Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain.

The preservation of rhinoceros semen is vital for captive breeding programs. While successful collection and cryopreservation of rhinoceros semen has been reported, the volume and quality of semen produced is often low due to the high viscosity associated with ejaculates collected via electroejaculation. Reducing semen viscosity would enable access to previously unusable spermatozoa from viscous fractions and could improve quality post-thaw. The enzyme papain successfully reduced the viscosity of camelid semen but has yet to be tested in wildlife species. This study assessed the influence of papain on the in vitro quality of rhinoceros spermatozoa during cryopreservation using advanced semen assessment. In experiment 1, the motility of spermatozoa from the viscous fraction of an ejaculate, either untreated or treated with papain and its inhibitor E-64 prior to cryopreservation, was assessed post-thaw. In experiment 2, spermatozoa from papain-treated viscous fractions were compared to spermatozoa frozen from untreated sperm-rich fractions pre-freeze, as well as after 0, 1.5 and 3 h of incubation post-thaw (37 °C). Papain significantly increased the quantity of spermatozoa collected from ejaculates, as well as the motility prior to freezing. Papain also improved the post-thaw motility, velocity, linearity and straightness of samples compared to sperm-rich samples, with no detriment to sperm viability, lipid membrane disorder, production of ROS or DNA integrity (p < 0.05). Results show the benefit of supplementing rhinoceros spermatozoa with papain prior to cryopreservation on sperm cryosurvival and demonstrates the potential of using papain to improve the success of cryopreservation protocols, not only for the rhinoceros, but also for other wildlife species.

BASELINE HEALTH AND DISEASE ASSESSMENT OF FOUNDER EASTERN QUOLLS (DASYURUS VIVERRINUS) DURING A CONSERVATION TRANSLOCATION TO MAINLAND AUSTRALIA.

We evaluated the health of 31 (eight males, 23 females) founder eastern quolls (Dasyurus viverrinus), translocated to a fenced reserve in the Australian Capital Territory between February 2016 and July 2017. Quolls were wild caught in Tasmania (16 animals) or captive bred at Mount Rothwell Biodiversity Interpretation Centre, Victoria (15 animals). Quolls were assessed for the presence of selected potential pathogens (Toxoplasma gondii, herpesviruses, Salmonella serovars, hemoprotozoa, and ectoparasites). We assessed the relationships among sex, provenance (captive or free ranging), T. gondii or herpesvirus infection, weight, and hematologic and biochemical variables. Six of 21 quolls (29%) tested were seropositive for antibodies to T. gondii. Seropositive quolls weighed significantly more and had significantly lower potassium levels, anion gaps, and urea and triglyceride levels than seronegative quolls had. Eighteen of 31 (58%) combined conjunctival-pharyngeal-cloacal swabs collected from quolls were PCR positive for a newly identified gammaherpesvirus, tentatively named dasyurid gammaherpesvirus 3. There were no significant differences among hematologic and biochemical variables or body weights from PCR-positive and PCR-negative quolls. Eighteen of 18 (100%) of rectal-swab samples were culture negative for Salmonella serovars. Three species of tick (Ixodes tasmani, Ixodes fecialis, and Ixodes holocyclus), two species of mite (Andreacus radfordi, one unidentified), and four species of flea (Pygiopsylla hoplia, Acanthopsylla rothschildi rothschildi, Uropsylla tasmanica, and Stephanocircus dasyuri), were detected on wild-caught quolls, whereas a fifth species of flea, Echidnophaga myremecobii, was detected only on captive-bred quolls. Five of 15 blood samples (33%) were positive for hemoprotozoan DNA via PCR, a novel Hepatozoon species, a novel Theileria species, Theileria paparinii, and Trypanosoma copemani were detected. Despite the presence of several potential pathogens known to be associated with disease in other marsupials, the quolls were considered to be in good general health, suitable for translocation, and a viable population was subsequently established.

Sequence analyses at mitochondrial and nuclear loci reveal a novel Theileria sp. and aid in the phylogenetic resolution of piroplasms from Australian marsupials and ticks.

