Multiplex and on-site PCR detection of swine diseases based on the microfluidic chip system.

BACKGROUND: At present, the process of inspection and quarantine starts with sampling at the customs port, continues with transporting the samples to the central laboratory for inspection experiments, and ends with the inspected results being fed back to the port. This process had the risks of degradation of biological samples and generation of pathogenic microorganisms and did not meet the rapid on-site detection demand because it took a rather long time. Therefore, it is urgently needed to develop a rapid and high-throughput detection assay of pathogenic microorganisms at the customs port. The aim of this study was to develop a microfluidic chip to rapidly detect swine pathogenic microorganisms with high-throughput and higher accuracy. Moreover, this chip will decrease the risk of spreading infection during transportation. RESULTS: A series of experiments were performed to establish a microfluidic chip. The resulting data showed that the positive nucleic acid of four swine viruses were detected by using a portable and rapid microfluidic PCR system, which could achieve a on-site real-time quantitative PCR detection. Furthermore, the detection results of eight clinical samples were obtained within an hour. The lowest concentration that amplified of this microfluidic PCR detection system was as low as 1 copies/µL. The results showed that the high specificity of this chip system in disease detection played an important role in customs inspection and quarantine during customs clearance. CONCLUSION: The microfluidic PCR detection system established in this study could meet the requirement for rapid detection of samples at the customs port. This chip could avoid the risky process of transporting the samples from the sampling site to the testing lab, and drastically reduce the inspection cycle. Moreover, it would enable parallel inspections on one chip, which greatly raised the efficiency of inspection.

Screening microchip sites to predict body temperature in young calves.

Thermal microchip sensors can automate body temperature measurements. The best site of implantation is still unknown, and the accuracy and precision of body temperature predictions based on microchip data need to be investigated. The aim of this study was to investigate the best site for microchip implant for monitoring body temperature in dairy calves. Seventeen calves were used (32.2 ± 5.2 kg of body weight) and the microchips were implanted four days after birth. The microchips were implanted at navel, ear and tail base (subcutaneous), neck (cleidocephalicus) and internal face of leg (gracilis) (intramuscular). Rectal temperature (RT, °C), obtained with a clinical thermometer, was considered as core temperature. Air temperature (AT), relative humidity (RH) and the temperature and humidity index (THI) were evaluated at the same time of rectal and microchip temperature measurements over 56 days. The range of AT, RH and THI was 7.6-34.4 °C, 17.5-99.0% and 50.6 to 91.5. The average for rectum, ear, neck, tail, leg, and navel were 38.7; 36.9; 38.0; 37.0, 37.8 and 37.0 °C. The intramuscular implantations had closest values to RT. The correlations between RT and ear, neck, tail, leg, and navel temperatures were 0.56, 0.60, 0.60, 0.53 e 0.48. The RT prediction based on microchip data had precision (rc) ranged between 0.49 and 0.60 and accuracy (Cb) between 0.79 and 0.88. The inclusion of AT, RH and THI as predictive variables in models decrease the mean absolute error (23%) and increase the precision (21.3%) and accuracy (10.2%). The Concordance Correlation Coefficient and root-mean-square error for equations using tail or neck microchips were 0.68 and 0.67, and 0.29 and 0.28 °C, respectively. The tail base is a promising site for microchip implantation to predict rectal temperature. The inclusion of air temperature as a predictive variable in the models is recommended.

Two novel protein chips for the detection of antibodies against porcine parvovirus.

BACKGROUND: PPV is one of the most important pathogens causing porcine reproductive disorder. It has been shown in clinical cases to be a commonly mixed infection with other important swine diseases which can aggravate the severity of the disease and bring serious economic losses to the pig industry. Serological methods, such as hemagglutination inhibition assays (HAI), serum neutralization (SN), and the modified direct complement-fixation (MDCF) test were utilized earlier, whereas the enzyme-linked immunosorbent assay (ELISA) is the most frequently applied assay to detect PPV-specific antibodies. RESULTS: We establish the visible protein chip and the cyanine dye 3 (Cy3)-labeled protein chip to detect the clinical serum from pigs. In this study, the recombinant protein VP2 of PPV was expressed in E.coli, purified with nickel magnetic beads, and then printed onto epoxy-coated glass slides for preparation of the protein chip. After a series of experiments, the conditions of antigen protein concentration, incubation time of primary antibody or secondary antibody, and optimal serum dilution fold were optimized, resulting in a successful visible protein chip and Cy3-labeled protein chip. The results showed that the positive serum, diluted up to 6000-fold, can be detected by the visible protein chip, and the positive serum, diluted up to 12,800-fold, can be detected by the Cy3-labeled protein chip, suggesting the high sensitivity of these protein chips. Moreover, the positive detection ratio, sensitivity, and specificity of these two kinds of protein chips were higher than those of commercial ELISA antibody detection kits. CONCLUSION: Overall, these two protein chips can be used to rapidly diagnose clinical samples with high throughput.

