GENOTYPE IDENTIFICATION OF TOXOPLASMA GONDII IN MACROPODS FROM A ZOOLOGICAL PARK IN FLORIDA, USA.

There are limited reports of the genetic characterization of Toxoplasma gondii infecting captive macropods in North America. A novel genotype, ToxoDB PCR-RFLP genotype 263, was reported from six wallabies at a zoological facility in Virginia, USA, prompting an investigation into the genotypes from T. gondii strains infecting macropods at a zoological park in Florida, USA. Cardiac muscle and/or lung samples from an agile wallaby (Macropus agilis, n = 1), red kangaroos (Macropus rufus, n = 8), red-necked wallaby (Macropus rufogriseus, n = 1), and a tammar wallaby (Macropus eugenii, n = 1) that died between 2014 and 2018 were collected. All 11 cases were confirmed to have died from systemic toxoplasmosis by histopathology and immunohistochemical staining. Multilocus PCR-RFLP genotyping of T. gondii was performed directly on tissue samples or on parasites isolated from myocardium by mouse bioassay. Two cases of toxoplasmosis were identified as the reported novel genotype, ToxoDB PCR-RFLP genotype 263, but no common source of exposure could be identified. Five cases were identified as genotype 2 (type III strain, haplogroup 3), and four cases were identified as genotype 216, which has been previously reported in North American wildlife. There were no overt differences in lesion severity or distribution related to genotype. These results suggest that the premise was contaminated with at least three genotypes of T. gondii causing systemic toxoplasmosis in macropods. The largest cluster of fatal toxoplasmosis in macropods in the study period occurred following severe rainfall flooding of the exhibit, suggesting the transmission of T. gondii by water and pointing out the importance of this transmission mechanism. In summary, our study revealed three T. gondii outbreaks that caused significant loss of macropods within 5 yr in a zoological facility in Florida. More studies are needed to understand transmission and prevention of toxoplasmosis in sensitive zoo animals.

EVALUATION OF NONINVASIVE OSCILLOMETRIC BLOOD PRESSURE MONITORING IN ANESTHETIZED BENNETT'S WALLABIES (MACROPUS RUFOGRISEUS).

The objective of this study was to determine the accuracy of a noninvasive oscillometric method in relation to invasively measured blood pressure in anesthetized Bennett's wallabies (Macropus rufogriseus) and also to compare the accuracy of two commonly used oscillometric blood pressure monitors (manufactured by Cardell and Datascope). Eleven animals were anesthetized, and each animal was instrumented with an arterial catheter in the right medial metatarsal artery connected to a pressure transducer to obtain invasive measurements of systolic (SAP), diastolic (DAP), and mean (MAP) arterial blood pressure as well as a pressure waveform. A cuff connected to an oscillometric device was placed on the base of the tail for noninvasive measurements. Paired data from noninvasive and invasive blood pressure measurements (SAP, DAP, and MAP) were obtained every 5 min for 60 min. Bland-Altman plots were used to compare invasive and noninvasive measurements and calculate bias and 95% limits of agreement for SAP, DAP, and MAP. For both monitors, the bias of SAP, DAP, and MAP was significant, although the bias of the Cardell was consistently lower than that of the Datascope for all parameters. Limits of agreement were wide for all parameters. In conclusion, when using an oscillometric blood pressure monitor on anesthetized Bennett's wallabies, trends in blood pressure may be monitored, although all displayed readings may not represent the true blood pressure measurement. Indirect measurements of blood pressure made with the oscillometric device cannot substitute for direct measurements.

Tall young females get ahead: size-specific fecundity in wild kangaroos suggests a steep trade-off with growth.

