[Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP].

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.

Direct Evidence of Powassan Virus Vertical Transmission in Ixodes scapularis in Nature.

Powassan virus (POWV) is a tick-borne flavivirus endemic in North America and Russia. Experimental infections with POWV have confirmed horizontal, transstadial, vertical, and cofeeding transmission routes for potential virus maintenance. In the field, vertical transmission has never been observed. During New York State tick-borne pathogen surveillance, POWV RNA and/or infectious POWV was detected in five pools of questing Ixodes scapularis larvae. Additionally, engorged female I. scapularis adults were collected from hunter-harvested white-tailed deer (Odocoileus virginianus) in a region with relatively high tick infection rates of POWV and allowed to oviposit under laboratory conditions. POWV RNA was detected in three female adult husks and one pool of larvae from a positive female. Infectious virus was isolated from all three RNA-positive females and the single positive larval pool. The detection of RNA and infectious virus in unfed questing larvae from the field and larvae from replete females collected from the primary tick host implicates vertical transmission as a potential mechanism for the maintenance of POWV in I. scapularis in nature, and elucidates the potential epidemiological significance of larval ticks in the transmission of POWV to humans.

Genetic architecture of inbreeding depression may explain its persistence in a population of wild red deer.

Inbreeding depression is of major concern in declining populations, but relatively little is known about its genetic architecture in wild populations, such as the degree to which it is composed of large or small effect loci and their distribution throughout the genome. Here, we combine fitness and genomic data from a wild population of red deer to investigate the genomic distribution of inbreeding effects. Based on the runs of homozygosity (ROH)-based inbreeding coefficient, FROH, we use chromosome-specific inbreeding coefficients (FROHChr) to explore whether the effect of inbreeding varies between chromosomes. Under the assumption that within an individual the probability of being identical-by-descent is equal across all chromosomes, we used a multi-membership model to estimate the deviation of FROHChr from the average inbreeding effect. This novel approach ensures effect sizes are not overestimated whilst maximising the power of our available dataset of >3000 individuals genotyped on >35,000 autosomal SNPs. We find that most chromosomes confer a minor reduction in fitness-related traits, which when these effects are summed, results in the observed inbreeding depression in birth weight, survival and lifetime breeding success. However, no chromosomes had a significant detrimental effect compared to the overall effect of inbreeding, indicating no major effect loci. We conclude that in this population, inbreeding depression is likely the result of multiple mildly or moderately deleterious mutations spread across all chromosomes, which are difficult to detect with statistical confidence. Such mutations will be inefficiently purged, which may explain the persistence of inbreeding depression in this population.

Genomic Analyses Capture the Human-Induced Demographic Collapse and Recovery in a Wide-Ranging Cervid.

The glacial cycles of the Quaternary heavily impacted species through successions of population contractions and expansions. Similarly, populations have been intensely shaped by human pressures such as unregulated hunting and land use changes. White-tailed and mule deer survived in different refugia through the Last Glacial Maximum, and their populations were severely reduced after the European colonization. Here, we analyzed 73 resequenced deer genomes from across their North American range to understand the consequences of climatic and anthropogenic pressures on deer demographic and adaptive history. We found strong signals of climate-induced vicariance and demographic decline; notably, multiple sequentially Markovian coalescent recovers a severe decline in mainland white-tailed deer effective population size (Ne) at the end of the Last Glacial Maximum. We found robust evidence for colonial overharvest in the form of a recent and dramatic drop in Ne in all analyzed populations. Historical census size and restocking data show a clear parallel to historical Ne estimates, and temporal Ne/Nc ratio shows patterns of conservation concern for mule deer. Signatures of selection highlight genes related to temperature, including a cold receptor previously highlighted in woolly mammoth. We also detected immune genes that we surmise reflect the changing land use patterns in North America. Our study provides a detailed picture of anthropogenic and climatic-induced decline in deer diversity and clues to understanding the conservation concerns of mule deer and the successful demographic recovery of white-tailed deer.