A comprehensive evaluation of microchips to measure temperature in dairy calves.

Elevated temperature is often an indicator of an immune response and used in the diagnosis of illness in dairy calves; however, measuring rectal temperature is labor intensive and often not measured daily on the farm. The objective of this study was to measure body temperature using a microchip and determine an appropriate implant site that would passively read body temperature in dairy calves. First, the precision of the temperature microchips and the rectal thermometer were tested ex vivo. Then, Holstein bull calves (n = 12) at 14 ± 12 d (mean ± SD) of age were implanted with microchips subcutaneously by the scutulum of the ear, subcutaneously in the upper scapula (SCAP), and intramuscularly in the trapezius muscle of the neck. One week after implantation, a temperature reading was taken for every microchip implant site using a radiofrequency ID (RFID) reader, as well as rectally and in the tympanic membrane using a digital thermometer every 60 min for 24 h in each calf (hereafter, the hourly study). Additionally, microchip readings and rectal temperatures were taken daily at 0800 h from 8 wk of age (n = 9; 57 ± 12 d of age) until 2 wk postweaning for a subset of the bull calves used in the hourly study (hereafter, the daily study). In the ex vivo trial, the microchip readings were very highly correlated with the rectal thermometer (r = 0.96), and the average coefficient of variation between microchip readings was very low (0.12 ± 0.03%). The relationships between the microchip readings within ear, SCAP, and neck and rectal and tympanic temperatures were analyzed using Pearson correlations and Bland-Altman plots. The ear and neck readings were strongly correlated for the hourly study [individual animal correlation; median (Q1, Q3), r = 0.78 (0.73, 0.84)] and for the daily study [r = 0.79 (0.73, 0.89)] across calves. However, rectal temperature was not significantly correlated with ear, SCAP, neck, or tympanic temperature for the daily and hourly studies. Results suggest that temperature microchips measure temperature appropriately, but temperature is dependent upon the implant site in calves, and temperature measured at ear, SCAP, and neck implant sites cannot be used to estimate rectal temperature. Future research should determine thresholds for fever that are specific to implant sites in calves.

Evidence that the stress hormone cortisol regulates biofilm formation differently among Flavobacterium columnare isolates.

The impact of cortisol on Flavobacterium columnare biofilm formation was explored. Firstly, the dynamics of biofilm formation by one highly (HV) and one low virulent (LV) F. columnare isolate with and without the stress hormone cortisol under microfluidic flow conditions was characterized. This to confirm that F. columnare cells could form biofilm under cortisol supplementation, and to compare the temporal and structural differences between different treatment groups. One trial revealed that in both isolates cell aggregates resembling biofilms occurred within 7-h post-inoculation. Consequently, cell clusters were sloughed away, followed by a rebuilding of bacterial cell aggregates, suggestive for a high spreading capacity. While the HV isolate revealed cell aggregates formed upstream at all time-points, for the LV isolate this was only seen upon cortisol supplementation. Secondly, the transcriptional effect of genes (gldK, gldL, gldM, gldN, sprA, sprE, sprT, and porV) belonging to the Type IX secretion system involved in gliding motility was investigated in planktonic and biofilm cells of a HV and LV isolate to which no, a low (LD) or high (HD) dose of cortisol was added. Significantly lower expression of gliding genes gldK, gldL, gldM and gldN, and of protein secretion regulator porV was seen in the LV isolate planktonic cells supplemented with a HD-cortisol. The LV isolate biofilm cells treated with the HD-cortisol showed a significant upregulation of sprT, encoding mobile surface adhesion important in bacterial colonization. This is the first evidence for the co-regulatory effect of cortisol on biofilm formation and F. columnare gliding gene expression.

