Silent threat in honey bee colonies: infection dynamics and molecular epidemiological assessment of black queen cell virus in Turkey.

Viruses can have devastating effects and cause epidemics in honey bee (Apis mellifera) colonies. Black queen cell virus (BQCV), which is one of the most common honey bee viruses, affects queen bee larvae and their pupae. This study provides information on the dynamics of BQCV infection in honey bees, using molecular diagnostics to investigate the effects of other pathogens and seasonal patterns that are considered relevant to the epidemiology of BQCV. The results showed a relatively high prevalence of the viruses studied. The prevalence of BQCV, acute bee paralysis virus, and deformed wing virus in worker bees was found to be 90%, 62%, and 84%, respectively. The prevalence of BQCV was 58% in larvae and pupae. Furthermore, the prevalence of Nosema ceranae was 46% in worker bees. Statistical analysis showed possible combined effects of BQCV and other examined viruses in terms of infection dynamics, while BQCV did not show seasonal variation. The BQCV isolates detected in this study were placed in a phylogenetic framework using sequence data from comprehensive sampling in previous studies. The analysis suggested that the Turkish strains of BQCV clustered together with Australian and European strains and consisted of homogeneous populations that had evolved from a common ancestor. This is the first report of BQCV infection dynamics in honey bees in Turkey.

High-Throughput, High-Resolution Interferometric Light Microscopy of Biological Nanoparticles.

Label-free, visible light microscopy is an indispensable tool for studying biological nanoparticles (BNPs). However, conventional imaging techniques have two major challenges: (i) weak contrast due to low-refractive-index difference with the surrounding medium and exceptionally small size and (ii) limited spatial resolution. Advances in interferometric microscopy have overcome the weak contrast limitation and enabled direct detection of BNPs, yet lateral resolution remains as a challenge in studying BNP morphology. Here, we introduce a wide-field interferometric microscopy technique augmented by computational imaging to demonstrate a 2-fold lateral resolution improvement over a large field-of-view (>100 × 100 µm2), enabling simultaneous imaging of more than 104 BNPs at a resolution of ∼150 nm without any labels or sample preparation. We present a rigorous vectorial-optics-based forward model establishing the relationship between the intensity images captured under partially coherent asymmetric illumination and the complex permittivity distribution of nanoparticles. We demonstrate high-throughput morphological visualization of a diverse population of Ebola virus-like particles and a structurally distinct Ebola vaccine candidate. Our approach offers a low-cost and robust label-free imaging platform for high-throughput and high-resolution characterization of a broad size range of BNPs.

Robust Visualization and Discrimination of Nanoparticles by Interferometric Imaging.

Single-molecule and single-nanoparticle biosensors are a growing frontier in diagnostics. Digital biosensors are those which enumerate all specifically immobilized biomolecules or biological nanoparticles, and thereby achieve limits of detection usually beyond the reach of ensemble measurements. Here we review modern optical techniques for single nanoparticle detection and describe the single-particle interferometric reflectance imaging sensor (SP-IRIS). We present challenges associated with reliably detecting faint nanoparticles with SP-IRIS, and describe image acquisition processes and software modifications to address them. Specifically, we describe a image acquisition processing method for the discrimination and accurate counting of nanoparticles that greatly reduces both the number of false positives and false negatives. These engineering improvements are critical steps in the translation of SP-IRIS towards applications in medical diagnostics.

Nanoparticle classification in wide-field interferometric microscopy by supervised learning from model.

Interference-enhanced wide-field nanoparticle imaging is a highly sensitive technique that has found numerous applications in labeled and label-free subdiffraction-limited pathogen detection. It also provides unique opportunities for nanoparticle classification upon detection. More specifically, the nanoparticle defocus images result in a particle-specific response that can be of great utility for nanoparticle classification, particularly based on type and size. In this work, we combine a model-based supervised learning algorithm with a wide-field common-path interferometric microscopy method to achieve accurate nanoparticle classification. We verify our classification schemes experimentally by blindly detecting gold and polystyrene nanospheres, and then classifying them in terms of type and size.

Label-Free and High-Throughput Detection of Biomolecular Interactions Using a Flatbed Scanner Biosensor.

Fluorescence based microarray detection systems provide sensitive measurements; however, variation of probe immobilization and poor repeatability negatively affect the final readout, and thus quantification capability of these systems. Here, we demonstrate a label-free and high-throughput optical biosensor that can be utilized for calibration of fluorescence microarrays. The sensor employs a commercial flatbed scanner, and we demonstrate transformation of this low cost (∼100 USD) system into an Interferometric Reflectance Imaging Sensor through hardware and software modifications. Using this sensor, we report detection of DNA hybridization and DNA directed antibody immobilization on label-free microarrays with a noise floor of ∼30 pg/mm2, and a scan speed of 5 s (50 s for 10 frames averaged) for a 2 mm × 2 mm area. This novel system may be used as a standalone label-free sensor especially in low-resource settings, as well as for quality control and calibration of microarrays in existing fluorescence-based DNA and protein detection platforms.

