Salmonella Typhimurium environmental reduction in a farrow-to-finish pig herd using a live attenuated Salmonella Typhimurium vaccine.

BACKGROUND: Salmonella Typhimurium is an important zoonotic pathogen in pigs, that can cause clinical disease. Many sow herds and finishing herds are infected with Salmonella, and therefore pose a threat for the contamination of pork and pork products and ultimately consumers. CASE PRESENTATION: This case study describes a farrow-to-finish pig herd, producing its own replacement gilts, which had experienced clinical outbreaks of salmonellosis since 2002. Outbreaks were characterised by profuse diarrhoea, dead pigs and high antimicrobial use (colistin sulphate). The aim of this study was to see whether using vaccination of sows and piglets with Salmoporc®, a live attenuated Salmonella Typhimurium vaccine, in combination with standard hygienic precautions, it was possible to reduce Salmonella Typhimurium to below the bacteriological detection limit. Monitoring of the presence of Salmonella was done using a total of 20 pooled faecal, sock and dust samples per herd visit in the period from September 2016 to October 2020. Within the first 10 months after the start of vaccination in August 2016, there was a rapid reduction in clinical symptoms, antimicrobial usage and the number of Salmonella-positive samples. During the winters of 2017/2018 and 2018/2019 the number of positive samples increased again, however with minimal need to use antimicrobials to treat the affected animals. In July 2019, only two samples from a corridor were positive. In September and November 2019 and in October 2020 all three samplings were completely negative for S. Typhimurium. CONCLUSIONS: This case, together with other longitudinal studies, can be seen as a proof of the principle that long term vaccination with a live attenuated S. Typhimurium vaccine can reduce the level of S. Typhimurium in the herd environment to very low levels within a farrow-to-finish herd initially suffering from clinical salmonellosis. Also, clinical symptoms indicating salmonellosis were no longer observed and antimicrobials to treat clinically diseased pigs were no longer needed.

Hierarchical assembly of metal nanoparticles, quantum dots and organic dyes using DNA origami scaffolds.

The self-assembly of nanoscale elements into three-dimensional structures with precise shapes and sizes is important in fields such as nanophotonics, metamaterials and biotechnology. Short molecular linkers have previously been used to create assemblies of nanoparticles, but the approach is limited to small interparticle distances, typically less than 10 nm. Alternatively, DNA origami can precisely organize nanoscale objects over much larger length scales. Here we show that rigid DNA origami scaffolds can be used to assemble metal nanoparticles, quantum dots and organic dyes into hierarchical nanoclusters that have a planet-satellite-type structure. The nanoclusters have a tunable stoichiometry, defined distances of 5-200 nm between components, and controllable overall sizes of up to 500 nm. We also show that the nanoscale components can be positioned along the radial DNA spacers of the nanostructures, which allows short- and long-range interactions between nanoparticles and dyes to be studied in solution. The approach could, in the future, be used to construct efficient energy funnels, complex plasmonic architectures, and porous, nanoengineered scaffolds for catalysis.

M1.3--a small scaffold for DNA origami .

The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides. Here we report the convenient preparation of a 704 nt fragment dubbed "M1.3" as a linear or cyclic scaffold and the assembly of small origami structures with just 15-24 staple strands. A typical M1.3 origami is large enough to be visualized by TEM, but small enough to show a cooperativity in its assembly and thermal denaturation that is reminiscent of oligonucleotide duplexes. Due to its medium size, M1.3 origami with globally modified staples is affordable. As a proof of principle, two origami structures with globally 5'-capped staples were prepared and were shown to give higher UV-melting points than the corresponding assembly with unmodified DNA. M1.3 has the size of a gene, not a genome, and may function as a model for gene-based nanostructures. Small origami with M1.3 as a scaffold may serve as a workbench for chemical, physical, and biological experiments.

Nucleic acid nanostructures for biomedical applications.

