The differentiation state of the Schwann cell progenitor drives phenotypic variation between two contagious cancers.

Contagious cancers are a rare pathogenic phenomenon in which cancer cells gain the ability to spread between genetically distinct hosts. Nine examples have been identified across marine bivalves, dogs and Tasmanian devils, but the Tasmanian devil is the only mammalian species known to have given rise to two distinct lineages of contagious cancer, termed Devil Facial Tumour 1 (DFT1) and 2 (DFT2). Remarkably, DFT1 and DFT2 arose independently from the same cell type, a Schwann cell, and while their ultra-structural features are highly similar they exhibit variation in their mutational signatures and infection dynamics. As such, DFT1 and DFT2 provide a unique framework for investigating how a common progenitor cell can give rise to distinct contagious cancers. Using a proteomics approach, we show that DFT1 and DFT2 are derived from Schwann cells in different differentiation states, with DFT2 carrying a molecular signature of a less well differentiated Schwann cell. Under inflammatory signals DFT1 and DFT2 have different gene expression profiles, most notably involving Schwann cell markers of differentiation, reflecting the influence of their distinct origins. Further, DFT2 cells express immune cell markers typically expressed during nerve repair, consistent with an ability to manipulate their extracellular environment, facilitating the cell's ability to transmit between individuals. The emergence of two contagious cancers in the Tasmanian devil suggests that the inherent plasticity of Schwann cells confers a vulnerability to the formation of contagious cancers.

CD4, CD8b, and Cytokines Expression Profiles in Peripheral Blood Mononuclear Cells Infected with Different Subtypes of KoRV from Koalas (Phascolarctos cinereus) in a Japanese Zoo.

Koala retrovirus (KoRV) poses a major threat to koala health and conservation, and currently has 10 identified subtypes: an endogenous subtype (KoRV-A) and nine exogenous subtypes (KoRV-B to KoRV-J). However, subtype-related variations in koala immune response to KoRV are uncharacterized. In this study, we investigated KoRV-related immunophenotypic changes in a captive koala population (Hirakawa zoo, Japan) with a range of subtype infection profiles (KoRV-A only vs. KoRV-A with KoRV-B and/or -C), based on qPCR measurements of CD4, CD8b, IL-6, IL-10 and IL-17A mRNA expression in unstimulated and concanavalin (Con)-A-stimulated peripheral blood mononuclear cells (PBMCs). Although CD4, CD8b, and IL-17A expression did not differ between KoRV subtype infection profiles, IL-6 expression was higher in koalas with exogenous infections (both KoRV-B and KoRV-C) than those with the endogenous subtype only. IL-10 expression did not significantly differ between subtype infection profiles but did show a marked increase-accompanying decreased CD4:CD8b ratio-in a koala with lymphoma and co-infected with KoRV-A and -B, thus suggesting immunosuppression. Taken together, the findings of this study provide insights into koala immune response to multiple KoRV subtypes, which can be exploited for the development of prophylactic and therapeutic interventions for this iconic marsupial species.

Evolution and lineage dynamics of a transmissible cancer in Tasmanian devils.

Devil facial tumour 1 (DFT1) is a transmissible cancer clone endangering the Tasmanian devil. The expansion of DFT1 across Tasmania has been documented, but little is known of its evolutionary history. We analysed genomes of 648 DFT1 tumours collected throughout the disease range between 2003 and 2018. DFT1 diverged early into five clades, three spreading widely and two failing to persist. One clade has replaced others at several sites, and rates of DFT1 coinfection are high. DFT1 gradually accumulates copy number variants (CNVs), and its telomere lengths are short but constant. Recurrent CNVs reveal genes under positive selection, sites of genome instability, and repeated loss of a small derived chromosome. Cultured DFT1 cell lines have increased CNV frequency and undergo highly reproducible convergent evolution. Overall, DFT1 is a remarkably stable lineage whose genome illustrates how cancer cells adapt to diverse environments and persist in a parasitic niche.

Prion Disease in Dromedary Camels, Algeria.

