A Salmonella Pullorum outbreak with neurological signs in adult layers and outbreak investigation using whole genome sequencing.

RESEARCH HIGHLIGHTS: Cerebral granulomas are associated with nervous signs in Salmonella Pullorum outbreak.Bone marrow is also a recommended tissue for isolation of Salmonella Pullorum.Rapid plate agglutination test detects Pullorum antibodies in a vaccinated flock.Phylogenetic analysis showed clonality of isolates within the outbreak.

Disentangling Hot Carrier Decay and the Nature of Low-n to High-n Transfer Processes in Quasi-Two-Dimensional Layered Perovskites.

Quasi-two-dimensional (2D) metal halide perovskites (MHPs) are promising photovoltaic (PV) materials because of their impressive optical and optoelectronic properties and improved stability compared to their 3D counterparts. The presence of domains with varying numbers of inorganic layers between the organic spacers (n-phases), each with different bandgaps, makes the photoinduced carrier dynamics in films of these materials complex and intriguing. Existing interpretations of the ultrafast femto- or picosecond spectroscopy data have been inconsistent, most of them focusing either on exciton/charge transfer from low-n to high-n phases or on hot carrier cooling, but not combined. Here, we present a comprehensive study of the carrier dynamics in the Dion-Jacobson type (PDMA)(MA)(n-1)PbnI(3n+1) (PDMA = 1,4-phenylenedimethylammonium, MA = methylammonium) perovskite, stoichiometrically prepared as ⟨n⟩ = 5. Within the film, a coexistence of various n-phases is observed instead of solely the n = 5 phase, resulting in an interesting energy landscape for the motion of excitons and charge carriers. We disentangle hot carrier cooling from exciton transfer between low-n and high-n phases using ultrafast time-resolved photoluminescence and transient absorption spectroscopy. Photophysical modeling by target analysis shows that carrier cooling occurring on a subpicosecond time scale is followed by exciton transfer from low-n into high-n phases in ca. 35 ps when the film is excited by 532 or 490 nm light. Carriers in the high-n phase are much longer lived and decay in a ns time window. Overall, our results provide a comprehensive understanding of the photophysics of this material, which helps to optimize quasi-2D MHP materials for a new generation of PV devices.

Quantification of Dark Protein Populations in Fluorescent Proteins by Two-Color Coincidence Detection and Nanophotonic Manipulation.

Genetically encoded visible fluorescent proteins (VFPs) are a key tool used to visualize cellular processes. However, compared to synthetic fluorophores, VFPs are photophysically complex. This photophysical complexity includes the presence of non-emitting, dark proteins within the ensemble of VFPs. Quantitative fluorescence microcopy approaches that rely on VFPs to obtain molecular insights are hampered by the presence of these dark proteins. To account for the presence of dark proteins, it is necessary to know the fraction of dark proteins (fdark) in the ensemble. To date, fdark has rarely been quantified, and different methods to determine fdark have not been compared. Here, we use and compare two different methods to determine the fdark of four commonly used VFPs: EGFP, SYFP2, mStrawberry, and mRFP1. In the first, direct method, we make use of VFP tandems and single-molecule two-color coincidence detection (TCCD). The second method relies on comparing the bright state fluorescence quantum yield obtained by photonic manipulation to the ensemble-averaged fluorescence quantum yield of the VFP. Our results show that, although very different in nature, both methods are suitable to obtain fdark. Both methods show that all four VFPs contain a considerable fraction of dark proteins. We determine fdark values between 30 and 60% for the different VFPs. The high values for fdark of these commonly used VFPs highlight that fdark has to be accounted for in quantitative microscopy and spectroscopy.

Manifestation of SARS-CoV-2 Infections in Mink Related to Host-, Virus- and Farm-Associated Factors, The Netherlands 2020.

SARS-CoV-2 outbreaks on 69 Dutch mink farms in 2020 were studied to identify risk factors for virus introduction and transmission and to improve surveillance and containment measures. Clinical signs, laboratory test results, and epidemiological aspects were investigated, such as the date and reason of suspicion, housing, farm size and distances, human contact structure, biosecurity measures, and presence of wildlife, pets, pests, and manure management. On seven farms, extensive random sampling was performed, and age, coat color, sex, and clinical signs were recorded. Mild to severe respiratory signs and general diseases such as apathy, reduced feed intake, and increased mortality were detected on 62/69 farms. Throat swabs were more likely to result in virus detection than rectal swabs. Clinical signs differed between virus clusters and were more severe for dark-colored mink, males, and animals infected later during the year. Geographical clustering was found for one virus cluster. Shared personnel could explain some cases, but other transmission routes explaining farm-to-farm spread were not elucidated. An early warning surveillance system, strict biosecurity measures, and a (temporary) ban on mink farming and vaccinating animals and humans can contribute to reducing the risks of the virus spreading and acquisition of potential mutations relevant to human and animal health.