X Chromosome-Specific Repeats in Non-Domestic Bovidae.

Repetitive sequences form a substantial and still enigmatic part of the mammalian genome. We isolated repetitive DNA blocks of the X chromosomes of three species of the family Bovidae: Kobus defassa (KDEXr sequence), Bos taurus (BTAXr sequence) and Antilope cervicapra (ACEXr sequence). The copy numbers of the isolated sequences were assessed using qPCR, and their chromosomal localisations were analysed using FISH in ten bovid tribes and in outgroup species. Besides their localisation on the X chromosome, their presence was also revealed on the Y chromosome and autosomes in several species. The KDEXr sequence abundant in most Bovidae species also occurs in distant taxa (Perissodactyla and Carnivora) and seems to be evolutionarily older than BTAXr and ACEXr. The ACEXr sequence, visible only in several Antilopini species using FISH, is probably the youngest, and arised in an ancestor common to Bovidae and Cervidae. All three repetitive sequences analysed in this study are interspersed among gene-rich regions on the X chromosomes, apparently preventing the crossing-over in their close vicinity. This study demonstrates that repetitive sequences on the X chromosomes have undergone a fast evolution, and their variation among related species can be beneficial for evolutionary studies.

Ancient and modern DNA track temporal and spatial population dynamics in the European fallow deer since the Eemian interglacial.

Anthropogenic factors have impacted the diversity and evolutionary trajectory of various species. This can be through factors such as pressure on population size or range, habitat fragmentation, or extensive manipulation and translocation. Here we use time-calibrated data to better understand the pattern and processes of evolution in the heavily manipulated European fallow deer (Dama dama). During the Pleistocene, fallow deer had a broad distribution across Europe and were found as far north as Britain during the Eemian interglacial. The last glacial period saw fallow deer retreat to southern refugia and they did not disperse north afterwards. Their recolonisation was mediated by people and, from northern Europe and the British Isles, fallow deer were transported around the world. We use ancient and modern mitochondrial DNA (mtDNA) and mitogenomic data from Eemian Britain to assess the pattern of change in distribution and lineage structure across Europe over time. We find founder effects and mixed lineages in the northern populations, and stability over time for populations in southern Europe. The Eemian sample was most similar to a lineage currently in Italy, suggesting an early establishment of the relevant refuge. We consider the implications for the integration of anthropogenic and natural processes towards a better understanding of the evolution of fallow deer in Europe.

DNA barcoding for the identification and authentication of medicinal deer (Cervus sp.) products in China.

Deer products from sika deer (Cervus nippon) and red deer (C. elaphus) are considered genuine and used for Traditional Chinese Medicine (TCM) materials in China. Deer has a very high economic and ornamental value, resulting in the formation of a characteristic deer industry in the prescription preparation of traditional Chinese medicine, health food, cosmetics, and other areas of development and utilization. Due to the high demand for deer products, the products are expensive and have limited production, but the legal use of deer is limited to only two species of sika deer and red deer; other wild deer are prohibited from hunting, so there are numerous cases of mixing and adulteration of counterfeit products and so on. There have been many reports that other animal (pig, cow, sheep, etc.) tissues or organs are often used for adulteration and confusion, resulting in poor efficacy of deer traditional medicine and trade fraud in deer products. To authenticate the deer products in a rapid and effective manner, the analysis used 22 deer products (antler, meat, bone, fetus, penis, tail, skin, and wool) that were in the form of blind samples. Total DNA extraction using a modified protocol successfully yielded DNA from the blind samples that was useful for PCR. Three candidate DNA barcoding loci, cox1, Cyt b, and rrn12, were evaluated for their discrimination strength through BLAST and phylogenetic clustering analyses. For the BLAST analysis, the 22 blind samples obtained 100% match identity across the three gene loci tested. It was revealed that 12 blind samples were correctly labeled for their species of origin, while three blind samples that were thought to originate from red deer were identified as C. nippon, and seven blind samples that were thought to originate from sika deer were identified as C. elaphus, Dama dama, and Rangifer tarandus. DNA barcoding analysis showed that all three gene loci were able to distinguish the two Cervus species and to identify the presence of adulterant species. The DNA barcoding technique was able to provide a useful and sensitive approach in identifying the species of origin in deer products.