When resources are limited, organisms face allocation conflicts. Indeterminate growth creates a persistent conflict with reproduction, as growth may enhance future reproduction, but diverts resources from current reproduction. Little is known about allocation trade-offs in mammals with indeterminate growth. We studied growth and reproduction in adult female eastern grey kangaroos (Macropus giganteus), an iteroparous mammal with indeterminate growth. Allocation trajectories varied with age and size: for 4-year-old females, fecundity increased from 30 to 82% from shortest to average-sized individuals. Older females had high fecundity regardless of size. The smallest females grew 30% more annually than average-sized females, but females that reached average size at an older age had lower growth rates. Environmental conditions affected allocation to size and reproduction. Rainy springs increased fecundity from 61 to 84% for females that had previously reproduced, but rainy winters reduced leg growth. Females in better relative condition grew 40% more than average, whereas most young of females below average relative condition failed to survive to 10 months of age. These results highlight an age-specific trade-off between growth and reproduction. Tall young females benefit from a smaller trade-off between somatic growth and early fecundity than shorter females of the same age, but older females appear to favor reproduction over growth regardless of size. Our study highlights how individual heterogeneity determines trade-offs between life-history components. We speculate that cohort effects affect age-specific reproductive success in this long-lived mammal.

Factors affecting the occurrence of enteritis in 10- to 12-month-old eastern grey kangaroos (Macropus giganteus).

Eastern grey kangaroos (Macropus giganteus) younger than 19 months at Kanazawa Zoological Gardens, Japan, frequently died of enteritis. The main cause of death was coccidiosis. This study aimed to reveal retrospectively the clinical features of enteritis and factors contributing to its high occurrence. In addition, haematological and serum biochemical parameters in kangaroos younger than 24 months were analyzed. The findings suggested that enteritis occurrence was higher in 10- to 12-month-old kangaroos than other ages and during seasons with high temperature and humidity than during seasons with low temperature and humidity. The haematological and serum biochemical analyses showed decreases in glucose, total cholesterol, calcium, and triglyceride levels at approximately 10 months of age. Joeys emerge from their mother's pouch at 8 months of age and permanently leave it at approximately 10 months of age. Subsequently, the joeys are exposed to the external environment and infectious agents. Although the joeys continue suckling until 13-19 months of age, the joeys start to eat the similar feed to the adult and their nutritional status may change at approximately 10 months of age. Seasonal influences, species behavior, and shifts in dietary composition are proposed to be contributory to the increased occurrence of enteritis between 10 and 12 months of age.

TREATMENT OF MYCOTIC RHINOSINUSITIS IN A BENNETT'S WALLABY ( MACROPUS RUFOGRISEUS) USING TOPICAL VORICONAZOLE SUSPENDED IN A REVERSE THERMODYNAMIC PLURONIC GEL.

An approximately 4-yr-old female Bennett's wallaby ( Macropus rufogriseus) was evaluated for chronic left-sided facial swelling and nasal discharge. Computed tomography, endoscopy, biopsy, mycologic culture, and panfungal polymerase chain reaction were consistent with destructive mycotic rhinosinusitis. The patient's infection was treated with a long-term injectable antibiotic, oral antifungal therapy, and multiple intranasal infusions of voriconazole suspended in a reverse thermodynamic pluronic gel. This case represents the first documented case of mycotic rhinosinusitis in a macropod and underlines the importance of advanced cross-sectional imaging in the diagnosis, monitoring, and management of nasal cavity disease in zoo animals.

Persistent infections support maintenance of a coronavirus in a population of Australian bats (Myotis macropus).

Understanding viral transmission dynamics within populations of reservoir hosts can facilitate greater knowledge of the spillover of emerging infectious diseases. While bat-borne viruses are of concern to public health, investigations into their dynamics have been limited by a lack of longitudinal data from individual bats. Here, we examine capture-mark-recapture (CMR) data from a species of Australian bat (Myotis macropus) infected with a putative novel Alphacoronavirus within a Bayesian framework. Then, we developed epidemic models to estimate the effect of persistently infectious individuals (which shed viruses for extensive periods) on the probability of viral maintenance within the study population. We found that the CMR data analysis supported grouping of infectious bats into persistently and transiently infectious bats. Maintenance of coronavirus within the study population was more likely in an epidemic model that included both persistently and transiently infectious bats, compared with the epidemic model with non-grouping of bats. These findings, using rare CMR data from longitudinal samples of individual bats, increase our understanding of transmission dynamics of bat viral infectious diseases.