Quantitative interferometric reflectance imaging for the detection and measurement of biological nanoparticles.

The sensitive detection and quantitative measurement of biological nanoparticles such as viruses or exosomes is of growing importance in biology and medicine since these structures are implicated in many biological processes and diseases. Interferometric reflectance imaging is a label-free optical biosensing method which can directly detect individual biological nanoparticles when they are immobilized onto a protein microarray. Previous efforts to infer bio-nanoparticle size and shape have relied on empirical calibration using a 'ruler' of particle samples of known size, which was inconsistent and qualitative. Here, we present a mechanistic physical explanation and experimental approach by which interferometric reflectance imaging may be used to not only detect but also quantitatively measure bio-nanoparticle size and shape. We introduce a comprehensive optical model that can quantitatively simulate the scattering of arbitrarily-shaped nanoparticles such as rod-shaped or filamentous virions. Finally, we optimize the optical design for the detection and quantitative measurement of small and low-index bio-nanoparticles immersed in water.

Corynebacterium cutis Lysate Treatment Can Increase the Efficacies of PPR Vaccine.

This study aimed to evaluate the effects of Peste des petits ruminants (PPR) vaccine on cytokine and antibody levels in sheep when administered alone or in combination with Corynebacterium cutis lysate (CCL). The PPR vaccine group received a single subcutaneous axillary injection of the PPR vaccine (1 mL containing tissue culture infectious dose (TCID)50 attenuated live PPRV, n = 6) and the combination treatment (1 mL CCL and 1 mL PPR vaccine, n = 6) groups received a single subcutaneous axillary injection of both CCL and PPR vaccine. Blood samples were collected from sheep before the treatment and at different points after treatment (1, 3, 7, 14, 21, and 28 days). Plasma and serum samples were evaluated for antibody percentage, levels of cytokines IL-6, IL-10, IFN-γ, IL-4, IL-12, and IL-18, oxidative stress marker Thiobarbituric acid reactive substances, and hematological and biochemical parameters. Maximum protective antibody levels reach 3-4 weeks after vaccine administration. The combination treatment resulted in significant changes in IFN-γ, IL-4, IL-12, and IL-18 cytokine levels. These changes were not evident when only the PPR vaccine was administered and antibody percentage against PPRV was short term in PPR vaccine group. In conclusion, combined usage of the PPR vaccine with CCL resulted in a heightened cytokine response, leading to improved antibody level against PPR virus. Repeated CCL treatments can lead to earlier vaccine potency, provide protective efficacy for a longer time, and increase passive immunity.

Serum Biochemistry of Lumpy Skin Disease Virus-Infected Cattle.

Lumpy skin disease is an economically important poxvirus disease of cattle. Vaccination is the main method of control but sporadic outbreaks have been reported in Turkey. This study was carried out to determine the changes in serum biochemical values of cattle naturally infected with lumpy skin disease virus (LSDV). For this study, blood samples in EDTA, serum samples, and nodular skin lesions were obtained from clinically infected animals (n = 15) whereas blood samples in EDTA and serum samples were collected from healthy animals (n = 15). A quantitative real-time PCR method was used to detect Capripoxvirus (CaPV) DNA in clinical samples. A real-time PCR high-resolution melt assay was performed to genotype CaPVs. Serum cardiac, hepatic, and renal damage markers and lipid metabolism products were measured by autoanalyzer. LSDV nucleic acid was detected in all samples which were obtained from clinically infected cattle. The results of serum biochemical analysis showed that aspartate aminotransferase, alkaline phosphatase, total protein, and creatinine concentrations were markedly increased in serum from infected animals. However, there were no significant differences in the other biochemical parameters evaluated. The results of the current study suggest that liver and kidney failures occur during LSDV infection. These findings may help in developing effective treatment strategies in LSDV infection.

Physical modeling of interference enhanced imaging and characterization of single nanoparticles.

Interferometric imaging schemes have gained significant interest due to their superior sensitivity over imaging techniques that are solely based on scattered signal. In this study, we outline the theoretical foundations of imaging and characterization of single nanoparticles in an interferometric microscopy scheme, examine key parameters that influence the signal, and benchmark the model against experimental findings.

Effects of inactive parapoxvirus ovis on cytokine levels in rats.

The aim of this study is to determine the effects of iPPOV on pro-inflammatory and anti-inflammatory cytokine levels in rats. iPPOV (1 ml/rat) was administered intraperitoneal route to 49 rats, except for 7 rats (Control, 0 group). Serum samples were collected from 7 rats at 1st, 2nd, 4th, 8th, 12th, 16th and 24th hr after treatments. Levels of TNF-α, IL-6, IL-12 and IL-10 were determined using ELISA. Administration of iPPOV stimulated TNF-α (16th and 24th hr) and IL-6 (12th, 16th and 24th hr) synthesis and caused fluctuations in IL-10 and IL-12 concentrations. In conclusion, increased cytokine levels could be attributed to immunomodulatory activity of iPPOV, however, detailed studies are required to fully understand effects of iPPOV on immune system.

