51 or RB51WboA vaccinated groups. Immunization with strain RB51WboAKF conferred enhanced protection against virulent B. abortus 2308, B. melitensis 16M and B. suis 1330 challenge when compared to the currently used vaccine strains. Our results suggest that strain RB51WboAKF has the potential to be a more efficacious vaccine than its parent strain in natural hosts. Public Health Service from the National Institute of Allergy and Infectious Diseases [AI065667-01A2] Public Health Service grant AI065667-01A2 from the National Institute of Allergy and Infectious Diseases, RV.

Mycobacterium bovis (M. bovis) BCG belongs to Mycobacterium tuberculosis complex (MTBC), highly relatedorganisms, which are 99.9 % similar at nucleotide level and phenotypically similar. Its differentiation from other membersof MTBC by conventional methods is laborious and time consuming. PCR provides a rapid alternative method fordifferentiation between members of MTBC. It depends on identification of region of difference (RD) which have beenlost during attenuation of M. bovis. Two different BCG strains (from two sources) were confirmed as a member of M.tuberculosis (MTB) complex (MTC) and as BCG strains by PCR using primers to a region of the 16S rRNA gene that isconserved in all mycobacteria and region of difference (RD1, RD4, RD9 and RD12) respectively. DNA of Mycobacteriumtuberculosis was used as a control to compare with BCG strains. The results showed that 16S rRNA gene was present inall tested strains, while the RD1, RD4, RD9 and RD12 were absent only in BCG strains

Mycobacterium bovis BCG is attenuated strain derived from M. bovis. It has been known to prevent childhood pulmonaryand meningeal tuberculosis and to treat superficial bladder cancer as immunotherapy. Administration of BCG has beenrestricted to immune-deficient individuals since 2007 because of its combined complications. Which treated withantituberculosis drugs based on assumption is fully responsive to these drugs. BCG Connaught showed moresusceptibility to the activity of isoniazid, ethambutol, kanamycin, ofloxacin, streptomycin, amikacin and capreomycinthan BCG Pasteur growing in J774A.1 cells. While the activity of rifampicin and levofloxacin was the same between thetwo tested strains. The spectrum of intracellular drug action can be ordered based on a decreasing order of inhibitoryactivity, as following rifampin > isoniazid > ethambutol > streptomycin > kanamycin> amikacin> ofloxacin>capreomycin> levofloxacin against BCG Connaught. While rifampin > isoniazid > ethambutol > kanamycin =streptomycin > ofloxacin> amikacin> levofloxacin> capreomycin for BCG Pasteur. There is a very limited publicationsdescribe the activity of antituberculosis drugs against intracellular growing BCG strains

SUMMARY Clostridial dermatitis, also referred to as cellulitis, is a disease of serious concern in turkeys in the United States whose incidence appears to have increased since its emergence in the early 1990s. The disease leads to increased mortality with the presence of fluid- and gasfilled lesions in the subcutaneous tissues of the breast, thigh, and tail-head. Management factors may contribute to the development of clostridial dermatitis. An analysis of mortality and health records of turkey flocks conducted over a one-year period for one commercial turkey operation indicates that breed, flock type, weight at processing, and stocking density affected the incidence of clostridial dermatitis. Season of placement, season of onset, prior health events, and prior vaccination and/or medication did not affect the incidence of clostridial dermatitis. Development of clostridial dermatitis, flock type, season of placement, and season of onset of clostridial dermatitis affected livability. Breed did not affect livability. In a survey of 8 commercial turkey companies, 639 of 3,398 market turkey flocks (18.8%) had developed clostridial dermatitis, and 239 of 967 farms (24.7%) raising market turkeys had at least one flockdevelopclostridialdermatitisoverthecourseofacalendaryear,withanestimatedincrease in the cost of production for flocks that developed clostridial dermatitis of 0.031 to 5.5 cents per kilogram of meat produced.

