Urine Antigen Detection as an Aid to Diagnose Invasive Aspergillosis.

Background: Establishing rapid diagnoses of invasive aspergillosis (IA) is a priority tests that detect galactomannan and ß-d-glucan are available, but are technically cumbersome and rely on invasive sampling (blood or bronchoalveolar lavage). Methods: We optimized a lateral flow dipstick assay using the galactofuranose-specific monoclonal antibody (mAb476), which recognizes urine antigens after Aspergillus fumigatus pulmonary infection in animals. Urine samples were obtained from a cohort of 78 subjects undergoing evaluation for suspected invasive fungal infections, and stored frozen until testing. Urine was processed by centrifugation through desalting columns and exposed to dipsticks. Reviewers blinded to clinical diagnoses graded results. Western blots were performed on urine samples from 2 subjects to characterize mAb476-reactive antigens. Results: Per-patient sensitivity and specificity for diagnosis of proven or probable IA in the overall cohort was 80% (95% confidence interval [CI], 61.4%-92.3%) and 92% (95% CI, 74%-99%), respectively. In the subgroup with cancer, sensitivity was 89.5% (95% CI, 66.7%-98.7%) and specificity was 90.9% (95% CI, 58.7%-99.8%); among all others, sensitivity and specificity were 63.6% (95% CI, 30.8%-89.1%) and 92.9% (95% CI, 66.1%-99.8%), respectively. Eliminating lung transplant recipients with airway disease increased sensitivity in the noncancer cohort (85.7% [95% CI, 42.1%-99.6%]). Semiquantitative urine assay results correlated with serum galactomannan indices. Western blots demonstrated mAb476-reactive antigens in urine from cases, ranging between 26 kDa and 35 kDa in size. Conclusions: Urine testing using mAb476 may be used as an aid to diagnose IA in high-risk patients.

Potential To Streamline Heterologous DNA Prime and NYVAC/Protein Boost HIV Vaccine Regimens in Rhesus Macaques by Employing Improved Antigens.

UNLABELLED: In a follow-up to the modest efficacy observed in the RV144 trial, researchers in the HIV vaccine field seek to substantiate and extend the results by evaluating other poxvirus vectors and combinations with DNA and protein vaccines. Earlier clinical trials (EuroVacc trials 01 to 03) evaluated the immunogenicity of HIV-1 clade C GagPolNef and gp120 antigens delivered via the poxviral vector NYVAC. These showed that a vaccination regimen including DNA-C priming prior to a NYVAC-C boost considerably enhanced vaccine-elicited immune responses compared to those with NYVAC-C alone. Moreover, responses were improved by using three as opposed to two DNA-C primes. In the present study, we assessed in nonhuman primates whether such vaccination regimens can be streamlined further by using fewer and accelerated immunizations and employing a novel generation of improved DNA-C and NYVAC-C vaccine candidates designed for higher expression levels and more balanced immune responses. Three different DNA-C prime/NYVAC-C+ protein boost vaccination regimens were tested in rhesus macaques. All regimens elicited vigorous and well-balanced CD8(+)and CD4(+)T cell responses that were broad and polyfunctional. Very high IgG binding titers, substantial antibody-dependent cellular cytotoxicity (ADCC), and modest antibody-dependent cell-mediated virus inhibition (ADCVI), but very low neutralization activity, were measured after the final immunizations. Overall, immune responses elicited in all three groups were very similar and of greater magnitude, breadth, and quality than those of earlier EuroVacc vaccines. In conclusion, these findings indicate that vaccination schemes can be simplified by using improved antigens and regimens. This may offer a more practical and affordable means to elicit potentially protective immune responses upon vaccination, especially in resource-constrained settings. IMPORTANCE: Within the EuroVacc clinical trials, we previously assessed the immunogenicity of HIV clade C antigens delivered in a DNA prime/NYVAC boost regimen. The trials showed that the DNA prime crucially improved the responses, and three DNA primes with a NYVAC boost appeared to be optimal. Nevertheless, T cell responses were primarily directed toward Env, and humoral responses were modest. The aim of this study was to assess improved antigens for the capacity to elicit more potent and balanced responses in rhesus macaques, even with various simpler immunization regimens. Our results showed that the novel antigens in fact elicited larger numbers of T cells with a polyfunctional profile and a good Env-GagPolNef balance, as well as high-titer and Fc-functional antibody responses. Finally, comparison of the different schedules indicates that a simpler regimen of only two DNA primes and one NYVAC boost in combination with protein may be very efficient, thus showing that the novel antigens allow for easier immunization protocols.

The Influence of Packaging on Palatability and Shelf Life Stability of Horse Treats.