Isolation and characterization of tannin-degrading bacteria from the rumen of wild Hokkaido sika deer (Cervus nippon yezoensis).

We isolated tannin-degrading bacteria from the rumen of wild Hokkaido sika deer and characterized their phylogeny and tannase activity in relation to sample sources. The condensed tannin level was higher in all deer rumen samples (n = 20) than in forage-fed cattle rumen samples (n = 6), whereas no hydrolyzable tannins were detected in any of the rumen samples. Rumen bacteria were enumerated on nonselective brain heart infusion (BHI) agar medium and then transferred onto tannic acid-containing BHI agar plates to screen for bacteria only showing growth (tannin-resistant bacteria) and those showing both growth and a clear zone (tannin-degrading bacteria). Summer samples provided only tannin-resistant bacteria, none of which showed tannin-degrading activity. Although winter samples also provided tannin-resistant bacteria, most isolates exhibited tannin-degrading activity. A total of 70 isolates exhibiting tannin-degrading activity were classified as Streptococcus bovis group based on 16S rRNA gene sequencing and further classified into two groups, either group A or group B. Group A consisted of isolates showing weak tannase activity, whereas group B included a majority of the isolates exhibiting high tannase activity. These results suggest that wild Hokkaido sika deer develop tannin-degrading Streptococcus in the rumen during winter, which allows access to woody food materials rich in tannins.

METTL3/YTHDC1-medicated m6A modification of circRNA3634 regulates the proliferation and differentiation of antler chondrocytes by miR-124486-5-MAPK1 axis.

BACKGROUND: The deer antler, a remarkable mammalian appendage, has a growth rate surpassing that of any other known osseous organ. Emerging evidence indicates that circRNA and MAPK1 play critical roles in chondrocytes. Thus, exploration of their functions in antler chondrocytes will help us to understand the mechanism regulating the rapid antler growth. METHODS: qRT-PCR, western blot, and immunohistochemistry were used to assess the expression of mRNAs and proteins. CCK-8, EdU, Cell migration, ALP activity detection, and ALP staining examined the effects of MAPK1 in antler chondrocytes. FISH, RIP, and luciferase assays were performed to evaluate the interactions among circRNA3634/MAPK1 and miR-124486-5. RIP and RAP assays proved the binding interaction between circRNA3634 and RBPs. Me-RIP was used to determine the m6A methylation modification of circRNA3634. RESULTS: This study revealed high MAPK1 expression in antler cartilage tissue. Overexpression of MAPK1 promoted the proliferation, migration, and differentiation of antler chondrocytes and increased the expression of MAPK3, RAF1, MEK1, RUNX2, and SOX9. The silencing of MAPK1 had the opposite effect. CircRNA3634 was found to act as a molecular sponge for miR-124486-5, leading to increased MAPK1 expression and enhanced proliferation and migration of antler chondrocytes through competitive miR-124486-5 binding. We discovered that METTL3 mediates m6A modification near the splicing site of circRNA3634 and is involved in the proliferation and differentiation of antler chondrocytes. The m6A reader YTHDC1 facilitated the nuclear export of circRNA3634 in an m6A-dependent manner. Our results indicate that m6A-modified circRNA3634 promotes the proliferation of antler chondrocytes by targeting MAPK1 and show that the nuclear export of circRNA3634 is related to the expression of YTHDC1, suggesting that circRNA3634 could represent a critical regeneration marker for the antler. CONCLUSIONS: Our results revealed a novel m6A-modified circRNA3634 promoted the proliferation and differentiation of antler chondrocytes by regulating MAPK1. The nuclear export of circRNA3634 was related to the expression of YTHDC1.

