Investigation into the characteristics, triggers and mechanism of apnoea and bradycardia in the anaesthetized platypus (Ornithorhynchus anatinus).

Health and conservation research on platypuses (Ornithorhynchus anatinus) may require anaesthesia to reduce stress and the risk of injury to both the animal and the researcher, as well as to facilitate examination and sample collection. Platypus anaesthesia can be difficult to manage, with reports of periods of apnoea and bradycardia described. This study investigated the conditions around sudden-onset apnoea and bradycardia in 163 field-anaesthetized platypuses as part of a health study. Anaesthesia was induced and maintained using isoflurane delivered in oxygen by face mask. Sudden-onset apnoea and bradycardia was observed in 19% of platypuses, occurring either at induction of anaesthesia, during recovery, or both. At induction, occurrence was more often recorded for adults (P = 0.19) and was correlated with low body temperature (P < 0.001), season (P = 0.06; greater incidence in summer) and longer pre-anaesthetic holding time (P = 0.16). At recovery, sudden-onset apnoea and bradycardia occurred only in platypuses that had been placed in dorsal recumbency as part of their examination, and correlated with poor body condition (P = 0.002), time in dorsal recumbency (P = 0.005), adults (P = 0.06), number of fieldworkers (P = 0.06) and females (P = 0.11). The sudden-onset apnoea and bradycardia we observed is likely to result from the irritant nature of isoflurane (stimulating the trigeminal nerve via nasal chemoreceptors). We propose that this mechanism is analogous to that of submersion of the face/nasal cavity in cold water during a natural dive response, but that the term 'nasopharyngeal response' would more appropriately describe the changes observed under isoflurane anaesthesia. Although we did not record any long-term adverse effects on platypuses that had undergone this response, the nasopharyngeal response could complicate the diagnosis of anaesthetic dose-dependent apnoea and bradycardia. Therefore, we suggest that these responses during anaesthesia of platypuses might be avoided by minimizing the stress around capture and handling, as well as reducing the time in dorsal recumbency.

Preliminary investigation into the prevalence of mucormycosis in the platypus (Ornithorhynchus anatinus) in three catchments in north-west Tasmania.

OBJECTIVE: To investigate the distribution and prevalence of mucormycosis in platypus (Ornithorhynchus anatinus) from the Inglis, Emu and Black-Detention catchment areas in north-west Tasmania. PROCEDURE: A field study was performed and resulted in the examination of 44 wild platypuses; in addition, one dead platypus and two live platypuses were examined after they were independently submitted to a local veterinary clinic. RESULTS: No cases of mucormycosis were conclusively diagnosed. One platypus with signs consistent with those previously described in cases of mucormycosis was captured in the Emu River catchment. However, laboratory tests did not provide a definitive diagnosis for the lesion. Two platypuses from the Inglis catchment area had signs very similar to those previously described in cases of mucormycosis, but laboratory tests found Corynebacterium ulcerans to be the likely cause of the cutaneous ulcers on one of these platypuses and an unidentified fungal agent to be the cause of a cutaneous nodule in the other. CONCLUSIONS: These findings do not prove that mucormycosis is absent from the populations studied. However, they may indicate that the prevalence of disease is low. The possibility that Mucor amphibiorum is present in a subclinical form in platypuses, or infecting another reservoir, is not excluded. The findings also suggest that caution should be exercised when diagnosing mucormycosis based on clinical findings alone and raise the possibility that some cases may have been incorrectly diagnosed.

Cutaneous papillomatosis and carcinomatosis in the Western barred bandicoot (Perameles bougainville).

A progressive wart-like syndrome in both captive and wild populations of the Western barred bandicoot (WBB) is hindering conservation efforts to prevent the extinction of this endangered marsupial. In this study, 42 WBBs exhibiting the papillomatosis and carcinomatosis syndrome were examined. The disease was characterized by multicentric proliferative lesions involving cutaneous and mucosal surfaces, which were seen clinically to increase in size with time. Grossly and histologically the smaller skin lesions resembled papillomas, whereas the larger lesions were most commonly observed to be squamous cell carcinomas. Large amphophilic intranuclear inclusion bodies were observed in hyperplastic conjunctival lesions of 8 WBBs under light microscopy. Conjunctival lesions from 2 WBBs examined using transmission electron microscopy contained a crystalline array of spherical electron-dense particles of 45-nm diameter, within the nucleus of conjunctival epithelial cells, consistent with a papillomavirus or polyomavirus. Conjunctival samples from 3 bandicoots that contained intranuclear inclusion bodies also demonstrated a positive immunohistochemical reaction after indirect immunohistochemistry for papillomavirus structural antigens. Ultrastructural and/or immunohistochemical evidence of an etiologic agent was not identified in the nonconjunctival lesions examined. Here we describe the gross, histopathologic, ultrastructural, and immunohistochemical findings of a papillomatosis and carcinomatosis syndrome recently identified in the WBB.

