Inactivation of Toxoplasma gondii in dry sausage and processed pork, and quantification of the pathogen in pig tissues prior to production.

Toxoplasma gondii is an important zoonotic foodborne parasite. Meat of infected animals appears to be a major source of infection in Europe. Pork is the most consumed meat in France, with dry sausages well represented. The risk of transmission via consumption of processed pork products is largely unknown, mainly since processing will affect viability but may not entirely inactivate all T. gondii parasites. We investigated the presence and concentration of T. gondii DNA in the shoulder, breast, ham, and heart of pigs orally inoculated with 1000 oocysts (n = 3) or tissue cysts (n = 3) and naturally infected pigs (n = 2), by means of magnetic capture qPCR (MC-qPCR). Muscle tissues of experimentally infected pigs were further used to evaluate the impact of manufacturing processes of dry sausages, including different concentrations of nitrates (0, 60, 120, 200 ppm), nitrites (0, 60, 120 ppm), and NaCl (0, 20, 26 g/kg), ripening (2 days at 16-24 °C) and drying (up to 30 days at 13 °C), by a combination of mouse bioassay, qPCR and MC-qPCR. DNA of T. gondii was detected in all eight pigs, including in 41.7% (10/24) of muscle samples (shoulder, breast and ham) and 87.5% (7/8) of hearts by MC-qPCR. The number of parasites per gram of tissue was estimated to be the lowest in the hams (arithmetic mean (M) = 1, standard deviation (SD) = 2) and the highest in the hearts (M = 147, SD = 233). However, the T. gondii burden estimates varied on the individual animal level, the tissue tested and the parasitic stage used for the experimental infection (oocysts or tissue cysts). Of dry sausages and processed pork, 94.4% (51/54) were positive for T. gondii by MC-qPCR or qPCR, with the mean T. gondii burden estimate equivalent to 31 parasites per gram (SD = 93). Only the untreated processed pork sample collected on the day of production was positive by mouse bioassay. The results suggest an uneven distribution of T. gondii in the tissues examined, and possibly an absence or a concentration below the detection limit in some of them. Moreover, the processing of dry sausages and processed pork with NaCl, nitrates, and nitrites has an impact on the viability of T. gondii from the first day of production. Results are valuable input for future risk assessments aiming to estimate the relative contribution of different sources of T. gondii human infections.

Systematic Review and Modelling of Age-Dependent Prevalence of Toxoplasma gondii in Livestock, Wildlife and Felids in Europe.

Toxoplasma gondii is a zoonotic parasite of importance to both human and animal health. The parasite has various transmission routes, and the meat of infected animals appears to be a major source of human infections in Europe. We aimed to estimate T. gondii prevalence in a selection of animal host species. A systematic literature review resulting in 226 eligible publications was carried out, and serological data were analyzed using an age-dependent Bayesian hierarchical model to obtain estimates for the regional T. gondii seroprevalence in livestock, wildlife, and felids. Prevalence estimates varied between species, regions, indoor/outdoor rearing, and types of detection methods applied. The lowest estimated seroprevalence was observed for indoor-kept lagomorphs at 4.8% (95% CI: 1.8-7.5%) and the highest for outdoor-kept sheep at 63.3% (95% CI: 53.0-79.3%). Overall, T. gondii seroprevalence estimates were highest within Eastern Europe, whilst being lowest in Northern Europe. Prevalence data based on direct detection methods were scarce and were not modelled but rather directly summarized by species. The outcomes of the meta-analysis can be used to extrapolate data to areas with a lack of data and provide valuable inputs for future source attribution approaches aiming to estimate the relative contribution of different sources of T. gondii human infection.

Comparison of a Commercial Enzyme-Linked Immunosorbent Assay (ELISA) with the Modified Agglutination Test (MAT) for the Detection of Antibodies against Toxoplasma gondii in a Cohort of Hunting Dogs.

