Seasonal and geographic variation in packed cell volume and selected serum chemistry of platypuses.

Platypuses (Ornithorhynchus anatinus) inhabit the permanent rivers and creeks of eastern Australia, from north Queensland to Tasmania, but are experiencing multiple and synergistic anthropogenic threats. Baseline information of health is vital for effective monitoring of populations but is currently sparse for mainland platypuses. Focusing on seven hematology and serum chemistry metrics as indicators of health and nutrition (packed cell volume (PCV), total protein (TP), albumin, globulin, urea, creatinine, and triglycerides), we investigated their variation across the species' range and across seasons. We analyzed 249 unique samples collected from platypuses in three river catchments in New South Wales and Victoria. Health metrics significantly varied across the populations' range, with platypuses from the most northerly catchment, having lower PCV, and concentrations of albumin and triglycerides and higher levels of globulin, potentially reflecting geographic variation or thermal stress. The Snowy River showed significant seasonal patterns which varied between the sexes and coincided with differential reproductive stressors. Male creatinine and triglyceride levels were significantly lower than females, suggesting that reproduction is energetically more taxing on males. Age specific differences were also found, with juvenile PCV and TP levels significantly lower than adults. Additionally, the commonly used body condition index (tail volume index) was only negatively correlated with urea, and triglyceride levels. A meta-analysis of available literature revealed a significant latitudinal relationship with PCV, TP, albumin, and triglycerides but this was confounded by variation in sampling times and restraint methods. We expand understanding of mainland platypuses, providing reference intervals for PCV and six blood chemistry, while highlighting the importance of considering seasonal variation, to guide future assessments of individual and population condition.

Haemolytic anaemia associated with Theileria sp. in an orphaned platypus.

CASE REPORT: The clinical and laboratory findings in an orphaned juvenile female platypus (Ornithorhynchus anatinus) that presented with a severe anaemia and tick infestation are reported. The animal developed a terminal septicaemia and died. Antemortem clinical pathology, postmortem histopathology and 18S rDNA sequencing supported a diagnosis of extravascular haemolytic anaemia secondary to Theileria ornithorhynchi infection. CONCLUSION: Although T. ornithorhynchi infection is common in the platypus, this is the first case in which it has been shown to cause a haemolytic anaemia in this species and molecular characterisation of the organism has been described. A review of the previous literature concerning T. ornithorhynchi and possible treatment options for future cases are discussed.

Hematologic, plasma biochemical, and other indicators of the health of Tasmanian platypuses (Ornithorhynchus anatinus): predictors of mucormycosis.

The fungal disease mucormycosis has affected Tasmanian platypuses for nearly three decades. We investigated the influences of mucormycosis on the hematologic, plasma biochemical, and other indicators of health in free-living platypuses across 18 Tasmanian river catchments. Live trapping enabled sampling of 161 (apparently) healthy and six ulcerated, mucormycosis-affected platypuses in 75 rivers and streams between January 2008 and June 2009. There were no obvious differences in any hematologic or biochemical measures between healthy and mucormycosis-affected platypuses. However, multivariate analysis revealed that ulceration was associated with living at higher altitudes, low tail fat content (high tail fat index), and low trypanosome load. There was evidence of overall lymphocytosis and monocytosis in animals from areas currently affected by mucormycosis, which suggests that some level of immune response to the introduced fungus is now widespread in disease-affected catchments. Animals from currently, historically, and possibly disease-affected catchments had lower neutrophil counts, mean cell volumes, plasma alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase levels, and higher plasma gamma-glutamyl transpeptidase and platelet counts compared to animals from catchments with no evidence of infection. Reference intervals were generated for all hematologic and biochemical measurements. Since this is the most comprehensive, systematic, and large-scale assessment of the health of the Tasmanian platypus to date, these references intervals should act as the standard against which future studies of platypuses in Tasmania should be compared.

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XVIII platypus (Ornithorhynchus anatinus) and saltwater crocodile (Crocodylus porosus).