Prions cause fatal and transmissible neurodegenerative diseases, including Creutzfeldt-Jakob disease in humans, scrapie in small ruminants, and bovine spongiform encephalopathy (BSE). After the BSE epidemic, and the associated human infections, began in 1996 in the United Kingdom, general concerns have been raised about animal prions. We detected a prion disease in dromedary camels (Camelus dromedarius) in Algeria. Symptoms suggesting prion disease occurred in 3.1% of dromedaries brought for slaughter to the Ouargla abattoir in 2015-2016. We confirmed diagnosis by detecting pathognomonic neurodegeneration and disease-specific prion protein (PrPSc) in brain tissues from 3 symptomatic animals. Prion detection in lymphoid tissues is suggestive of the infectious nature of the disease. PrPSc biochemical characterization showed differences with BSE and scrapie. Our identification of this prion disease in a geographically widespread livestock species requires urgent enforcement of surveillance and assessment of the potential risks to human and animal health.

MHC diversity and female age underpin reproductive success in an Australian icon; the Tasmanian Devil.

Devil Facial Tumour Disease (DFTD), a highly contagious cancer, has decimated Tasmanian devil (Sarcophilus harrisii) numbers in the wild. To ensure its long-term survival, a captive breeding program was implemented but has not been as successful as envisaged at its launch in 2005. We therefore investigated the reproductive success of 65 captive devil pair combinations, of which 35 produced offspring (successful pairs) whereas the remaining 30 pairs, despite being observed mating, produced no offspring (unsuccessful pairs). The devils were screened at six MHC Class I-linked microsatellite loci. Our analyses revealed that younger females had a higher probability of being successful than older females. In the successful pairs we also observed a higher difference in total number of heterozygous loci, i.e. when one devil had a high total number of heterozygous loci, its partner had low numbers. Our results therefore suggest that devil reproductive success is subject to disruptive MHC selection, which to our knowledge has never been recorded in any vertebrate. In order to enhance the success of the captive breeding program the results from the present study show the importance of using young (2-year old) females as well as subjecting the devils to MHC genotyping.

Using Gene Transcription to Assess Ecological and Anthropological Stressors in Brown Bears.

Increasingly, population- and ecosystem-level health assessments are performed using sophisticated molecular tools. Advances in molecular technology enable the identification of synergistic effects of multiple stressors on the individual physiology of different species. Brown bears (Ursus arctos) are an apex predator; thus, they are ideal candidates for detecting potentially ecosystem-level systemic perturbations using molecular-based tools. We used gene transcription to analyze 130 brown bear samples from three National Parks and Preserves in Alaska. Although the populations we studied are apparently stable in abundance and exist within protected and intact environments, differences in transcript profiles were noted. The most prevalent differences were among locations. The transcript patterns among groups reflect the influence of environmental factors, such as nutritional status, disease, and xenobiotic exposure. However, these profiles also likely represent baselines for each unique environment by which future measures can be made to identify early indication of population-level changes due to, for example, increasing Arctic temperatures. Some of those environmental changes are predicted to be potentially positive for brown bears, but other effects such as the manifestation of disease or indirect effects of oceanic acidification may produce negative impacts.

Identification of novel and differentially expressed MicroRNAs in goat enzootic nasal adenocarcinoma.

BACKGROUND: MicroRNAs (miRNAs) post-transcriptionally regulate a variety of genes involved in eukaryotic cell growth, development, metabolism and other biological processes, and numerous miRNAs are implicated in the initiation and progression of cancer. Enzootic nasal adenocarcinoma (ENA), an epithelial tumor induced in goats and sheep by enzootic nasal tumor virus (ENTV), is a chronic, progressive, contact transmitted disease. METHODS: In this work, small RNA Illumina high-throughput sequencing was used to construct a goat nasal miRNA library. This study aimed to identify novel and differentially expressed miRNAs in the tumor and para-carcinoma nasal tissues of Nanjiang yellow goats with ENA. RESULTS: Four hundred six known miRNAs and 29 novel miRNAs were identified. A total of 116 miRNAs were significantly differentially expressed in para-carcinoma nasal tissues and ENA (54 downregulated; 60 upregulated; two only expressed in control group); Target gene prediction and functional analysis revealed that 6176 non-redundancy target genes, 1792 significant GO and 97 significant KEGG pathway for 121 miRNAs (116 significant expression miRNAs and five star sequence) were predicted. GO and KEGG pathway analysis revealed the majority of target genes in ENA are involved in cell proliferation, signal transduction and other processes associated with cancer. CONCLUSIONS: This is the first large-scale identification of miRNAs in Capra hircus ENA and provides a theoretical basis for investigating the complicated miRNA-mediated regulatory networks involved in the pathogenesis and progression of ENA.