Toxoplasmosis is a zoonotic disease, caused by the protozoan Toxoplasma gondii, affecting most warm-blooded animals. Assessing the seroprevalence of T. gondii in different animal species gives a good estimate of the global circulation of the parasite and the risk for human infections. However, the seroprevalence of T. gondii in dogs is not studied as much as other species, despite their close contact with wildlife and humans in rural or urban environments and evidence that dogs can also be a potential source for human contaminations. A commercial enzyme-inked immunosorbent assay (ELISA) kit to detect anti-T. gondii antibodies in sera of hunting dogs potentially naturally infected, was compared to the modified agglutination test (MAT), used as the reference method. The ELISA presented a sensitivity of 76.5% (CI 95%: 60.0-87.6) and a specificity of 87.7% (CI 95%: 76.7-93.9) and a substantial agreement with the MAT for the detection of canine anti-T. gondii antibodies. Both tests can therefore be used widely for epidemiology studies on T. gondii infections in dogs. With a mean seroprevalence of T. gondii infection in hunting dogs from northern Algeria of 36.8% (CI 95%: 34.9-38.7), this study also highlights the importance of T. gondii seroprevalence studies in companion animals to assess infectious risk for human populations.

Blood biomarkers for canine cancer, from human to veterinary oncology.

In recent decades, interest in circulating tumour biomarkers is increasing both in human and veterinary oncology. An ideal tumour biomarker would allow early diagnosis of neoplasia, identify it specifically, accurately, establish a prognosis and predict its behaviour, especially regarding different therapeutic solutions. It would also allow to monitor its evolution over time and all this in a non-invasive and inexpensive way. Actually, no biomarkers meeting all of these criteria have been identified in veterinary medicine, particularly due to a lack of specificity of the main protein tumour biomarkers studied to date. However, great hope is currently placed in biomarkers grouped under the name of liquid biopsy, which could prove to be effective tools for common clinical use in the near future. This review gives an update on blood cancer biomarkers studied in dogs, such as ions, proteins, nucleic acids and also circulating cells, of which some might become more prominent in the coming years to help improve the management of animal care.

Constitutive IFNα Protein Production in Bats.

Bats are the only mammals with self-powered flight and account for 20% of all extant mammalian diversity. In addition, they harbor many emerging and reemerging viruses, including multiple coronaviruses, several of which are highly pathogenic in other mammals, but cause no disease in bats. How this symbiotic relationship between bats and viruses exists is not yet fully understood. Existing evidence supports a specific role for the innate immune system, in particular type I interferon (IFN) responses, a major component of antiviral immunity. Previous studies in bats have shown that components of the IFN pathway are constitutively activated at the transcriptional level. In this study, we tested the hypothesis that the type I IFN response in bats is also constitutively activated at the protein level. For this, we utilized highly sensitive Single Molecule (Simoa) digital ELISA assays, previously developed for humans that we adapted to bat samples. We prospectively sampled four non-native chiroptera species from French zoos. We identified a constitutive expression of IFNα protein in the circulation of healthy bats, and concentrations that are physiologically active in humans. Expression levels differed according to the species examined, but were not associated with age, sex, or health status suggesting constitutive IFNα protein expression independent of disease. These results confirm a unique IFN response in bat species that may explain their ability to coexist with multiple viruses in the absence of pathology. These results may help to manage potential zoonotic viral reservoirs and potentially identify new anti-viral strategies.

Burden and regional distribution of Toxoplasma gondii cysts in the brain of COBB 500 broiler chickens following chronic infection with 76K strain.