Klossiella quimrensis (Apicomplexa: Klossiellidae) causes renal coccidiosis in western barred bandicoots Perameles bougainville (Marsupialia: Peramelidae) in Western Australia.

Previous studies have described a range of Klossiella species parasitic in marsupial hosts. Klossiella quimrensis is the etiologic agent of renal coccidiosis in the peramelid marsupial hosts Isoodon obesulus and Perameles gunnii in Eastern Australia, but there is no previous report of klossiellosis in Western Australian peramelids. This study describes klossiellosis diagnosed by histology of renal tissue sections collected during necropsy of 20 Perameles bougainville between 2000 and 2005. Sporonts, sporoblasts, and macrogametes were identified within parasitophorous vacuoles of epithelial cells located near the renal corticomedullary junction. The prevalence of renal coccidiosis in P. bougainville diagnosed by renal histology is estimated at 30%. Only a single unsporulated sporocyst was detected by examination of cystocentesis-collected urine, indicating that microscopic evaluation of urine samples is an insensitive diagnostic test for detection of K. quimrensis in P. bougainville. This infection in P. bougainville is indirectly associated with mild multifocal interstitial lymphohistiocytic nephritis and is likely to be only minimally pathogenic in otherwise healthy individuals. Our study also extends the host and geographic range of K. quimrensis to include P. bougainville and Western Australia.

Cryptococcosis in Gilbert's and long-nosed potoroo.

Two cases of fatal cryptococcosis are described, one of Cryptococcus neoformans infection in a Gilbert's potoroo (Potorous gilbertii) and one of Cryptococcus gattii infection in a long-nosed potoroo (Potorous tridactylus). The diagnoses were confirmed by culture and specific immunohistochemistry, respectively. The long-nosed potoroo tested positive using the latex cryptococcal antigen test (LCAT), whereas the Gilbert's potoroo had a negative LCAT result despite having advanced disease of some duration. In both cases, the clinical presentation was a progressive neurologic disease associated with a central nervous system infection. Pulmonary infection was also observed in the long-nosed potoroo. Specific treatment with antifungal agents was unsuccessful in the long-nosed potoroo.

Are bone scans necessary in men with low prostate specific antigen levels following localized therapy?

PURPOSE: The prostate specific antigen level at which to recommend a bone scan after treatment of early prostate cancer is controversial. We identified the incidence of bone metastases at varying prostate specific antigen levels in asymptomatic men following radical prostatectomy, radiation therapy and watchful waiting. MATERIALS AND METHODS: Data were obtained from the Early Prostate Cancer trial comparing placebo with bicalutamide in addition to standard care for localized prostate cancer. As part of the trial patients were required to have routine bone scans regardless of prostate specific antigen levels. The prostate specific antigen levels were divided into subgroups and the incidence of positive bone scans was calculated for each group. RESULTS: The incidence of positive bone scans in patients treated with watchful waiting and given bicalutamide or placebo was low (0.7% to 3.2%) at prostate specific antigen levels less than 20 ng/ml. At greater than this level the sample sizes were smaller but there was a significant increase in the incidence of positive bone scans. In the groups treated with radiation therapy or radical prostatectomy, regardless of the addition of bicalutamide, the incidence of positive bone scans was low (0.2% to 1.4%) at prostate specific antigen levels less than 5 ng/ml. The sample sizes were smaller at prostate specific antigen levels greater than 5 ng/ml so the results are harder to interpret. CONCLUSIONS: Bone scans can be confidently eliminated in the followup of patients with early prostate cancer after standard care of those with prostate specific antigen levels less than 5 ng/ml. This level can be increased to 20 ng/ml with caution in those patients treated with watchful waiting.

Watchful waiting for prostate cancer: a review article.