Quantification of the Retention and Disassembly of Virus Particles by a PEI-Functionalized Microfiltration Membrane.

Monitoring the performance of polymer-functionalized surfaces that aim at removing and inactivating viruses is typically labor-intensive and time-consuming. This hampers the development and optimization of such surfaces. Here we present experiments of low complexity that can be used to characterize and quantify the antiviral properties of polymer-functionalized surfaces. We showcase our approach on polyethylenimine (PEI)-coated poly(ether sulfone) (PES) microfiltration membranes. We use a fluorescently labeled model virus to quantify both virus removal and inactivation. We directly quantify the log removal of intact viruses by this membrane using single particle counting. Additionally, we exploit the change in photophysical properties upon disassembly of the virus to show that viruses are inactivated by the PEI coating. Although only a small fraction of intact viruses can pass the membrane, a considerable fraction of inactivated, disassembled viruses are found in the filtrate. Fluorescence microscopy experiments show that most of the viruses left behind on the microfiltration membrane are in the inactivated, disassembled state. Combined, our fluorescence microscopy and spectroscopy experiments show that not only does the model virus adsorb to the PEI coating on the membrane but also the interaction with PEI results in the disassembly of the virus capsid.

SARS-CoV-2 infection in cats and dogs in infected mink farms.

Animals like mink, cats and dogs are susceptible to SARS-CoV-2 infection. In the Netherlands, 69 out of 127 mink farms were infected with SARS-CoV-2 between April and November 2020 and all mink on infected farms were culled after SARS-CoV-2 infection to prevent further spread of the virus. On some farms, (feral) cats and dogs were present. This study provides insight into the prevalence of SARS-CoV-2-positive cats and dogs in 10 infected mink farms and their possible role in transmission of the virus. Throat and rectal swabs of 101 cats (12 domestic and 89 feral cats) and 13 dogs of 10 farms were tested for SARS-CoV-2 using PCR. Serological assays were performed on serum samples from 62 adult cats and all 13 dogs. Whole Genome Sequencing was performed on one cat sample. Cat-to-mink transmission parameters were estimated using data from all 10 farms. This study shows evidence of SARS-CoV-2 infection in 12 feral cats and 2 dogs. Eleven cats (18%) and two dogs (15%) tested serologically positive. Three feral cats (3%) and one dog (8%) tested PCR-positive. The sequence generated from the cat throat swab clustered with mink sequences from the same farm. The calculated rate of mink-to-cat transmission showed that cats on average had a chance of 12% (95%CI 10%-18%) of becoming infected by mink, assuming no cat-to-cat transmission. As only feral cats were infected it is most likely that infections in cats were initiated by mink, not by humans. Whether both dogs were infected by mink or humans remains inconclusive. This study presents one of the first reports of interspecies transmission of SARS-CoV-2 that does not involve humans, namely mink-to-cat transmission, which should also be considered as a potential risk for spread of SARS-CoV-2.

Adaptation, spread and transmission of SARS-CoV-2 in farmed minks and associated humans in the Netherlands.

In the first wave of the COVID-19 pandemic (April 2020), SARS-CoV-2 was detected in farmed minks and genomic sequencing was performed on mink farms and farm personnel. Here, we describe the outbreak and use sequence data with Bayesian phylodynamic methods to explore SARS-CoV-2 transmission in minks and humans on farms. High number of farm infections (68/126) in minks and farm workers (>50% of farms) were detected, with limited community spread. Three of five initial introductions of SARS-CoV-2 led to subsequent spread between mink farms until November 2020. Viruses belonging to the largest cluster acquired an amino acid substitution in the receptor binding domain of the Spike protein (position 486), evolved faster and spread longer and more widely. Movement of people and distance between farms were statistically significant predictors of virus dispersal between farms. Our study provides novel insights into SARS-CoV-2 transmission between mink farms and highlights the importance of combining genetic information with epidemiological information when investigating outbreaks at the animal-human interface.