Toxoplasmosis is a worldwide zoonosis caused by the obligate intracellular apicomplexan parasite Toxoplasma gondii (T. gondii). Chickens are ground-feeders and represent, especially if free-range, important intermediate hosts in the epidemiology of toxoplasmosis and are used as sentinels of environmental contamination with T. gondii oocysts. Until now, little is known about the burden and regional distribution of T. gondii cysts in the chicken brain. It was therefore the aim of this study to investigate the abundance and specific distribution of T. gondii cysts within the chicken brain following chronic infection with a type II strain (76 K) of T. gondii. A total of 29 chickens were included in the study and divided into control group (n = 9) and two different infection groups, a low dose (n = 10) and a high dose (n = 10) group, which were orally inoculated with 1500 or 150,000 T. gondii oocysts per animal, respectively. Seroconversion was detected in the majority of chickens of the high dose group, but not in the animals of the low dose and the control group. Moreover, T. gondii DNA was detected most frequently in the brain and more frequently in the heart than in liver, spleen, thigh and pectoral muscle using qPCR analysis. The number of T. gondii cysts, quantified in the chicken brain using histological analysis, seems to be considerably lower as compared to studies in rodents, which might explain why T. gondii infected chickens very rarely, if at all, develop neurological deficits. Similar to observations in mice, in which no lateralisation for T. gondii cysts was reported, T. gondii cysts were distributed nearly equally between the left and right chicken brain hemispheres. When different brain regions (fore-, mid- and hindbrain) were compared, all T. gondii cysts were located in the forebrain with the overwhelming majority of these cysts being present in the telencephalic pallium and subpallium. More studies including different strains and higher doses of T. gondii are needed in order to precisely evaluate its cyst burden and regional distribution in the chicken brain. Together, our findings provide insights into the course of T. gondii infection in chickens and are important to understand the differences of chronic T. gondii infection in the chicken and mammalian brain.

Infection with Toxocara canis Inhibits the Production of IgE Antibodies to α-Gal in Humans: Towards a Conceptual Framework of the Hygiene Hypothesis?

α-Gal syndrome (AGS) is a type of anaphylactic reaction to mammalian meat characterized by an immunoglobulin (Ig)E immune response to the oligosaccharide α-Gal (Galα1-3Galß1-4GlcNAc-R). Tick bites seems to be a prerequisite for the onset of the allergic disease in humans, but the implication of non-tick parasites in α-Gal sensitization has also been deliberated. In the present study, we therefore evaluated the capacity of helminths (Toxocara canis, Ascaris suum, Schistosoma mansoni), protozoa (Toxoplasma gondii), and parasitic fungi (Aspergillus fumigatus) to induce an immune response to α-Gal. For this, different developmental stages of the infectious agents were tested for the presence of α-Gal. Next, the potential correlation between immune responses to α-Gal and the parasite infections was investigated by testing sera collected from patients with AGS and those infected with the parasites. Our results showed that S. mansoni and A. fumigatus produce the terminal α-Gal moieties, but they were not able to induce the production of specific antibodies. By contrast, T. canis, A. suum and T. gondii lack the α-Gal epitope. Furthermore, the patients with T. canis infection had significantly decreased anti-α-Gal IgE levels when compared to the healthy controls, suggesting the potential role of this nematode parasite in suppressing the allergic response to the glycan molecule. This rather intriguing observation is discussed in the context of the 'hygiene hypothesis'. Taken together, our study provides new insights into the relationships between immune responses to α-Gal and parasitic infections. However, further investigations should be undertaken to identify T. canis components with potent immunomodulatory properties and to assess their potential to be used in immunotherapy and control of AGS.

Evaluation of immunogenicity and protection of the Mic1-3 knockout Toxoplasma gondii live attenuated strain in the feline host.

Toxoplasmosis is a zoonotic disease caused by the parasite Toxoplasma gondii. Up to a third of the global human population is estimated to carry a T. gondii infection, which can result in severe complications in immunocompromised individuals and pregnant women. Humans and animals can become infected by ingesting either tissue cysts containing T. gondii bradyzoites, from raw or undercooked meat, or sporulated oocysts from environmental sources. T. gondii oocysts are released in the faeces of cats and other felids, which are the parasite's definitive hosts, leading to environmental contamination. Therefore, vaccination of the feline host against T. gondii is an interesting strategy to interrupt the parasitic life cycle and subsequently limit contamination of intermediate hosts. With this goal in mind, we tested in cats, an attenuated live strain of T. gondii deleted for the Mic1 and Mic3 genes (Mic1-3KO) that was previously shown to be an efficient vaccine candidate in mouse and sheep models. Subcutaneous or oral vaccination routes induced a high specific antibody titer in the cat sera, indicating that the Mic1-3KO strain is immunogenic for cats. To assess protection induced by the vaccine candidate strain, we followed oocysts shedding by vaccinated cats, after oral challenge with a T. gondii wild-type strain. Surprisingly, a high antibody titer did not prevent cats from shedding oocysts from the challenge strain, regardless of the vaccination route. Our results show that the Mic1-3KO vaccine candidate is immunogenic in the feline host, is well tolerated and safe, but does not confer protection against oocysts shedding after natural infection with wild type T. gondii. This result highlights the particular relationship between T. gondii and its unique definitive host, which indicates the need for further investigations to improve vaccination strategies to limit environmental and livestock contaminations.

