Comparison of Direct and Indirect Toxoplasma gondii Detection and Genotyping in Game: Relationship and Challenges

The importance of game as a source of Toxoplasma gondii (T. gondii) infection in humans is largely unknown. New data on the presence of T. gondii in game hunted in the Federal State of Brandenburg, Germany, were obtained by direct and indirect detection (ELISA). DNA extracted either directly (5 g heart or foreleg muscle, DE) or after acid pepsin digestion (50 g heart, PD) or enriched by magnetic capture (50 g heart, MC) was examined by real-time PCR (qPCR). ELISA revealed seroprevalences of 20% in wild boar (Sus scrofa), 11% in roe deer (Capreolus capreolus) and 6% in red deer (Cervus elaphus). T. gondii DNA was detected by at least one direct detection method in 12% of wild boar, 6% of roe deer, 2% of fallow deer (Dama dama) and 2% of red deer. In both, positive wild boar and roe deer, T. gondii type II specific alleles were the most prevalent, as assessed by PCR-restriction fragment length polymorphism. The highest proportion of positive animals was detected by MC qPCR, followed by PD qPCR with a similar proportion of positive findings. Investigation of 50 g of heart muscle revealed a significantly higher proportion of positive qPCR results than analysis of 5 g (p = 0.048). An association between seropositivity and direct detection was evident in wild boar and roe deer (p < 0.001). Infectivity of T. gondii DNA–positive samples was confirmed by bioassay (4/4), providing evidence that game could represent a relevant source of viable T. gondii posing a risk for human infection.

Future trends in magnetic source device design for magnetic targeted drug delivery system

Magnetic targeted drug delivery systems can improve drug utilization and reduce drug side effects. There are still many difficulties to be overcome in clinical practice. The main problems include providing large enough magnetic capture particles to concentrate drug-loaded magnetic micro-nanoparticles (MNPs) to the lesion site. The existing research focuses on the development of targeted carriers, and the magnet device has not formed a mature research system. Based on the recent development of magnet devices in recent years, this paper proposes the research direction of potential magnet devices. From the dynamic behavior of the particles under the magnetic field, the magnetic system and the mathematical model design direction of the optimized magnet are developed. This article describes the obstacles encountered in drug targeting in the study of magnet devices and summarizes the potential development of future magnet devices.

An Enhanced Lateral Flow Assay Based on Aptamer–Magnetic Separation and Multifold AuNPs for Ultrasensitive Detection of Salmonella Typhimurium in Milk

In this paper, a novel and ultrasensitive lateral flow assay (LFA) based on aptamer–magnetic separation, and multifold Au nanoparticles (AuNPs) was developed for visual detecting Salmonella enterica ser. Typhimurium (S. Typhimurium). The method realized magnetic enrichment and signal transduction via magnetic separation and achieved signal amplification through hybridizing AuNPs–capture probes and AuNPs–amplification probes to form multifold AuNPs. Two different thiolated single-strand DNA (ssDNA) on the AuNPs–capture probe played different roles. One was combined with the AuNPs–amplification probe on the conjugate pad to achieve enhanced signals. The other was connected to transduction ssDNA1 released by aptamer–magnetic capture of S. Typhimurium, and captured by the T-line, forming a positive signal. This method had an excellent linear relationship ranging from 8.6 × 102 CFU/mL to 8.6 × 107 CFU/mL with the limit of detection (LOD) as low as 8.6 × 100 CFU/mL in pure culture. In actual samples, the visual LOD was 4.1 × 102 CFU/mL, which did not carry out nucleic acid amplification and pre-enrichment, increasing three orders of magnitudes than unenhanced assays with single–dose AuNPs and no magnetic separation. Furthermore, the system showed high specificity, having no reaction with other nontarget strains. This visual signal amplificated system would be a potential platform for ultrasensitive monitoring S. Typhimurium in milk samples.