As part of a programme of comparative measurements of Pd (diffusional water permeability) the RBCs (red blood cells) from an aquatic monotreme, platypus (Ornithorhynchus anatinus), and an aquatic reptile, saltwater crocodile (Crocodylus porosus) were studied. The mean diameter of platypus RBCs was estimated by light microscopy and found to be approximately 6.3 microm. Pd was measured by using an Mn2+-doping 1H NMR (nuclear magnetic resonance) technique. The Pd (cm/s) values were relatively low: approximately 2.1 x 10(-3) at 25 degrees C, 2.5 x 10(-3) at 30 degrees C, 3.4 x 10(-3) at 37 degrees C and 4.5 at 42 degrees C for the platypus RBCs and approximately 2.8 x 10(-3) at 25 degrees C, 3.2 x 10(-3) at 30 degrees C, 4.5 x 10(-3) at 37 degrees C and 5.7 x 10(-3) at 42 degrees C for the crocodile RBCs. In parallel with the low water permeability, the Ea,d (activation energy of water diffusion) was relatively high, approximately 35 kJ/mol. These results suggest that "conventional" WCPs (water channel proteins), or AQPs (aquaporins), are probably absent from the plasma membranes of RBCs from both the platypus and the saltwater crocodile.

Body temperature-related structural transitions of monotremal and human hemoglobin.

In this study, temperature-related structural changes were investigated in human, duck-billed platypus (Ornithorhynchus anatinus, body temperature T(b) = 31-33 degrees C), and echidna (Tachyglossus aculeatus, body temperature T(b) = 32-33 degrees C) hemoglobin using circular dichroism spectroscopy and dynamic light scattering. The average hydrodynamic radius (R(h)) and fractional (normalized) change in the ellipticity (F(obs)) at 222 +/- 2 nm of hemoglobin were measured. The temperature was varied stepwise from 25 degrees C to 45 degrees C. The existence of a structural transition of human hemoglobin at the critical temperature T(c) between 36-37 degrees C was previously shown by micropipette aspiration experiments, viscosimetry, and circular dichroism spectroscopy. Based on light-scattering measurements, this study proves the onset of molecular aggregation at T(c). In two different monotremal hemoglobins (echidna and platypus), the critical transition temperatures were found between 32-33 degrees C, which are close to the species' body temperature T(b). The data suggest that the correlation of the structural transition's critical temperature T(c) and the species' body temperature T(b) is not mere coincidence but, instead, is a more widespread structural phenomenon possibly including many other proteins.

Plasma glucocorticoid concentrations in free-ranging platypuses (Ornithorhynchus anatinus): response to capture and patterns in relation to reproduction.

Energy demands in the platypus are likely to increase in the breeding season, which occurs from winter to early spring. Glucocorticoids, which play a major role in energy mobilisation, were measured in consecutive blood samples from free-ranging adults at approximately monthly intervals throughout the year. Glucose and free fatty acids were also measured in some samples. Plasma concentrations of glucocorticoids rose significantly during the first 30 min after capture, accompanied by a rise in free fatty acids, but no corresponding increase in glucose concentrations. We observed a strong pattern in plasma glucocorticoids in samples collected within 15 min of capture (indicative of pre-disturbance concentrations) in different phases of the annual reproductive cycle, with significantly higher levels in both males and females in the breeding season compared to the non-breeding season. These data, and the decline in tail fat stores that occur towards the end of the mating period (around October), suggest that platypus experience high-energy demands during this phase of reproduction. Plasma glucocorticoid concentrations in females sampled during the lactation period (October-February) were relatively low, and similar to those in females sampled in the non-breeding, non-lactation period (March-June). The latter requires further investigation as results may have been influenced by sampling limitations.

Serological responses against the pathogenic dimorphic fungus Mucor amphibiorum in populations of platypus (Ornithorhynchus anatinus) with and without ulcerative mycotic dermatitis.

Mucor amphibiorum, a dimorphic fungus, causes ulcerative dermatitis and systemic infections in the platypus Ornithorhynchus anatinus in some river systems in Tasmania but apparently not in other regions of Australia. As yet there are no suitable tests for population surveys, nor for detection of internal lesions in live animals. Consequently, immunoglobulins were purified from the serum of platypuses and anti-immunoglobulin antisera were prepared in rabbits in order to develop an enzyme-linked immunosorbent assay (ELISA) for anti-M. amphibiorum antibodies. Antigens from plate-grown cultures resulted in greater signal-to-noise ratios in indirect ELISA than those from broth-grown cultures. Platypuses with clinical ulcerative dermatitis had elevated anti-Mucor antibody levels compared to apparently unaffected individuals. Seroconversion was observed in one animal coincident with the development of cutaneous ulcers. The results suggested that platypuses in affected rivers were exposed to M. amphibiorum at a higher frequency than the occurrence of clinical disease. Some platypuses from New South Wales had elevated antibody levels but these increased significantly with age suggesting exposure to cross-reactive antigens, although exposure to M. amphibiorum cannot be excluded. Further studies are warranted to determine factors that result in progression from infection to disease, the occurrence of the fungus in areas where disease has not been observed and the specificity of antigen used in ELISA.

