ADVANCED DIAGNOSTIC IMAGING AND PATHOLOGIC FINDINGS OF THYROID LESIONS IN TWO SHORT-BEAKED ECHIDNAS (TACHYGLOSSUS ACULEATUS).

Two zoo-maintained short-beaked echidnas (Tachyglossus aculeatus) had long histories of intermittent anorexia and lethargy. Case 1 presented with a recurrence of these signs after transfer to another facility and died shortly after arrival. A focal area of hyperattenuation within the paratracheal tissue of the cranial mediastinum was noted antemortem on CT. Postmortem, this corresponded with severe thyroid follicular hyperplasia with lymphoplasmacytic thyroiditis. Additional findings included a systemic fungal infection without an inflammatory response, suggesting underlying factors such as torpor or immunosuppression. In Case 2, an intrathoracic mass was identified during a preshipment examination. CT confirmed a contrast-enhanced mass compressing the cranial vena cava and right atrium, and the animal was euthanized. The mass was diagnosed histologically as thyroid adenocarcinoma. These cases report thyroiditis and thyroid adenocarcinoma in echidna and describe the use of IV contrast and CT as a diagnostic aid in this species.

Dynamic Evolution of Retroviral Envelope Genes in Egg-Laying Mammalian Genomes.

Independently acquired envelope (env) genes from endogenous retroviruses have contributed to the placental trophoblast cell-cell fusion in therian mammals. Egg-laying mammals (monotremes) are an important sister clade for understanding mammalian placental evolution, but the env genes in their genomes have yet to be investigated. Here, env-derived open reading frames (env-ORFs) encoding more than 400 amino acid lengths were searched in the genomes of two monotremes: platypus and echidna. Only two env-ORFs were present in the platypus genome, whereas 121 env-ORFs were found in the echidna genome. The echidna env-ORFs were phylogenetically classified into seven groups named env-Tac1 to -Tac7. Among them, the env-Tac1 group contained only a single gene, and its amino acid sequence showed high similarity to those of the RD114/simian type D retroviruses. Using the pseudotyped virus assay, we demonstrated that the Env-Tac1 protein utilizes echidna sodium-dependent neutral amino acid transporter type 1 and 2 (ASCT1 and ASCT2) as entry receptors. Moreover, the Env-Tac1 protein caused cell-cell fusion in human 293T cells depending on the expression of ASCT1 and ASCT2. These results illustrate that fusogenic env genes are not restricted to placental mammals, providing insights into the evolution of retroviral genes and the placenta.

Investigating the utility of using fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas (Tachyglossus aculeatus).

This study demonstrates the utility of the analysis of fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas. Over three breeding seasons daily fecal samples were collected from female echidnas (n = 8) that were monitored continuously by video surveillance to confirm key reproductive events. Fecal progesterone metabolite concentrations were elevated above baseline values (448.0 ± 156.3 ng/g) during pregnancy and the luteal phase. However, compared to plasma progesterone the rise in fecal progesterone metabolite concentrations after copulation was delayed (3.3 ± 0.4 versus 8.3 ± 0.6 days, respectively), such that pregnancy was more reliably detected in its latter half when using fecal samples. Mating and oviposition were observed for 14 of the 19 pregnancies resulting in an estimated gestation of 16.7 ± 0.2 days (range 16.0-18.1 d). The estrogen enzyme-immunoassays tested (n = 3) in this study were not suitable for the fecal samples of the echidna. Fecal progesterone metabolites are an effective tool for confirming the timing and occurrence of estrous cycles in captive echidna colonies and can assist zookeepers in identifying possible causes of sub-optimal reproductive success without the unnecessary stress of repeated capture and anaesthesia for blood collection.

Optimizing captive short-beaked echidna (Tachyglossus aculeatus) fecal sample identification and hormonal analysis.