We review the current developments of DNA-based nanostructures for drug delivery, immunotherapy, diagnostics and molecular biology. DNA is a powerful building block, which by the nature of predictable base pairing, allows the creation of molecular scaffolds, cages and multifunctional carriers with nanoscale dimensions. These engineered constructs have unsurpassed structural qualities such as full control over size, shape and dispersity. Site-specific surface modification enables the presentation of biomolecules at defined distances and stochiometries, which allows tailored cell targeting and substance delivery on demand. As the first successful in vivo applications of DNA nanostructures have recently been demonstrated, we now expect a burst of biomedical studies involving this rapidly progressing technology.

Cellular immunostimulation by CpG-sequence-coated DNA origami structures.

To investigate the potential of DNA origami constructs as programmable and noncytotoxic immunostimulants, we tested the immune responses induced by hollow 30-helix DNA origami tubes covered with up to 62 cytosine-phosphate-guanine (CpG) sequences in freshly isolated spleen cells. Unmethylated CpG sequences that are highly specific for bacterial DNA are recognized by a specialized receptor of the innate immune system localized in the endosome, the Toll-like receptor 9 (TLR9). When incubated with oligonucleotides containing CpGs, immune cells are stimulated through TLR9 to produce and secrete cytokine mediators such as interleukin-6 (IL-6) and interleukin-12p70 (IL-12p70), a process associated with the initiation of an immune response. In our studies, the DNA origami tube built from an 8634 nt long variant of the commonly used single-stranded DNA origami scaffold M13mp18 and 227 staple oligonucleotides decorated with 62 CpG-containing oligonucleotides triggered a strong immune response, characterized by cytokine production and immune cell activation, which was entirely dependent on TLR9 stimulation. Such decorated origami tubes also triggered higher immunostimulation than equal amounts of CpG oligonucleotides associated with a standard carrier system such as Lipofectamine. In the absence of CpG oligonucleotides, cytokine production induced by the origami tubes was low and was not related to TLR9 recognition. Fluorescent microscopy revealed localization of CpG-containing DNA origami structures in the endosome. The DNA constructs showed in contrast to Lipofectamine no detectable toxicity and did not affect the viability of splenocytes. We thus demonstrate that DNA origami constructs represent a delivery system for CpG oligonucleotides that is both efficient and nontoxic.

A structurally variable hinged tetrahedron framework from DNA origami.

Nanometer-sized polyhedral wire-frame objects hold a wide range of potential applications both as structural scaffolds as well as a basis for synthetic nanocontainers. The utilization of DNA as basic building blocks for such structures allows the exploitation of bottom-up self-assembly in order to achieve molecular programmability through the pairing of complementary bases. In this work, we report on a hollow but rigid tetrahedron framework of 75 nm strut length constructed with the DNA origami method. Flexible hinges at each of their four joints provide a means for structural variability of the object. Through the opening of gaps along the struts, four variants can be created as confirmed by both gel electrophoresis and direct imaging techniques. The intrinsic site addressability provided by this technique allows the unique targeted attachment of dye and/or linker molecules at any point on the structure's surface, which we prove through the superresolution fluorescence microscopy technique DNA PAINT.

Single-molecule FRET ruler based on rigid DNA origami blocks.

Fluorescence resonance energy transfer (FRET) has become a work-horse for distance measurements on the nanometer scale and between single molecules. Recent model systems for the FRET distance dependence such as polyprolines and dsDNA suffered from limited persistence lengths and sample heterogeneity. We designed a series of rigid DNA origami blocks where each block is labeled with one donor and one acceptor at distances ranging between 2.5 and 14 nm. Since all dyes are attached in one plane to the top surface of the origami block, static effects of linker lengths cancel out in contrast to commonly used dsDNA. We used single-molecule spectroscopy to compare the origami-based ruler to dsDNA and found that the origami blocks directly yield the expected distance dependence of energy transfer since the influence of the linkers on the donor-acceptor distance is significantly reduced. Based on a simple geometric model for the inter-dye distances on the origami block, the Förster radius R(0) could directly be determined from the distance dependence of energy transfer yielding R(0)=5.3±0.3 nm for the Cy3-Cy5 pair.