Horse treat packaging may be composed of materials including plastic and paper which protect the product from the environment to improve shelf life. Objectives of this research were to 1) assess the impact of packaging on shelf life of horse treats and 2) evaluate the impact of packaging on horse preferences. Three packaging treatments (control, poly, and paper) were examined at five time points over a 12 month period. Treatments were analyzed for moisture, water activity, mold, yeast, pH, and volatile organic acids. Horse preference testing evaluated first treatment sniffed, consumed, and finished as well as number of treats consumed. Significance was set at P < .05 and trends at P < .10. Moisture content and water activity increased in all treatments (P < .01) from month 0 to month 12, with paper packaging providing a greater fluctuation and containing visible mold at month 12 (P < .01). No difference was observed for first treatment sniffed, consumed, or finished during preference testing. However a trend (P = .09) for the period∗treatment interaction was observed for number of treats consumed, with poly increasing while paper decreased. These data indicate that packaging impacts shelf life and horse preference of treats.

Palatability of Horse Treats: Comparing the Preferences of Horses and Humans.

Despite its importance to product development, few data compare preferences for horses with human consumers. The objectives of this research were to compare treat preferences of horses against horse owners. Product A was a disk-shaped cinnamon-flavored flax-based treat, and product B was a textured apple-flavored oat-based treat. Horses were presented with two treat products in a paired preference test which comprised separate olfaction and consumption periods. Consumers evaluated the treats separately for purchase intent as well as hedonic testing of sensory attributes. No difference was observed for first product sniffed, consumed, or finished during the horse preference test. However, moderate positive correlations were observed between first product sniffed and consumed (P = .01, ф = 0.40) as well as first product consumed and finished (P < .01, ф = 0.48). Horse owners rated product A lower in appearance, texture, size, and purchase intent (P < .01) than product B. These results indicate that consumer testing for animal food should be considered during product development.

Multi-wavelength Spatial LED illumination based detector for in vitro detection of Botulinum Neurotoxin A Activity.

A portable and rapid detection system for the activity analysis of Botulinum Neurotoxins (BoNT) is needed for food safety and bio-security applications. To improve BoNT activity detection, a previously designed portable charge-coupled device (CCD) based detector was modified and equipped with a higher intensity more versatile multi-wavelength spatial light-emitting diode (LED) illumination, a faster CCD detector and the capability to simultaneously detect 30 samples. A FITC/DABCYL Förster Resonance Energy Transfer (FRET)-labeled peptide substrate (SNAP-25), with BoNT-A target cleavage site sequence was used to measure BoNT-A light chain (LcA) activity through the FITC fluorescence increase that occurs upon peptide substrate cleavage. For fluorescence excitation, a multi-wavelength spatial LED illuminator was used and compared to our previous electroluminescent (EL) strips. The LED illuminator was equipped with blue, green, red and white LEDs, covering a spectrum of 450-680 nm (red 610-650 nm, green 492-550 nm, blue 450-495 nm, and white LED 440-680 nm). In terms of light intensity, the blue LED was found to be ~80 fold higher than the previously used blue EL strips. When measuring the activity of LcA the CCD detector limit of detection (LOD) was found to be 0.08 nM LcA for both the blue LED (2 s exposure) and the blue EL (which require ≥60 s exposure) while the limits of quantitation (LOQ) is about 1 nM. The LOD for white LED was higher at 1.4 nM while the white EL was not used for the assay due to a high variable background. Unlike the weaker intensity EL illumination the high intensity LED illumination enabled shorter exposure times and allowed multi-wavelength illumination without the need to physically change the excitation strip, thus making spectrum excitation of multiple fluorophores possible increasing the versatility of the detector platform for a variety of optical detection assays.

The Influence of Topically Applied Oil-Based Palatants on Eating Behavior in Horses.

Palatants may be added to equine feed and medication either during or after manufacturing to enhance product acceptance. Prior studies have examined a variety of palatants but results have been limited and inconsistent. Therefore, the objective of this research was to evaluate topically applied oil-based palatants on feeding preferences in horses. Stock-type horses (n = 10) were used in this paired preference test across a two-phase study. Phase one compared six palatants (banana, anise, peppermint, apple, spearmint, and orange) to a control (corn oil), whereas phase two compared preference among palatants (anise, apple, and peppermint). Feeding stocks were utilized and horses were allowed 15 seconds for olfaction followed by 3 minutes for consumption. Variables recorded included first diet sniffed and consumed, first action, aversive behaviors, excessive salivation, and consumption. Each trial was also video-recorded and number of chews were counted. Data were analyzed using chi-square and t tests in SAS version 9.4 with P < .05 established as significant. Findings from phase one reveal excessive salivation was observed less frequently (P < .05) for most palatants with the exception of peppermint and orange. Orange negatively impacted palatability indicated by less consumption when compared with the control (P = .02), although there was no impact on chews per gram. No difference between control and treatment diets for first sniff or first consumed was observed when analyzed individually in either phase one or two, although there was a moderate positive correlation (ф = 0.39, P = .04) between olfaction and consumption during the peppermint and anise comparison. Consumption as the first action was consistent across all trials (P < .05). Anise was preferred over apple and peppermint as indicated by higher total consumption (P < .05) in phase two. These data indicate that oil-based palatants can affect feed preferences in horses with increased palatability from anise and decreased palatability from orange flavors.