Brucella abortus live vaccines have been used successfully to control bovine brucellosis worldwide for decades. However, due to some limitations of these live vaccines, efforts are being made for the development of new safer and more effective vaccines that could also be used in other susceptible species. In this context, understanding the protective immune responses triggered by B. abortus is critical for the development of new vaccines. Such understandings will enhance our knowledge of the host/pathogen interactions and enable to develop methods to evaluate potential vaccines and innovative treatments for animals or humans. At present, almost all the knowledge regarding B. abortus specific immunological responses comes from studies in mice. Active participation of macrophages, dendritic cells, IFN-γ producing CD4(+) T-cells and cytotoxic CD8(+) T-cells are vital to overcome the infection. In this review, we discuss the characteristics of the immune responses triggered by vaccination versus infection by B. abortus, in different hosts.

The present investigation is an attempt to address the effect of P2O5 on the optical properties, cross link density (nc) as well as theoretical parameters like Poisson’s ratio and bulk modulus for (25−x)%CaO−x%P2O5–60%SiO2–5%B2O3–10%ZnO (x = 5, 10, 15, 20) novel sol–gel glasses. The optical basicity, oxide polarisability, direct/indirect band gaps and theoretical elastic constants have been calculated for all the glasses. In addition to this, the cross-link density, average force stretching constant and number of network bonds per unit volume (nb) have been calculated. The values of optical basicity and polarisabilty have allowed the same trend for all these glasses. The cross-link density decreases when the P2O5 content increases from 5 to 20 %.

Oncolytic virotherapy is a promising novel approach that overcomes the limitations posed by radiation and chemotherapy. In this study, the oncolytic efficacy of a recombinant Newcastle disease virus (rNDV) BC-KLQL-GFP, against prostate cancer stem-like/tumor initiating cells was evaluated. Xenograft derived prostaspheres (XPS) induced tumor more efficiently than monolayer cell derived prostaspheres (MCPS) in nude mice. Primary and secondary XPS show enhanced self-renewal and clonogenic potential compared to MCPS. XPS also expressed embryonic stem cell markers, such as Nanog, CD44 and Nestin. Further, prostate specific antigen (PSA) activated recombinant Newcastle Disease Virus (rNDV) was selectively cytotoxic to tumor derived DU145 prostaspheres. An effective concentration (EC 50 ) of 0.11–0.14 multiplicity of infection was sufficient to cause prostasphere cell death in serum free culture. DU145 tumor xenograft derived prostaspheres were used as tumor surrogates as they were enriched for a putative tumor initiating cell population. PSA activated rNDV was efficient in inducing cell death of cells and prostaspheres derived from primary xenografts ex-vivo , thus signifying a potential in vivo efficacy. The EC 50 (∼0.1 MOI) for cytolysis of tumor initiating cells was slightly higher than that was required for the parental cell line, but within the therapeutic margin for safety and efficacy.

Tuberculosis (TB) is a deadly infectious disease; approximately 2 billion people are currently latently infected with the causative agent Mycobacterium tuberculosis. Approximately 8 million new active cases and 2 million deaths due to TB are recorded annually.1 Rifampin (Rif) is a vital first-line TB treatment drug. Its effectiveness is hampered by the high dose required (600 mg 1×/day) and by its moderate, variable bioavailability. These issues can be explained by Rif instability at gastric pH, limited solubility at neutral pH, polymorphism, and stimulation of its own metabolism. To overcome these obstacles, we developed new cellulose-based oral drug delivery systems aiming to increase and make more consistent Rif solubility and bioavailability. Amorphous solid dispersions (ASDs) of Rif with cellulose ω-carboxyalkanoates (cellulose acetate suberate, cellulose acetate propionate adipate, and cellulose acetate butyrate sebacate) were prepared and compared with crystalline Rif (negative) and carboxymethyl cellulose acetate butyrate ASD (positive) controls. Cellulose ω-carboxyalkanoate ASDs prevented acid-catalyzed degradation in conditions mimicking the acidic stomach and provided complete release of intact Rif at intestinal pH. Rif incorporation into ASD in these novel cellulose derivative matrices creates the potential for convenient, robust, consistent, and high Rif oral bioavailability for treatment of TB.