The order Piroplasmida encompasses two main families: Babesiidae and Theileriidae, containing tick-borne pathogens of veterinary and medical importance worldwide. While only three genera (Babesia, Cytauxzoon and Theileria) comprising piroplasm parasites are currently recognised, phylogenetic studies at the 18S rRNA (18S) gene suggest that these organisms represent at least ten lineages, one of which comprises the relatively unique and highly diverse Theileria spp. from Australian marsupials and ticks. As an alternative to analysing 18S sequences alone, sequencing of mitochondrial genes has proven to be useful for the elucidation of evolutionary relationships amongst some groups of piroplasms. This research aimed to characterise piroplasms from Australian native mammals and ticks using multiple genetic markers (18S, cytochrome c, oxidase subunit III (cox3) and cytochrome B (cytB)) and microscopy. For this, nearly complete piroplasm-18S sequences were obtained from 32 animals belonging to six marsupial species: eastern bettong (Bettongia gaimardi), eastern quoll (Dasyurus viverrinus), eastern grey kangaroo (Macropus giganteus), swamp wallaby (Wallabia bicolor), quokka (Setonix brachyurus) and Gilbert's potoroo (Potorous gilbertii). The organisms detected represented eight novel Theileria genotypes, which formed five sub-clades within the main marsupial clade containing previously reported Australian marsupial and tick-derived Theileria spp. A selection of both novel and previously described Australian piroplasms at the 18S were also successfully characterised, for the first time, at the cox3 and cytB loci, and corroborated the position of Australian native theilerias in a separate, well-supported clade. Analyses of the cox3 and cytB genes also aided in the taxonomic resolution within the clade of Australian Piroplasmida. Importantly, microscopy and molecular analysis at multiple loci led to the discovery of a unique piroplasm species that clustered with the Australian marsupial theilerias, for which we propose the name Theileria lupei n. sp.

CALVARIAL OSTEOSARCOMA WITH CEREBRAL COMPRESSION IN A FREE-RANGING POWERFUL OWL (NINOX STRENUA).

A free-ranging powerful owl (Ninox strenua) presented in a dull state with extensive bruising of the skin overlying the skull, due to suspected trauma. Supportive care was provided, which resulted in the return to a normal state, although intermittent subtle neurologic abnormalities remained. One month from original presentation, intermittent episodes of head turning and a possible seizure were noted, but behavior and appetite were otherwise normal. The owl was referred to Taronga Wildlife Hospital for evaluation. On presentation, the owl exhibited severe neurologic abnormalities, prompting euthanasia. At necropsy the dorsum of the skull exhibited marked osseous proliferation, extending ventrally and compressing the cerebrum. The skull was radiographed and submitted for histopathology. A diagnosis of osteoblastic osteosarcoma was made. This is the first report of a calvarial osteosarcoma in a powerful owl.

CLINICOPATHOLOGIC CORRELATES OF FASCIOLIASIS IN TWO EASTERN GREY KANGAROOS (MACROPUS GIGANTEUS).

Infection with the introduced trematode Fasciola hepatica was associated with anemia, mild to moderate azotemia, hypoalbuminemia, and elevated liver enzymes and creatine kinase values in two free-ranging eastern grey kangaroos (Macropus giganteus). Both kangaroos were euthanized because of the severity of clinical signs associated with infection. Histopathologic changes included severe cholangiohepatitis, biliary hyperplasia, and fibrosis. Hepatic, splenic, and intestinal amyloidosis was present in one kangaroo and hepatic abscessation in the other; neither histologic change has been reported in macropodids with fascioliasis previously.

Prevalence and Clinical Significance of Herpesvirus Infection in Populations of Australian Marsupials.

