The use of echocardiography as a health assessment tool in green sea turtles (Chelonia mydas).

There are limited techniques available to assess the health of sea turtles as physical examination has little correlation to clinical findings, and blood reference intervals are broad and provide limited prognostic significance. Advances in the portability of ultrasound machines allow echocardiography to be increasingly used in the health assessments of wild animals. This study performed blood analysis and echocardiograms on 11 green sea turtles upon admission to a rehabilitation clinic and six animals before release. Significant differences were seen between groups, with admission animals having significantly smaller diameters of the cavum arteriosum at systole and diastole, smaller E-waves and an increased fractional shortening. Pre-release animals displayed significant increases in the maximum blood velocities of both the pulmonary artery and the left aorta. Significant negative correlations were seen between fractional shortening and uric acid and between the velocity time integral of the pulmonary artery and urea. The pulmonary artery velocity time integral was also significantly correlated to the E wave. Furthermore, there was asynchrony between the cavum arteriosum and the cavum pulmonale and the detection of a parasitic granuloma in the ventricular outflow tract of one animal. Overall, the results suggest that cardiac function in stranded green sea turtles is significantly impaired and that echocardiography has applications in the health assessments of green sea turtles.

Brucellosis associated with stillbirth in a bottlenose dolphin in Australia.

A captive adult female bottlenose dolphin presented with stillbirth. The placenta appeared oedematous. No other gross lesions were evident in the placenta or the stillborn calf. Histopathology revealed mild multifocal placentitis and foetal encephalitis. Brucella sp. was isolated from lung, liver, spleen and kidney. Sequence and phylogenetic analysis demonstrated this organism to be most similar to Brucella ceti sequence type (ST) 27. Brucella sp. DNA was detected in formalin-fixed paraffin-embedded placenta and brain by real-time PCR using primers targeting the IS711 gene. Immunohistochemical staining revealed Brucella sp. antigen in placental inflammation. This is the first report of isolation of Brucella sp. from a marine mammal in the Southern Hemisphere and the first report of marine Brucella-associated disease in Australia.

Evaluation of respiratory vapour and blubber samples for use in endocrine assessments of bottlenose dolphins (Tursiops spp.).

Blubber and respiratory vapour ('blow') are now commonly used for endocrine studies on cetaceans, primarily because they can be obtained using minimally invasive methods. For many species, these samples have yet to be validated for these purposes. The objective of this study was to examine the performance of blow and blubber hormone monitoring, relative to serum hormone monitoring, for evaluating the reproductive and adrenal condition of captive bottlenose dolphins (Tursiops spp.). Eighteen bottlenose dolphins were sampled five times for serum and blow and twice for blubber throughout a one-year period. Concentrations of progesterone, testosterone, oestradiol and cortisol were measured in each sample type. Hormone levels were examined in relation to dolphin age, sex, reproductive status, season, time of sample collection (morning/afternoon) and collection type (in- or out-of-water sampling). Patterns in hormone levels were similar for serum and blubber. For instance, in both sample types, progesterone levels were significantly higher in pregnant (serum: 34.10 ±â€¯8.64 ng/mL; blubber: 13.01 ±â€¯0.72 ng/g) than in non-pregnant females (serum: 0.32 ±â€¯0.09 ng/mL; blubber: 1.17 ±â€¯0.10 ng/g). This pattern was not detected in blow, primarily because seawater contamination, nylon sampling materials and variable sample volumes influenced measured concentrations. In addition, the respiratory water content of a blow sample is known to affect measured hormone levels. Two methods were trialled to control for variability in sample volumes and dilution: (1) normalising blow hormone concentrations relative to urea nitrogen levels (a potential endogenous standard), and (2) measuring the relative proportions (i.e. ratios) of blow hormones. These correction measures had little influence on blow hormone results. Further refinement of blow hormone monitoring methods is required before they can be used for reproductive or adrenal assessments of bottlenose dolphins. Blubber, on the other hand, should be a suitable proxy for serum when attempting to classify pregnancy status and male maturity in these species.

Epizootics of Streptococcus agalactiae infection in captive rays from Queensland, Australia.