Occupational and environmental exposure to SARS-CoV-2 in and around infected mink farms.

OBJECTIVE: Unprecedented SARS-CoV-2 infections in farmed minks raised immediate concerns regarding transmission to humans and initiated intensive environmental investigations to assess occupational and environmental exposure. METHODS: Air sampling was performed at infected Dutch mink farms, at farm premises and at nearby residential sites. A range of other environmental samples were collected from minks' housing units, including bedding materials. SARS-CoV-2 RNA was analysed in all samples by quantitative PCR. RESULTS: Inside the farms, considerable levels of SARS-CoV-2 RNA were found in airborne dust, especially in personal inhalable dust samples (approximately 1000-10 000 copies/m3). Most of the settling dust samples tested positive for SARS-CoV-2 RNA (82%, 75 of 92). SARS-CoV-2 RNA was not detected in outdoor air samples, except for those collected near the entrance of the most recently infected farm. Many samples of minks' housing units and surfaces contained SARS-CoV-2 RNA. CONCLUSIONS: Infected mink farms can be highly contaminated with SARS-CoV-2 RNA. This warns of occupational exposure, which was substantiated by considerable SARS-CoV-2 RNA concentrations in personal air samples. Dispersion of SARS-CoV-2 to outdoor air was found to be limited and SARS-CoV-2 RNA was not detected in air samples collected beyond farm premises, implying a negligible risk of environmental exposure to nearby communities. Our occupational and environmental risk assessment is in line with whole genome sequencing analyses showing mink-to-human transmission among farm workers, but no indications of direct zoonotic transmission events to nearby communities.

Optimizing fluorophore density for single virus counting: a photophysical approach.

In health and environmental research, it is often necessary to quantify the concentrations of single (bio) nanoparticles present at very low concentrations. Suitable quantification approaches that rely on counting and tracking of single fluorescently labelled (bio) nanoparticles are often challenging since fluorophore self-quenching limits the maximum particle brightness. Here we study how the number of labels per nanoparticle influences the total brightness of fluorescently labelled cowpea chlorotic mottle virus (CCMV). We analyze in detail the photophysical interplay between the fluorophores on the virus particles. We deduce that the formation of dark aggregates and energy transfer towards these aggregates limits the total particle brightness that can be achieved. We show that by carefully selecting the number of fluorescent labels per CCMV, and thus minimizing the negative effects on particle brightness, it is possible to quantify fluorescently labelled CCMV concentrations down to fM concentrations in single particle counting experiments.

Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans.

Animal experiments have shown that nonhuman primates, cats, ferrets, hamsters, rabbits, and bats can be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition, SARS-CoV-2 RNA has been detected in felids, mink, and dogs in the field. Here, we describe an in-depth investigation using whole-genome sequencing of outbreaks on 16 mink farms and the humans living or working on these farms. We conclude that the virus was initially introduced by humans and has since evolved, most likely reflecting widespread circulation among mink in the beginning of the infection period, several weeks before detection. Despite enhanced biosecurity, early warning surveillance, and immediate culling of animals in affected farms, transmission occurred between mink farms in three large transmission clusters with unknown modes of transmission. Of the tested mink farm residents, employees, and/or individuals with whom they had been in contact, 68% had evidence of SARS-CoV-2 infection. Individuals for which whole genomes were available were shown to have been infected with strains with an animal sequence signature, providing evidence of animal-to-human transmission of SARS-CoV-2 within mink farms.

Clinical and Pathological Findings in SARS-CoV-2 Disease Outbreaks in Farmed Mink (Neovison vison).

SARS-CoV-2, the causative agent of COVID-19, caused respiratory disease outbreaks with increased mortality in 4 mink farms in the Netherlands. The most striking postmortem finding was an acute interstitial pneumonia, which was found in nearly all examined mink that died at the peak of the outbreaks. Acute alveolar damage was a consistent histopathological finding in mink that died with pneumonia. SARS-CoV-2 infections were confirmed by detection of viral RNA in throat swabs and by immunohistochemical detection of viral antigen in nasal conchae, trachea, and lung. Clinically, the outbreaks lasted for about 4 weeks but some animals were still polymerase chain reaction-positive for SARS-CoV-2 in throat swabs after clinical signs had disappeared. This is the first report of the clinical and pathological characteristics of SARS-CoV-2 outbreaks in mink farms.