The objectives of this study were to develop a fecal marking protocol to distinguish male from female samples during the echidna breeding season and to determine if normalizing fecal progesterone metabolite data for inorganic content improves the detection of biologically relevant changes in metabolite concentrations. Over a period of 6 weeks, four echidnas were provided with green food coloring powder mixed into 20 g of their regular feed with the dose adjusted weekly by 0.05 g. The proportion of organic (feces) versus inorganic matter (sand) in the fecal samples of three echidnas was determined by combustion of organic matter. Hormonal data was then expressed as metabolite concentration per total dry mass (with sand) of extracted sample versus metabolite concentration per total mass of organic material (without sand). The optimal dose of food coloring powder was 0.30 g: this was excreted in the feces of all echidnas within 24 h of consumption with color present for two consecutive days. Correction for inorganic content (sand) did not significantly affect variability of fecal progesterone metabolite levels (mean CV ± SE with sand: 142.3 ± 13.3%; without sand: 127.0 ± 14.4%; W = 6, p = .2500), or the magnitude of change from basal to elevated fecal progesterone metabolite concentrations (mean ± SE with sand: 8.4 ± 1.7; without sand: 6.6 ± 0.5, W = 10, p = .1250). Furthermore, progesterone metabolite concentrations before and after correction for sand contamination correlated strongly (r = .92, p = < .001). These methods will facilitate future reproductive endocrinology studies of echidna and other myrmecophagous species.

Can spines tell a story? Investigation of echidna spines as a novel sample type for hormone analysis in monotremes.

The short-beaked echidna (Tachyglossus aculeatus) is a monotreme endemic to Australia and New Guinea, and is the most widespread native mammal in Australia. Despite its abundance, there are considerable gaps in our understanding of echidna life history such as reproductive cycles in both sexes, patterns of stress physiology, and possible seasonal changes in metabolism. Slow-growing integumentary sample types comprised of keratin (hair, claw, etc.) have been used in other wildlife to assess these questions via analysis of longitudinal patterns in steroid and thyroid hormones that are deposited in these tissues as they grow. Hairs and spines comprise the pelage of echidnas, the spines being keratinized structures homologous to hair. Thus, echidna spines could be a viable sample type for hormone analysis contributing to a better understanding of the biology of echidnas. The aim of this work was to determine whether steroid hormones are detectable in echidna spines, to perform assay validations, and to establish a protocol for extracting and quantifying hormones in echidna spines using commercially available assay kits. We also inspected cross-sectioned spines using light and electron microscopy for any evidence of annual growth markers that might enable inferences about spine growth rate. Corticosterone, progesterone, estradiol, and testosterone were detectable in all samples, and echidna spine extract passed standard assay validations (parallelism and accuracy), indicating that commercially available assay kits can quantify hormones accurately in this sample type. No visible growth marks were identified in the spines and thus spine growth rate is currently unknown. Echidna spines show promise as a novel matrix from which hormones can be quantified; next steps should involve determination of spine annual growth rate, possible seasonal changes in growth rate, and persistence of spines over time in order to perform physiological validations, i.e., relationship between physiological status and hormone concentrations in spines.

Plasma progesterone secretion during gestation of the captive short-beaked echidna.

This study describes the progesterone profile during pregnancy in sexually mature female captive short-beaked echidnas (Tachyglossus aculeatus aculeatus). Echidnas were monitored daily by video surveillance to confirm key reproductive behaviour. Plasma samples were collected and pouch morphology was assessed three times a week. The pouch of the female echidna only develops during gestation and it was possible to create a four-stage grading system using the most distinguishable characteristics of pouch development. Maximum pouch development was associated with declining progesterone concentrations, with the pouch closing in a drawstring-like manner at oviposition. Control of pouch development in pregnant echidnas is not yet clear but later pouch development is associated with a decrease in progesterone and pouch closure and may be under mechanical influences of the egg or young in the pouch. The length of pregnancy was 16.7 ± 0.2 days with a 15.1 ± 1.0 days luteal phase followed by an incubation period in the pouch. Eggs could be detected in utero at least 4 days before oviposition. Plasma progesterone peaked at 10.5 ± 0.9 ng/mL within 12 days of mating but then declined to basal levels within 1 day of oviposition and remained basal throughout egg incubation, confirming that progesterone is elevated throughout pregnancy and that gestation does not extend beyond the luteal phase. After the loss of an egg or pouch young, most females entered a second oestrous cycle and ovulated, suggesting echidnas are seasonally polyoestrous. The duration of the luteal phase in the echidna corresponds with that observed in other mammals.

