Measuring the benefit of a defensive trait: Vigilance and survival probability.

Defensive traits are hypothesized to benefit prey by reducing predation risk from a focal predator but come at a cost to the fitness of the prey. Variation in the expression of defensive traits is seen among individuals within the same population, and in the same individual in response to changes in the environment (i.e., phenotypically plastic responses). It is the relative magnitude of the cost and benefit of the defensive trait that underlies the defensive trait expression and its consequences to the community. However, whereas the cost has received much attention in ecological research, the benefit is seldom examined. Even in a defensive trait as extensively studied as vigilance, there are few studies of the purported benefit of the behavior, namely that vigilance enhances survival. We examined whether prey vigilance increased survival and quantified that benefit in a natural system, with white-tailed deer (Odocoileus virginianus) experiencing unmanipulated levels of predation risk from Florida panther (Puma concolor coryi). Deer that spent more time vigilant (as measured by head position using camera trap data) had a higher probability of survival. Indeed, an individual deer that was vigilant 75% of the time was more than three times as likely to be killed by panthers over the course of a year than a deer that was vigilant 95% of the time. Our results therefore show that within-population variation in the expression of a defensive trait has profound consequences for the benefit it confers. Our results provide empirical evidence supporting a long-held but seldom-tested hypothesis, that vigilance is a behavior that reduces the probability of predation and quantifies the benefit of this defensive trait. Our work furthers an understanding of the net effects of a trait on prey fitness and predator-prey interactions, within-population variation in traits, and predation risk effects.

Early antibody responses to lipid antigens in red deer infected with Mycobacterium bovis.

THE PROBLEM: Early and rapid diagnosis of bovine tuberculosis remains an issue of great interest. AIM: The aim of this study was to evaluate the use of synthetic lipid antigens for diagnosis of tuberculosis in red deer (Cervus elaphus). The proposition: Synthetic mycolic acid derivatives, identical to components of mycobacterial cells, bind to antibodies to lipids produced in active human tuberculosis. Experimental infection studies in red deer (Cervus elaphus) allow the evaluation of such antigens for the serodiagnosis of bovine tuberculosis. RESULTS: Antibody levels in plasma from deer experimentally infected with Mycobacterium bovis were evaluated in ELISA using synthetic antigens based on several classes of mycolic acid, using protein G as conjugate. All antigens gave significantly increased responses 60 days post-infection, when all animals had active disease. A significantly increased response was also observed with four antigens 15 days after infection. CONCLUSION: ELISA using synthetic lipid antigens not only detects antibodies in the plasma of deer experimentally infected with M. bovis, but a strong response occurs early in the infection. With a full analysis of responses with naturally infected animals, this may offer a useful supplement to current diagnostic methods.

Genetic investigation of glycosylphosphatidylinositol (GPI) anchored Bd37 orthologs in Babesia divergens group and potential use of recombinant protein for ecological survey in deer.

The Babesia divergens/B. capreoli group includes parasites with confirmed or possible zoonotic potential to cause human babesiosis. Currently, diagnostic antigen of the group has not been established. In this study, we investigated the ortholog of Bd37, a (GPI)-anchored major merozoite surface protein of B. divergens sensu stricto, in the Asia lineage of the group. From two genomic isolates from sporozoites/sporoblasts stage, three Bd37 gene variants, namely Bd37 JP-A, JP-B, and JP-C, were isolated with 62.3-64.1% amino acid sequences identity. Discriminative blood direct PCR revealed that Bd37 JP-A was encoded in all parasites infecting wild sika deer examined (n=22). While Bd37 JP-B and JP-C genes were randomly detected in 12 and 11 specimens, respectively. Sequencing of all JP-A variants revealed that the gene was polymorphic with low ratio of non-synonymous to synonymous substitutions (dN/dS) and that a highly polymorphic region was not related to predicted B-cell epitopes. A recombinant JP-A-based ELISA showed an overall positive rate of 13.9% in sika deer in Japan from north (Hokkaido) to south (Kyushu island) across 24 prefectures (n=360). This positive rate was twice as high as that examined by 18S rRNA-based PCR (6.8%). The geographical trends in infection rates were consistent. This study demonstrated that direct examination was informative for revealing genetic background and selecting antigen candidates. Bd37 orthologs may serve diagnostic purposes in combination with indirect fluorescence assay, which requires biological isolates.