Surveys of eleven species of wild and zoo birds and feeding experiments in white-tailed eagles reveal differences in the composition of the avian gut microbiome based on dietary habits between and within species.

The composition of the gut microbiome varies due to dietary habits. We investigated influences of diet on the composition of the gut microbiome using the feces of 11 avian species, which consumed grain-, fish- and meat-based diets. We analyzed gut microbiome diversity and composition by next-generation sequencing (NGS) of 16S ribosomal RNA. The grain-diet group had higher gut microbiome diversity than the meat- and fish-diet group. The ratio of Bacteroidetes and Firmicutes phyla was higher in the grain-diet group than in the meat- and fish-diet groups. The grain-diet group had a higher ratio of Veillonellaceae than the meat-diet group and a higher ratio of Eubacteriaceae than the fish-diet habit group. To clarify the influence of diet within the same species, white-tailed eagles (Haliaeetus albicilla, n=6) were divided into two groups, and given only deer meat or fish for approximately one month. The composition of the gut microbiome of individuals in both groups were analyzed by NGS. There were indications of fluctuation in the levels of some bacteria (Lactobacillus, Coriobacteriales, etc.) in each diet group. Moreover, one individual for each group which switched each diet in last week changed to each feature of composition of bacterial flora. The above results show that the composition of the gut microbiome differ depending on diet, even within the same species.

Comparative genomics and genome-wide SNPs of endangered Eld's deer provide breeder selection for inbreeding avoidance.

Eld's deer, a conserved wildlife species of Thailand, is facing inbreeding depression, particularly in the captive Siamese Eld's deer (SED) subspecies. In this study, we constructed genomes of a male SED and a male Burmese Eld's deer (BED), and used genome-wide single nucleotide polymorphisms to evaluate the genetic purity and the inbreeding status of 35 SED and 49 BED with limited pedigree information. The results show that these subspecies diverged approximately 1.26 million years ago. All SED were found to be purebred. A low proportion of admixed SED genetic material was observed in some BED individuals. Six potential breeders from male SED with no genetic relation to any female SED and three purebred male BED with no relation to more than 10 purebred female BED were identified. This study provides valuable insights about Eld's deer populations and appropriate breeder selection in efforts to repopulate this endangered species while avoiding inbreeding.

Evolutionary insights on critically endangered Kashmir red deer or hangul (Cervus hanglu hanglu) through a mitogenomic lens.

Background: The Kashmir red deer or Hangul (Cervus hanglu hanglu) is the only Tarim red deer species endemic to India. With a current estimated population size of fewer than 200 individuals, this critically endangered species is confined to the greater Dachigam landscape in Jammu and Kashmir. Poaching, habitat loss and fragmentation, resource competition with livestock, and small population size are the major conservation challenges for this species. Methods: Blood sampling was conducted from two wild Hangul individuals during radio-collaring operations at Dachigam National Park, Kashmir in 2013 and 2020, respectively. Using next-generation sequencing approach, we sequenced the 16,351 bp long mitogenome of two wild-caught Hangul individuals (1 M:1 F at ~14× and ~10× coverage, respectively) from Dachigam National Park. Results: The annotated sequences were identical with an AT-rich composition, including 13 protein-coding genes (11,354 bp), 22 tRNA genes (1,515 bp), two ribosomal genes (2,526 bp) and a non-coding control region (917 bp) in a conserved order like other red deer species. Bayesian phylogenetic reconstruction of the red deer complex revealed two major groups: the elaphoid and the wapitoid clades. Hangul formed a distinct clade with its other subspecies C. hanglu yarkandensis and is sister to the Hungarian red deer (C. elaphus hippelaphus). Divergence time analyses suggested that the Tarim deer species group separated ~1.55 Mya from their common ancestors and Hangul diverged ~0.75 Mya from closely related C. yarkandensis, corroborating with the known paleobiogeographic events related to refugia during glaciations in the Pleistocene era. This study provides baseline information on Hangul mitogenome for further research on phylogeography and other population parameters and helps in developing suitable conservation plans for this species.