SARS-CoV-2 infection in farmed minks, the Netherlands, April and May 2020.

Respiratory disease and increased mortality occurred in minks on two farms in the Netherlands, with interstitial pneumonia and SARS-CoV-2 RNA in organ and swab samples. On both farms, at least one worker had coronavirus disease-associated symptoms before the outbreak. Variations in mink-derived viral genomes showed between-mink transmission and no infection link between the farms. Inhalable dust contained viral RNA, indicating possible exposure of workers. One worker is assumed to have attracted the virus from mink.

A cardiomyocyte show of force: A fluorescent alpha-actinin reporter line sheds light on human cardiomyocyte contractility versus substrate stiffness.

Cardiovascular disease is often associated with cardiac remodeling, including cardiac fibrosis, which may lead to increased stiffness of the heart wall. This stiffness in turn may cause subsequent failure of cardiac myocytes, however the response of these cells to increased substrate stiffness is largely unknown. To investigate the contractile response of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to increased substrate stiffness, we generated a stable transgenic human pluripotent stem cell line expressing a fusion protein of α-Actinin and fluorescent mRubyII in a previously characterized NKX2.5-GFP reporter line. Cardiomyocytes differentiated from this line were subjected to a substrate with stiffness ranging from 4 kPa to 101 kPa, while contraction of sarcomeres and bead displacement in the substrate were measured for each single cardiomyocyte. We found that sarcomere dynamics in hPSC-CMs on polyacrylamide gels of increasing stiffness are not affected above physiological levels (21 kPa), but that contractile force increases up to a stiffness of 90 kPa, at which cell shortening, deducted from bead displacement, is significantly reduced compared to physiological stiffness. We therefore hypothesize that this discrepancy may be the cause of intracellular stress that leads to hypertrophy and consequent heart failure in vivo.

Quantitative Determination of Dark Chromophore Population Explains the Apparent Low Quantum Yield of Red Fluorescent Proteins.

The fluorescence quantum yield of four representative red fluorescent proteins mCherry, mKate2, mRuby2, and the recently introduced mScarlet was investigated. The excited state lifetimes were measured as a function of the distance to a gold mirror in order to control the local density of optical states (LDOS). By analyzing the total emission rates as a function of the LDOS, we obtain separately the emission rate and the nonradiative rate of the bright states. We thus obtain for the first time the bright state quantum yield of the proteins without interference from dark, nonemitting states. The bright state quantum yields are considerably higher than previously reported quantum yields that average over both bright and dark states. We determine that mCherry, mKate2, and mRuby2 have a considerable fraction of dark chromophores up to 45%, which explains both the low measured quantum yields of red emitting proteins reported in the literature and the difficulties in developing high quantum yield variants of such proteins. For the recently developed bright mScarlet, we find a much smaller dark fraction of 14%, accompanied by a very high quantum yield of the bright state of 81%. The presence of a considerable fraction of dark chromophores has implications for numerous applications of fluorescent proteins, ranging from quantitative fluorescence microscopy to FRET studies to monitoring protein expression levels. We recommend that future optimization of red fluorescent proteins should pay more attention to minimizing the fraction of dark proteins.

Floating and Tether-Coupled Adhesion of Bacteria to Hydrophobic and Hydrophilic Surfaces.

Models for bacterial adhesion to substratum surfaces all include uncertainty with respect to the (ir)reversibility of adhesion. In a model, based on vibrations exhibited by adhering bacteria parallel to a surface, adhesion was described as a result of reversible binding of multiple bacterial tethers that detach from and successively reattach to a surface, eventually making bacterial adhesion irreversible. Here, we use total internal reflection microscopy to determine whether adhering bacteria also exhibit variations over time in their perpendicular distance above surfaces. Streptococci with fibrillar surface tethers showed perpendicular vibrations with amplitudes of around 5 nm, regardless of surface hydrophobicity. Adhering, nonfibrillated streptococci vibrated with amplitudes around 20 nm above a hydrophobic surface. Amplitudes did not depend on ionic strength for either strain. Calculations of bacterial energies from their distances above the surfaces using the Boltzman equation showed that bacteria with fibrillar tethers vibrated as a harmonic oscillator. The energy of bacteria without fibrillar tethers varied with distance in a comparable fashion as the DLVO (Derjaguin, Landau, Verwey, and Overbeek)-interaction energy. Distance variations above the surface over time of bacteria with fibrillar tethers are suggested to be governed by the harmonic oscillations, allowed by elasticity of the tethers, piercing through the potential energy barrier. Bacteria without fibrillar tethers "float" above a surface in the secondary energy minimum, with their perpendicular displacement restricted by their thermal energy and the width of the secondary minimum. The distinction between "tether-coupled" and "floating" adhesion is new, and may have implications for bacterial detachment strategies.