The myeloid derived suppressor cells: Who are they? Can they be used as a diagnostic tool to investigate metastasis in veterinary medicine?

Myeloid-derived suppressor cells (MDSCs) are key players in immunosuppression mechanisms that lead to tumor escape and metastasis formation. Studies on these cells in many cancer types using human patients and murine models, have greatly increased since their discovery in 1980s. MDSCs are now defined as different subpopulations with specific phenotypes in mice and humans with clear immunosuppressive capacities, which are summarized in this review. Current knowledge on these cells have allowed comparative studies and MDSCs have also recently been identified in dogs. As in other species, canine MDSCs have immunosuppressive activities and their number is increased in blood of metastasis-bearing dogs. Circulating MDSCs could therefore represent a new biomarker for cancer progression in both veterinary and human medicine. Further characterization of these cells in other cancer-suffering animal species would also be of great interest.

Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances.

If misregulated, macrophage (Mϕ)-T cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-Mϕ (GM-CSF)- and Mϕ colony-stimulating factor (M-CSF)-dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, marked by folate receptor ß (FRß), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRß+CD39+CD73+ Mϕs, which boosts adenosine production and curtails the dominance of proinflammatory Mϕs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the Mϕ extracellular purine metabolism as a novel checkpoint in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in immune-mediated diseases.

Rapid multiplex DNA amplification on an inexpensive microdevice for human identification via short tandem repeat analysis.

Forensic DNA analysis requires several steps, including DNA extraction, PCR amplification, and separation of PCR fragments. Intuitively, there are numerous situations where it would be beneficial to speed up the overall DNA analysis process; in this work, we focus on the most time-consuming component in the analysis pipeline, namely the polymerase chain reaction (PCR). Primers were specially designed to target 10 human genomic loci, all yielding amplicons shorter than 350 bases, for ease of downstream integration with on-board microchip electrophoresis. Primer concentrations were adjusted specifically for microdevice amplification, resulting in well-balanced short tandem repeat (STR) profiles. Furthermore, studies were performed to push the limits of the DNA polymerase to achieve rapid, multiplexed PCR on various substrates, including transparent and black polyethylene terephthalate (Pe), and with two distinct adhesives, toner and heat sensitive adhesive (HSA). Rapid STR-based multiplexed PCR amplification is demonstrated in 15 min on a Pe microdevice using a custom-built system for fluid flow control and thermocycling for the full 10-plex, and in 10 min for a smaller multiplex consisting of six core CODIS loci plus Amelogenin with amplicons shorter than 200bp. Lastly, preliminary studies indicate the capability of this PCR microdevice platform to be integrated with both upstream DNA extraction, and downstream microchip electrophoresis. This, coupled to the use of reagents that are compatible with lyophilization (lyo-compatible) for PCR, represents the potential for a fully integrated rotationally-driven microdevice for complete forensic DNA analysis.

Warfarin genotyping with hybridization-induced aggregation on a poly(ethylene terephthalate) microdevice.