Microtubule search-and-capture model evaluates the effect of chromosomal volume conservation on spindle assembly during mitosis.

Variation in the chromosome numbers can arise from the erroneous mitosis or fusion and fission of chromosomes. While the mitotic errors lead to an increase or decrease in the overall chromosomal substance in the daughter cells, fission and fusion keep this conserved. Variations in chromosome numbers are assumed to be a crucial driver of speciation. For example, the members of the muntjac species are known to have very different karyotypes with the chromosome numbers varying from 2n=70+3B in the brown brocket deer to 2n=46 in the Chinese muntjac and 2n=6/7 in the Indian muntjac. The chromosomal content in the nucleus of these closely related mammals is roughly the same and various chromosome fusion and fission pathways have been suggested as the evolution process of these karyotypes. Similar trends can also be found in lepidoptera and yeast species which show a wide variation of chromosome numbers. The effect of chromosome number variation on the spindle assembly time and accuracy is still not properly addressed. We computationally investigate the effect of conservation of the total chromosomal substance on the spindle assembly during prometaphase. Our results suggest that chromosomal fusion pathways aid the microtubule-driven search and capture of the kinetochore in cells with monocentric chromosomes. We further report a comparative analysis of the site and percentage of amphitelic captures, dependence on cell shape, and position of the kinetochore in respect to chromosomal volume partitioning.

Epidemiology Update of Hepatitis E Virus (HEV) in Uruguay: Subtyping, Environmental Surveillance and Zoonotic Transmission.

Hepatitis E Virus (HEV) infection is an emergent zoonotic disease of increasing concern in developed regions. HEV genotype 3 (HEV-3) is mainly transmitted through consumption of contaminated food in high-income countries and is classified into at least 13 subtypes (3a-3n), based on p-distance values from complete genomes. In Latin America, HEV epidemiology studies are very scant. Our group has previously detected HEV3 in clinical cases, swine, wild boars, captive white-collared peccaries, and spotted deer from Uruguay. Herein, we aimed to provide novel insights and an updated overview of the molecular epidemiology of zoonotic HEV in Uruguay, including data from wastewater-based surveillance studies. A thorough analysis of HEV whole genomes and partial ORF2 sequences from Uruguayan human and domestic pig strains showed that they formed a separate monophyletic cluster with high nucleotide identity and exhibited p-distance values over the established cut-off (0.093) compared with reference subtypes' sequences. Furthermore, we found an overall prevalence of 10.87% (10/92) in wastewater, where two samples revealed a close relationship with humans, and animal reservoirs/hosts isolates from Uruguay. In conclusion, a single, new HEV-3 subtype currently circulates in different epidemiological settings in Uruguay, and we propose its designation as 3o along with its reference sequence.

Range-wide and temporal genomic analyses reveal the consequences of near-extinction in Swedish moose.

Ungulate species have experienced severe declines over the past centuries through overharvesting and habitat loss. Even if many game species have recovered thanks to strict hunting regulation, the genome-wide impacts of overharvesting are still unclear. Here, we examine the temporal and geographical differences in genome-wide diversity in moose (Alces alces) over its whole range in Sweden by sequencing 87 modern and historical genomes. We found limited impact of the 1900s near-extinction event but local variation in inbreeding and load in modern populations, as well as suggestion of a risk of future reduction in genetic diversity and gene flow. Furthermore, we found candidate genes for local adaptation, and rapid temporal allele frequency shifts involving coding genes since the 1980s, possibly due to selective harvesting. Our results highlight that genomic changes potentially impacting fitness can occur over short time scales and underline the need to track both deleterious and selectively advantageous genomic variation.

Deer antlers: the fastest growing tissue with least cancer occurrence.