Interferometric Reflectance Imaging Sensor (IRIS)--A Platform Technology for Multiplexed Diagnostics and Digital Detection.

Over the last decade, the growing need in disease diagnostics has stimulated rapid development of new technologies with unprecedented capabilities. Recent emerging infectious diseases and epidemics have revealed the shortcomings of existing diagnostics tools, and the necessity for further improvements. Optical biosensors can lay the foundations for future generation diagnostics by providing means to detect biomarkers in a highly sensitive, specific, quantitative and multiplexed fashion. Here, we review an optical sensing technology, Interferometric Reflectance Imaging Sensor (IRIS), and the relevant features of this multifunctional platform for quantitative, label-free and dynamic detection. We discuss two distinct modalities for IRIS: (i) low-magnification (ensemble biomolecular mass measurements) and (ii) high-magnification (digital detection of individual nanoparticles) along with their applications, including label-free detection of multiplexed protein chips, measurement of single nucleotide polymorphism, quantification of transcription factor DNA binding, and high sensitivity digital sensing and characterization of nanoparticles and viruses.

An 8-year longitudinal sero-epidemiological study of bovine leukaemia virus (BLV) infection in dairy cattle in Turkey and analysis of risk factors associated with BLV seropositivity.

Enzootic bovine leukosis (EBL) which is caused by bovine leukaemia virus (BLV) has an important economic impact on dairy herds due to reduced milk production and restrictions on livestock exports. This study was conducted to determine the BLV infection status in Central Anatolia Region of Turkey, an important milk production centre, and to examine the risk factors such as purchasing cattle, increasing cattle age, cattle breed and herd size associated with transmission of BLV infection. To estimate the rate of BLV infection, a survey for specific antibodies in 28,982 serum samples from animals belonging to 1116 different herds situated in Central Anatolia Region of Turkey were tested from January 2006 to December 2013. A generalized mixed linear model was used to evaluate the risk factors that influenced BLV seroprevalence. Antibodies against BLV were detected in 431 (2.28 %) of 18,822 Holstein and 29 (0.28 %) of 10,160 Brown Swiss cows. Among 1116 herds, 132 herds (11.82 %) had one or more positive animals. Also results of our study show that the prevalence of BLV infection increased from 2006 to 2011, and it tends to reduce with BLV control programme. Furthermore, we found positive associations between percentage of seropositive animal and increasing cattle age, herd size, cattle breed and purchased cattle. Age-specific prevalence showed that BLV prevalence increased with age. These factors should be taken into consideration for control of BLV infection.

Nanoscale characterization of DNA conformation using dual-color fluorescence axial localization and label-free biosensing.

Quantitative determination of the density and conformation of DNA molecules tethered to the surface can help optimize and understand DNA nanosensors and nanodevices, which use conformational or motional changes of surface-immobilized DNA for detection or actuation. We present an interferometric sensing platform that combines (i) dual-color fluorescence spectroscopy for precise axial co-localization of two fluorophores attached at different nucleotides of surface-immobilized DNA molecules and (ii) independent label-free quantification of biomolecule surface density at the same site. Using this platform, we examined the conformation of DNA molecules immobilized on a three-dimensional polymeric surface and demonstrated simultaneous detection of DNA conformational change and binding in real-time. These results demonstrate that independent quantification of both surface density and molecular nanoscale conformation constitutes a versatile approach for nanoscale solid-biochemical interface investigations and molecular binding assays.

The serological and virological investigation of canine adenovirus infection on the dogs.

Two types of Canine Adenovirus (CAVs), Canine Adenovirus type 1 (CAV-1), the virus which causes infectious canine hepatitis, and Canine Adenovirus type 2 (CAV-2), which causes canine infectious laryngotracheitis, have been found in dogs. In this study, blood samples taken from 111 dogs, which were admitted to the Internal Medicine Clinic of Selcuk University, Faculty of Veterinary Medicine, with clinical symptoms. Seventy-seven dogs were sampled from Isparta and Burdur dog shelters by random sampling, regardless of the clinical findings. Dogs showed a systemic disease, characterized by fever, diarrhea, vomiting, oculonasal discharge, conjunctivitis, severe moist cough, signs of pulmonary disease and dehydration. Two dogs had corneal opacity and photophobia. In serological studies, 188 serum samples were investigated on the presence of CAV antibodies by ELISA. Total 103 (103/188-54.7%) blood samples were detected to be positive for CAV antibodies by ELISA. However, 85 (85/188-45.2%) blood samples were negative. Blood leukocyte samples from dogs were processed and inoculated onto confluent monolayers of MDCK cells using standard virological techniques. After third passage, cells were examined by direct immunoflourescence test for virus isolation. But positive result was not detected. In conclusion, this study clearly demonstrates the high prevalence of CAV infection in dogs.

Smart interface materials integrated with microfluidics for on-demand local capture and release of cells.

Stimuli responsive, smart interface materials are integrated with microfluidic technologies creating new functions for a broad range of biological and clinical applications by controlling the material and cell interactions. Local capture and on-demand local release of cells are demonstrated with spatial and temporal control in a microfluidic system.