Sika Deer antler protein antagonizes the inflammatory response and oxidative damage induced by jellyfish venom.

The investigation into specific treatments for jellyfish stings has consistently presented a significant medical challenge. Sika Deer antler protein (DAP), a valuable component of traditional Chinese medicine (TCM) known for its various pharmacological properties, has been widely utilized for the prevention and treatment of numerous diseases. In this study, proteome analysis and biological activity assays of DAP identified 94 distinct protein components and demonstrated its capability to scavenge free radicals. Moreover, administration of 50 mg/kg DAP notably enhanced survival rates in mice, mitigated increases in hematological indicators and inflammatory markers (IL-6, IL-1ß, and TNF-α), and alleviated pathological abnormalities induced by jellyfish venom. Additionally, DAP intervention significantly decreased the hemolysis rate and improved the viability of RAW264.7 cells, while reducing cell apoptosis and oxidative stress. Transcriptome analysis and western blotting of RAW264.7 cells further confirmed that DAP inhibited the activation of the NF-κB and MAPK signaling pathways. Overall, DAP effectively countered the toxicity of jellyfish venom by reducing oxidative damage and inflammatory response, highlighting the potential of TCM in treating jellyfish stings.

Dynamic basis of lipopolysaccharide export by LptB2FGC.

Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB2FG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptB2FG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptB2FG and LptB2FGC in micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored LPS binding and release using laser-induced liquid bead ion desorption mass spectrometry. The ß-jellyroll domain of LptF stably interacts with the LptG and LptC ß-jellyrolls in both the apo and vanadate-trapped states. ATP binding at the cytoplasmic side is allosterically coupled to the selective opening of the periplasmic LptF ß-jellyroll domain. In LptB2FG, ATP binding closes the nucleotide binding domains, causing a collapse of the first lateral gate as observed in structures. However, the second lateral gate, which forms the putative entry site for LPS, exhibits a heterogeneous conformation. LptC binding limits the flexibility of this gate to two conformations, likely representing the helix of LptC as either released from or inserted into the transmembrane domains. Our results reveal the regulation of the LPS entry gate through the dynamic behavior of the LptC transmembrane helix, while its ß-jellyroll domain is anchored in the periplasm. This, combined with long-range ATP-dependent allosteric gating of the LptF ß-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm.

Dependence of Lyme disease incidence in children on environmental factors in Wielkopolska Province (West-Central Poland).

Introduction and Objective. Lyme borreliosis (LB) causes hundreds of thousands of new human infections worldwide annually. This is the first study connecting the LB risk to children with environmental factors. Materials and Method. The potential impacts were assessed of environmental factors (deer density in forests, coverage of potential broadleaved forest plant communities, urbanization index) on the number of LB cases in children. Analysis covered the medical records of 196 children diagnosed with LB (ICD- A69.2) from 1 January 2012 - 30 October 2021 in Wielkopolska Province (Poland). Results. All examined factors were positively correlated with LB cases. The highest correlation with the number of patients diagnosed with LB was presented by the degree of urbanization (percentage of the population living in cities in the total inhabitants of the study region). The number of cases was much higher in the second research period (2017-2021). Conclusions. The number of LB cases in children is increasing as the coverage of potential broadleaved forest plant communities increases. The number of cases among males is positively correlated with the coverage. Deer density is positively correlated with the number of LB cases among children - the higher the deer density, the greater the risk of LB infection. LB cases in children are positively correlated with the urbanization index - the more people that live in cities, the greater the risk to children of LB infection.

Analysis of the genetic diversity of Tahe red deer and population structure of Cervus elaphu/hanglu/canadensis.