Cutaneous botryomycosis in a free-living short-beaked echidna (Tachyglossus aculeatus).

Botryomycosis is a chronic bacterial infection of the skin and viscera caused by nonbranching bacteria such as Staphylococcus spp. It results in the formation of chronic, suppurative and often granulomatous lesions that may resemble multiple abscesses. It has been diagnosed worldwide in both humans and animals. It is a rare disease, usually requiring surgical excision combined with long-term antibiotics to achieve a good resolution. A wild echidna (Tachyglossus aculeatus) was presented with a soft tissue mass at the base of the right distal beak with multiple discharging fistulas arising from it. The mandible appeared to be shortened and deviated to the right, and the animal was unable to close its beak. The echidna was in poor body condition and was euthanased on welfare grounds. Histopathology of the lesions demonstrated the classical botryoid lesions with the characteristic Splendore-Hoeppli reaction surrounding the bacterial colonies, in a background of extensive granulation tissue. Radiographs revealed mild mandibular osteomyelitis, yet haematology and biochemistry results were nonspecific. This disease has not previously been reported in a wild monotreme.

Functional evaluation of a monotreme-specific antimicrobial protein, EchAMP, against experimentally induced mastitis in transgenic mice.

EchAMP, the tenth most abundant transcript expressed in the mammary gland of echidna, has in vitro broad-spectrum antibacterial effects. However, the effects of EchAMP on mastitis, a condition where inflammation is triggered following mammary gland infection, has not been investigated. To investigate the impact of EchAMP against mastitis, EchAMP transgenic mice were generated. In antibacterial assays, the whey fractions of milk from transgenic mice significantly reduced growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa compared with whey fractions from wildtype mice. Furthermore, a mastitis model created by infecting mammary gland with these four bacterial strains displayed a significant reduction in bacterial load in transgenic mice injected with S. aureus and B. subtilis. On further confirmation, histomorphologic analysis showed absence of necrosis and cell infiltration in the mammary glands of transgenic mice. To understand the role of EchAMP against inflammation, we employed an LPS-injected mastitis mouse model. LPS is known to induce phopshorylation of NF-κB and MAPK pathways, which in turn activate downstream proinflammatory signaling mediators, to promote inflammation. In LPS-treated EchAMP transgenic mice, phosphorylation levels of NF-κB, p38 and ERK1/2 were significantly downregulated. Furthermore, in mammary gland of transgenic mice, there was a significant downregulation of mRNA levels of proinflammatory cytokines, namely TNF-α, IL-6 and IL-1ß. Taken together, these data suggest that EchAMP has an antiinflammatory response and is effective against S. aureus and B. subtilis. We suggest that EchAMP may be a potential prophylactic protein against mastitis in dairy animals by expressing this gene in their mammary gland.

Structural and mechanistic insights into EchAMP: A antimicrobial protein from the Echidna milk.