Phylogenetic relationships of Trypanosoma chelodina and Trypanosoma binneyi from Australian tortoises and platypuses inferred from small subunit rRNA analyses.

Trypanosome infections are often difficult to detect by conventional microscopy and their pleomorphy often confounds differential diagnosis. Molecular techniques are now being used to diagnose infections and to determine phylogenetic relationships between species. Complete small subunit rRNA gene sequences were determined for isolates of Trypanosoma chelodina from the Brisbane River tortoise (Emydura signata), the saw-shelled tortoise (Elseya latisternum), and the eastern snake-necked tortoise (Chelodina longicollis) from southeast Queensland, Australia. Partial sequence data were also obtained for T. binneyi from a platypus (Ornithorhynchus anatinus) from Tasmania. Phylogenetic relationships between T. chelodina, T. binneyi and other species were examined by maximum parsimony and likelihood methods. The Australian tortoise and platypus trypanosomes did not exhibit any close phylogenetic relationships with those of mammals, reptiles or amphibians, but were closely related to each other, and to fish trypanosomes. This contra-indicates their co-evolution with their vertebrate hosts but does not exclude co-evolution with different groups of invertebrate vectors, notably insects and leeches.

Haematological, serum biochemical and serological features of platypuses with and without mycotic granulomatous dermatitis.

OBJECTIVE: To determine whether there are haematological, serum biochemical and serological differences between platypuses (Ornithorhynchus anatinus) with and without granulomatous dermatitis due to Mucor amphibiorum infection. An additional objective was to establish reference haematological and serum biochemical ranges for the species in Tasmania. DESIGN: A clinicopathological and serological study. ANIMALS: A total of 37 free-living adult platypuses captured from streams and dams in Northern Tasmania were used in the clinicopathological study. Twenty-seven were clinically normal and 10 had mycotic granulomatous dermatitis. A total of 22 platypuses (20 adult and 2 juvenile) were used for the serosurvey. Eighteen were captured from streams in Northern Tasmania, and four were submitted for necropsy. RESULTS: Platypuses with mycotic ulcerative dermatitis had significantly smaller packed red cell volumes, haemoglobin concentrations, lymphocyte counts, serum cholesterol and calcium concentrations, and higher serum globulin and potassium concentrations than clinically normal animals. The lymphopenia and hyperkalaemia were thought to be clinically significant. Numbers of Trypanosoma binneyi in blood smears were similar between the two groups. Diseased platypuses had higher concentrations of serum antibody against Mucor amphibiorum as determined by ELISA compared to clinically normal platypuses. CONCLUSION: Platypuses affected by mycotic granulomatous dermatitis showed haematological and serum biochemical changes when compared to clinically normal animals from the same Tasmanian sites. A serological survey may be a useful method for detecting the prevalence of exposure to Mucor amphibiorum and humoral immunity in platypus populations both in Tasmania and the mainland of Australia.

A sedimentation equilibrium study of platypus insulin: the HB10D mutant does not associate beyond dimer.

An extensive study of the self-association patterns of zinc-free synthetic native and mutant (HB10D) platypus insulin in solution (pH = 7.0; I = 0.1 M; 25 degrees C) has been undertaken using the method of sedimentation equilibrium. The data was fitted to a mathematical equation describing the indefinite duoisodesmic (IDI) model of self-association [A.E. Mark, P.D. Jeffrey, Biol. Chem. Hoppe-Slayer, 371 (1990) 1165]. From this the relevant association constants, KA and KB, describing the polymerising system were calculated. This information allows the calculation of the complex distribution of odd and even numbered polymeric species within the insulin system in solution. In the studies on the self-association of the synthetic native and mutant platypus insulin, each was compared with bovine insulin as well as with each other. It is concluded that there is some reduction in the extent of the self-association of native platypus insulin compared to bovine insulin. A reduction, in specifically the dimer-dimer interaction, is indicated by the higher KA and lower KB values. HB10D platypus insulin shows a dramatic reduction in self-association compared to native platypus and to bovine insulin. Analysis of the self-association pattern yielding a KB value of effectively zero suggests that the substitution of an aspartic acid residue for a histidine at B10 virtually abolishes its dimer-dimer interaction. Platypus insulin has essentially the same biological activity as that of porcine (submitted for publication) but a somewhat lower self-association, while the introduction of one amino acid in a critical region increases the activity twofold while abolishing self-association beyond dimer.