Herpesviruses have been reported in several marsupial species, but molecular classification has been limited to four herpesviruses in macropodids, a gammaherpesvirus in two antechinus species (Antechinus flavipes and Antechinus agilis), a gammaherpesvirus in a potoroid, the eastern bettong (Bettongia gaimardi) and two gammaherpesviruses in koalas (Phascolarctos cinereus). In this study we examined a range of Australian marsupials for the presence of herpesviruses using molecular and serological techniques, and also assessed risk factors associated with herpesvirus infection. Our study population included 99 koalas (Phascolarctos cinereus), 96 eastern grey kangaroos (Macropus giganteus), 50 Tasmanian devils (Sarcophilus harrisii) and 33 common wombats (Vombatus ursinius). In total, six novel herpesviruses (one alphaherpesvirus and five gammaherpesviruses) were identified in various host species. The overall prevalence of detection of herpesvirus DNA in our study population was 27.2% (95% confidence interval (CI) of 22.6-32.2%), but this varied between species and reached as high as 45.4% (95% CI 28.1-63.7%) in common wombats. Serum antibodies to two closely related macropodid herpesviruses (macropodid herpesvirus 1 and 2) were detected in 44.3% (95% CI 33.1-55.9%) of animals tested. This also varied between species and was as high as 92% (95% CI 74.0-99.0%) in eastern grey kangaroos. A number of epidemiological variables were identified as positive predictors for the presence of herpesvirus DNA in the marsupial samples evaluated. The most striking association was observed in koalas, where the presence of Chlamydia pecorum DNA was strongly associated with the presence of herpesvirus DNA (Odds Ratio = 60, 95% CI 12.1-297.8). Our results demonstrate the common presence of herpesviruses in Australian marsupials and provide directions for future research.

THADEUA GREENI-ASSOCIATED DERMATITIS IN AN EASTERN BETTONG (BETTONGIA GAIMARDI).

An adult female bettong (Bettongia gaimardi) presented with extensive alopecia and dermatitis affecting the ventral and lateral aspects of the neck and thorax. Microscopic examination of skin scrapings collected from the affected area revealed large numbers of the dermanyssid mite Thadeua greeni. A histopathologic diagnosis of chronic proliferative and hyperkeratotic perivascular dermatitis with intralesional mites was returned. Treatment with a combination of topical fipronil and parenteral ivermectin weekly for 3 wk resulted in the resolution of clinical signs and apparent elimination of the mite.

Health evaluation of free-ranging eastern bettongs (Bettongia gaimardi) during translocation for reintroduction in Australia.

Sixty (19 male, 41 female) free-ranging adult eastern bettongs (Bettongia gaimardi) were captured in Tasmania and translocated to the Australian Capital Territory between July 2011 and September 2012 for reintroduction into fenced, predator-proof reserves. The bettongs were anesthetized for physical examination and screened for selected diseases during translocation. Reference ranges for hematologic and biochemical parameters were determined. Two bettongs had detectable antibodies to the alphaherpesviruses macropodid herpesvirus 1 and macropodid herpesvirus 2 by serum neutralization assay. A novel gammaherpesvirus was detected, via PCR, from pooled swabs collected from the nasal, conjunctival, and urogenital tract mucosa of four other bettongs. Sera from 59 bettongs were negative for antibodies to Toxoplasma gondii as assessed by both the modified agglutination test and the direct agglutination test (n = 53) or by the modified agglutination test only (n = 6). Rectal swabs from 14 bettongs were submitted for bacterial culture and all were negative for Salmonella serovars. Ectoparasites identified on the bettongs included fleas (Pygiopsylla zethi, Stephanocircus harrisoni), a louse (Paraheterodoxous sp.), mites (Guntheria cf. pertinax, Haemolaelaps hatteni, a suspected protonymph of Thadeua sp., Cytostethum tasmaniense, Cytostethum intermedium, Cytostethum thetis, Cytostethum wallabia), and ticks (Ixodes cornuatus, Ixodes trichosuri, Ixodes tasmani). An intraerythrocytic organism morphologically consistent with a Theileria species was identified in blood smears from four bettongs. These data provide baseline health and disease information for free-ranging eastern bettongs that can be used for the conservation management of both the source and translocated populations.

