First insight into strongylid nematode diversity and anthelmintic treatment effectiveness in beef cattle in the Czech Republic explored by HTS metagenomics.

Parasitic diseases and mitigation of their effects play an important role in the health management of grazing livestock worldwide, with gastrointestinal strongylid nematodes being of prominent importance. These helminths typically occur in complex communities, often composed of species from numerous strongylid genera. Detecting the full diversity of strongylid species in non-invasively collected faecal samples is nearly impossible using conventional methods. In contrast, high-throughput amplicon sequencing (HTS) can effectively identify co-occurring species. During the four-year project, we collected and analysed faecal samples from beef cattle on >120 farms throughout the Czech Republic. Strongylids were the predominant nematodes, detected in 56% of the samples, but at a low level of infection. The apparent limitations in identifying strongylid taxa prompted this pilot study on a representative group of samples testing positive for strongylids using ITS-2 metabarcoding. The most widespread genera parasitizing Czech cattle were Ostertagia (O. ostertagi) and Oesophagostomum spp., followed by Trichostrongylus and Cooperia, while Bunostomum, Nematodirus and Chabertia were present only in a minority. As comparative material, 21 samples of cattle from the Danube Delta in Romania were used, which, in contrast, were dominated by Haemonchus placei. Finally, the effect of ivermectin treatment was tested at two Czech farms. After treatment with the anthelmintic, there was a shift in the strongylid communities, with a dominance of Cooperia and Ostertagia.

Serum and cerebrospinal fluid phosphorylated neurofilament heavy subunit as a marker of neuroaxonal damage in tick-borne encephalitis.

Extensive axonal and neuronal loss is the main cause of severe manifestations and poor outcomes in tick-borne encephalitis (TBE). Phosphorylated neurofilament heavy subunit (pNF-H) is an essential component of axons, and its detection in cerebrospinal fluid (CSF) or serum can indicate the degree of neuroaxonal damage. We examined the use of pNF-H as a biomarker of neuroaxonal injury in TBE. In 89 patients with acute TBE, we measured CSF levels of pNF-H and 3 other markers of brain injury (glial fibrillary acidic protein, S100B and ubiquitin C-terminal hydrolase L1) and compared the results to those for patients with meningitis of other aetiology and controls. Serum pNF-H levels were measured in 80 patients and compared with findings for 90 healthy blood donors. TBE patients had significantly (P<0.001) higher CSF pNF-H levels than controls as early as hospital admission. Serum pNF-H concentrations were significantly higher in samples from TBE patients collected at hospital discharge (P<0.0001) than in controls. TBE patients with the highest peak values of serum pNF-H, exceeding 10 000 pg ml-1, had a very severe disease course, with coma or tetraplegia. Patients requiring intensive care had significantly higher serum pNF-H levels than other TBE patients (P<0.01). Elevated serum pNF-H values were also observed in patients with incomplete recovery (P<0.05). Peak serum pNF-H levels correlated positively with the duration of hospitalization (P=0.005). Measurement of pNF-H levels in TBE patients might be useful for assessing disease severity and determining prognosis.

Taxa-specific activity loss and mortality patterns in freshwater trematode cercariae under subarctic conditions.

Cercarial activity and survival are crucial traits for the transmission of trematodes. Temperature is particularly important, as faster depletion of limited cercarial energy reserves occurs at high temperatures. Seasonal climate conditions in high latitude regions may be challenging to complete trematode life cycle during the 6-month ice-free period, but temperature effects on the activity and survival of freshwater cercariae have not been previously identified. After experimentally simulating natural subarctic conditions during warmer and colder months (13 and 6°C), a statistical approach identifying changes in the tendency of cercarial activity loss and mortality data was used to detect differences in three trematode genera, represented by four taxa (Diplostomum spp., Apatemon spp., small- and large-sized Plagiorchis spp.). A strong temperature-dependent response was identified in both activity loss and mortality in all taxa, with Diplostomum spp. cercariae showing the most gradual changes compared to other taxa. Furthermore, whilst activity loss and mortality dynamics could not be divided into 'fish- vs invertebrate-infecting cercariae' groups, the detected taxa-specific responses in relation to life-history traits indicate the swimming behaviour of cercariae and energy allocation among larvae individuals as the main drivers. Cercariae exploit the short transmission window that allows a stable continuance of trematodes' life cycles in high-latitude freshwater ecosystems.

Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern.

BACKGROUND: The emergence of new SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) that harbor mutations in the viral S protein raised concern about activity of current vaccines and therapeutic antibodies. Independent studies have shown that mutant variants are partially or completely resistant against some of the therapeutic antibodies authorized for emergency use. METHODS: We employed hybridoma technology, ELISA-based and cell-based S-ACE2 interaction assays combined with authentic virus neutralization assays to develop second-generation antibodies, which were specifically selected for their ability to neutralize the new variants of SARS-CoV-2. FINDINGS: AX290 and AX677, two monoclonal antibodies with non-overlapping epitopes, exhibit subnanomolar or nanomolar affinities to the receptor binding domain of the viral Spike protein carrying amino acid substitutions N501Y, N439K, E484K, K417N, and a combination N501Y/E484K/K417N found in the circulating virus variants. The antibodies showed excellent neutralization of an authentic SARS-CoV-2 virus representing strains circulating in Europe in spring 2020 and also the variants of concern B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). In addition, AX677 is able to bind Omicron Spike protein just like the wild type Spike. The combination of the two antibodies prevented the appearance of escape mutations of the authentic SARS-CoV-2 virus. Prophylactic administration of AX290 and AX677, either individually or in combination, effectively reduced viral burden and inflammation in the lungs, and prevented disease in a mouse model of SARS-CoV-2 infection. INTERPRETATION: The virus-neutralizing properties were fully reproduced in chimeric mouse-human versions of the antibodies, which may represent a promising tool for COVID-19 therapy. FUNDING: The study was funded by AXON Neuroscience SE and AXON COVIDAX a.s.

Spiroplasma Isolated From Third-Generation Laboratory Colony Ixodes persulcatus Ticks.

Spiroplasma are vertically-transmitted endosymbionts of ticks and other arthropods. Field-collected Ixodes persulcatus have been reported to harbour Spiroplasma, but nothing is known about their persistence during laboratory colonisation of this tick species. We successfully isolated Spiroplasma from internal organs of 6/10 unfed adult ticks, belonging to the third generation of an I. persulcatus laboratory colony, into tick cell culture. We screened a further 51 adult male and female ticks from the same colony for presence of Spiroplasma by genus-specific PCR amplification of fragments of the 16S rRNA and rpoB genes; 100% of these ticks were infected and the 16S rRNA sequence showed 99.8% similarity to that of a previously-published Spiroplasma isolated from field-collected I. persulcatus. Our study shows that Spiroplasma endosymbionts persist at high prevalence in colonised I. persulcatus through at least three generations, and confirms the usefulness of tick cell lines for isolation and cultivation of this bacterium.

Cercarial behaviour alters the consumer functional response of three-spined sticklebacks.

Free-living parasite life stages may contribute substantially to ecosystem biomass and thus represent a significant source of energy flow when consumed by non-host organisms. However, ambient temperature and the predator's own infection status may modulate consumption rates towards parasite prey. We investigated the combined effects of temperature and predator infection status on the consumer functional response of three-spined sticklebacks towards the free-living cercariae stages of two common freshwater trematode parasites (Plagiorchis spp., Trichobilharzia franki). Our results revealed genera-specific functional responses and consumption rates towards each parasite prey: Type II for Plagiorchis spp. and Type III for T. franki, with an overall higher consumption rate on T. franki. Elevated temperature (13°C) increased the consumption rate on Plagiorchis spp. prey for sticklebacks with mild cestode infections (<5% fish body weight) only. High consumption of cercarial prey by sticklebacks may impact parasite population dynamics by severely reducing or even functionally eliminating free-living parasite life stages from the environment. This supports the potential role of fish as biocontrol agents for cercariae with similar dispersion strategies, in instances where functional response relationships have been established. Our study demonstrates how parasite consumption by non-host organisms may be shaped by traits inherent to parasite transmission and dispersal, and emphasises the need to consider free-living parasite life stages as integral energy resources in aquatic food webs.