As earlier detection of prostate cancer increases because of prostate-specific antigen (PSA) testing, appropriate use for watchful waiting warrants re-evaluation. We have drawn together the significant watchful waiting literature and used it to evaluate the use of watchful waiting in the PSA era. We conducted literature searches for studies examining outcomes of watchful waiting and examined new literature emerging about the use of PSA for the follow-up of watchful waiting patients. Watchful waiting has the potential to play an increasingly important role in prostate cancer as less advanced disease is detected and methods are refined for identifying low-risk patients.

Calcium extrusion is critical for cardiac morphogenesis and rhythm in embryonic zebrafish hearts.

Calcium entry into myocytes drives contraction of the embryonic heart. To prepare for the next contraction, myocytes must extrude calcium from intracellular space via the Na+/Ca2+ exchanger (NCX1) or sequester it into the sarcoplasmic reticulum, via the sarcoplasmic reticulum Ca2+-ATPase2 (SERCA2). In mammals, defective calcium extrusion correlates with increased intracellular calcium levels and may be relevant to heart failure and sarcoplasmic dysfunction in adults. We report here that mutation of the cardiac-specific NCX1 (NCX1h) gene causes embryonic lethal cardiac arrhythmia in zebrafish tremblor (tre) embryos. The tre ventricle is nearly silent, whereas the atrium manifests a variety of arrhythmias including fibrillation. Calcium extrusion defects in tre mutants correlate with severe disruptions in sarcomere assembly, whereas mutations in the L-type calcium channel that abort calcium entry do not produce this phenotype. Knockdown of SERCA2 activity by morpholino-mediated translational inhibition or pharmacological inhibition causes embryonic lethality due to defects in cardiac contractility and morphology but, in contrast to tre mutation, does not produce arrhythmia. Analysis of intracellular calcium levels indicates that homozygous tre embryos develop calcium overload, which may contribute to the degeneration of cardiac function in this mutant. Thus, the inhibition of NCX1h versus SERCA2 activity differentially affects the pathophysiology of rhythm in the developing heart and suggests that relative levels of NCX1 and SERCA2 function are essential for normal development.

Cryptosporidium muris infection in bilbies (Macrotis lagotis).

Cryptosporidiosis is an enteric disease of animals and humans that can be fatal in immunocompromised individuals. There is no known effective treatment for cryptosporidiosis. Bilbies are threatened marsupials and are bred in captivity as part of a recovery program to re-introduce this species to the southwest of Western Australia. Cryptosporidium muris infection was detected in the faeces of bilbies at a captive breeding colony. Stress associated with a high density of bilbies in enclosures may have predisposed some of the bilbies to infection with C. muris. C. muris has been described in mice and was found in the faeces of one mouse trapped in the breeding enclosures. It is likely the bilbies acquired the infection from mice by faecal contamination of food and water. The infection cleared within 2 months from some bilbies, however others remained infected for 6 months and treatment was attempted with dimetridazole. Subsequently the parasite was no longer be detectable in the faeces.

The slow mo mutation reduces pacemaker current and heart rate in adult zebrafish.

Genetic studies in zebrafish have focused on embryonic mutations, but many physiological mechanisms continue to mature after embryogenesis. We report here that zebrafish homozygous for the mutation slow mo can be raised to adulthood. In the embryo, the slow mo gene is needed to regulate heart rate, and its mutation causes a reduction in pacemaker current (I(h)) and slowing of heart rate (bradycardia). The homozygous adult slow mo fish continues to manifest bradycardia, without other evident ill effects. Patch-clamp analysis of isolated adult cardiomyocytes reveals that I(h) has chamber-specific properties such that the atrial current density of I(h) is far greater than the ventricular current density of I(h). I(h) is markedly diminished in cardiomyocytes from both chambers of slow mo mutant fish. Thus I(h) continues to be a critical determinant of pacemaker rate even after adult neural and humoral influences have developed. It is clear that zebrafish may be used for genetic dissection of selected physiological mechanisms in the adult.

Speciation and intrasubspecific variation of Bornean orangutans, Pongo pygmaeus pygmaeus.

Mitochondrial DNA control region sequences of orangutans (Pongo pygmaeus) from six different populations on the island of Borneo were determined and analyzed for evidence of regional diversity and were compared separately with orangutans from the island of Sumatra. Within the Bornean population, four distinct subpopulations were identified. Furthermore, the results of this study revealed marked divergence, supportive evidence of speciation between Sumatran and Bornean orangutans. This study demonstrates that, as an entire population, Bornean orangutans have not experienced a serious genetic bottleneck, which has been suggested as the cause of low diversity in humans and east African chimpanzees. Based on these new data, it is estimated that Bornean and Sumatran orangutans diverged approximately 1.1 MYA and that the four distinct Bornean populations diverged 860,000 years ago. These findings have important implications for management, breeding, and reintroduction practices in orangutan conservation efforts.

