Measuring the Asian seabass (Lates calcarifer) neutrophil respiratory burst activity by the dihydrorhodamine-123 reduction flow cytometry assay in whole blood.

The neutrophil oxidative respiratory burst response is a key component of the innate immune system responsible for killing microbial pathogens. Since fish rely on the innate immune system for health, monitoring the respiratory burst activity may be an effective means of gauging fish health status. Here we report that the respiratory burst of Asian seabass neutrophils can be measured in whole blood by the dihydrorhodamine (DHR)-123 reduction assay and flow cytometry. Neutrophils responded to phorbol myristate acetate (PMA) in a concentration dependent manner with significant respiratory burst activity at 100-1000 nM. Other known neutrophil agonists, such as bacterial lipopolysaccharide, tumor necrosis factor, the tripeptide f-met-leu-phe and zymosan, did not induce a significant DHR reduction. Thus, the findings enable us to propose that the DHR-123 flow cytometry whole blood assay, incorporating PMA as a stimulator, would not only facilitate future studies into fish blood neutrophil research but provides a simple, rapid and reliable assay for gauging fish natural immunity status and health.

Genetic diversity and population structure of the endangered marsupial Sarcophilus harrisii (Tasmanian devil).

The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction because of a contagious cancer known as Devil Facial Tumor Disease. The inability to mount an immune response and to reject these tumors might be caused by a lack of genetic diversity within a dwindling population. Here we report a whole-genome analysis of two animals originating from extreme northwest and southeast Tasmania, the maximal geographic spread, together with the genome from a tumor taken from one of them. A 3.3-Gb de novo assembly of the sequence data from two complementary next-generation sequencing platforms was used to identify 1 million polymorphic genomic positions, roughly one-quarter of the number observed between two genetically distant human genomes. Analysis of 14 complete mitochondrial genomes from current and museum specimens, as well as mitochondrial and nuclear SNP markers in 175 animals, suggests that the observed low genetic diversity in today's population preceded the Devil Facial Tumor Disease disease outbreak by at least 100 y. Using a genetically characterized breeding stock based on the genome sequence will enable preservation of the extant genetic diversity in future Tasmanian devil populations.

Evolution of a contagious cancer: epigenetic variation in Devil Facial Tumour Disease.

The emergence of Devil Facial Tumour Disease (DFTD), a highly contagious cancer, is driving Tasmanian devils (Sarcophilus harrisii) to extinction. The cancer is a genetically and chromosomally stable clonal cell line which is transmitted by biting during social interactions. In the present study, we explore the Devil Facial Tumour (DFT) epigenome and the genes involved in DNA methylation homeostasis. We show that tumour cells have similar levels of methylation to peripheral nerves, the tissue from which DFTD originated. We did not observe any strain or region-specific epimutations. However, we revealed a significant increase in hypomethylation in DFT samples over time (p < 0.0001). We propose that loss of methylation is not because of a maintenance deficiency, as an upregulation of DNA methyltransferase 1 gene was observed in tumours compared with nerves (p < 0.005). Instead, we believe that loss of methylation is owing to active demethylation, supported by the temporal increase in MBD2 and MBD4 (p < 0.001). The implications of these changes on disease phenotypes need to be explored. Our work shows that DFTD should not be treated as a static entity, but rather as an evolving parasite with epigenetic plasticity. Understanding the role of epimutations in the evolution of this parasitic cancer will provide unique insights into the role of epigenetic plasticity in cancer evolution and progression in traditional cancers that arise and die with their hosts.

Wound healing of experimental equine skin wounds and concurrent microbiota in wound dressings following topical propylene glycol gel treatment.