The Tahe red deer is currently the largest breeding population of antlered Cervus elaphus in China. It has unique characteristics such as drought and roughage tolerance, high antler yield and early sexual maturity. It is a high-quality provenance for cultivating high-yield Cervus elaphus breeds and is also the subject of study on the origin, evolution, and classification of Cervus elaphus. The breeding quantity of Tahe red deer has decreased significantly in recent years due to the influence of feeding conditions and consumer market. This has resulted in a serious threat to its genetic resources. To provide a scientific theoretical basis for the protection of the Tahe red deer population, we performed PCR amplification and direct sequencing of the AMELY2, DBY and SRY genes of the Y chromosome, and the ND1, COX1, ATP6, ND5, Cyt b and D-loop regions of the mtDNA, and analysed their genetic diversity and population genetic structure. The results showed high haplotype diversity and low nucleotide diversity in both the Y chromosome and mtDNA genes. The phylogenetic tree and haplotype network diagram, constructed using white-lipped deer as the outgroup, indicate that Tahe red deer has two distinct ancestral types. The phylogenetic tree, based on the Cyt b gene and D-loop region, reveals that the Cervus elaphus/hanglu/canadensis is divided into three clades: western, central, and eastern. Tahe red deer, C.h.bactrianus, and C.h.hanglu are clustered in the central clade. The study results indicate that Tahe red deer has low genetic diversity and two distinct ancestor types. It is speculated that the central clade is either the earliest differentiation from the ancestor species or the closest to it.

GFI-YOLOv8: Sika Deer Posture Recognition Target Detection Method Based on YOLOv8.

As the sika deer breeding industry flourishes on a large scale, accurately assessing the health of these animals is of paramount importance. Implementing posture recognition through target detection serves as a vital method for monitoring the well-being of sika deer. This approach allows for a more nuanced understanding of their physical condition, ensuring the industry can maintain high standards of animal welfare and productivity. In order to achieve remote monitoring of sika deer without interfering with the natural behavior of the animals, and to enhance animal welfare, this paper proposes a sika deer individual posture recognition detection algorithm GFI-YOLOv8 based on YOLOv8. Firstly, this paper proposes to add the iAFF iterative attention feature fusion module to the C2f of the backbone network module, replace the original SPPF module with AIFI module, and use the attention mechanism to adjust the feature channel adaptively. This aims to enhance granularity, improve the model's recognition, and enhance understanding of sika deer behavior in complex scenes. Secondly, a novel convolutional neural network module is introduced to improve the efficiency and accuracy of feature extraction, while preserving the model's depth and diversity. In addition, a new attention mechanism module is proposed to expand the receptive field and simplify the model. Furthermore, a new pyramid network and an optimized detection head module are presented to improve the recognition and interpretation of sika deer postures in intricate environments. The experimental results demonstrate that the model achieves 91.6% accuracy in recognizing the posture of sika deer, with a 6% improvement in accuracy and a 4.6% increase in mAP50 compared to YOLOv8n. Compared to other models in the YOLO series, such as YOLOv5n, YOLOv7-tiny, YOLOv8n, YOLOv8s, YOLOv9, and YOLOv10, this model exhibits higher accuracy, and improved mAP50 and mAP50-95 values. The overall performance is commendable, meeting the requirements for accurate and rapid identification of the posture of sika deer. This model proves beneficial for the precise and real-time monitoring of sika deer posture in complex breeding environments and under all-weather conditions.

Chronic wasting disease effects on a breeding season behavior in White-tailed Deer (Odocoileus virginianus).