The aim of this study was to describe two epizootics of high mortalities from infection with Streptococcus agalactiae, occurring in captive rays held in a marine display aquarium in south-east Queensland, Australia, in 2009 and 2010. Five different species of rays were affected, including mangrove whiprays (Himantura granulata), estuary rays (Dasyatis fluviorum), eastern shovelnose rays (Aptychotrema rostrata), white-spotted eagle rays (Aetobatus narinari) and blue-spotted mask rays (Neotrygon kuhlii). This report describes the history of both epizootics including collection, quarantine and husbandry of rays, the disease epizootics, clinico-pathological features of the disease, antimicrobial therapy, autogenous vaccine production, and laboratory studies including clinical and histopathology, bacteriology, PCR, molecular serotyping and sequencing of the bacterium S. agalactiae.

Piroxicam and doxycycline treatment for an oral squamous cell carcinoma in an inshore bottlenose dolphin (Tursiops aduncus).

BACKGROUND: The virus family Papillomaviridae has been documented in a wide range of animal species and can cause benign and malignant proliferative lesions. The presence of concurrent lingual papillomas and squamous cell carcinomas (SCC) in cetaceans has also been documented in both wild and captive populations, suggesting malignant transformation of benign papilloma to SCC may occur in this species. CASE REPORT: In 2008, a 38-year-old captive male inshore bottlenose dolphin (Tursiops aduncus) was diagnosed with papillomatous lesions on the intermandibular frenulum rostral to the tongue and an infiltrative SCC of the soft palate following biopsy and histological analysis. A treatment regimen of piroxicam and doxycycline was initiated with misoprostol as a gastroprotectant. The treatment resulted in a marked reduction in tumour size and reversible hepatotoxicosis. Subsequent biopsies revealed the presence of SCC in the oral cavity; however, the disease remains stable at the time of writing. CONCLUSION: To the best of our knowledge, this case is the first report of piroxicam and doxycycline used to treat SCC in a bottlenose dolphin. The treatment was successful in reducing the clinical presentation of the disease.

Effect of cryopreservation on the sperm DNA fragmentation dynamics of the bottlenose dolphin (Tursiops truncatus).

Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species-specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post-thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post-thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax(®)). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES-TRIS-fructose buffer (TTF), an egg-yolk-free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p < 0.05); this difference was not apparent in any other seminal characteristic. While there was no difference observed in SDF between fresh and frozen-thawed sperm using the different cryopreservation protocols immediately after thawing (T0), frozen-thawed spermatozoa incubated at 37°C showed an increase in the rate of SDF after 24 h. Sperm frozen in the LP1(℗) buffer had higher levels (p < 0.05) of DNA fragmentation after 24- and 48-h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen-thawed samples.

Validation of a field based chromatin dispersion assay to assess sperm DNA fragmentation in the bottlenose dolphin (Tursiops truncatus).

Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management of captive dolphin populations, including evaluation of sperm DNA quality. This study validated a customized sperm chromatin dispersion test (SCDt) for the bottlenose dolphin (Tursiops truncatus) as a means of assessing sperm DNA damage both in the field and in the laboratory. After performing the SCDt, two different sperm morphotypes were identified: (i) sperm with fragmented DNA showed large haloes of dispersed DNA fragments emerging from a compact sperm nucleoid core and (ii) sperm containing non-fragmented DNA displayed small compact haloes surrounded by a dense core of non-dispersed DNA and protein complex. Estimates of sperm DNA fragmentation by means of SCDt were directly comparable to results obtained following a two-tailed comet assay and showed a significant degree of correlation (r = 0.961; p < 0.001). This investigation also revealed that the SCDt, with minor modifications to the standard protocol, can be successfully conducted in the field using a LED florescence microscopy obtaining a high correlation (r = 0.993; p = 0.01) between the data obtained in the laboratory and in the field.

Intestinal volvulus in cetaceans.