Hematological changes in the platypus (Ornithorhynchus anatinus) following capture.

Blood samples were collected from the bill sinus of nine free-living platypuses (Ornithorhynehus anatinus) within 12 min of capture of each and again after 1 to 12 hr, New South Wales, Australia, 1981 to 1988. In seven animals which were not anesthetized, there was a significant (P < 0.01) fall in lymphocyte count between the two samples. The reduction ranged from 10 to 58% of the initial lymphocyte count and caused a significant reduction in the total white cell count (P < 0.05). Both the neutrophil and the lymphocyte counts increased in two platypuses which were anesthetized with ether prior to collection of the second blood sample. We propose that the peripheral blood lymphocyte count is a simple means of monitoring the stress response of non-anesthetized, newly-captured platypuses and may be a useful adjunct to behavioral observation.

Comparative erythrocyte metabolism in marsupials and monotremes.

Concentrations of ATP and DPG, activities of 10 enzymes and the glycolytic rates were measured in the erythrocytes of 11 species of marsupials and two species of monotremes. Mean DPG concentrations were greater in the erythrocytes of marsupials than those of eutherian mammals. The opposite is true of ATP. Significant findings from the results of enzyme activities were: high activity of hexokinase (7.39 +/- 0.82 EU/g Hb) in the short-beaked echidna, pyruvate kinase (37.49 +/- 1.0 EU/g) Hb in bridled nailtail wallaby and glucose-6-phosphate dehydrogenase (G6PD; 41.66 +/- 1.24 EU/g Hb) in black-striped wallaby. About 6- to 7-fold difference in the activity of G6PD levels between the two species of wombats was confirmed. Glucose phosphate isomerase activity was also shown to be twice as high in the red cells of the common wombat compared with those of the southern hairy nosed wombat. There were wide variations in the glycolytic rate among the species examined.

Glucocorticoids in the blood plasma of the platypus Ornithorynchus anatinus.

Blood samples were obtained from two male and two female platypuses at various times after capture and anaesthesia for other experimental purposes. In samples obtained during ketamine-xylazine or pregnanediol anaesthesia 15-24 h after capture, the concentration of total glucocorticoids, measured as 'cortisol equivalent' in a radioligand assay, was 207-620 nmol/l. In samples taken 14-35 h after injection of dexamethasone (0.2 mg/kg) total glucocorticoid concentration was 79-88 nmol/l. Individual glucocorticoids were isolated on columns of Sephadex LH-20 and measured separately against appropriate standards. In all except two haemolysed samples obtained from a male that died 25 h after capture, the major glucocorticoid behaved as cortisol, contributing 77-94% of the total. The remainder was made up of varying proportions of substances behaving as corticosterone, 11-deoxycortisol and cortisone. In the haemolysed samples from the moribund animal the major reactive substance, contributing 52-54% of the total, behaved as cortisone. The total adrenal gland weight of this animal was 747 mg, compared with 200-286 mg in two others, suggesting preceding exposure to stress. Equilibrium dialysis and polyacrylamide gel electrophoresis (PAGE) revealed no evidence for a transcortin-like glucocorticoid- and progesterone-binding protein in platypus plasma. However, as in the echidna, there was a heat-labile, high-capacity binding system migrating with albumin on PAGE. Glucose was undetectable in the plasma of the moribund animal and only 1.7-2.8 mmol/l in the initial plasma samples from the others. In two animals, injection of glucose i.p. and dexamethasone i.m. was followed by an increase in the plasma concentration of glucose to the range 3.8-9.9 mmol/l and commencement of normal swimming and feeding activity for the next 36-48 h.