BACKGROUND: Antibiotic resistance is a problem that necessitates the identification of new antimicrobial molecules. Milk is known to have molecules with antimicrobial properties (AMPs). Echidna Antimicrobial Protein (EchAMP) is one such lactation specific AMP exclusively found in the milk of Echidna, an egg-laying mammal geographically restricted to Australia and New Guinea. Previous studies established that EchAMP exhibits substantial bacteriostatic activity against multiple bacterial genera. However, the subsequent structural and functional studies were hindered due to the unavailability of pure protein. RESULTS: In this study, we expressed EchAMP protein using a heterologous expression system and successfully purified it to >95% homogeneity. The purified recombinant protein exhibits bacteriolytic activity against both Gram-positive and Gram-negative bacteria as confirmed by live-dead staining and scanning electron microscopy. Structurally, this AMP belongs to the family of intrinsically disordered proteins (IDPs) as deciphered by the circular-dichroism, tryptophan fluorescence, and NMR spectroscopy. Nonetheless, EchAMP has the propensity to acquire structure with amphipathic molecules, or membrane mimics like SDS, lipopolysaccharides, and liposomes as again observed through multiple spectroscopic techniques. CONCLUSIONS: Recombinant EchAMP exhibits broad-spectrum bacteriolytic activity by compromising the bacterial cell membrane integrity. Hence, we propose that this intrinsically disordered antimicrobial protein interact with the bacterial cell membrane and undergoes conformational changes to form channels in the membrane resulting in cell lysis. GENERAL SIGNIFICANCE: EchAMP, the evolutionarily conserved, lactation specific AMP from an oviparous mammal may find application as a broad-spectrum antimicrobial against pathogens that affect mammary gland or otherwise cause routine infections in humans and livestock.

Soft tissue sarcoma in a short-beaked echidna (Tachyglossus aculeatus).

CASE REPORT: An adult male short-beaked echidna in poor body condition was found with a 25 × 12 mm round, ulcerated and bleeding mass on the left side of the face at the base of the beak. The animal responded well to initial supportive care and was referred to a specialist wildlife centre for further assessment and treatment. Clinical pathology showed moderate neutrophilia, mild anaemia, mild elevation in liver enzymes (ALT, AST and ALP) and mild azotaemia. Initial clinical differential diagnoses for the facial mass favoured an inflammatory rather than a neoplastic lesion, based on previous reports. Examination of an incisional biopsy identified a malignant spindle cell proliferation (sarcoma) not amenable to complete surgical excision. The animal was euthanased on humane grounds. Immunohistochemical assessment of the mass showed it to be negative for cytokeratin, desmin, smooth muscle actin, periaxin and MAC387 antibody labelling. Definitive histogenesis was undetermined and a final diagnosis of poorly differentiated sarcoma, unlikely to be of muscle, Schwann cell or histiocytic origin, was made. CONCLUSION: Reports of neoplasia in prototherian mammals (monotremes) are rare. To the authors' knowledge this is the first report of such a tumour in a monotreme species and the first immunohistochemical characterisation of a stromal tumour in these animals. The malignant nature of this tumour contrasts with a previous report of benign neoplasia (fibroma) associated with the beak. Although rare, malignant neoplasia should be included in the differential diagnoses of mass lesions in monotremes, despite inflammatory or traumatic mass lesions being more commonly reported.

Metastatic mineralisation caused by vitamin D intoxication in hand-raised short-beaked echidnas (Tachyglossus aculeatus).

CASE REPORT: Two cases of metastatic mineralisation caused by dietary excess of vitamin D are reported in hand-raised short-beaked echidna (Tachyglossus aculeatus) puggles at the Australian Wildlife Health Centre. Oversupplementation was attributed to excessive levels of vitamin D in Wombaroo Echidna Milk Replacer fed to the puggles. No information exists regarding the naturally occurring vitamin D levels in echidna milk, but, given the low serum levels that have been observed in free-ranging animals, it is likely to be low. CONCLUSION: The vitamin D concentration in Wombaroo Echidna Milk Replacer has been reduced.

Digestibility of a new diet for captive short-beaked echidnas (Tachyglossus aculeatus).

Short-beaked echidnas (Tachyglossus aculeatus) are myrmecophages, or ant and termite insectivore specialists, and replicating their exact diet in captivity is problematic. Diets for captive animals often incorporate raw meat, eggs and cat food mixed together with water, and vitamin and mineral supplements. These diets have promoted a number of health problems in captive echidnas, such as gastritis, cystitis, gut impaction, obesity, and diarrhea. A manufactured diet was designed and three echidnas from two zoos were transitioned onto this diet to assess the acceptability and digestibility of this diet for echidnas. The new "test" diet was readily accepted by the echidnas with a 1 week transition period. Daily digestible energy intake was 280 kJ kg-0.75 d-1 , similar to another myrmecophagous species. Digestibility values were above 74% for all macronutrients. It was determined that this diet was an acceptable replacement for the previous diets and it was decided that the remaining echidnas at both institutions would be transitioned to the new diet. The diet will also be used for wild echidnas being rehabilitated in the zoo hospitals prior to release and commercially available within Australia. Further data are being collected to assess the use of this diet for seasonal weight management, transitioning hand-reared puggles and effects on gastrointestinal tract health. Zoo Biol. 36:56-61, 2017. © 2017 Wiley Periodicals, Inc.