The seminiferous cycle of the rhinoceros.

The seminiferous cycle of the rhinoceros was investigated for the first time using testicular tissue from captive white (Ceratotherium simum, n=2) and black (Diceros bicornis, n=1) rhinoceroses. Stages of the seminiferous epithelial cycle were characterised using the tubular morphology method and relative frequencies of each stage determined. This method allowed for the identification of eight stages of cellular associations characteristic of the seminiferous cycle in white and black rhinoceroses. The eight stages of the seminiferous cycle observed closely approximated the stages previously described for the domestic horse (Equus caballus). Premeiotic (stages I-III), meiotic (stage IV) and postmeiotic (stages V-VIII) represented 44.8%, 5.3% and 49.9% of the seminiferous cycle respectively.

Toxoplasmosis in macropodids: a review.

Toxoplasmosis is a well-described disease entity that causes significant morbidity and mortality in both captive and free-ranging macropodids. The clinical presentation of toxoplasmosis in macropodids is variable, which reflects the multiple body systems affected by this disease. Animals may die without exhibiting premonitory signs or after the acute development of nonspecific signs of illness. In more chronic cases, clinical signs include neurologic deficits, blindness, respiratory signs, and, in some cases, diarrhea. Histologic lesions can be extensive and affect the pulmonary parenchyma, cardiac and skeletal muscle, lymph nodes, spleen, gastrointestinal tract, adrenal glands, pancreas, central nervous system, liver, and kidney. An antemortem diagnosis can be challenging, although a range of serologic tests are available. Treatment is frequently unrewarding, although recent evidence suggests that the anti-protozoal drug atovaquone may be effective in treating acute cases and eliminating infection. Attempts to vaccinate macropodids against toxoplasmosis have been unsuccessful, and preventive measures are limited to preventing exposure to sporulated oocysts in the environment.

Investigation and diagnosis of nontuberculous mycobacteriosis in a captive herd of aoudad (Ammotragus lervia).

An epizootic of nontuberculous mycobacteriosis occurred in a captive herd of aoudad (Ammotragus lervia) over a period of 18 mo. Each of the affected animals was subject to a thorough postmortem examination that included histopathology, tissue concentration and acid-fast staining, aerobic and anaerobic bacterial culture, mycobacterial culture, and real-time polymerase chain reaction specific for Mycobacterium tuberculosis DNA. Histopathologic lesions consistent with pulmonary mycobacteriosis, including the presence of acid-fast bacteria, were identified in two captive adult male aoudad. M. avium was isolated in culture from the pulmonary parenchyma, and M. parafortuitum was isolated from a mesenteric lymph node of a third animal, an adult female, euthanized subsequent to an illness characterized by progressive dyspnea and tachypnea. M. intracellulare was isolated within the bronchial lymph node of a fourth aoudad, an adult female that was euthanized due to chronic weight loss. Diagnostic testing of the 34 individuals in the herd included collection of blood for an interferon-gamma assay, intradermal tuberculin testing, and radiometric fecal culture for M. avium subsp. paratuberculosis. On the basis of this investigation, mycobacteriosis associated with M. bovis, M. tuberculosis, and/or M. avium subsp. paratuberculosis was ruled out and nontuberculous mycobacteriosis was confirmed in this herd.

Ulnar metaphyseal osteochondrosis in seven captive bred cheetahs (Acinonyx jubatus).

Distal ulna metaphyseal osteochondrosis was identified in seven captive bred cheetahs raised in Australia between 1984 and 2005. The disorder was characterized by bilateral carpal valgus conformation. In the metaphyseal region of the distal ulnae, an osteolucent defect that appeared as a proximal extension of the lucent physis was identified radiographically between 6 and 10 months of age. Ulna ostectomy was done to correct the angular limb deformity. Histologically, changes were identified in the osteolucent lesion that resembled osteochondrosis. We propose that the condition is probably familial and/or dietary in origin.