An Assessment of Decontamination Strategies for Materials Commonly Used in Canine Equipment.

Working canines are frequently exposed to hazardous environments with a high potential for contamination. Environmental contamination may occur in many ways. Contamination may be chemical, biological, radiological, or nuclear. Examples may include a pipeline rupture following an earthquake, microbiological contamination of floodwaters, or exposure to toxic industrial chemical such as hydrogen chloride, ammonia, or toluene. Evidence to support effective methods for decontamination of equipment commonly used by working canines is lacking. Recent work has identified decontamination protocols for working canines, but little data are available to guide the decontamination of equipment used during tactical operations. The objective of our work was to investigate the effects of cleanser, cleaning method, and material type on contaminant reduction for tactical canine equipment materials using an oil-based contaminant as a surrogate for toxic industrial chemical exposure. A contaminant was applied, and effectiveness was represented as either success (= 50% contaminant reduction) or failure (< 50% contaminant reduction). A two-phase study was used to investigate cleanser, method of cleaning, and material types for effective contaminant reduction. In phase 1, Simple Green® cleanser had a higher frequency (P = .0075) of failure, but method and material did not affect contamination reduction (P > .05). In phase 2, Dawn® (P = .0004) and Johnson's® (P = .0414) successfully reduced contamination. High-pressure cleaning (HPC) resulted in successful decontamination (P < .0001). These novel data demonstrate potential techniques for reduction of contaminants on tactical canine equipment.

Miniaturized 96-well ELISA chips for staphylococcal enterotoxin B detection using portable colorimetric detector.

A previously developed fluorescence sensing platform, combining spatial illumination using electroluminescence (EL) semiconductor strips with charge coupled device (CCD)-based detection (EL-CCD), was adapted to a new 96-well chip for colorimetric immunological assays, enhancing the capabilities of the EL-CCD platform. The modified system was demonstrated using a colorimetric-based enzyme linked immunosorbent assay (ELISA) for detection of staphylococcal enterotoxin B (SEB). Limits of detection (LODs) of 3.9 ng/mL (+/-2.4 ng/mL) SEB were determined with the ELISA chip measured using the EL-CCD platform, following a standard 4-h ELISA protocol. The LODs were comparable to those obtained using standard 96-well ELISA plates measured using a standard laboratory 96-well plate reader. The miniature 96-well ELISA chip however required as little as 5-microL samples, representing a tenfold reduction in sample volume compared to a standard 96-well ELISA plates. The ELISA chip also demonstrated detection of SEB spiked into various food matrices (milk, mushrooms, and mayonnaise) using limited-to-no sample preparation, with LODs ranging from 3.9 to 18.5 ng/mL depending on the matrix. The EL-CCD platform is versatile, capable of multi-mode detection (e.g., fluorescent and colorimetric along with solution and solid phase assays), and could readily be applied to other field portable or point-of-care applications.

Effect of Road Transport on the Equine Cecal Microbiota.

The effects of travel stress on the equine cecal microbiota are poorly understood. We hypothesized that travel would affect the equine cecal microbiota. Cecally-cannulated horses (n = 6) were randomly assigned to one of two groups, travel (n = 3) and control (n = 3). Horses received a basal diet (Strategy, Purina Animal Nutrition) with 1.2% body weight mixed grass/alfalfa. Travel horses were transported to an unfamiliar location, stalled to simulate weekend horse show conditions, and then returned to the Southern Illinois University Equine Center. Control horses remained at the equine center for the entire study. Cecal fluid was collected on a 6-hour rotating schedule, four times daily throughout the 6-day study. Data were analyzed using mixed models in SAS with P < .05. Cecal bacterial DNA was extracted, followed by 16S RNA sequencing and then analyzed using QIIME 1.8.0. Averages of sequence data were reported by phase (baseline, transportation, post-travel). Although there were no effects of travel associated with ß-diversity (P > .05), analysis of α-diversity measures indicated an effect within the travel group during the transportation phase as compared with baseline (P < .05). Interestingly, α-diversity was also affected for control horses in the return phase when compared to baseline. This may be due to the disruption of the return of the travel group. In addition, we identified multiple taxa affected by travel at both the genus and phylum level. Continued profiling of equine gastrointestinal microbiota is necessary to improve our understanding of equine microbial dysbiosis.