Active cation transport and Na+K+Mg ATPase of the monotreme erythrocytes.

The erythrocytes of the echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus), which are practically devoid of intracellular ATP content (1), were examined for active Rb86 influx and for the presence of Na+K+Mg ATPase. We found that intact erythrocytes of both species possess the ability to actively transport cations. Ouabain sensitive Rb86 influx in the echidna was approximately 0.17 mumoles/ml cells x hr, whereas the platypus exhibited a higher value of 0.43 mumoles/ml cells x hr. Surprisingly, ouabain sensitive Na+K+Mg ATPase activity of isolated membranes was high amounting to some 15 to 25 fold higher than the human erythrocyte counterpart determined under identical conditions. These findings suggest that a trace amount of ATP is sufficient to maintain active cation transport across the monotreme cell membranes.

Erythrocyte phosphates and hemoglobin function in monotremes and some marsupials.

Hematologic values, red blood cell (RBC) organic phosphate composition, hemoglobin function, and hemoglobin composition have been determined on blood from the monotremes, the duckbill platypus and the echidna, and three species of marsupials, the Tasmanian devil, the wallaby, and the brush-tail possum. Blood from the platypus had a RBC count of 8.63 X 10(6)/mm3, a mean corpuscular volume of 49.1 millemicron3, and a white blood cell count of 26.0 X 10(3)/mm3. The RBCs from the monotremes and the three marsupials exhibited hemoglobin polymorphism, each with three hemoglobin components. Addition of ATP, 2,3-bisphosphoglycerate (2,3-P2-glycerate), or inositol pentakisphosphate (inositol-P5) to phosphate-free hemoglobin from each species decreased hemoglobin oxygen affinity; the order of effect of these compounds was ATP less than 2,3-P2-glycerate less than inositol-P5. The RBCs of all species had concentrations of 2,3-P2-glycerate ranging from 6.02 mumol/ml RBCs in the wallaby to 10.39 mumol/ml RBCs in the possum. The RBCs from the three species of marsupials had concentrations of ATP ranging from 0.24 mumol/ml RBCs in the possum to 0.80 mumol/ml RBCs in the Tasmanian devil. The level of ATP in RBCs of the platypus and echidna were 0.06 and 0.03 mumol/ml RBCs, respectively.

Thyroid hormone levels in an egg-laying mammal, the platypus Ornithorhynchus anatinus.

Serum thyroxine (T4) and triiodothyronine (T3) levels were measured in wild-caught platypuses in southeastern Australia in summer (February) and late winter (September). There was little seasonal difference in thyroid hormone levels. Males had lower levels of both T4 and T3 than did females, but this was only statistically significant for T3. Juveniles had higher levels than adults. The levels of thyroxine ranged from 37 to 136 nmol . liter-1, and triiodothyronine from 0.3 to 1.7 nmol . liter-1. These thyroxine levels are much higher than found in marsupials or the other egg-laying mammal, the echidma, and it is suggested that they may be associated with the platypus' relatively high metabolism and growth rate.

Studies on monotreme proteins. VI. Amino acid sequence of the beta-chain of haemoglobin from the platypus, Ornithorhynchus anatinus.

The amino acid sequence of the 146 residues of the beta-chain of the major haemoglobin from the platypus has been determined. The soluble peptides derived from the chain by tryptic digestion were isolated by paper ionophoresis and chromatography. The amino acid sequences were determined by the dansyl-Edman procedure or by further digestion with other enzymes. The tryptic peptides were aligned by homology with other beta-globins. There were 14 changes in sequence compared with echidna beta-chain. The number of changes in sequence compared with human beta-chain is 34 which is less than the 39 changes between human and platypus alpha-chains. Generally there are more changes between beta-chains; there are only three other examples reported where there are more changes between alpha-chains than beta-chains, these are of echidna, rabbit and dog globins. By comparison with the 'contact sites' in horse haemoglobin there is one change in beta-haem contacts, three changes in beta1-alpha1 contacts and no changes in beta2-alpha1 contacts. The date of divergence of the monotremes from the other mammals was estimated at 132 +/- 33 million years, based on the number of amino acid differences between species and allowing for multiple mutations during the evolutionary period. This estimate differs widely from the estimate given by similar treatment of the alpha-chain sequences and the significance of this discrepancy to the validity of the method is discussed.