CD63 Promotes Hemocyte-Mediated Phagocytosis in the Clam, Paphia undulata.

As one of the surface membrane proteins of tetraspanin family, CD63 plays a crucial role in cellular trafficking and endocytosis, which also is associated with activation of a wide variety of immune cells. Here, the homolog of CD63 was characterized from one marine mollusk, Paphia undulata, which is designated as Pu-CD63. The complete cDNA of Pu-CD63 is 1,738 bp in length with an open reading frame (ORF) of 849 bp, encoding a 282 amino acid protein with four putative hydrophobic transmembrane helixes. Bioinformatic analysis revealed that Pu-CD63 contains one putative YXXØ consensus motif of "110-YVII-113" and one N-glycosylation site "155-NGT-157" within the large extracellular loop (LEL) region, supporting its conserved function in plasma membrane and endosomal/lysosomal trafficking. Moreover, temporal expression profile analysis demonstrates a drastic induction in the expression of CD63 in hemocytes after pathogenic challenge with either V. parahaemolyticus or V. alginolyticus. By performing dsRNA-mediate RNAi knockdowns of CD63, a dramatic reduction in hemocytes phagocytic activity to pathogenic Vibrio is recorded by flow cytometry, revealing the definite role of Pu-CD63 in promoting hemocyte-mediated phagocytosis. Therefore, our work has greatly enhanced our understanding about primitive character of innate immunity in marine mollusk.

Vertebrate Host Susceptibility to Heartland Virus.

Heartland virus (HRTV) is a recently described phlebovirus initially isolated in 2009 from 2 humans who had leukopenia and thrombocytopenia. Serologic assessment of domestic and wild animal populations near the residence of 1 of these persons showed high exposure rates to raccoons, white-tailed deer, and horses. To our knowledge, no laboratory-based assessments of viremic potential of animals infected with HRTV have been performed. We experimentally inoculated several vertebrates (raccoons, goats, chickens, rabbits, hamsters, C57BL/6 mice, and interferon-α/ß/γ receptor-deficient [Ag129]) mice with this virus. All animals showed immune responses against HRTV after primary or secondary exposure. However, neutralizing antibody responses were limited. Only Ag129 mice showed detectable viremia and associated illness and death, which were dose dependent. Ag129 mice also showed development of mean peak viral antibody titers >8 log10 PFU/mL, hemorrhagic hepatic lesions, splenomegaly, and large amounts of HRTV antigen in mononuclear cells and hematopoietic cells in the spleen.

Widespread transmission of independent cancer lineages within multiple bivalve species.

Most cancers arise from oncogenic changes in the genomes of somatic cells, and while the cells may migrate by metastasis, they remain within that single individual. Natural transmission of cancer cells from one individual to another has been observed in two distinct cases in mammals (Tasmanian devils and dogs), but these are generally considered to be rare exceptions in nature. The discovery of transmissible cancer in soft-shell clams (Mya arenaria) suggested that this phenomenon might be more widespread. Here we analyse disseminated neoplasia in mussels (Mytilus trossulus), cockles (Cerastoderma edule), and golden carpet shell clams (Polititapes aureus) and find that neoplasias in all three species are attributable to independent transmissible cancer lineages. In mussels and cockles, the cancer lineages are derived from their respective host species; however, unexpectedly, cancer cells in P. aureus are all derived from Venerupis corrugata, a different species living in the same geographical area. No cases of disseminated neoplasia have thus far been found in V. corrugata from the same region. These findings show that transmission of cancer cells in the marine environment is common in multiple species, that it has originated many times, and that while most transmissible cancers are found spreading within the species of origin, cross-species transmission of cancer cells can occur.