Intestinal volvulus was recognized as the cause of death in 18 cetaceans, including 8 species of toothed whales (suborder Odontoceti). Cases originated from 11 institutions from around the world and included both captive (n = 9) and free-ranging (n = 9) animals. When the clinical history was available (n = 9), animals consistently demonstrated acute dullness 1 to 5 days prior to death. In 3 of these animals (33%), there was a history of chronic gastrointestinal illness. The pathological findings were similar to those described in other animal species and humans, and consisted of intestinal volvulus and a well-demarcated segment of distended, congested, and edematous intestine with gas and bloody fluid contents. Associated lesions included congested and edematous mesentery and mesenteric lymph nodes, and often serofibrinous or hemorrhagic abdominal effusion. The volvulus involved the cranial part of the intestines in 85% (11 of 13). Potential predisposing causes were recognized in most cases (13 of 18, 72%) but were variable. Further studies investigating predisposing factors are necessary to help prevent occurrence and enhance early clinical diagnosis and management of the condition.

Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia.

Ninety-three giant Queensland grouper, Epinephelus lanceolatus (Bloch), were found dead in Queensland, Australia, from 2007 to 2011. Most dead fish occurred in northern Queensland, with a peak of mortalities in Cairns in June 2008. In 2009, sick wild fish including giant sea catfish, Arius thalassinus (Rüppell), and javelin grunter, Pomadasys kaakan (Cuvier), also occurred in Cairns. In 2009 and 2010, two disease epizootics involving wild stingrays occurred at Sea World marine aquarium. Necropsy, histopathology, bacteriology and PCR determined that the cause of deaths of 12 giant Queensland grouper, three wild fish, six estuary rays, Dasyatis fluviorum (Ogilby), one mangrove whipray, Himantura granulata (Macleay), and one eastern shovelnose ray, Aptychotrema rostrata (Shaw), was Streptococcus agalactiae septicaemia. Biochemical testing of 34 S. agalactiae isolates from giant Queensland grouper, wild fish and stingrays showed all had identical biochemical profiles. The 16S rRNA gene sequences of isolates confirmed all isolates were S. agalactiae; genotyping of selected S. agalactiae isolates showed the isolates from giant Queensland grouper were serotype Ib, whereas isolates from wild fish and stingrays closely resembled serotype II. This is the first report of S. agalactiae from wild giant Queensland grouper and other wild tropical fish and stingray species in Queensland, Australia.

Fatal cetacean morbillivirus infection in an Australian offshore bottlenose dolphin (Tursiops truncatus).

A juvenile offshore bottlenose dolphin (Tursiops truncatus) was found stranded with neurological signs and unable to swim or float unassisted. It subsequently died, succumbing to a combination of severe pneumonia and encephalitis. Morbillivirus serum neutralisation test serology was positive (titre 1:16) for cetacean morbillivirus and negative for both phocine distemper virus and canine distemper virus. There was concurrent thymic and lymph node lymphoid depletion and necrosis, together with intranuclear and intracytoplasmic acidophilic viral inclusion bodies and multinucleate syncytia within multiple organs. Paramyxovirus capsids were identified in lung sections via electron microscopy and morbillivirus antigen was demonstrated within sections of lung, thymus and brain by immunohistochemistry. Reverse transcription-polymerase chain reaction for morbillivirus nucleoprotein (N) and phosphoprotein (P) genes were positive and phylogenetic gene product sequence analysis revealed 98% and 94% sequence identity to dolphin morbillivirus, respectively. To the authors' knowledge, this is the first report of a cetacean mortality due to morbillivirus infection occurring in the southern hemisphere. Morbillivirus infection should be included in the differential diagnosis of stranded live or dead cetaceans in Australian waters, particularly if animals display neurological signs.

Studies of male reproduction in captive African wild dogs (Lycaon pictus).