USE OF ORAL FLUOXETINE FOR THE TREATMENT OF ABNORMAL AGGRESSION IN TWO RED-NECKED WALLABIES (MACROPUS RUFOGRISEUS).

: Abnormal inter- and intraspecies aggression, perceived to be anxiety related, was identified in two male red-necked wallabies (Macropus rufogriseus) housed within a zoo. Aggressive episodes were directed at conspecifics, other exhibit animals, and, in one case, human caretakers. The clinical use of oral fluoxetine (0.5 mg/kg po bid) for a period of approximately 4 mo was effective in eliminating aggression towards humans and other animals in these two individuals. There was no evidence of recrudescence of aggression in either case following discontinuation of therapy for up to 3 yr posttreatment. Other than a period of mild transient sedation in one animal, side effects were not noted with fluoxetine treatment in these cases. Additional studies on the pharmacokinetics and side effects of fluoxetine treatment for anxiety behaviors are warranted in wallabies.

CRYPTOCOCCUS NEOFORMANS VAR. GRUBII-ASSOCIATED RENAL AMYLOIDOSIS CAUSING PROTEIN-LOSING NEPHROPATHY IN A RED KANGAROO (MACROPUS RUFUS).

A 10-year-old male castrated red kangaroo (Macropus rufus) presented with mandibular swelling. Examination findings included pitting edema with no dental disease evident on examination or radiographs. The results of blood work were moderate azotemia, hypoalbuminemia, and severely elevated urine protein:creatinine ratio (9.9). Radiographs showed an interstitial pattern of the caudal right lung, and an abdominal ultrasound demonstrated scant effusion. Symptomatic and empirical therapy with antibiotics, anti-inflammatory drugs, and an angiotensin-converting enzyme (ACE) inhibitor did not resolve clinical signs. Due to poor prognosis and declining quality of life, euthanasia was elected. Necropsy revealed chronic granulomatous pneumonia of the caudal right lung lobe with intralesional Cryptococcus, identified as C. neoformans var. grubii by DNA sequencing. Severe bilateral glomerular and tubulointerstitial amyloidosis induced protein-losing nephropathy, leading to tri-cavitary effusion, subcutaneous edema, and cachexia. The authors speculate that renal amyloidosis was associated with chronic cryptococcal pneumonia in this red kangaroo.

NEWLY DESCRIBED TOXOPLASMA GONDII STRAIN CAUSES HIGH MORTALITY IN RED NECKED WALLABIES (MACROPUS RUFOGRISEUS) IN A ZOO.

This manuscript describes an outbreak of fatal toxoplasmosis in wallabies. Ten adult red necked wallabies (Macropus rufogriseus) were imported from New Zealand to the Virginia Zoo. Agglutination testing upon admission into quarantine showed all animals to be negative for antibodies to Toxoplasma gondii. Nine of these wallabies died from acute toxoplasmosis within 59-565 (average 224) days after being moved onto exhibit. Clinical signs included lethargy, diarrhea, tachypnea, and ataxia that progressed rapidly; death without premonitory signs occurred in one case. Histopathologic examination revealed interstitial pneumonia, encephalomyelitis, myositis, enteritis, and myocarditis. The diagnosis was confirmed through serologic, histopathologic, and polymerase chain reaction (PCR) testing. Multilocus PCR-RFLP (restriction fragment length polymorphism) genotyping revealed that the first six animals were infected by a previously undiscovered Toxoplasma gondii genotype, designated as ToxoDB PCR-RFLP genotype No. 263. These six cases survived for an average of 118 days on exhibit before succumbing to toxoplasmosis. The other three wallabies were infected with a Toxoplasma gondii strain of ToxoDB PCR-RFLP genotype No. 4, which is a common strain type circulating in wild animals in North America. These three cases survived for an average of 435 days on exhibit before succumbing to toxoplasmosis. The outbreaks of toxoplasmosis in these wallabies are likely from two different sources. Furthermore, the results highlight Toxoplasma gondii PCR-RFLP genotyping in parasite diagnosis and understanding parasite transmission and potential mitigation procedures.