Warfarin, a commonly prescribed oral anticoagulant, is burdened by a narrow therapeutic index and high inter-individual variability in response, making it the second leading cause of drug-related emergency room visits. Since genetic factors contribute significantly to warfarin sensitivity, a genotype-guided dosing strategy may reduce the occurrence of adverse events. While numerous methods have been demonstrated for warfarin genotyping, the specifications of most assays with respect to turnaround time and cost are not ideal for routine testing. Here, we present a unique method for warfarin genotyping based on multiplex PCR coupled with Hybridization-induced Aggregation (HIA), a bead-based technique for sequence-specific detection. A multiplex allele-specific PCR reaction was used to generate products corresponding to 3 genetic variants associated with warfarin sensitivity [CYP2C9 *2, CYP2C9 *3, and VKORC1 (1173C>T)] and an internal control product. The products were detected simultaneously on a poly(ethylene terephthalate) (PeT) microdevice using HIA, which provided genotyping results in approximately 15 minutes following PCR. The genotyping results of 23 patient DNA samples using this approach were in 100% concordance with the results of a validated test (WARFGENO test, ARUP laboratories). Additionally, the PCR reaction was successfully transferred to a PeT chip, which provided accurate genotyping results from patient DNA samples in under an hour. This work demonstrates a simple, rapid, and affordable approach to warfarin genotyping based on multiplex allele-specific PCR coupled with HIA detection. By demonstrating both chemistries on PeT microdevices, we show the potential for integration on a single device for sample-to-answer genotyping.

A centrifugal microfluidic device with integrated gold leaf electrodes for the electrophoretic separation of DNA.

Current conventional methods utilized for forensic DNA analysis are time consuming and labor-intensive requiring large and expensive equipment and instrumentation. While more portable Rapid DNA systems have been developed, introducing them to a working laboratory still necessitates a high cost of initiation followed by the recurrent cost of the devices. This has highlighted the need for an inexpensive, rapid and portable DNA analysis tool for human identification in a forensic setting. In order for an integrated DNA analysis system such as this to be realized, device operations must always be concluded by a rapid separation of short-tandem repeat (STR) DNA fragments. Contributing to this, we report the development of a unique, multi-level, centrifugal microdevice that can perform both reagent loading and DNA separation. The fabrication protocol was inspired by the print, cut and laminate (PCL) technique described previously by our group, and in accordance, offers a rapid and inexpensive option compared with existing approaches. The device comprises multiple polyester-toner fluidic layers, a cyclic olefin copolymer separation domain and integrated gold leaf electrodes. All materials are commercially-available and complement the PCL process in a way that permits fabrication of increasingly sought after single-use devices. All reagents, including a viscous sieving matrix, are loaded centrifugally, eliminating external pneumatic pumping, and the sample is separated in <5 minutes using an effective separation length of only 4 cm (reagent loading to completed separation, is <37 minutes). The protocol for gold leaf electrode manufacture yielded up to 30 electrodes for less than $3 (cost of a 79 mm × 79 mm gold leaf sheet) and when using a device combining these electrodes and centrifugal reagent/polymer loading, the electrophoretic separation of STR fragments with two base resolution was demonstrated. This exemplary performance makes the device an ideal candidate for further integration and development of an inexpensive, portable and rapid forensic human identification system.

Microfluidic enzymatic DNA extraction on a hybrid polyester-toner-PMMA device.

To date, the forensic community regards solid phase extraction (SPE) as the most effective methodology for the purification of DNA for use in short tandem repeat (STR) polymerase chain reaction (PCR) amplification. While a dominant methodology, SPE protocols generally necessitate the use of PCR inhibitors (guanidine, IPA) and, in addition, can demand timescales of up to 30 min due to the necessary load, wash and elution steps. The recent discovery and characterization of the EA1 protease has allowed the user to enzymatically extract (not purify) DNA, dramatically simplifying the task of producing a PCR-ready template. Despite this, this procedure has yet to make a significant impact on microfluidic technologies. Here, we describe a microfluidic device that implements the EA1 enzyme for DNA extraction by incorporating it into a hybrid microdevice comprising laminated polyester (Pe) and PMMA layers. The PMMA layer provides a macro-to-micro interface for introducing the biological sample into the microfluidic architecture, whilst also possessing the necessary dimensions to function as the swab acceptor. Pre-loaded reagents are then introduced to the swab chamber centrifugally, initiating DNA extraction at 75 °C. The extraction of DNA occurs in timescales of less than 3 min and any external hardware associated with the transportation of reagents by pneumatic pumping is eliminated. Finally, multiplexing is demonstrated with a circular device containing eight separate chambers for the simultaneous processing of eight buccal swab samples. The studies here provide DNA concentrations up to 10 ng µL(-1) with a 100% success rate in less than 3 minutes. The STR profiles generated using these extracted samples demonstrate that the DNA is of PCR forensic-quality and adequate for human identification.