Species-specific patterns in cercarial emergence of Diplostomum spp. from snails Radix lagotis.

The cercarial emergence patterns of three species of Diplostomum (Diplostomum 'mergi', Diplostomum spathaceum and Diplostomum parviventosum) parasitizing freshwater first intermediate host Radix lagotis sampled in Most Lake, Czech Republic, were studied under various experimental conditions, i.e. field, laboratory and incubator, and seasons, i.e. spring, summer and autumn. We discovered unexpected daily periodicity-dependent species-specific emergence patterns among the three Diplostomum spp. depending on experimental conditions. At the same time, the intraspecific variation of D. spathaceum cercarial release in response to seasonal conditions was observed. We found that a complex array of mechanisms can affect Diplostomum species-specific patterns in cercarial emergence, of which behavioural characteristics of fish related to reproduction and feeding processes are considered the most important factors. This might represent a specific adaptive evolutionary mechanism to maximise transmission success while avoiding competition for host resources. Our results contribute to a better understanding of ecological and epidemiological aspects with respect to specific adaptive strategies compartmentalised among species of Diplostomum and consequences for infection risk in fish hosts.

Temperature does not influence functional response of amphipods consuming different trematode prey.

Direct consumption on free-living cercariae stages of trematodes by non-host organisms interferes with trematode transmission and leads to reduced infections in the next suitable hosts. Consumer functional responses provide a useful tool to examine relationships between consumption rates and ecologically relevant prey densities, whilst also accounting for abiotic factors that likely influence consumption rates. We investigated how temperature influences the consumer functional response of the amphipod Gammarus lacustris towards the cercariae of three freshwater trematodes (Diplostomum, Apatemon and Trichobilharzia). Amphipods displayed different functional responses towards the parasites, with Type II responses for Diplostomum and Type I responses for Apatemon prey. Temperature did not alter the consumption rate of the amphipod predator. Trichobilharzia was likely consumed at similar proportions as Diplostomum; however, this could not be fully evaluated due to low replication. Whilst Type II responses of invertebrate predators are common to various invertebrate prey types, this is the first time a non-filter feeding predator has been shown to exhibit Type I response towards cercarial prey. The prey-specific consumption patterns of amphipods were related to cercarial distribution in the water column rather than to the size of cercariae or temperature influence. The substantial energy flow into food webs by non-host consumer organisms highlights the importance of understanding the mechanisms that modulate functional responses and direct predation in the context of parasitic organisms.

Unraveling an FNR based regulatory circuit in Paracoccus denitrificans using a proteomics-based approach.

The switch from aerobic to anaerobic respiration in the bacterium Paracoccus denitrificans is orchestrated by the action of three FNR-type transcription regulators FnrP, NNR and NarR, which are sensors for oxygen, nitric oxide and nitrite, respectively. In this work, we analyzed the protein composition of four strains (wild type, FnrP-, NNR- and NarR-mutant strains) grown aerobically, semiaerobically and semiaerobically in the presence of nitrate to discover the global role of FNR-family transcription regulators using proteomics, with data validation at the transcript and genome levels. Expression profiles were acquired using two-dimensional gel electrophoresis for 737 protein spots, in which 640 proteins were identified using mass spectrometry. The annotated 2-D proteome map provided the most comprehensive coverage of P. denitrificans proteome available to-date and can be accessed on-line at http://www.mpiib-berlin.mpg.de/2D-PAGE/. Our results revealed several types of regulation under the conditions tested: (1) FnrP-controlled regulation of nitrous oxide reductase, UspA and OmpW as confirmed at protein, transcript and DNA level (position of FNR boxes). (2) Proteins regulated via additional regulators, including proteins involved in NNR and NarR regulons: nitrate reductase beta-subunit, TonB-dependent receptors, nitrite reductase, a TenA-type transcription regulator, and an unknown protein with an alpha/beta hydrolase fold. (3) Proteins whose expression was affected mainly by the growth condition. This group contains SSU ribosomal protein S305 / sigma(54) modulation protein, and two short-chain reductase-dehydrogenase proteins.