Implementation of assisted breeding in the captive African wild dog is restricted by a current lack of knowledge on their reproductive physiology and the apparent difficulty of effectively manipulating the complex social dynamic of the pack in order to conduct reproductive procedures. In this study, we describe protocols for the safe and repeated capture and restraint of the African wild dog (n=7) as well as techniques for assessment of male reproductive function, semen collection and preservation. In a serendipitous finding, captive African wild dogs appeared to display significant seasonal change in male reproduction. Testicular volume and tone, spermatorrhea and the ability to collect semen by electroejaculation all increased significantly during late summer and then subsequently declined by early spring. While there were no detectable seasonal changes in testosterone concentration in the population as whole, the alpha-dominant male in both years of the study, had a highly elevated testosterone concentration compared to subordinate males. Semen collection by electroejaculation during the late summer was most effective in peri-pubertal males (15 months) when all seven electroejaculates were of adequate quality for cryopreservation. In the second breeding season (27 months), there were numerous changes in the pack hierarchy and electroejaculation was not as successful (3/7). The characteristics of electroejaculated semen collected in the breeding season are described for seven animals including the first descriptions and incidence of sperm abnormalities in the species. Semen (n=7) was frozen using a Tris-citrate fructose buffer and final egg yolk and glycerol concentration of 20% and 4%, respectively. Sperm were loaded into 0.25 mL straws, frozen in liquid nitrogen vapor and then thawed at 37 degrees C. Initial post-thaw survival of spermatozoa was encouraging (% motile: 31.8+/-5.8%; rate: 2.8+/-0.3; % intact plasma membranes: 33.4+/-5.3% and the % of damaged acrosomes: 4.4+/-1.5%) but following 2 h incubation at 37 degrees C, post-thaw survival declined markedly.

Actin localisation and the effect of cytochalasin D on the osmotic tolerance of cauda epididymidal kangaroo spermatozoa.

This study examined the hypothesis that filamentous actin associated with the complex cytoskeleton of the kangaroo sperm head and tail may be contributing to lack of plasma membrane plasticity and a consequent loss of membrane integrity during cryopreservation. In the first study, the distribution of G and F actin within Eastern Grey Kangaroo (EGK, Macropus giganteus) cauda epididymidal spermatozoa was successfully detected using DNAse-FITC and a monoclonal F-actin antibody (ab205, Abcam), respectively. G-actin staining was most intense in the acrosome but was also observed with less intensity over the nucleus and mid-piece. F-actin was located in the sperm nucleus but was not discernable in the acrosome or sperm tail. To investigate whether cytochalasin D (a known F-actin depolymerising agent) was capable of improving the osmotic tolerance of EGK cauda epididymal spermatozoa, sperm were incubated in hypo-osmotic media (61 and 104 mOsm) containing a range of cytochalasin D concentrations (0-200 microM). Cytochalasin D had no beneficial effect on plasma membrane integrity of sperm incubated in hypo-osmotic media. However, when EGK cauda epididymidal sperm were incubated in isosmotic media, there was a progressive loss of sperm motility with increasing cytochalasin D concentration. The results of this study indicated that the F-actin distribution in cauda epididymidal spermatozoa of the EGK was surprisingly different from that of the Tammar Wallaby (M. eugenii) and that cytochalasin-D does not appear to improve the tolerance of EGK cauda epididymidal sperm to osmotically induced injury.

An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa.

The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to investigate reasons for their respective post-thaw survival by examining the sperm's response to a range of osmotic media and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting a degree of structural inflexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5 microM), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of cell volume changes that typically occur during cryopreservation. In experiment 1, koala (n = 4) and wombat (n = 4) spermatozoa packaged in 0.25 mL straws were cryopreserved using two freezing rates (fast-3 cm above liquid N2 interface; slow-6 degrees C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris-citrate glucose buffer with 15% (v/v) egg yolk. Wombat spermatozoa showed better (P < 0.01) post-thaw survival (% motile, % intact plasma membranes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala spermatozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410 mOsmol/kg) than wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneficial effect on sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw survival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely to be associated with F-actin induced plasma membrane inflexibility.

Effects of p-chloromercuribenzene sulfonate on water transport across the marsupial erythrocyte membrane.