An overview of the lagomorph immune system and its genetic diversity.

Our knowledge of the lagomorph immune system remains largely based upon studies of the European rabbit (Oryctolagus cuniculus), a major model for studies of immunology. Two important and devastating viral diseases, rabbit hemorrhagic disease and myxomatosis, are affecting European rabbit populations. In this context, we discuss the genetic diversity of the European rabbit immune system and extend to available information about other lagomorphs. Regarding innate immunity, we review the most recent advances in identifying interleukins, chemokines and chemokine receptors, Toll-like receptors, antiviral proteins (RIG-I and Trim5), and the genes encoding fucosyltransferases that are utilized by rabbit hemorrhagic disease virus as a portal for invading host respiratory and gut epithelial cells. Evolutionary studies showed that several genes of innate immunity are evolving by strong natural selection. Studies of the leporid CCR5 gene revealed a very dramatic change unique in mammals at the second extracellular loop of CCR5 resulting from a gene conversion event with the paralogous CCR2. For the adaptive immune system, we review genetic diversity at the loci encoding antibody variable and constant regions, the major histocompatibility complex (RLA) and T cells. Studies of IGHV and IGKC genes expressed in leporids are two of the few examples of trans-species polymorphism observed outside of the major histocompatibility complex. In addition, we review some endogenous viruses of lagomorph genomes, the importance of the European rabbit as a model for human disease studies, and the anticipated role of next-generation sequencing in extending knowledge of lagomorph immune systems and their evolution.

Transcriptome sequencing and development of an expression microarray platform for liver infection in adenovirus type 5-infected Syrian golden hamsters.

The Syrian golden hamster is an attractive animal for research on infectious diseases and other diseases. We report here the sequencing, assembly, and annotation of the Syrian hamster transcriptome. We include transcripts from ten pooled tissues from a naïve hamster and one stimulated with lipopolysaccharide. Our data set identified 42,707 non-redundant transcripts, representing 34,191 unique genes. Based on the transcriptome data, we generated a custom microarray and used this new platform to investigate the transcriptional response in the Syrian hamster liver following intravenous adenovirus type 5 (Ad5) infection. We found that Ad5 infection caused a massive change in regulation of liver transcripts, with robust up-regulation of genes involved in the antiviral response, indicating that the innate immune response functions in the host defense against Ad5 infection of the liver. The data and novel platforms developed in this study will facilitate further development of this important animal model.

Yeast Surface Display of Two Proteins Previously Shown to Be Protective Against White Spot Syndrome Virus (WSSV) in Shrimp.

Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7) and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV) could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1) promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7) and partial VP28 (pVP28) were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA) using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against WSSV by oral administration.

Computational prediction of disease microRNAs in domestic animals.

BACKGROUND: The most important means of identifying diseases before symptoms appear is through the discovery of disease-associated biomarkers. Recently, microRNAs (miRNAs) have become highly useful biomarkers of infectious, genetic and metabolic diseases in human but they have not been well studied in domestic animals. It is probable that many of the animal homologs of human disease-associated miRNAs may be involved in domestic animal diseases. Here we describe a computational biology study in which human disease miRNAs were utilized to predict orthologous miRNAs in cow, chicken, pig, horse, and dog. RESULTS: We identified 287 human disease-associated miRNAs which had at least one 100% identical animal homolog. The 287 miRNAs were associated with 359 human diseases referenced in 2,863 Pubmed articles. Multiple sequence analysis indicated that over 60% of known horse mature miRNAs found perfect matches in human disease-associated miRNAs, followed by dog (50%). As expected, chicken had the least number of perfect matches (5%). Phylogenetic analysis of miRNA precursors indicated that 85% of human disease pre-miRNAs were highly conserved in animals, showing less than 5% nucleotide substitution rates over evolutionary time. As an example we demonstrated conservation of human hsa-miR-143-3p which is associated with type 2 diabetes and targets AKT1 gene which is highly conserved in pig, horse and dog. Functional analysis of AKT1 gene using Gene Ontology (GO) showed that it is involved in glucose homeostasis, positive regulation of glucose import, positive regulation of glycogen biosynthetic process, glucose transport and response to food. CONCLUSIONS: This data provides the animal and veterinary research community with a resource to assist in generating hypothesis-driven research for discovering animal disease-related miRNA from their datasets and expedite development of prophylactic and disease-treatment strategies and also influence research efforts to identify novel disease models in large animals. Integrated data is available for download at http://agbase.hpc.msstate.edu/cgi-bin/animal_mirna.cgi.