Wildlife disease outbreaks can lead to population declines, which are usually attributed to increased direct or indirect mortality. Alternatively, behavior associated with sickness can lead to social isolation, potentially decreasing fitness of affected individuals. A useful case study to examine this dynamic is chronic wasting disease (CWD), a neurological disease of cervids, known to affect behavior and movement. In this study, we monitored scraping, a White-tailed Deer (Odocoileus virginianus; WTD) breeding season behavior, in an area of high CWD prevalence to determine if this reproductive behavior is affected by CWD. At 107 scrape sites, we detected 3,063 scrape interactions and 218 unique bucks. Bucks engaged with scrapes most often, performing 73% of interactions-compared to 23% by does, and 4% by fawns. Twenty-one bucks captured on camera traps at scrape sites were harvested through recreational hunting, 13 testing CWD-positive and 8 CWD not-detected. We found no significant effect of CWD status on specific scraping behaviors. There may, however, have been population-level effects, with shifts toward greater proportions of scraping by yearling bucks and during daylight hours compared to findings from past studies.

Report of Giardia duodenalis in a non-captive chital Axis axis (Erxleben 1777) in Santa Catarina, South of Brazil.

Giardiasis is an infection of the small intestine by protozoa of the genus Giardia, which has a wide range of susceptible hosts, including domestic and wild animals and humans. Giardia is a zoonotic agent and represents one of the main human parasites, with high prevalence and great importance in public health. This report aims to describe the parasitism of a non-captive Chital deer (Axis axis) by Giardia duodenalis. The animal, after being rescued by soldiers from the 2nd Platoon of the 2nd Company of the 2nd Battalion of the Environmental Military Police of the State of Santa Catarina, was sent to the Wildlife Studies Center (NEVS) of the University of Western Santa Catarina (UNOESC). During clinical care, an exposed fracture in the left pelvic limb and signs of acute respiratory failure were found. Fecal samples were collected for later parasitological diagnosis. Two techniques were applied: centrifugal flotation with zinc sulfate, to diagnose parasites of the gastrointestinal system, and Baermann, to search for parasitism in the respiratory tract. The investigation revealed the presence of Giardia duodenalis. The animal died on the same day of its arrival due to a cardiorespiratory arrest. The presence of this parasite in an invasive exotic deer species highlights its epidemiological importance, as it can act as a source of infection and spread the disease to humans and other animals.

Giardíase é uma infecção do intestino delgado por protozoários do gênero Giardia, o qual apresenta uma ampla variedade de hospedeiros suscetíveis, incluindo animais domésticos, selvagens e também humanos. Giardia é um agente zoonótico, e representa um dos principais parasitas de humanos, com alta prevalência e grande importância em saúde pública. Este relato tem como objetivo descrever o parasitismo de um indivíduo de cervo Chital (Axis axis) de vida livre por Giardia duodenalis. O animal, após ser resgatado por soldados do 2º Pelotão da 2ª Companhia do 2º Batalhão da Polícia Militar Ambiental do Estado de Santa Catarina, foi encaminhado ao Núcleo de Estudos em Vida Selvagem (NEVS) da Universidade do Oeste de Santa Catarina (UNOESC). Durante o atendimento clínico foi constatada fratura exposta no membro pélvico esquerdo e sinais de insuficiência respiratória aguda. Amostras de fezes foram coletadas para posterior diagnóstico parasitológico. Para isso, foram usadas duas técnicas: centrífugo-flutuação em sulfato de zinco, com o objetivo de diagnosticar parasitas do sistema gastrointestinal, e Baermann, com o objetivo de pesquisar parasitas do trato respiratório. Os achados revelaram presença de cistos de Giardia duodenalis. O animal morreu no mesmo dia de sua chegada, devido a uma parada cardiorrespiratória. A presença desse parasito em uma espécie de cervídeo exótico invasor destaca sua importância epidemiológica, pois o mesmo pode atuar como fonte de infecção e de disseminação da doença para o ser humano e/ou outros animais.

The ancient mitochondrial genome of a Siberian roe deer (Capreolus pygargus) from Jartai Pass Site in Xinjiang, China, and its phylogenetic relationships.