Hematologic and serum biochemical reference values for free-ranging northern hairy-nosed wombats.

Hematologic and serum biochemistry values were determined for 31 adult (21 male and 10 female) and four subadult male northern hairy-nosed wombats (Lasiorhinus krefftii) from the only existing population in Epping Forest National Park, Australia. Blood samples were obtained from free-ranging northern hairy-nosed wombats during trapping for population census and health and reproductive assessment in 1999. Hematologic and biochemical values were compared between adult males and adult females, and between adult and subadult wombats. Values were also compared with those previously published for southern hairy-nosed (Lasiorhinus latifrons) and common (Vombatus ursinus) wombats. The values from this study were used to create reference intervals, and they make up the first comprehensive hematologic and biochemical study for this highly endangered species.

Spotted black snake (Pseudechis guttatus) envenomation in a maned wolf (Chrysocyon brachyurus).

Envenomation by a spotted black snake (Pseudechis guttatus), following multiple bites on the buccal mucosa of a captive maned wolf (Chrysocyon brachyurus), caused the animal's collapse, hemolysis, rhabdomyolysis, local tissue necrosis, hepatic and renal failure, and subsequent death. The wolf died despite intensive supportive care including antivenom administration, fluid support, and a blood transfusion. Gross necropsy findings included myocardial and intestinal hemorrhage, pulmonary congestion, hepatomegaly, and splenomegaly. Microscopic examination of formalin-fixed tissues demonstrated pulmonary and abdominal visceral hemorrhage, acute nephrosis with casts, multifocal hepatic necrosis, and splenic congestion.

Anesthesia and use of a sling system to facilitate transvaginal laparoscopy in a black rhinoceros (Diceros bicornis minor).

Transvaginal laparoscopy to allow assessment of ovarian pathology and to attempt retrieval of oocytes was facilitated in a captive, female black rhinoceros (Diceros bicornis minor) through the use of a sling on two separate occasions. Following induction of anesthesia with an opioid-based combination, the rhinoceros was intubated and maintained on isoflurane in oxygen. The use of the sling and volume controlled inhalation anesthesia allowed for maintenance of appropriate anatomic positioning, analgesia, and insufflation of the abdominal cavity for laparoscopy during both procedures.

Morbidity and mortality associated with Arcanobacterium pyogenes in a group of captive blackbuck (Antilope cervicapra).

Arcanobacterium pyogenes was associated with necrotizing pneumonia; mandibular osteomyelitis; peritonitis; and hepatic, pulmonary, renal, and subcutaneous abscessation in a group of captive blackbuck (Antilope cervicapra). Males were more frequently (73.3%) affected than females. Infection with A. pyogenes was fatal or necessitated euthanasia in 15 of 16 (93.7%) cases. Deaths associated with A. pyogenes occurred most frequently (60%) during winter.

Comparison of etorphine-detomidine and medetomidine-ketamine anesthesia in captive addax (Addax nasomaculatus).

Thirty-five anesthetic events involving 15 captive addax (Addax nasonzaculatus) were performed between August 1998 and February 2002 using a combination of etorphine (33.7 +/- 7.9 microg/kg) and detomidine (21.9 +/- 4.6 microg/ kg) or a combination of medetomidine (57.4 +/- 8.6 microg/kg) and ketamine (1.22 +/- 0.3 microg/kg), with or without supplemental injectable or inhalant anesthetic agents. Etorphine-detomidine anesthesia was antagonized with diprenorphine (107.1 +/- 16.4 microg/kg) and atipamezole (100.9 +/- 42.4 microg/kg). Medetomidine-ketamine anesthesia was antagonized with atipamezole (245.3 +/- 63.4 microg/kg). Animals became recumbent within 5 min when the combination of etorphine and detomidine was used and within 11 min when the combination of medetomidine and ketamine was used. Both drug combinations were suitable for use as primary immobilizing agents producing short-duration restraint and analgesia. Bradycardia was noted with both combinations. Further investigation of the cardiopulmonary effects of both combinations is warranted.