Development of a Multiplex PCR and Magnetic DNA Capture Assay for Detecting Six Species Pathogens of the Genera Anaplasma and Ehrlichia in Canine, Bovine, Caprine and Ovine Blood Samples from Grenada, West Indies

Infections with tick-borne pathogens belonging to Anaplasma/Ehrlichia in various vertebrate hosts are a persistent problem resulting in nonspecific clinical signs during early infection. Diagnosis of single and multi-infections with these pathogens, causing diseases in companion/agricultural animals and people, remains a challenge. Traditional methods of diagnosis, such as microscopy and serology, have low sensitivity and specificity. Polymerase chain reaction (PCR) assays are widely used to detect early-phase infections, since these have high sensitivity and specificity. We report the development and validation of an assay involving PCR followed by magnetic capture method using species-specific oligonucleotides to detect six Anaplasma/Ehrlichia species pathogens in canine, bovine, caprine, and ovine blood samples. Overall, the assay application to 455 samples detected 30.1% (137/455) positives for one or more out of six screened pathogens. Single-pathogen infections were observed in 94.9% (130/137) of the positive samples, while co-infections were detected in 5.1% (7/137). Anaplasma marginale infection in cattle had the highest detection rate (34.4%), followed by canines positive for Anaplasma platys (16.4%) and Ehrlichia canis (13.9%). The assay aided in documenting the first molecular evidence for A. marginale in cattle and small ruminants and Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in the Caribbean island of Grenada.

Recent Advances in Molecular Diagnostics of Fungal Plant Pathogens: A Mini Review

Phytopathogenic fungal species can cause enormous losses in quantity and quality of crop yields and this is a major economic issue in the global agricultural sector. Precise and rapid detection and identification of plant infecting fungi are essential to facilitate effective management of disease. DNA-based methods have become popular methods for accurate plant disease diagnostics. Recent developments in standard and variant polymerase chain reaction (PCR) assays including nested, multiplex, quantitative, bio and magnetic-capture hybridization PCR techniques, post and isothermal amplification methods, DNA and RNA based probe development, and next-generation sequencing provide novel tools in molecular diagnostics in fungal detection and differentiation fields. These molecular based detection techniques are effective in detecting symptomatic and asymptomatic diseases of both culturable and unculturable fungal pathogens in sole and co-infections. Even though the molecular diagnostic approaches have expanded substantially in the recent past, there is a long way to go in the development and application of molecular diagnostics in plant diseases. Molecular techniques used in plant disease diagnostics need to be more reliable, faster, and easier than conventional methods. Now the challenges are with scientists to develop practical techniques to be used for molecular diagnostics of plant diseases. Recent advancement in the improvement and application of molecular methods for diagnosing the widespread and emerging plant pathogenic fungi are discussed in this review.

Rapid and Reliable Detection of Echinococcus multilocularis from Faeces Using Droplet Digital PCR

Purpose Alveolar echinococcosis is a severe helminthic disease in humans caused by larvae of the fox tapeworm Echinococcus multilocularis. Austria is considered an endemic area with hotspots having up to 45% prevalence (Bagó et al. in Proceedings of the Zoo and Wildlife Health Conference 2019, Berlin, p. 91, 2019). At our facility, we have registered a notifiable increase of animals submitted for the diagnosis of E. multilocularis since 2016. Therefore, we investigated high throughput diagnostic methods to provide rapid and reliable results in comparison with our current method. Methods We have developed and compared a novel method of detection using droplet digital PCR (ddPCR) combined with previous target specific extraction according to Maas et al. (Vet Parasitol 230:20–24, 2016), with our current macroscopic method “Shaking in a Vessel Technique” (SVT) by Duscher et al. (Parasitol Res 95(1):40–42, 2005). We investigated 77 wild canids (72 red foxes, 5 golden jackals) using both methods. The data were analyzed using a non-Bayesian approach, applying bootstrapping to create confidentiality intervals. Results Sensitivity for droplet digital PCR was 90.51% with the 95% credibility interval ranging from 82.50 to 96.92%, whereas mean sensitivity for SVT was 92.04% with a 95% credibility interval ranging from 84.75% to 98.36%. Additionally, a non-linear regression similar to R2 could be pointed out between the counted worms and the results gathered from ddPCR. Conclusion Magnetic capture extraction followed by ddPCR shows strong potential as a high throughput method for diagnosing E. multilocularis prevalence in diverse canid populations as well as infection intensities of individual animals, giving valuable epidemiological insights of the distribution amongst wild canids as an alternative to conventional qPCR or macroscopic methods.