Enhancing Methotrexate Tolerance with Folate Tagged Liposomes in Arthritic Mice.

Methotrexate is the first line of treatment of rheumatoid arthritis. Since many patients become unresponsive to methotrexate treatment, only very expensive biological therapies are effective and increased methotrexate tolerance strategies need to be identified. Here we propose the encapsulation of methotrexate in a new liposomal formulation using a hydrophobic fragment of surfactant protein conjugated to a linker and folate to enhance their tolerance and efficacy. In this study we aim to evaluate the efficiency of this system to treat rheumatoid arthritis, by targeting folate receptor ß present at the surface of activated macrophages, key effector cells in this pathology. The specificity of our liposomal formulation to target folate receptor ß was investigated both in vitro as in vivo using a mouse model of arthritis (collagen-induced arthritis in DBA/1J mice strain). In both systems, the liposomal constructs were shown to be highly specific and efficient in targeting folate receptor ß. These liposomal formulations also significantly increase the clinical benefit of the encapsulated methotrexate in vivo in arthritic mice, together with reduced expression of CD39 and CD73 ectonucleotidases by joint-infiltrating macrophages. Thus, our formulation might be a promising cost effective way to treat rheumatoid arthritis and delay or reduce methotrexate intolerance.

Upholding the T cell immune-regulatory function of CD31 inhibits the formation of T/B immunological synapses in vitro and attenuates the development of experimental autoimmune arthritis in vivo.

CD31, a trans-homophilic inhibitory receptor expressed on both T- and B-lymphocytes, drives the mutual detachment of interacting leukocytes. Intriguingly, T cell CD31 molecules relocate to the immunological synapse (IS), where the T and B cells establish a stable interaction. Here, we show that intact CD31 molecules, which are able to drive an inhibitory signal, are concentrated at the periphery of the IS but are excluded from the center of the IS. At this site, were the cells establish the closest contact, the CD31 molecules are cleaved, and most of the extracellular portion of the protein, including the trans-homophilic binding sites, is shed from the cell surface. T cells lacking CD31 trans-homophilic binding sites easily establish stable interactions with B cells; at the opposite, CD31 signaling agonists inhibit T/B IS formation as well as the ensuing helper T cell activation and function. Confocal microscopy and flow cytometry analysis of experimental T/B IS shows that the T cell inhibitory effects of CD31 agonists depend on SHP-2 signaling, which reduces the phosphorylation of ZAP70. The analysis of synovial tissue biopsies from patients affected by rheumatoid arthritis showed that T cell CD31 molecules are excluded from the center of the T/B cell synapses in vivo. Interestingly, the administration of CD31 agonists in vivo significantly attenuated the development of the clinical signs of collagen-induced arthritis in DBA1/J mice. Altogether, our data indicate that the T cell co-inhibitory receptor CD31 prevents the formation of functional T/B immunological synapses and that therapeutic strategies aimed at sustaining CD31 signaling will attenuate the development of autoimmune responses in vivo.

Mini-FLOTAC for counting Toxoplasma gondii oocysts from cat feces--comparison with cell counting plates.