Best Practice Approach for Assessment of Microchip-associated Tumors in Preclinical Safety Studies: Position of the Registry of Industrial Toxicology Animal-data (RITA).

Microchip (passive radio-frequency identification device) implantation is a common and widely employed means of animal identification in laboratory animal facilities. However, these devices have been associated with tumors of the skin and subcutis in rodents. While microchip-associated tumors are rare, they pose a challenge for accurate diagnosis and documentation in preclinical toxicity studies. Documentation of these tumors should differentiate microchip-associated lesions with spontaneously occurring or test article-induced tumors. Standardizing criteria for microchip-associated lesions will aid the diagnostic process and allow for preclinical regulatory standardization. To this end, the Registry of Industrial Toxicology Animal-data have developed clear recommendations for diagnosis and documentation of microchip-associated lesions.

Point-of-care devices for physiological measurements in field conditions. A smorgasbord of instruments and validation procedures.

Point-of-care (POC) devices provide quick diagnostic results that increase the efficiency of patient care. Many POC devices are currently available to measure metabolites, blood gases, hormones, disease biomarkers or pathogens in samples as diverse as blood, urine, feces or exhaled breath. This diversity is potentially very useful for the comparative physiologist in field studies if proper validation studies are carried out to justify the accuracy of the devices in non-human species under different conditions. Our review presents an account of physiological parameters that can be monitored with POC devices and surveys the literature for suitable quantitative and statistical procedures for comparing POC measurements with reference "gold standard" procedures. We provide a set of quantitative tools and report on different correlation coefficients (Lin's Concordance Correlation Coefficient or the more widespread Pearson correlation coefficient), describe the graphical assessment of variation using Bland-Altman plots and discuss the difference between Model I and Model II regression procedures. We also report on three validation datasets for lactate, glucose and hemoglobin measurements in birds using the newly proposed procedures. We conclude the review with a haphazard account of future developments in the field, emphasizing the interest in lab-on-a-chip devices to carry out more complex experimental measurements than the ones currently available in POC devices.

Experimental system to detect a labeled cell monolayer in a microfluidic environment.

PURPOSE: To investigate the feasibility of detecting a living cell monolayer labeled with gadoterate (Gd-DOTA) in a microfluidic environment, by micromagnetic resonance imaging (MRI) in a 2.35T small-animal system. The development of new targeted contrast agents (CAs) requires proof-of-concept studies in order to establish the detectability of the CA and to predict the role of biodistribution in its uptake mechanisms. A promising approach is to carefully mimic the in vivo pharmacokinetic context with reduced experimental complexity compared to in vivo situations. MATERIALS AND METHODS: A dedicated experimental system was built by combining a microfluidic slide and a radiofrequency probe based on a 6 mm diameter multiturn transmission-line resonator. Adherent KB cells were incubated with different concentrations of Gd. MRI data were acquired at 2.35T with a 3D gradient echo and a resolution of 12.4 µm perpendicular to the cell layer. The longitudinal relaxation rate, R1 , was measured as a function of the amount of Gd internalized by the cells. RESULTS: R1 measurements for different Gd concentrations per cell were performed using data with an signal-to-noise ratio (SNR) of 100. Relaxation-rate variations ΔR1 of 0.035 s(-1) were measured. A quenching effect was observed at Gd concentrations above 20 fmol/cell. CONCLUSION: Our results suggest that this dedicated experimental system is suitable for specifically assessing new high-relaxivity targeted CAs under real-time uptake conditions.

Technical note: A portable on-chip assay system for absorbance and plasmonic detection of protein hormone in milk.

This paper reports a portable device and method to extract and detect protein hormone in milk samples. Recombinant protein hormone spiked into milk samples was extracted by solid-phase extraction, and detection was carried out using the plasmonic property of gold nanoislands deposited on a glass substrate. Trace levels of hormone spiked in milk were analyzed by their optical absorbance property using a microfluidic chip. We built a portable assay system using disposable lab-on-chip devices. The proposed method is able to detect spiked recombinant protein hormone in milk at concentrations as low as 5ng/mL.

The GENOTEND chip: a new tool to analyse gene expression in muscles of beef cattle for beef quality prediction.