Deer antlers are a bony organ solely able to acquired distinct unique attributes during evolution and all these attributes are against thus far known natural rules. One of them is as the fastest animal growing tissue (2 cm/day), they are remarkably cancer-free, despite high cell division rate. Although tumor-like nodules on the long-lived castrate antlers in some deer species do occur, but they are truly benign in nature. In this review, we tried to find the answer to this seemingly contradictory phenomenon based on the currently available information and give insights into possible clinic application. The antler growth center is located in its tip; the most intensive dividing cells are resident in the inner layer of reserve mesenchyme (RM), and these cells are more adopted to osteosarcoma rather than to normal bone tissues in gene expression profiles but acquire their energy mainly through aerobic oxidative phosphorylation pathway. To counteract propensity of neoplastic transformation, antlers evolved highly efficient apoptosis exactly in the RM, unparalleled by any known tissues; and annual wholesale cast to jettison the corps. Besides, some strong cancer suppressive genes including p53 cofactor genes and p53 regulator genes are highly positively selected by deer, which would have certainly contributed to curb tumorigenesis. Thus far, antler extracts and RM cells/exosomes have been tried on different cancer models either in vitro or in vivo, and all achieved positive results. These positive experimental results together with the anecdotal folklore that regular consumption of velvet antler is living with cancer-free would encourage us to test antlers in clinic settings.

Characterization of complete mitochondrial genome of three Horse flies of the genus Tabanus (Diptera: Tabanidae): comparative analysis.

BACKGROUND: Tabanidae (Horse-Flies or Deer-Flies) are one of the most economically important as well as medically significant haematophagous insect family within the order Diptera. Members of this group are also responsible for the mortality of substantial number of live-stock every year. Due to their pathogen transmission potential and vector competencies makes them an important insect group to study. Till now, mitochondrial genome of 18 species of tabanids were available. METHODS AND RESULTS: The complete mitogenome of three species T. diversifrons (15,809 bp), T. rubidus (15,878 bp) and T. tenens (15,872 bp) were generated by Next generation sequencing method. They consist 37 genes, with a positive AT skew and a negative GC skew. The gene order of these three species is similar to the typical gene arrangement of infra-order Tabanomorpha. Most of the tRNAs showed typical clover-leaf secondary structure except trnS1, which lacks the DHU arm. The sliding window analysis showed that the nad4L is the most conserved while atp8, and nad6 are the most variable genes. Moreover, the ratios of non-synonymous to synonymous substitution rates indicated that all PCGs under the purifying selection. Phylogeny revealed Chrysops and Haematopota are monophyletic while species of Hybomitra are nested within the polyphyletic clade of Tabanus. T. diversifrons exhibits sister relationship with Atylotus miser. Two morphologically divergent species T. rubidus and T. tenens are found to be genetically similar and indistinguishable by mitochondrial genome. CONCLUSIONS: The hypervariable genes like atp8 and nad6 can be used as molecular markers for the identification of recently diverged lineages of family Tabanidae. Further, to address uncertainties arising from the two morphological divergent species, it is imperative to obtain data from nuclear gene markers.

Systemic epigenome-wide association study of elk treponeme-associated hoof disease.

Treponeme-associated hoof disease (TAHD) is an emerging disease of elk (Cervus canadensis) in the U.S. Pacific West. Because environmental epigenetics is the primary molecular process that mediates environmental factor impacts on a host organism and disease, the role of epigenetics in TAHD etiology was examined. The current study was designed to examine potential effects of TAHD on systemic epigenetic modifications in infected elk over a range of TAHD lesion severity. Leg tendons that contain predominantly fibroblast connective tissue cells were used to isolate fibroblast cells for epigenetic analysis in unaffected and TAHD-positive male and female Roosevelt and Rocky Mountain elk. Differential DNA methylation regions (DMRs) between the unaffected and TAHD-positive elk were identified for both female and male elk. The presence of TAHD was associated with alteration of the connective tissue cell epigenetics, and DMR associated genes identified. Therefore, the infected elk were found to have a systemic epigenetic alteration that was associated with the disease, despite pathology being generally limited to feet. If the elk germline epigenetics is altered then generational transmission of susceptibility to TAHD may impact subsequent generations through epigenetic inheritance. This first study of epigenetic changes associated with disease in elk suggests that TAHD promotes a systemic effect on the elk epigenetics which could exert health impacts on the elk.