Monotreme glucagon-like peptide-1 in venom and gut: one gene - two very different functions.

The importance of Glucagon like peptide 1 (GLP-1) for metabolic control and insulin release sparked the evolution of genes mimicking GLP-1 action in venomous species (e.g. Exendin-4 in Heloderma suspectum (gila monster)). We discovered that platypus and echidna express a single GLP-1 peptide in both intestine and venom. Specific changes in GLP-1 of monotreme mammals result in resistance to DPP-4 cleavage which is also observed in the GLP-1 like Exendin-4 expressed in Heloderma venom. Remarkably we discovered that monotremes evolved an alternative mechanism to degrade GLP-1. We also show that monotreme GLP-1 stimulates insulin release in cultured rodent islets, but surprisingly shows low receptor affinity and bias toward Erk signaling. We propose that these changes in monotreme GLP-1 are the result of conflicting function of this peptide in metabolic control and venom. This evolutionary path is fundamentally different from the generally accepted idea that conflicting functions in a single gene favour duplication and diversification, as is the case for Exendin-4 in gila monster. This provides novel insight into the remarkably different metabolic control mechanism and venom function in monotremes and an unique example of how different selective pressures act upon a single gene in the absence of gene duplication.

Formation and Dissociation of Sperm Bundles in Monotremes.

Because monotremes are the earliest offshoot of the mammalian lineage, the platypus and short-beaked echidna were studied as model animals to assess the origin and biological significance of adaptations considered unique to therian mammals: epididymal sperm maturation and subsequent capacitation. We show that spermatozoa from both species assemble into bundles of approximately 100 cells during passage through the epididymis and that an epididymal protein-secreted protein, acidic, cysteine-rich (osteonectin; SPARC)-is involved in bundle formation. The bundles persisted during incubation in vitro for at least 1 h under conditions that capacitate therian spermatozoa, and then underwent a time-dependent dissociation to release spermatozoa capable of fertilization. Only after this dissociation could the spermatozoa bind to the perivitelline membrane of a hen's egg, display an altered form of motility reminiscent of hyperactivation, and be induced to undergo an acrosome reaction. It is concluded that the development of sperm bundles in the monotreme epididymis mandates that they require a time-dependent process to be capable of fertilizing an ovum. However, because this functional end point was achieved without overt changes in protein tyrosine phosphorylation (a hallmark of capacitation in therians), it is concluded that the process in monotremes is distinctly different from capacitation in therian mammals.

Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.).

Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. ß-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and ß-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or ß-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution.

The mongoose, the pheasant, the pox, and the retrovirus.

Paleovirology is the study of ancient viruses. The existence of a paleovirus can sometimes be detected by virtue of its accidental insertion into the germline of different animal species, which allows one to date when the virus actually existed. However, the ancient and the modern often connect, as modern viruses have unexpected origins that can be traced to ancient infections. The genomes of two species of mongooses and an egg-laying mammal called an echidna show that a virus currently present in poultry, the reticuloendotheliosis virus (REV), is actually of ancient exotic mammalian origin. REV apparently spread to poultry through a circuitous route involving the isolation of malaria parasites from a pheasant from Borneo housed at the Bronx Zoo that was contaminated with REV. Repeated passage of this virus in poultry adapted the virus to its new host. At some point, the virus got inserted into another virus, called fowlpox virus, which has spread back into the wild. Although REV may still exist somewhere in a mammalian host, its modern form links an 8 million-year-old infection of the ancestor of a mongoose to a virus that now is circulating in wild birds through malaria studies in the mid-20(th) century. These lessons of ancient and modern viruses have implications for modern human pandemics from viral reservoirs and for human interventions that may come with unintended consequences.