BACKGROUND: Previous research programmes have described muscle biochemical traits and gene expression levels associated with beef tenderness. One of our results concerning the DNAJA1 gene (an Hsp40) was patented. This study aims to confirm the relationships previously identified between two gene families (heat shock proteins and energy metabolism) and beef quality. RESULTS: We developed an Agilent chip with specific probes for bovine muscular genes. More than 3000 genes involved in muscle biology or meat quality were selected from genetic, proteomic or transcriptomic studies, or from scientific publications. As far as possible, several probes were used for each gene (e.g. 17 probes for DNAJA1). RNA from Longissimus thoracis muscle samples was hybridised on the chips. Muscles samples were from four groups of Charolais cattle: two groups of young bulls and two groups of steers slaughtered in two different years. Principal component analysis, simple correlation of gene expression levels with tenderness scores, and then multiple regression analysis provided the means to detect the genes within two families (heat shock proteins and energy metabolism) which were the most associated with beef tenderness. For the 25 Charolais young bulls slaughtered in year 1, expression levels of DNAJA1 and other genes of the HSP family were related to the initial or overall beef tenderness. Similarly, expression levels of genes involved in fat or energy metabolism were related with the initial or overall beef tenderness but in the year 1 and year 2 groups of young bulls only. Generally, the genes individually correlated with tenderness are not consistent across genders and years indicating the strong influence of rearing conditions on muscle characteristics related to beef quality. However, a group of HSP genes, which explained about 40% of the variability in tenderness in the group of 25 young bulls slaughtered in year 1 (considered as the reference group), was validated in the groups of 30 Charolais young bulls slaughtered in year 2, and in the 21 Charolais steers slaughtered in year 1, but not in the group of 19 steers slaughtered in year 2 which differ from the reference group by two factors (gender and year). When the first three groups of animals were analysed together, this subset of genes explained a 4-fold higher proportion of the variability in tenderness than muscle biochemical traits. CONCLUSION: This study underlined the relevance of the GENOTEND chip to identify markers of beef quality, mainly by confirming previous results and by detecting other genes of the heat shock family as potential markers of beef quality. However, it was not always possible to extrapolate the relevance of these markers to all animal groups which differ by several factors (such as gender or environmental conditions of production) from the initial population of reference in which these markers were identified.

A dog oviduct-on-a-chip model of serous tubal intraepithelial carcinoma.

Ovarian cancer is the fifth cause of cancer-related mortality in women, with an expected 5-year survival rate of only 47%. High-grade serous carcinoma (HGSC), an epithelial cancer phenotype, is the most common malignant ovarian cancer. It is known that the precursors of HGSC originate from secretory epithelial cells within the Fallopian tube, which first develops as serous tubal intraepithelial carcinoma (STIC). Here, we used gene editing by CRISPR-Cas9 to knock out the oncogene p53 in dog oviductal epithelia cultured in a dynamic microfluidic chip to create an in vitro model that recapitulated human STIC. Similar to human STIC, the gene-edited oviduct-on-a-chip, exhibited loss of cell polarization and had reduced ciliation, increased cell atypia and proliferation, with multilayered epithelium, increased Ki67, PAX8 and Myc and decreased PTEN and RB1 mRNA expression. This study provides a biomimetic in vitro model to study STIC progression and to identify potential biomarkers for early detection of HGSC.

Improved bovine embryo production in an oviduct-on-a-chip system: prevention of poly-spermic fertilization and parthenogenic activation.

The oviduct provides the natural micro-environment for gamete interaction, fertilization and early embryo development in mammals, such as the cow. In conventional culture systems, bovine oviduct epithelial cells (BOEC) undergo a rapid loss of essential differentiated cell properties; we aimed to develop a more physiological in vitro oviduct culture system capable of supporting fertilization. U-shaped chambers were produced using stereo-lithography and mounted with polycarbonate membranes, which were used as culture inserts for primary BOECs. Cells were grown to confluence and cultured at an air-liquid interface for 4 to 6 weeks and subsequently either fixed for immune staining, incubated with sperm cells for live-cell imaging, or used in an oocyte penetration study. Confluent BOEC cultures maintained polarization and differentiation status for at least 6 weeks. When sperm and oocytes were introduced into the system, the BOECs supported oocyte penetration in the absence of artificial sperm capacitation factors while also preventing polyspermy and parthenogenic activation, both of which occur in classical in vitro fertilization systems. Moreover, this "oviduct-on-a-chip" allowed live imaging of sperm-oviduct epithelium binding and release. Taken together, we describe for the first time the use of 3D-printing as a step further on bio-mimicking the oviduct, with polarized and differentiated BOECs in a tubular shape that can be perfused or manipulated, which is suitable for live imaging and supports in vitro fertilization.