Introduction: Topical wound treatments rely on carrier formulations with little to no biological impact. The potential for a common vehicle, a propylene glycol (PG) gel, to affect wound healing measures including microbiota is not known. Microbiome characterization, based on next generation sequencing methods is typically performed on tissue or directly obtained wound fluid samples. The utility for primary wound dressings to characterize equine wound microbiota in the context of topical treatments is currently unknown. This investigation reports the topical effect of an 80% PG based gel on wound healing and microbiota in wound dressings. Methods: Experiments were performed in six mature horses utilizing a surgical, distal limb wound model, histology of sequential wound biopsies, photographic wound measurements and microbiota profiling via 16s rRNA sequencing of wound dressing samples. Experimental wounds were surveyed for 42 days and either treated (Day 7, 14, 21 and 28; at 0.03 ml/cm2) or unexposed to the PG gel. Wound surface area, relative and absolute microbial abundances, diversity indices and histologic parameters were analyzed in the context of the experimental group (treatment; control) using qualitative or quantitative methods depending on data characteristics. Results: Compared to controls, treatment slowed the wound healing rate (17.17 ± 4.27 vs. 18.56 ± 6.3 mm2/day), delayed the temporal decline of polymorphonucleated cells in wound beds and operational taxonomic units (OTU) in wound dressings and lowered alpha-diversity indices for microbiota in primary wound dressing. Relative abundances of OTUs were in line with those previously reported for equine wounds. Clinical outcomes 42 days post wounding were considered similar irrespective of PG gel exposure. Discussion: Results highlight the potential for vehicle exposure to alter relevant wound outcome measures, imposing the need for stringent experimental control measures. Primary wound dressings may represent an alternate sample source for characterization of the wound microbiome alleviating the need for additional interventions. Further studies are warranted to contrast the microbiome in wound dressings against that present on wound surfaces to conclude on the validity of this approach.

Development and validation of a TaqMan PCR assay for the Australian abalone herpes-like virus.

The recent emergence of a herpes-like virus in both farmed and wild populations of abalone in Victoria, Australia, has been associated with high mortality rates in animals of all ages. Based on viral genome sequence information, a virus-specific real-time TaqMan assay was developed for detection and identification of the abalone herpes-like virus (AbHV). The assay was shown to be specific as it did not detect other viruses from either the Herpesvirales or the Iridovirales orders which have genome sequence similarities. However, the TaqMan assay was able to detect DNA from the Taiwanese abalone herpes-like virus, suggesting a relationship between the Taiwanese and Australian viruses. In addition, the assay detected < 300 copies of recombinant plasmid DNA per reaction. Performance characteristics for the AbHV TaqMan assay were established using 1673 samples from different abalone populations in Victoria and Tasmania. The highest diagnostic sensitivity and specificity were 96.7 (95% CI: 82.7 to 99.4) and 99.7 (95% CI: 99.3 to 99.9), respectively, at a threshold cycle (C(T)) value of 35.8. The results from 2 separate laboratories indicated good repeatability and reproducibility. This molecular assay has already proven useful in confirming presumptive diagnosis (based on the presence of ganglioneuritis) of diseased abalone in Victorian waters as well as being a tool for surveillance of wild abalone stocks in other parts of Australia.

Assessment of cellular immune responses of healthy and diseased Tasmanian devils (Sarcophilus harrisii).

The Tasmanian devil (TD) (Sarcophilus harrisii) is under threat from devil facial tumour disease (DFTD), a cancer that is transmitted between devils by direct cell implantation. As no devil is known to have rejected the tumour allograft, an understanding of the immune status of this species is essential to help explain the unique infectious nature of this cancer. We analysed differential white blood cell counts, the phagocytic response of neutrophils as well as mitogen-induced lymphocyte proliferation. Devils analysed included healthy TDs kept in captivity, healthy devils from disease-free and diseased areas as well as diseased devils. Neutrophils isolated from the peripheral blood of healthy devils showed competent phagocytosis and peripheral blood mononuclear cells from healthy and diseased devils proliferated in response to Con A, PHA and PWM stimulation. Although a wide range of responses was observed and relatively high doses of mitogens were required, there was no significant difference between males and females, adults and juveniles or between normal and diseased animals, suggesting that transmission of DFTD is not a consequence of a severely impaired immune system. As lymphocytes from all TDs appear to require strong stimulation for activation, this threshold may contribute to all devils being susceptible to DFTD.

Dental Pulp Stem Cells - Exploration in a Novel Animal Model: the Tasmanian Devil (Sarcophilus harrisii).

Dental pulp stem cells (DPSC) are a heterogeneous population of highly proliferative stem cells located in the soft inner pulp tissue of the tooth. Demonstrated to have an affinity for neural differentiation, DPSC have been reported to generate functional Schwann cells (SC) through in vitro differentiation. Both DPSC and SC have neural crest origins, recently a significant population of DPSC have been reported to derive from peripheral nerve-associated glia. The predisposition DPSC have towards the SC lineage is not only a very useful tool for neural regenerative therapies in the medical field, it also holds great promise in the veterinary field. Devil Facial Tumour (DFT) is a clonally transmissible cancer of SC origin responsible for devastating wild populations of the Tasmanian devil. Very few studies have investigated the healthy Tasmanian devil SC (tdSC) for comparative studies between tdSC and DFT cells, and the development and isolation of a tdSC population is yet to be undertaken. A Tasmanian devil DPSC model offers a promising new outlook for DFT research, and the link between SC and DPSC may provide a potential explanation as to how a cancerous SC initially arose in a single Tasmanian devil to then go on to infect others as a parasitic clonal cell line. In this review we explore the current role of DPSC in human regenerative medicine, provide an overview of the Tasmanian devil and the devastating effect of DFT, and highlight the promising potential DPSC techniques pose for DFT research and our current understanding of DFT.