An HIV gp120-CD4 Immunogen Does Not Elicit Autoimmune Antibody Responses in Cynomolgus Macaques.

A promising concept for human immunodeficiency virus (HIV) vaccines focuses immunity on the highly conserved transition state structures and epitopes that appear when the HIV glycoprotein gp120 binds to its receptor, CD4. We are developing chimeric antigens (full-length single chain, or FLSC) in which gp120 and CD4 sequences are flexibly linked to allow stable intrachain complex formation between the two moieties (A. DeVico et al., Proc Natl Acad Sci U S A 104:17477-17482, 2007, doi:10.1073/pnas.0707399104; T. R. Fouts et al., J Virol 74:11427-11436, 2000, doi:10.1128/JVI.74.24.11427-11436.2000). Proof of concept studies with nonhuman primates show that FLSC elicited heterologous protection against simian-human immunodeficiency virus (SHIV)/simian immunodeficiency virus (SIV) (T. R. Fouts et al., Proc Natl Acad Sci U S A 112:E992-E999, 2016, doi:10.1073/pnas.1423669112), which correlated with antibodies against transition state gp120 epitopes. Nevertheless, advancement of any vaccine that comprises gp120-CD4 complexes must consider whether the CD4 component breaks tolerance and becomes immunogenic in the autologous host. To address this, we performed an immunotoxicology study with cynomolgus macaques vaccinated with either FLSC or a rhesus variant of FLSC containing macaque CD4 sequences (rhFLSC). Enzyme-linked immunosorbent assay (ELISA) binding titers, primary CD3(+) T cell staining, and temporal trends in T cell subset frequencies served to assess whether anti-CD4 autoantibody responses were elicited by vaccination. We find that immunization with multiple high doses of rhFLSC did not elicit detectable antibody titers despite robust responses to rhFLSC. In accordance with these findings, immunized animals had no changes in circulating CD4(+) T cell counts or evidence of autoantibody reactivity with cell surface CD4 on primary naive macaque T cells. Collectively, these studies show that antigens using CD4 sequences to stabilize transition state gp120 structures are unlikely to elicit autoimmune antibody responses, supporting the advancement of gp120-CD4 complex-based antigens, such as FLSC, into clinical testing.

Virological and molecular characterization of a simian human immunodeficiency virus (SHIV) encoding the envelope and reverse transcriptase genes from HIV-1.

Simian-human immunodeficiency virus encoding both reverse transcriptase (RT) and envelope genes of HIV-1 (RT Env SHIV) is important for evaluating biomedical prevention modalities for HIV/AIDS. We describe virological characterization of a clade B RT Env SHIV following infection of macaques via multiple routes. In vivo passage of the RT Env SHIV through Indian rhesus macaque enhanced infectivity. Expanded virus had minimal envelope heterogeneity and was inhibited by NNRTIs and CCR5 antagonists. Infection of macaques with RT Env SHIV via mucosal or intravenous routes resulted in stable infection accompanied by peak plasma viremia of approximately 5×10(6) copies/ml that was controlled beyond set point. Molecular homogeneity of the virus was maintained following in vivo passage. Inhibition of RT Env SHIV by RT and entry inhibitors and ease of in vivo transmission make it a useful model for testing the efficacy of combinations of entry and RT inhibitors in nonhuman primates.

A fluorescence detection platform using spatial electroluminescent excitation for measuring botulinum neurotoxin A activity.

Current biodetection illumination technologies (laser, LED, tungsten lamp, etc.) are based on spot illumination with additional optics required when spatial excitation is required. Herein we describe a new approach of spatial illumination based on electroluminescence (EL) semiconductor strips available in several wavelengths, greatly simplifying the biosensor design by eliminating the need for additional optics. This work combines EL excitation with charge-coupled device (CCD) based detection (EL-CCD detector) of fluorescence for developing a simple portable detector for botulinum neurotoxin A (BoTN-A) activity analysis. A Förster Resonance Energy Transfer (FRET) activity assay for BoTN-A was used to both characterize and optimize the EL-CCD detector. The system consists of two modules: (1) the detection module which houses the CCD camera and emission filters, and (2) the excitation and sample module, containing the EL strip, the excitation filter and the 9-well sample chip. The FRET activity assay used in this study utilized a FITC/DABCYL-SNAP-25 peptide substrate in which cleavage of the substrate by BoTN-A, or its light chain derivative (LcA), produced an increase in fluorescence emission. EL-CCD detector measured limits of detection (LODs) were similar to those measured using a standard fluorescent plate reader with valves between 0.625 and 1.25 nM (31-62 ng/ml) for LcA and 0.313 nM (45 ng/ml) for the full toxin, BoTN-A. As far as the authors are aware this is the first demonstration of phosphor-based EL strips being used for the spatial illumination/excitation of a surface, coupled with CCD for point of care detection.