Separation of motile sperm for in vitro fertilization from frozen-thawed bull semen using progesterone induction on a microchip.

This study presents a novel method for the separation of motile sperm from non-progressive motile and immotile sperm and in vitro Fertilization (IVF). This separation of bull sperm was accomplished by inducing chemotaxis along a progesterone release agent in a 7.5-mm microchannel microchip composed of a biocompatible polydimethysiloxane layer and a glass gradient. The selected sperm was applied directly for IVF. In the first experiment, we tested the effect of different lengths of microchannnel (5mm, 7.5mm and 10mm) on quality parameter of separated sperm. The results showed that separated sperm using 7.5-mm microchannel chip were improved in sperm motility, swimming velocity, and beat frequency compared with other groups. In the second experiment, a medium containing sperm from swim-up method and outlet reservoir of our 7.5-mm microchannel chip was collected and mitochondrial activity of the sperm was determined by fluorescence microscopy. The sperm from the microchip had higher mitochondria activity (47.6%±6.0%) than the sperm from the swim-up method (23.6%±4.7%) (P<0.05). There were significant differences in rate of acrosome intactness between the swim-up method and the microchip (36.0%±4.1% vs. 66.8±2.1%, respectively, P<0.05). In the third experiment, we compared sperm penetration in the microchip-IVF system with a standard IVF method (droplet-IVF). The microchip-IVF group had the highest percentages of oocytes penetrated (82.2%±1.6% vs. 63.5%±2.4%) and monospermic oocytes (67.8%±3.4% vs. 42.4%±1.5%). In addition, early developmental competence of oocytes to the blastocyst stage was higher when the oocytes were inseminated in the microchip-IVF system compared with those inseminated in a standard droplet-IVF system. These results demonstrate that our microchip based on a sperm chemotaxis system is useful for motile sperm separation from frozen-thawed bull semen for IVF. Therefore, the optimized microchip system provides a good opportunity to sort motile bull sperm for IVF.

Identification of microchips through diagnostic imaging helps to determine the longevity of the golden lancehead (Bothrops insularis) in captivity / Identificação de microchips por meio de diagnóstico por imagem auxilia a determinar a longevidade da jararaca-ilhoa (Bothrops insularis) em cativeiro

As serpentes vindas da natureza e encaminhadas para centros de reabilitação ou de pesquisa têm uma idade incerta. Na natureza, esses animais, muitas vezes atingem um tamanho corpóreo menor do que os indivíduos cativos devido a uma menor frequência de alimentação. Assim, a idade de uma cobra recém-chegada da natureza é geralmente estimada com base em seu tamanho corpóreo, o qual é comparado com dados em cativeiro. A utilização dos meios de diagnóstico por imagem tem auxiliado a medicina na análise das serpentes em cativeiro, mediante avaliação da estrutura óssea, dos órgãos e de alterações. Este trabalho relata o uso de diagnóstico por imagem (radiografia e ultrassonografia) para identificar a presença de microchips e, consequentemente, estimar a idade de uma Bothrops insularis em cativeiro.(AU)

Make it spin: individual trapping of sperm for analysis and recovery using micro-contact printing.

In this article, we describe the development of a high throughput platform to spatially manipulate viable sperm for motility measurements and recovery of the best single sperm for fertilization purposes. Micro-contact printing was used to pattern islands of adhesive proteins (fibronectin) separated by sperm repellent species (Pluronic acid F-127) on commercially available polystyrene substrates. Following washing, arrays of viable single sperm were captured onto the islands demonstrating for the first time that sperm can be trapped by micro-contact printing with patterning efficiency of 90% while retaining 100% viability. These were then subjected to motility analysis whilst remaining spatially confined to the islands. Single sperm motility was assessed (n = 37) by software analysis measuring the number of rotations per second (degrees s⁻¹). The assignment of array coordinates allows the more active single sperm to be easily identified and recovered by a simple micromanipulator pipette aspiration step with automated possibility for assisted reproductive technologies or further quality correlation analysis. Taken together, we show for the first time a technique to simultaneously screen thousands of viable single sperm for motility assessment while retaining the ability for single species recovery for enhanced fertilization purposes.