The ancient mitochondrial genome of a Siberian roe deer (Capreolus pygargus) coded as NJ26S from Jartai Pass Site was obtained by high throughput sequencing. The damage pattern demonstrated the authenticity and reliability of the ancient DNA data. The length of the mitogenome was 16,357 bp, which contained 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one control region. The total base composition of the mitochondrial genome is 28.17% A, 25.01% T, 11.89% G, 19.72% C, and 15.21% missing data with an AT composition of 53.18%. A maximum-likelihood phylogenetic tree was recovered including other roe deer sequences under the TIM2 + I + G4 model. This study presents molecular evidence indicating the presence of Capreolus pygargus in the Xinjiang Uygur Autonomous Region in China more than 3,000 years ago.

Exploring protein structural ensembles: Integration of sparse experimental data from electron paramagnetic resonance spectroscopy with molecular modeling methods.

Under physiological conditions, proteins continuously undergo structural fluctuations on different timescales. Some conformations are only sparsely populated, but still play a key role in protein function. Thus, meaningful structure-function frameworks must include structural ensembles rather than only the most populated protein conformations. To detail protein plasticity, modern structural biology combines complementary experimental and computational approaches. In this review, we survey available computational approaches that integrate sparse experimental data from electron paramagnetic resonance spectroscopy with molecular modeling techniques to derive all-atom structural models of rare protein conformations. We also propose strategies to increase the reliability and improve efficiency using deep learning approaches, thus advancing the field of integrative structural biology.

The role of wild red deer on soil physical properties in a Mediterranean ecosystem: insights from a Portuguese mountain.

In this study we aimed to assess the role of wild red deer, along with other ungulates such as roe deer and wild boar, in the soil's physical properties, namely soil penetration resistance and depth (used as a proxy for soil compaction), hydraulic conductivity (a proxy for water infiltration), and the proportion of soil stable aggregates. Results showed that, at the density level found in our study area, red deer have a neutral effect at the soil level, not causing significant soil compaction or significantly influencing measured soil functions.

Relationship between Plasma and Saliva Urea Nitrogen Concentrations in New Zealand Red Deer Calves (Cervus elaphus).

Red deer (Cervus elaphus), like other ruminants, excrete approximately 70% of the nitrogen they ingest. Developing ways in which to reduce the rate of loss, such as manipulating the diet or selecting for efficiency of growth, requires close monitoring of the plasma urea N (PUN) concentration which, in turn, requires a simple, safe, and reliable method for collecting samples. Saliva is easier to collect than blood, but the relationship between the salivary urea N (SUN) and the PUN is not known for red deer. This was therefore evaluated in two strains of mixed-sex red deer calves (Cervus elaphus): a phenotype with a high seasonality of growth (H, n = 10) and a phenotype with a low seasonality of growth (L, n = 13). Both phenotypes were divided into two groups, which were each offered one of two forage-based diets ad libitum: a medium-quality diverse treatment and a low-quality perennial ryegrass-white clover treatment. Blood and saliva samples for the determination of the PUN and SUN were collected at dawn every four weeks for five months (April to September 2022). There was a strong linear relationship between the PUN and SUN in the pooled sample (R2 = 0.65, p < 0.001). The estimations of the PUN were significantly improved by adding diet and the date of sampling into the model (p < 0.001), but not phenotype (p > 0.75). SUN represents a reliable index of the PUN, and collecting saliva therefore represents a simple and inexpensive alternative to collecting blood samples in studies of nitrogen metabolism in red deer.

Temporal Characterization of Prion Shedding in Secreta of White-Tailed Deer in Longitudinal Study of Chronic Wasting Disease, United States.

Chronic wasting disease (CWD) affects cervids in North America, Asia, and Scandinavia. CWD is unique in its efficient spread, partially because of contact with infectious prions shed in secreta. To assess temporal profiles of CWD prion shedding, we collected saliva, urine, and feces from white-tailed deer for 66 months after exposure to low oral doses of CWD-positive brain tissue or saliva. We analyzed prion seeding activity by using modified amyloid amplification assays incorporating iron oxide bead extraction, which improved CWD detection and reduced false positives. CWD prions were detected in feces, urine, and saliva as early as 6 months postinfection. More frequent and consistent shedding was observed in deer homozygous for glycine at prion protein gene codon 96 than in deer expressing alternate genotypes. Our findings demonstrate that improved amplification methods can be used to identify early antemortem CWD prion shedding, which might aid in disease surveillance of cervids.