MicroRNAs: history, biogenesis, and their evolving role in animal development and disease.

The discovery of microRNAs (miRNAs) in 1993 followed by developments and discoveries in small RNA biology have redefined the biological landscape by significantly altering the longstanding dogmas that defined gene regulation. These small RNAs play a significant role in modulation of an array of physiological and pathological processes ranging from embryonic development to neoplastic progression. Unique miRNA signatures of various inherited, metabolic, infectious, and neoplastic diseases have added a new dimension to the studies that look at their pathogenesis and highlight their potential to be reliable biomarkers. Also, altering miRNA functionality and the development of novel in vivo delivery systems to achieve targeted modulation of specific miRNA function are being actively pursued as novel approaches for therapeutic intervention in many diseases. Here we review the current body of knowledge on the role of miRNAs in development and disease and discuss future implications.

Beyond H&E: integration of nucleic acid-based analyses into diagnostic pathology.

Veterinary pathology of infectious, particularly viral, and neoplastic diseases has advanced significantly with the advent of newer molecular methodologies that can detect nucleic acid of infectious agents within microscopic lesions, differentiate neoplastic from nonneoplastic cells, or determine the suitability of a targeted therapy by detecting specific mutations in certain cancers. Polymerase chain reaction-based amplification of DNA or RNA and in situ hybridization are currently the most commonly used methods for nucleic acid detection. In contrast, the main methodology used for protein detection within microscopic lesions is immunohistochemistry. Other methods that allow for analysis of nucleic acids within a particular cell type or individual cells, such as laser capture microdissection, are also available in some laboratories. This review gives an overview of the factors that influence the accurate analysis of nucleic acids in formalin-fixed tissues, as well as of different approaches to detect such targets.

Not lost in translation: how study of diseases in our pets can benefit them and us.

Practice-changing medical discovery requires preclinical and clinical assessment be carried out using appropriate disease models. There is growing awareness of companion animals with naturally-occurring disease as such models. They offer significant advantages over more traditional in vivo models of induced disease. This review describes current efforts to promote translation of discoveries between human and veterinary medicine in order to more rapidly and efficiently make progress in improving the health of all human and animal patients.

Gene selection and cancer type classification of diffuse large-B-cell lymphoma using a bivariate mixture model for two-species data.

: A bivariate mixture model utilizing information across two species was proposed to solve the fundamental problem of identifying differentially expressed genes in microarray experiments. The model utility was illustrated using a dog and human lymphoma data set prepared by a group of scientists in the College of Veterinary Medicine at North Carolina State University. A small number of genes were identified as being differentially expressed in both species and the human genes in this cluster serve as a good predictor for classifying diffuse large-B-cell lymphoma (DLBCL) patients into two subgroups, the germinal center B-cell-like diffuse large B-cell lymphoma and the activated B-cell-like diffuse large B-cell lymphoma. The number of human genes that were observed to be significantly differentially expressed (21) from the two-species analysis was very small compared to the number of human genes (190) identified with only one-species analysis (human data). The genes may be clinically relevant/important, as this small set achieved low misclassification rates of DLBCL subtypes. Additionally, the two subgroups defined by this cluster of human genes had significantly different survival functions, indicating that the stratification based on gene-expression profiling using the proposed mixture model provided improved insight into the clinical differences between the two cancer subtypes.