Genetic steps to organ laterality in zebrafish.

All internal organs are asymmetric along the left-right axis. Here we report a genetic screen to discover mutations which perturb organ laterality. Our particular focus is upon whether, and how, organs are linked to each other as they achieve their laterally asymmetric positions. We generated mutations by ENU mutagenesis and examined F3 progeny using a cocktail of probes that reveal early primordia of heart, gut, liver and pancreas. From the 750 genomes examined, we isolated seven recessive mutations which affect the earliest left-right positioning of one or all of the organs. None of these mutations caused discernable defects elsewhere in the embryo at the stages examined. This is in contrast to those mutations we reported previously (Chen et al., 1997) which, along with left-right abnormalities, cause marked perturbation in gastrulation, body form or midline structures. We find that the mutations can be classified on the basis of whether they perturb relationships among organ laterality. In Class 1 mutations, none of the organs manifest any left-right asymmetry. The heart does not jog to the left and normally leftpredominant BMP4 in the early heart tube remains symmetric. The gut tends to remain midline. There frequently is a remarkable bilateral duplication of liver and pancreas. Embryos with Class 2 mutations have organotypic asymmetry but, in any given embryo, organ positions can be normal, reversed or randomized. Class 3 reveals a hitherto unsuspected gene that selectively affects laterality of heart. We find that visceral organ positions are predicted by the direction of the preceding cardiac jog. We interpret this as suggesting that normally there is linkage between cardiac and visceral organ laterality. Class 1 mutations, we suggest, effectively remove the global laterality signals, with the consequence that organ positions are effectively symmetrical. Embryos with Class 2 mutations do manifest linkage among organs, but it may be reversed, suggesting that the global signals may be present but incorrectly orientated in some of the embryos. That laterality decisions of organs may be independently perturbed, as in the Class 3 mutation, indicates that there are distinctive pathways for reception and organotypic interpretation of the global signals.

The genetic basis of cardiac function: dissection by zebrafish (Danio rerio) screens.

The vertebrate heart differs from chordate ancestors both structurally and functionally. Genetic units of form, termed 'modules', are identifiable by mutation, both in zebrafish and mouse, and correspond to features recently acquired in evolution, such as the ventricular chamber or endothelial lining of the vessels and heart. Zebrafish (Danio rerio) genetic screens have provided a reasonably inclusive set of such genes. Normal cardiac function may also be disrupted by single-gene mutations in zebrafish. Individual mutations may perturb contractility or rhythm generation. The zebrafish mutations which principally disturb cardiac contractility fall into two broad phenotypic categories, 'dilated' and 'hypertrophic'. Interestingly, these correspond to the two primary types of heart failure in humans. These disorders of early cardiac function provide candidate genes to be examined in complex human heart diseases, including arrhythmias and heart failure.

Microsatellite DNA variation in Bornean orangutans (Pongo pygmaeus).

Orangutans (Pongo pygmaeus) on the islands of Borneo and Sumatra are considered two separate subspecies. However, the genetic relationships between isolated populations on Borneo are not clear. This study determined the extent of variation within the Bornean subspecies of orangutan, using microsatellite DNA analysis. Blood samples were collected from 96 individuals of known origin from East, West and Central Kalimantan. Human microsatellite primer pairs located at human map position D2S141, D4S431, D 11S925, D16S420 and D17S791 were suitable for use in primates. D4S431 appeared monomorphic for all orangutans. In three cases (D2S141 East and West and D16S420 West), a highly significant excess of homozygous allele frequencies was detected, but with other primer pairs no significant difference in allele frequencies occurred. We conclude that the divergence between the different populations on Borneo is less than the variation within the populations. There was also evidence that inbreeding occurred within the populations.

A new group of hepadnaviruses naturally infecting orangutans (Pongo pygmaeus).