Alternating access of a bacterial homolog of neurotransmitter: sodium symporters determined from AlphaFold2 ensembles and DEER spectroscopy.

Neurotransmitter:sodium symporters (NSSs) play critical roles in neural signaling by regulating neurotransmitter uptake into cells powered by sodium electrochemical gradients. Bacterial NSSs orthologs, including MhsT from Bacillus halodurans, have emerged as model systems to understand the structural motifs of alternating access in NSSs and the extent of conservation of these motifs across the family. Here, we apply a computational/experimental methodology to illuminate the conformational landscape of MhsT alternating access. Capitalizing on our recently developed method, Sampling Protein Ensembles and Conformational Heterogeneity with AlphaFold2 (SPEACH_AF), we derived clusters of MhsT models spanning the transition from inward-facing to outward-facing conformations. Systematic application of double electron-electron resonance (DEER) spectroscopy revealed ligand-dependent movements of multiple structural motifs that underpin MhsT's conformational cycle. Remarkably, comparative DEER analysis in detergent micelles and lipid nanodiscs highlights the profound effect of the environment on the energetics of conformational changes. Through experimentally derived selection of collective variables, we present a model of ion and substrate-powered transport by MhsT consistent with the conformational cycle derived from DEER. Our findings not only advance the understanding of MhsT's function but also uncover motifs of conformational dynamics conserved within the broader context of the NSS family and within the LeuT-fold class of transporters. Importantly, our methodological blueprint introduces an approach that can be applied across a diverse spectrum of transporters to describe their conformational landscapes.

Haemolysis overestimates plasma oxidative stress biomarkers in free-ranging roe deer.

Quantifying oxidative stress has garnered extensive interest in evolutionary ecology and physiology since proposed as a mediator of life histories. However, while the theoretical framework of oxidative stress ecology is well-supported by laboratory-based studies, results obtained in wild populations on oxidative damage and antioxidant biomarkers have shown inconsistent trends. We propose that red blood cell lysis could be a source of bias affecting measurements of oxidative stress biomarkers, distorting the conclusions drawn from them. Using an experimental approach consisting of enriching plasma from roe deer with lysed red blood cells, we show that the values of commonly used oxidative stress biomarkers linearly increase with the degree of haemolysis - assayed by haemoglobin concentration. This result concerns oxidized proteins (carbonyls) and lipids (TBARS), as well as enzymatic (superoxide dismutase) and non-enzymatic (trolox assay, OXY assay) antioxidant markers. Based on 707 roe deer blood samples collected in the field, we next show that the occurrence of haemolysis in plasma samples is negatively related to age. Finally, we illustrate that considering the variance explained by age-related haemolysis improves explanatory models for inter-individual variability in plasma oxidative stress biomarkers, without substantially altering the estimates of the parameters studied here. Our results raise the question of the veracity of the conclusions if the degree of haemolysis in plasma is not considered in animal models such as roe deer, for which the occurrence and severity of haemolysis vary according to individual characteristics. We recommend measuring and controlling for the degree of haemolysis be considered in future studies that investigate the causes and consequences of oxidative stress in ecophysiological studies.

Variation in the prion protein gene (PRNP) open reading frame sequence in French cervids.