Analysis of Some Biochemical Properties of Recombinant Siberian Roe Deer (Capreolus pygargus) Chymosin Obtained in the Mammalian Cell Culture (CHO-K1).

Structure of the chymosin gene of Siberian roe deer (Capreolus pygargus) was established for the first time and its exon/intron organization was determined. Coding part of the chymosin gene of C. pygargus was reconstructed by the Golden Gate method and obtained as a DNA clone. Comparative sequence analysis of the roe deer, cow, and one-humped camel prochymosins revealed a number of amino acid substitutions at the sites forming the substrate-binding cavity of the enzyme and affecting the S4 and S1' + S3' specificity subsites. Integration vector pIP1 was used to construct a plasmid pIP1-Cap in order to express recombinant roe deer prochymosin gene in CHO-K1 cells. CHO-K1-CYM-Cap pool cells were obtained, allowing synthesis and secretion of recombinant prochymosin into the culture fluid. As a result of zymogen activation, a recombinant roe deer chymosin was obtained and its total milk-clotting activity was estimated to be 468.4 ± 11.1 IMCU/ml. Yield of the recombinant roe deer chymosin was 500 mg/liter or ≈468,000 IMCU/liter, which exceeds the yields of genetically engineered chymosins in most of the expression systems used. Basic biochemical properties of the obtained enzyme were compared with the commercial preparations of recombinant chymosins from one-humped camel (Camelus dromedarius) and cow (Bos taurus). Specific milk-clotting activity of the recombinant chymosin of C. pygargus was 938 ± 22 IMCU/mg, which was comparable to that of the reference enzymes. Non-specific proteolytic activity of the recombinant roe deer chymosin was 1.4-4.5 times higher than that of the cow and camel enzymes. In terms of coagulation specificity, recombinant chymosin of C. pygargus occupied an intermediate position between the genetically engineered analogs of B. taurus and C. dromedarius chymosins. Thermostability threshold of the recombinant roe deer chymosin was 55°C. At 60°C, the enzyme retained <1% of its initial milk-clotting activity, and its complete thermal inactivation was observed at 65°C.

Genetic modulation of CWD prion propagation in cervid PrP Drosophila.

Chronic wasting disease is a fatal prion condition of cervids such as deer, elk, moose and reindeer. Secretion and excretion of prion infectivity from North American cervids with this condition causes environmental contamination and subsequent efficient lateral transmission in free-ranging and farmed cervids. Variants of cervid PrP exist that affect host susceptibility to chronic wasting disease. Cervid breeding programmes aimed at increasing the frequency of PrP variants associated with resistance to chronic wasting disease may reduce the burden of this condition in animals and lower the risk of zoonotic disease. This strategy requires a relatively rapid and economically viable model system to characterise and support selection of prion disease-modifying cervid PrP variants. Here, we generated cervid PrP transgenic Drosophila to fulfil this purpose. We have generated Drosophila transgenic for S138 wild type cervid PrP, or the N138 variant associated with resistance to chronic wasting disease. We show that cervid PrP Drosophila accumulate bona fide prion infectivity after exposure to cervid prions. Furthermore, S138 and N138 PrP fly lines are susceptible to cervid prion isolates from either North America or Europe when assessed phenotypically by accelerated loss of locomotor ability or survival, or biochemically by accumulation of prion seeding activity. However, after exposure to European reindeer prions, N138 PrP Drosophila accumulated prion seeding activity with slower kinetics than the S138 fly line. These novel data show that prion susceptibility characteristics of cervid PrP variants are maintained when expressed in Drosophila, which highlights this novel invertebrate host in modelling chronic wasting disease.