Microscopy of the echidna sublingual glands.

The secretory units and duct system of the echidna sublingual glands exhibit subtle architectural modifications to accommodate the viscous secretion produced by these glands. The glands are compound tubular glands, the secretory units of which are elongate with open lumina and consist only of mucous cells. Closely packed spindle-shaped myoepithelial cells invest the secretory units, but are absent around the ducts. The branched secretory tubules open into an abbreviated duct system characterized by wide lumina. Striated ducts normally associated with the second portion of the intralobular duct system are absent. The duct system shows the most obvious modification of general salivary gland architecture presumably to accommodate the viscous secretion propelled from the secretory units by surrounding myoepithelial cells.

Utilisation of Pangolin (Manis sps) in traditional Yorubic medicine in Ijebu province, Ogun State, Nigeria.

BACKGROUND: Concern about the use of endangered and threatened species in traditional medicine escalated as populations of many species plummeted because of poaching for the medicinal trade. Nigeria is known for a long and valued tradition of using wild animals and plants for medicinal purposes. Despite this, studies on medicinal animals are still scarce when compared to those focusing on medicinal plants. Utilisation of wild animals in traditional Yorubic medical practices was indiscriminate as it involved threatened species. By touting the medicinal properties of these species, traditional medicine fuel continuing demand, thereby subjecting such species to further threats. This paper examined the use and commercialisation of pangolins for traditional medicinal purposes amongst the Ijebus, South-western Nigeria, and the implications of this utilisation for the conservation of this species. METHODS: Traditional Yorubic medical practitioners (tymps) (16) and dealers in traditional medicinal ingredients (56) in public markets in Ijebu province, Nigeria, were interviewed using open-ended questionnaires. The dynamic stock movement of pangolins in the stalls of dealers was also monitored to determine quantity of pangolins sold into the traditional Yorubic medicinal practices. Specific conditions treated and the parts required were also documented. RESULTS: A total of 178 whole pangolin carcasses were sold into traditional medical practices. Above 55% of respondents had just primary education, over 90% of respondents were not aware of either the conservation status of this species or the existence of any legal machinery regulating its trade and utilisation, while 14% admitted to giving contracts to hunters for deliberate search for this animal when needed. More than 98% of respondents have no other means of livelihood. The trade was female dominated while the healing practice had more males. Pangolins were used in various preparations to treat a total of 42 conditions. These include infertility, gastro-intestinal disorders, safe parturition, stomach ulcers, rheumatism and fibroid. Traditional Yorubic medicine also accommodated some situations that are out of the range of conventional medicine like boosting sales, conferring invisibility, removing bad luck, appeasing/wading off witches cum evil forces and money rituals. Some of these situations specifically require juvenile, or even pregnant female animals. CONCLUSION: Traditional Yorubic medical practices eats deep into the reproductive base of the species, presently listed in Appendix II of CITES and Schedule I of the Nigerian Decree 11 (1985), both of which recommended strict control in sales and utilisation of this species. Its numerous medicinal values, folk culture and financial benefits of these activities are the main factors promoting the commercialisation and use of this species. Pharmacological studies on the various preparations are required to identify the bioactive compounds in them. There is a need for improved and urgent measures to conserve populations of this species in-situ. Massive education and enlightenment is urgently needed for the populace to have the necessary awareness and orientation about the conservation of this species.

Splenic lymphoma in a short-beaked echidna (Tachyglossus aculeatus).

A 23-year-old, male short-beaked echidna (Tachyglossus aculeatus) housed at a North American zoo was successfully treated for flea-associated anaemia, but subsequently died. Cause of death was presumptive septicaemia secondary to splenic lymphoma. This is only the fifth case of neoplasia reported in this monotreme species, and the first from outside of Australia.