The recent emergence of chronic wasting disease (CWD) in Europe has become a new public health risk for monitoring of wild and farmed cervids. This disease, due to prions, has proliferated in North America in a contagious manner. In several mammalian species, polymorphisms in the prion protein gene (PRNP) play a crucial role in the susceptibility to prions and their spread. To obtain a reliable picture of the distribution of PRNP polymorphisms in the two most common cervid species in France, we sequenced the open reading frame (ORF) of this gene in 2114 animals, 1116 roe deer (Capreolus capreolus) and 998 red deer (Cervus elaphus). Selection criteria such as historical origin, spatial distribution and sex ratio have been integrated to establish this sample collection. Except for one heterozygous animal with a non-synonymous mutation at codon 37 (G37A), all the 1116 French roe deer were monomorphic. Red deer showed greater variation with two non-synonymous substitutions (T98A; Q226E), three synonymous substitutions (codons 21, 78 and 136) and a new 24pb deletion (Δ69-77). We found significant regional variations between French regions in the frequency of the identified substitutions. After cloning of the PRNP ORF from animals presenting multiple non-synonymous polymorphisms, we identified six haplotypes and obtained a total of twelve genotypes. As in other European countries, we highlighted the apparent homogeneity of PRNP in the French roe deer and the existence of a greater diversity in the red deer. These results were in line with European phylogeographic studies on these two species.

Theileria orientalis Ikeda infection detected in red deer but not dogs or horses in New Zealand.

AIMS: To determine whether evidence for infection with Theileria orientalis (Ikeda) could be identified in samples of commercial red deer (Cervus elaphus), horses, and working farm dogs in New Zealand. METHODS: Blood samples were collected during October and November 2019 from a convenience sample of red deer (n = 57) at slaughter. Equine blood samples (n = 50) were convenience-sampled from those submitted to a veterinary pathology laboratory for routine testing in January 2020. Blood samples, collected for a previous study from a convenience sample of Huntaway dogs (n = 115) from rural regions throughout the North and South Islands of New Zealand between August 2018 and December 2020, were also tested. DNA was extracted and quantitative PCR was used to detect the T. orientalis Ikeda major piroplasm surface protein (MPSP) gene. A standard curve of five serial 10-fold dilutions of a plasmid carrying a fragment of the T. orientalis MPSP gene was used to quantify the number of T. orientalis organisms in the samples. MPSP amplicons obtained by end-point PCR on positive samples were isolated and subjected to DNA sequencing. The resulting sequences were compared to previously published T. orientalis sequences. RESULTS: There were 6/57 (10%) samples positive for T. orientalis Ikeda from the deer and no samples positive for T. orientalis Ikeda from the working dogs or horses. The mean infection intensity for the six PCR-positive deer was 5.1 (min 2.2, max 12.4) T. orientalis Ikeda organisms/µL. CONCLUSIONS AND CLINICAL RELEVANCE: Red deer can potentially sustain low infection intensities of T. orientalis Ikeda and could act as reservoirs of infected ticks. Further studies are needed to determine whether naïve ticks feeding on infected red deer can themselves become infected. ABBREVIATIONS: Cq: Quantification cycle; LOQ: Limits of quantification; MPSP: Major piroplasm surface protein; qPCR: Quantitative polymerase chain reaction.

Pulse EPR Methods in the Angstrom to Nanometre Scale Shed Light on the Conformational Flexibility of a Fluoride Riboswitch.

Riboswitches control gene regulation upon external stimuli such as environmental factors or ligand binding. The fluoride sensing riboswitch from Thermotoga petrophila is a complex regulatory RNA proposed to be involved in resistance to F- cytotoxicity. The details of structure and dynamics underpinning the regulatory mechanism are currently debated. Here we demonstrate that a combination of pulsed electron paramagnetic resonance (ESR/EPR) spectroscopies, detecting distances in the angstrom to nanometre range, can probe distinct regions of conformational flexibility in this riboswitch. PELDOR (pulsed electron-electron double resonance) revealed a similar preorganisation of the sensing domain in three forms, i.e. the free aptamer, the Mg2+-bound apo, and the F--bound holo form. 19F ENDOR (electron-nuclear double resonance) was used to investigate the active site structure of the F--bound holo form. Distance distributions without a priori structural information were compared with in silico modelling of spin label conformations based on the crystal structure. While PELDOR, probing the periphery of the RNA fold, revealed conformational flexibility of the RNA backbone, ENDOR indicated low structural heterogeneity at the ligand binding site. Overall, the combination of PELDOR and ENDOR with sub-angstrom precision gave insight into structural organisation and flexibility of a riboswitch, not easily attainable by other biophysical techniques.