Cell microencapsulation is a technique to treat a wide range of diseases through the continuous and controlled delivery of therapeutic products. This technique can also treat multiple diseases in the absence of immunosuppression. Over the past few years, the quality of life of patients has improved remarkably as a direct result of microencapsulation technology, as this technology eliminates the requirement of an immunosuppressant. However, much additional research needs to be conducted in order to commercialize and clinically apply more widely this life-saving technology.

Outer membrane vesicles (OMVs) are released from the outer membrane of Gram-negative bacteria. Moreover, Gram-positive bacteria also produce membrane-derived vesicles. As OMVs transport several bacterial components, especially from the cell envelope, their interaction with the host cell, with other bacteria or as immunogens, have been studied intensely. Several functions have been ascribed to OMVs, especially those related to the transport of virulence factors, antigenic protein composition, and development as acellular vaccines. In this work, we review some of the recent findings about OMVs produced by specific pathogenic bacterial species.

The aims of this study were to address the protective immune response induced by S19 vaccination (n=10) and RB51 revaccination, in pregnant (n=9) and non-pregnant (n=10) S19 calfhood-vaccinated cattle as follows: evaluate the in vitro CD4(+) and CD8(+) T-lymphocytes specific proliferation, and in vitro expression of IFN-γ by CD4(+) and CD8(+) T-cells and IL-4 by CD4(+), CD8(+) and CD21(+) lymphocytes subset. Upon in vitro stimulation with γ-irradiated Brucella abortus 2308, blood mononuclear cells from S19 vaccinated and RB51 revaccinated cows exhibited significantly higher proliferation of CD4(+) and CD8(+) T-lymphocytes and CD4(+)IFN-γ(+) T-cells compared to non-vaccinated animals. RB51 revaccination, regardless of the pregnancy status, did not enhance the proliferation of CD4(+) or CD8(+) T-cells nor IFN-γ or IL-4 production. Data from the present study suggest that cattle's cellular immune response induced after brucellosis vaccination and revaccination is due to CD4(+) and CD8(+) T-lymphocytes, being CD4(+) T-cells the main source of IFN-γ.

Escherichia coli and Salmonella enterica species are Gram-negative bacteria that have recently shown acquired resistance to multiple antimicrobials. These resistant strains, referred to as multidrug resistant (MDR), are not effectively inhibited by current antimicrobials and therefore cause a range of untreatable infections. Peptide nucleic acids (PNA) have been proposed as an alternative antimicrobial treatment. These synthetic nucleic acid mimics are effective through steric inhibition of bacterial transcription and translation. This study examines the effect of PNA on MDR strains of E. coli and S. enterica serovar Typhimurium (S. Typhimurium). Kirby-Bauer disk diffusion susceptibility and minimum inhibitory concentration determinations (MIC) were completed to show bacterial resistance levels to current antimicrobials. Using S. Typhimurium specific dnaK PNA, MIC was determined to assess the effectiveness of PNA on MDR strains of E. coli and S. Typhimurium. Results showed that while the dnaK PN...

The activity of antituberculosis drugs (streptomycin sulfate, isoniazid, pyrazinamid, and clarithromycin) embedded in biodegradable nanofibers against Mycobacterium avium has been studied by broth dilution assay and by agar plate assay. These drugs have also been embedded in electrospun polyvinyl alcohol (PVA), polyethylene oxide (PEO), and polycaprolacton (PCL) nanofibers to design a new single tablet containing first-line antituberculosis drugs. Our results show that antituberculosis drugs are active at tiny amounts (up to 300 µg mL−1 of solvent). However, within polymer matrices, high amounts of drugs are required to avoid unwanted weak interactions within PEO and PCL matrices. The successful design of a single tablet containing required amounts of antituberculosis drugs is essential for the full treatment of tuberculosis in patients with HIV. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 4009–4016, 2014.

Fluorescent protein biosensors are typically implemented via genetic encoding which makes the examination of scarce cell samples impractical. By directly delivering the protein form of the biosensor into cells using electroporation, we detected intracellular molecular activity with the sample size down to ∼100 cells with high spatiotemporal resolution.

Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

Brucellosis is one of the most common zoonotic diseases worldwide. Almost 500,000 new human cases occur each year; yet there is no vaccine for human use. Moreover, there is no universal Brucella vaccine that would provide protection against all pathogenic species of Brucella. We generated a rough, live-attenuated B. neotomae strain by deleting the wboA gene encoding a glycosyltransferase. This strain lacks the O-side chain in its lipopolysaccharide (LPS) and thus the vaccinated animals can be differentiated serologically from the field-infected animals. We tested the efficacy of rough B. neotomae strain to stimulate dendritic cells compared to the smooth wild type strain. Based on TNF-α production, our data suggests that a significantly higher stimulation was obtained when dendritic cells were stimulated with the rough vaccine strain compared to the smooth wild type B. neotomae. Furthermore, the rough mutant was cleared from mice within 6 weeks even at a dose as high as 2 x 108 CFU. Vaccinated mice showed significantly higher level of protection against a virulent B. suis 1330 challenge compared to the control mice. Antibody titers in the mice and cytokine production by the splenocytes from the vaccinated mice showed a Th1 mediated immune response that correlated with the protection.

Brucellosis is a neglected bacterial zoonotic disease in many countries affecting both humans and animals. The aim of this paper is to review published reports of the seroprevalence of brucellosis in humans and animals (cattle, buffalo, sheep, goats and dogs) in Bangladesh. The prevalence studies are based primarily on the following serological tests: rose bengal plate agglutination test (RBT), plate agglutination test (PAT), tube agglutination test (TAT), mercaptoethanol agglutination test (MET), standard tube agglutination test (STAT), slow agglutination test (SAT), milk ring test (MRT), indirect enzyme-linked immunosorbant assay (I-ELISA), competitive ELISA (C-ELISA) and fluorescent polarization assay (FPA). Seroprevalences of brucellosis were found to be affected by the sensitivity and specificity of serological tests employed. Brucellosis prevalence varied based on occupations of people (2.5-18.6%) and species of animals (3.7% in cattle, 4.0% in buffalo, 3.6% in goats and 7.3% in sheep). The prevalence of brucellosis in humans was reported in livestock farmers (2.6-21.6%), milkers (18.6%), butchers (2.5%) and veterinarians (5.3-11.1%) who have direct contact with animal and its products or who consume raw milk. According to published reports brucellosis does affect people and livestock of Bangladesh. There is an immediate need for a concerted effort to control and eradicate brucellosis from domesticated animals in Bangladesh.

Brucella is amongst the top 5 causes of zoonotic disease worldwide. Infection is through ingestion, inhalation or contact exposure. Brucella is characterized as a class B pathogen by Centers of Disease Control and Prevention (CDC). Currently, there are no efficacious vaccines available in people. Currently available USDA approved vaccines for animals include B. abortus strain RB51 and B. melitensis Rev1. Protection is mediated by a strong innate and CD4 Th1, CD8 Tc1 immune response. If protective vaccines can be developed, disease in people and animals can be controlled. While strain RB51 protects in cattle, and against intraperitoneal challenge in mice, it does not protect against respiratory challenge. Therefore, we assessed the efficacy of strain RB51 combined with different TLR agonists, and O-side chain from LPS, to enhance protection against respiratory challenge with strain 2308. We hypothesized that TLR agonists and O-side chain would enhance protection. Strains RB51 with TLR2 agonist, RB51 with TLR4 agonist and strain 19 provided significant protection in the lung. Protection using strain RB51 with TLR agonists was associated with increased IgG2a and IgG1 in the (bronchoalveolar lavage) BAL and serum, and increased IgA (serum). Splenocytes from strain RB51 with TLR2 vaccinated mice up-regulated antigen specific interferon-gamma and TNF-alpha production. Vaccination and challenge resulted in significant increases in activated dendritic cells (DCs), and increased CD4 and CD8 cells in the BAL. Overall, this study demonstrates the ability of TLR agonists 2 and 4 to up-regulate strain RB51 mediated protection in the lung to respiratory challenge against strain 2308.