The effects of exposure of red blood cells (RBC) of three species of marsupial to a mercury-containing sulfhydryl-modifying reagent, p-chloromercuribenzene sulfonate (PCMBS), on the water diffusional permeability ( P (d)) of their membranes were monitored by using an Mn(2+)-doping (1)H nuclear magnetic resonance (NMR) technique at 400 MHz. For koala ( Phascolarctos cinereus), RBC the maximal inhibition was reached at 37 degrees C in 60 min with 1 mmol.l(-1) PCMBS or in 15-30 min with 2 mmol. l(-1) PCMBS. In contrast, in the case of red kangaroo ( Macropus rufus) or swamp wallaby ( Wallabia bicolor) RBC, maximal inhibition required an incubation of 90 min at 37 degrees C with 2 mmol.l(-1) PCMBS. For the RBC of all three species the value of maximal inhibition was very high, being 50-70% when measured at 25 degrees C, 60-80% at 30 degrees C and 60-70% at 37 degrees C. The lowest values of P (d) appeared to be around 2 x 10(-3)-3 x 10(-3) cm.s(-1) in the temperature range of 25-37 degrees C. The mean value of the activation energy of water diffusion ( E (a,d)) was approximately 20-25 kJ.mol(-1) for control and approximately 40 kJ.mol(-1) for PCMBS-inhibited RBCs. These results show that marsupial RBC have a basal permeability to water similar to that previously reported for human RBC, but a higher value of the PCMBS-inhibitable water permeability. This indicates that the higher water permeability of marsupial RBC compared with human RBC is associated with a higher fraction of protein-mediated water permeability.

Respiratory diseases, diagnostics, and treatment of marsupials.

Primary respiratory disease is uncommon in marsupials, but generalized disease often involves the respiratory tract in these animals. Other disease entities may spread to involve the respiratory system secondarily. Treatment is challenging because most animals are severely compromised on presentation. Bacteria, viruses, parasites, fungi and yeasts, trauma, and neoplasia all can affect the respiratory systems of marsupials. Some of these conditions are only incidental findings, whereas others are pathogenic. Many therapies and diagnostic procedures are extrapolated from those in companion animal medicine and surgery. Despite the problems associated with the diagnosis and treatment of respiratory disease in marsupials, new therapeutic agents and diagnostic techniques are making these tasks easier and ultimately more successful.

Experimental reproduction of viral chorioretinitis in kangaroos.

OBJECTIVE: To investigate whether preparations containing Wallal and/or Warrego viruses could cause disease when inoculated subcutaneously into captive kangaroos. DESIGN AND PROCEDURE: Four groups of two kangaroos, seronegative to both Wallal and Warrego virus, were each inoculated with wild Wallal virus, cultured Wallal virus, wild Warrego virus, or wild Warrego virus followed by wild Wallal virus after 3 weeks. A single uninoculated animal served as a control. Animals were monitored weekly under anaesthesia, examined ophthalmoscopically (including fundic photography), and samples collected for haematological and serum biochemical analysis, virus isolation, PCR and serological examination for antibodies against Wallal and Warrego viruses. Animals inoculated with cultured Wallal virus were killed at week 10, and remaining kangaroos were reinoculated with cultured Wallal virus at week 12. RESULTS: Virus was isolated from the blood of two kangaroos 2 weeks after inoculation with Wallal virus preparations, and from a third kangaroo 2 weeks after reinoculation. By 3 weeks after inoculation, all kangaroos given Wallal virus preparations had seroconverted to Wallal virus and one had seroconverted to Warrego virus. Fundic changes were detected in the three viraemic kangaroos 4 or more weeks after inoculation, and lesions were present in the eye and brain typical of those seen in field cases of chorioretinitis. No other kangaroos had lesions. Wallal virus was identified by PCR and immunohistochemical analysis in the retina of one affected animal and orbivirus-like particles were seen by electron microscopy in the remains of retinal cells. CONCLUSION: The condition of chorioretinitis was reproduced in three of eight kangaroos by inoculation with preparations containing Wallal virus.

Fungal pneumonia in a captive black rhinoceros.

A captive black rhinoceros (Diceros bicornis) with a hoof abscess was treated with long-term antibiotic therapy. After 9 months of treatment, there was rapid deterioration, marked weight loss and reluctance to stand. Profuse, bilateral epistaxis developed accompanied by collapse and the animal was euthanased. Necropsy revealed pulmonary aspergillosis with concurrent Pseudomonas aeruginosa infection. Though a well-recognized disease of black rhinoceros, fungal pneumonia has not been reported in this species in Australia. The cost and efficacy of treatment have been questioned, however, prophylactic antifungal drug administration will be considered in any further cases of chronic, debilitating illness in black rhinoceros at Western Plains Zoo.