A high prevalence (42.6%) of hepatitis B virus (HBV) infection was suspected in 195 formerly captive orangutans due to a large number of serum samples which cross-reacted with human HBV antigens. It was assumed that such viral infections were contracted from humans during captivity. However, two wild orangutans were identified which were HBV surface antigen positive, indicating that HBV or related viruses may be occurring naturally in the orangutan populations. Sequence analyses of seven isolates revealed that orangutans were infected with hepadnaviruses but that these were clearly divergent from the six known human HBV genotypes and those of other nonhuman hepadnaviruses reported. Phylogenetic analyses revealed geographic clustering with Southeast Asian genotype C viruses and gibbon ape HBV. This implies a common origin of infection within this geographic region, with cross-species transmission of hepadnaviruses among hominoids.

Platynosomum fastosum in ex-captive orangutans from Indonesia.

The liver fluke Platynosomum fastosum was identified upon necropsy of three ex-captive orangutans (Pongo pygmaeus) which had been part of a rehabilitation program for reintroduction to the wild. This trematode has not been reported in orangutans previously and is commonly found in cats in Southeast Asia. Cross infection from cats via intermediate hosts, to orangutans kept in captivity as pets, could explain their presence in the latter. Although P. fastosum caused intrahepatic and bile duct damage, death of the hosts could not be attributed solely to the presence of the liver fluke infection.

"Physiological genomics": mutant screens in zebrafish.

Large-scale mutagenesis screens have proved essential in the search for genes that are important to development in the fly, worm, and yeast. Here we present the power of large-scale screening in a vertebrate, the zebrafish Danio rerio, and propose the use of this genetic system to address fundamental questions of vertebrate developmental physiology. As an example, we focus on zebrafish mutations that reveal single genes essential for normal development of the cardiovascular system. These single gene mutations disrupt specific aspects of rate, rhythm, conduction, or contractility of the developing heart.

Seroprevalence of specific viral infections in confiscated orangutans (Pongo pygmaeus).

A serological survey of confiscated orangutans was conducted to determine the prevalence of specific viral infections cross reacting with human viruses. Antibodies specific for human hepatitis A (HAV) and B (HBV) viruses, herpes simplex viruses (HSV), and human T-lymphotropic virus (HTLV types I and II), as well as for the simian type D retroviruses (SRV types 1 to 3) and simian immunodeficiency virus (SIV) were tested in samples from 143 orangutans. Results revealed a high prevalence of potential pathogens. The most prevalent viral infection found was HBV (59.4% prevalence) of which 89.4% of infected individuals seroconverted to the non-infectious state and 10.6% remained as chronic carriers. Antibodies to HAV, HSV, HTLV-1, and SRV were also detected but at a lower prevalence. There was no evidence of lentiviral infections in this group of animals. The results confirm the importance of quarantine and the need for diagnostic differentiation of virus infections to determine if they are of human origin or unique orangutan viruses.

Characterization of a simian T-lymphotropic virus from a wild-caught orang-utan (Pongo pygmaeus) from Kalimantan, Indonesia.

In a recent serological survey among 143 ex-captive orang-utans two individuals were found that reacted positive in an ELISA detecting antibodies which cross-react with human T-lymphotropic virus type I (HTLV-I) antigens. Infection of both animals with an HTLV-I or simian T-lymphotropic virus (STLV)-like virus was confirmed by Western blot analysis. A third wild-caught animal, which was not part of the original serological survey, was also found to be infected with an HTLV-related virus in a diagnostic PCR assay and Western blot assay. Nucleotide sequence analysis of the 709 bp PCR fragment from the tax/rex region of the HTLV/STLV genome confirmed infection of orang-utans with an STLV similar to but clearly distinct from other Asian STLVs.

Left-right pattern of cardiac BMP4 may drive asymmetry of the heart in zebrafish.

The first evident break in left-right symmetry of the primitive zebrafish heart tube is the shift in pattern of BMP4 expression from radially symmetric to left-predominant. The midline heart tube then 'jogs' to the left and subsequently loops to the right. We examined 279 mutations, affecting more than 200 genes, and found 21 mutations that perturb this process. Some cause BMP4 to remain radially symmetric. Others randomize the asymmetric BMP4 pattern. Retention of BMP4 symmetry is associated with failure to jog: right-predominance of the BMP4 pattern is associated with reversal of the direction of jogging and looping. Raising BMP4 diffusely throughout the heart, via sonic hedgehog injection, or the blocking of its action by injection of a dominant negative BMP4 receptor, prevent directional jogging or looping. The genes crucial to directing cardiac asymmetry include a subset of those needed for patterning the dorsoventral axis and for notochord and ventral spinal cord development. Thus, the pattern of cardiac BMP4 appears to be in the pathway by which the heart interprets lateralizing signals from the midline.