ERBB3: A potential serum biomarker for early detection and therapeutic target for devil facial tumour 1 (DFT1).

Devil Facial Tumour 1 (DFT1) is one of two transmissible neoplasms of Tasmanian devils (Sarcophilus harrisii) predominantly affecting their facial regions. DFT1's cellular origin is that of Schwann cell lineage where lesions are evident macroscopically late in the disease. Conversely, the pre-clinical timeframe from cellular transmission to appearance of DFT1 remains uncertain demonstrating the importance of an effective pre-clinical biomarker. We show that ERBB3, a marker expressed normally by the developing neural crest and Schwann cells, is immunohistohemically expressed by DFT1, therefore the potential of ERBB3 as a biomarker was explored. Under the hypothesis that serum ERBB3 levels may increase as DFT1 invades local and distant tissues our pilot study determined serum ERBB3 levels in normal Tasmanian devils and Tasmanian devils with DFT1. Compared to the baseline serum ERBB3 levels in unaffected Tasmanian devils, Tasmanian devils with DFT1 showed significant elevation of serum ERBB3 levels. Interestingly Tasmanian devils with cutaneous lymphoma (CL) also showed elevation of serum ERBB3 levels when compared to the baseline serum levels of Tasmanian devils without DFT1. Thus, elevated serum ERBB3 levels in otherwise healthy looking devils could predict possible DFT1 or CL in captive or wild devil populations and would have implications on the management, welfare and survival of Tasmanian devils. ERBB3 is also a therapeutic target and therefore the potential exists to consider modes of administration that may eradicate DFT1 from the wild.

Doxorubicin and carboplatin trials in Tasmanian devils (Sarcophilus harrisii) with Tasmanian devil facial tumor disease.

The devil facial tumor disease (DFTD) is having a devastating impact on Tasmanian devils (Sarcophilus harrisii) (devils) in the wild. Only a single study has been published regarding treatment of DFTD, where vincristine was not found to be an effective chemotherapeutic agent. In the current study, devils were treated with escalating dosages of carboplatin (8-26 mg/kg) (n = 13) and doxorubicin (0.75-1.0 mg/kg) (n = 5). Dosages for carboplatin (20 mg/kg) and doxorubicin (1.0 mg/kg) were identified as maximally tolerated dosages. Limiting toxicities for carboplatin were anorexia and weight loss (gastrointestinal signs) and azotemia. Limiting toxicities for doxorubicin were neutropenia, anorexia and weight loss. None of the treated devils responded to either drug, suggesting that, based on the clonality of this tumour, it is unlikely that either drug individually or in combination would be effective treatments for DFTD. These results, however, provide valuable information for practitioners who may choose to treat other neoplastic diseases in the devil or other marsupials. In addition, these results show that even drugs that are metabolized and excreted in the same manner can be tolerated to different degrees by the same species.

Anthropogenic selection enhances cancer evolution in Tasmanian devil tumours.

The Tasmanian Devil Facial Tumour Disease (DFTD) provides a unique opportunity to elucidate the long-term effects of natural and anthropogenic selection on cancer evolution. Since first observed in 1996, this transmissible cancer has caused local population declines by >90%. So far, four chromosomal DFTD variants (strains) have been described and karyotypic analyses of 253 tumours showed higher levels of tetraploidy in the oldest strain. We propose that increased ploidy in the oldest strain may have evolved in response to effects of genomic decay observed in asexually reproducing organisms. In this study, we focus on the evolutionary response of DFTD to a disease suppression trial. Tumours collected from devils subjected to the removal programme showed accelerated temporal evolution of tetraploidy compared with tumours from other populations where no increase in tetraploid tumours were observed. As ploidy significantly reduces tumour growth rate, we suggest that the disease suppression trial resulted in selection favouring slower growing tumours mediated by an increased level of tetraploidy. Our study reveals that DFTD has the capacity to rapidly respond to novel selective regimes and that disease eradication may result in novel tumour adaptations, which may further imperil the long-term survival of the world's largest carnivorous marsupial.