Outbreaks of canine distemper in Dutch and Belgian mink farms.

BACKGROUND: Vaccination of farmed minks against canine distemper virus (CDV) has proved to be very effective. In the Netherlands, vaccination of farmed minks against CDV was mandatory until the closure of the local agricultural product boards at the end of 2014. OBJECTIVES: To describe the first documented outbreaks of CD in Dutch mink farms since the closure of the agricultural product boards, as well as an outbreak in Belgium, with special attention to genotyping of the isolates. METHODS: A full post-mortem was performed on three carcasses per submission from farms A-C and on two carcasses from farm D. Molecular detection with subsequent typing was performed on eleven samples originating from four different farms. To assess genetic diversity partial sequences of the H gene of CDV were compared based on phylogenetic analysis. RESULTS: In 2017, there was a sudden series of CD outbreaks affecting four mink farms in the Netherlands (A-C) and Belgium (D). Gross, histologic and immunohistochemical findings were similar. There was a degree of genetic similarity between the viruses on farms A and D (98.5%) and between the viruses on farms B and C (97.3%), but the viruses from farms A and D belonged to a different clade than the viruses from farms B and C. Higher mortalities were reported in white and pastel minks. CONCLUSIONS: Findings indicated that the difference in severity of the outbreaks was partially related to the genetic composition of the farm populations. Vaccination against CDV on Dutch and Belgian mink farms seems warranted.

Postvaccination wounds associated predominantly with Arcanobacterium phocae in mink (Neovison vison) at three mink farms.

BACKGROUND: The emerging skin disease fur animal epidemic necrotic pyoderma (FENP) has been attributed to infection with Arcanobacterium phocae (ABP). The exact pathogenesis and risk factors of FENP have yet to be elucidated. ANIMALS: Three mink from each of three different mink farms (A-C) with postvaccination skin wounds at the vaccination site and six mink from an unaffected mink farm (D) that had used the same vaccine batch and vaccination site (hind leg). METHODS AND RESULTS: All mink from farms A-C had severe necrotizing to necropurulent dermatitis where they were vaccinated intramuscularly in the hind leg. ABP was the sole bacterium cultured from six of nine wounds. Using 16S-23S rDNA intergenic spacer region and BOX-PCR, the ABP isolates from these wounds were indistinguishable from isolates originating from several cases of FENP. CONCLUSIONS AND CLINICAL IMPORTANCE: This is the first report of FENP-like lesions at the site of vaccination, in the days following the procedure, associated with ABP. At farms with FENP vaccination, procedures should be considered carefully.

Monitoring the Switching of Single BSA-ATTO 488 Molecules Covalently End-Attached to a pH-Responsive PAA Brush.

We describe a novel combination of a responsive polymer brush and a fluorescently labeled biomolecule, where the position of the biomolecule can be switched from inside to outside the brush and vice versa by a change in pH. For this, we grafted ultrathin, amino-terminated poly(acrylic acid) brushes to glass and silicon substrates. Individual bovine serum albumin (BSA) molecules labeled with fluorophore ATTO 488 were covalently end-attached to the polymers in this brush using a bis-N-succinimidyl-(pentaethylene glycol) linker. We investigated the dry layer properties of the brush-protein ensemble, and it is swelling behavior using spectroscopic ellipsometry. Total internal reflection fluorescence (TIRF) microscopy enabled us to study the distance-dependent switching of the fluorescently labeled protein molecules. The fluorescence emission from the labeled proteins ceased (out-state) when the polymer chains stretched away from the interface under basic pH conditions, and fluorescence recurred (in-state) when the chains collapsed under acidic conditions. Moreover, TIRF allowed us to study the fluorescence switching behavior of fluorescently labeled BSA molecules down to the single-molecule level, and we demonstrate that this switching is fast but that the exact intensity during the in-state is the result of a more random process. Control experiments verify that the switching behavior is directly correlated to the responsive behavior of the polymer brush. We propose this system as a platform for switchable sensor applications but also as a method to study the swelling and collapse of individual polymer chains in a responsive polymer brush.