Drug delivery systems refers to systems designed to transport or deliver drugs to treat a certain infected organ. Generally, these systems include loading of the drug and effective substances on a carrier (nanofibers, nanoparticles, nanorods, capsule, or matrix) and delivering these substances to the infected organs. The drugs can be used in the form of dispersion, emulsion, bandages, gels, creams, or as tablets. Developing controllable drug delivery systems allow them to be utilized in wide medical applications as wound dressings, skin treatments, or to treat internal infected organs. In this work, tuberculosis drugs (pyrazinamide, isoniazid, clarithromycin, and streptomycin) are loaded on poly(e-caprolactone), poly(ethylene oxide), and poly(vinyl alcohol) nanofibers to design single tablets suitable for HIV patients. The activity of these drugs loaded on the nanofiber is tested against Mycobacterium avium through broth dilution assay and by agar plate assay. The minimal inhibitory concentration and the minimal bactericidal concentration are also studied.

This work reports the distribution of an oral dose of Salmonella enterica serovar Enteritidis (SE) in C57Bl/6-Bcgr mice, to study its pathogenesis in a latent carrier animal. Mice orally inoculated with a high dose of SE developed a latent infection characterized by the absence of clinical symptoms in which the cecum is functioning as a “strategic site” of SE proliferation, releasing bacteria into feces intermittently over the 4-week study. A sequence of disruptions occurred in the small intestine at 1 day postinculation (PI). The microvilli exhibited different degrees of degeneration, which were reversible as the cells became vacuolated. From 2 days PI, SE was detected in the mononuclear phagocytic system, and an exponential growth of the remaining bacteria in tissues was observed until 4 days PI. The production of interferon gamma from 3 days PI is restricting the SE growth, and a plateau phase was observed from 4 to 15 days PI. A recurrence of the bacterial growth in tissue occurred from 15 to 28 days PI, especially in the cecum. Increasing our knowledge about the host–pathogen interaction of adapted pathogens with the ability to develop latency is essential for the development of an efficient strategy for Salmonella control.

Trypanosoma cruzi was demonstrated in blood smears and heart tissue from a 5-year old, female, English Cocker Spaniel that had never been outside of the state of Virginia, USA. Plasma from the dog was positive in a commercially available immunochromatographic dipstick assay for T. cruzi and negative in an immunochromatographic dipstick assay for visceral Leishmania spp. The plasma from the dog had an indirect immunofluorescent antibody titer of 1:800 against epimastigotes of T. cruzi while the titer was 1:50 against promastigotes of L. infantum. The parasite was isolated from the blood in vitro from the dog (TcVT-1 isolate) and used to experimentally infect female C3H and ICR mice. The parasite was nonpathogenic for experimentally inoculated mice. DNA was isolated from parasites grown in vitro and used to determine that the genotype of T. cruzi present in the dog was genotype TcIV. This genotype is common in raccoons, Procyon lotor, in North America and suggests that raccoons may serve as reservoirs for canine infection.

Magnetic block ionomer complexes (MBICs) containing a combination of therapeutic and imaging agents are of great interest for delivering drugs and tracking their biodistribution with magnetic resonance imaging. With an aim to develop nanocarriers with high drug loadings that integrate magnetic nanoparticles into one system, we herein report antibiotic-laden MBICs based on assembly of poly(ethylene oxide-b-acrylate) (PEO-b-PAA) ionomers with nanomagnetite and gentamicin. The polymer was bound to the magnetic nanoparticle surfaces via ligand adsorption of the PAA block, thereby creating a double corona structure with a nonionic PEO shell and an ionic region rich in PAA. The portion of carboxylates that were not bound to the magnetite provided binding sites for drug loading via ionic complexation. PEO was chosen as a block copolymer segment to improve biocompatibility and aid in dispersion through interparticle steric repulsion. Intensity average diameters increased from 34 to 62 nm upon adding the drug, sug...