Oocysts of Toxoplasma gondii represent one of the most common environmental contaminants causing the zoonotic infection toxoplasmosis. The aim of the present study was to compare the Mini-FLOTAC device with traditional cell counting plates (Kova Slide) for the detection of T. gondii oocysts from feline feces. Two types of experiments were performed: (i) purified oocysts were counted in different dilutions and (ii) specific pathogen free T. gondii-negative cat feces was inoculated with numbers of purified oocysts and counting was performed directly from feces. Our analysis showed a thousand times higher sensitivity of Mini-FLOTAC (5 × 10(2) oocysts) compared to Kova Slide (5 × 10(5) oocysts). Also, when compared by McNemar's test, counting of the purified oocysts showed a higher sensitivity of Mini-FLOTAC compared to Kova Slide, for a dilution of 10(3) oocysts/ml (chi(2) = 6.1; P < 0.05). A better sensitivity was also found with Mini-FLOTAC in dilutions of 10(5) and 10(4) oocysts/ml, when counted from feces (chi(2) = 4.2 and 8.1, respectively, P < 0.05). Our results show that Mini-FLOTAC is more sensitive than traditional methods of T. gondii oocysts detection and quantification is more accurate. Furthermore, Mini-FLOTAC simplicity and cost effectiveness allow it to be used with light microscopes in any laboratory or field conditions. We therefore recommend its use for regular screening. Further studies are needed to validate Mini-FLOTAC for the detection of oocysts in soil and water samples in field conditions.

An integrated sample-in-answer-out microfluidic chip for rapid human identification by STR analysis.

A fully integrated microfluidic chip for human identification by short tandem repeat (STR) analysis that includes a unique enzymatic liquid preparation of the DNA, microliter non-contact PCR, and a polymer that allows a high-resolution separation within a compact microchip footprint has been developed. A heat-activated enzyme that digests biological materials is employed to generate the target yield of DNA from a buccal swab or FTA paper. The microfluidic architecture meters an aliquot of the liberated DNA and mixes it with the PCR reagents prior to non-contact IR-mediated PCR amplification. The products of PCR amplification are mixed with a sizing standard (ladder) and the 18-plex STR amplicons are separated in an effective length (Leff) of just 7 cm. The development, optimization and integration of each of these processes within the microfluidic chip are described. The device is able to generate genetic profiles in approximately 2 hours that match the profiles from the conventional processes performed using separate conventional instruments. Analysis is performed on a single plastic microchip with a size similar to that of a 96-well plate and only a few mm thick with no pretreatment of any of the functional domains. This is significant advancement in terms of ease of fabrication over glass microdevices or polymeric systems assembled from multiple components. Consequently, this fully integrated sample-in-answer-out microchip is an important step toward generation of a rapid micro-total analysis system for point-of-collection human identification based on genetic analysis.

DNA analysis using an integrated microchip for multiplex PCR amplification and electrophoresis for reference samples.

A system that automatically performs the PCR amplification and microchip electrophoretic (ME) separation for rapid forensic short tandem repeat (STR) forensic profiling in a single disposable plastic chip is demonstrated. The microchip subassays were optimized to deliver results comparable to conventional benchtop methods. The microchip process was accomplished in sub-90 min compared with >2.5 h for the conventional approach. An infrared laser with a noncontact temperature sensing system was optimized for a 45 min PCR compared with the conventional 90 min amplification time. The separation conditions were optimized using LPA-co-dihexylacrylamide block copolymers specifically designed for microchip separations to achieve accurate DNA size calling in an effective length of 7 cm in a plastic microchip. This effective separation length is less than half of other reports for integrated STR analysis and allows a compact, inexpensive microchip design. This separation quality was maintained when integrated with microchip PCR. Thirty samples were analyzed conventionally and then compared with data generated by the microfluidic chip system. The microfluidic system allele calling was 100% concordant with the conventional process. This study also investigated allelic ladder consistency over time. The PCR-ME genetic profiles were analyzed using binning palettes generated from two sets of allelic ladders run three and six months apart. Using these binning palettes, no allele calling errors were detected in the 30 samples demonstrating that a microfluidic platform can be highly consistent over long periods of time.

New insights into antigen encounter by B cells.

Antibody production by B lymphocytes is crucial during immune responses to pathogens and represents a major target in vaccination and immunotherapy strategies. To produce antibodies, B lymphocytes need to be activated in the lymphoid tissues by their cognate antigens, potentially in their native form. In recent years, novel insights have revealed the different ways for antigens to reach the B cell compartment. In this review, we discuss the various cell types that might serve as transport intermediates, with a focus on cellular mechanisms in dendritic cells.