Phylogenetic relationships of species of the oesophageal parasitic nematode genera Cyclostrongylus and Spirostrongylus (Strongyloidea: Chabertiidae: Cloacininae) with their wallaby hosts (Marsupialia: Macropodidae).

A phylogeny for seven species of Cyclostrongylus and the monotypic genus Spirostrongylus (Nematoda: Chabertiidae), all highly host specific parasites of the oesophagi of wallabies (Marsupialia: Macropodidae), was constructed using sequence data for the first and second internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. There was no evidence for co-speciation, or for the sympatric or synxenic speciation of Cyclostrongylus alatus and Cyclostrongylus perplexus, both of which are parasites of Macropus rufogriseus. Rather, host switching, correlating with geographical distributions, appeared to provide some explanation of the pattern of speciation observed.

Individual heterogeneity and offspring sex affect the growth-reproduction trade-off in a mammal with indeterminate growth.

Reproduction can lead to a trade-off with growth, particularly when individuals reproduce before completing body growth. Kangaroos have indeterminate growth and may always face this trade-off. We combined an experimental manipulation of reproductive effort and multi-year monitoring of a large sample size of marked individuals in two populations of eastern grey kangaroos to test the predictions (1) that reproduction decreases skeletal growth and mass gain and (2) that mass loss leads to reproductive failure. We also tested if sex-allocation strategies influenced these trade-offs. Experimental reproductive suppression revealed negative effects of reproduction on mass gain and leg growth from 1 year to the next. Unmanipulated females, however, showed a positive correlation between number of days lactating and leg growth over periods of 2 years and longer, suggesting that over the long term, reproductive costs were masked by individual heterogeneity in resource acquisition. Mass gain was necessary for reproductive success the subsequent year. Although mothers of daughters generally lost more mass than females nursing sons, mothers in poor condition experienced greater mass gain and arm growth if they had daughters than if they had sons. The strong links between individual mass changes and reproduction suggest that reproductive tactics are strongly resource-dependent.

Spatial dynamics of the bacterial community structure in the gastrointestinal tract of red kangaroo (Macropus rufus).

The quantification and community of bacteria in the gastrointestinal (GI) tract (stomach, jejunum, ileum, cecum, colon and rectum) of red kangaroos (Macropus rufus) were examined by using real-time PCR and paired-end Illumina sequencing. The quantification of bacteria showed that the number of bacteria in jejunum and rectum was significantly lower than that in colon and cecum (P < 0.05). A total of 1,872,590 sequences was remained after quality-filtering and 50,948 OTUs were identified at the 97 % similarity level. The dominant phyla in the GI tract of red kangaroos were identified as Actinobacteria, Bacteroidetes and Firmicutes. At the level of genus, the samples from different parts of GI tract clustered into three groups: stomach, small intestine (jejunum and ileum) and large intestine (cecum and rectum). Prevotella (29.81 %) was the most dominant genus in the stomach and significantly (P < 0.05) higher than that in other parts of GI tract. In the small intestine, Bifidobacterium (33.04, 12.14 %) and Streptococcus (22.90, 19.16 %) were dominant genera. Unclassified Ruminococcaceae was the most dominant family in large intestine and the total relative abundance of unclassified bacteria was above 50 %. In identified genera, Dorea was the most important variable to discriminate large intestine and it was significantly higher in cecum than in stomach, small intestine and colon (P < 0.05). Bifidobacterium (21.89 %) was the only dominant genus in colon. Future work on culture in vitro and genome sequencing of those unidentified bacteria might give us insight into the function of these microorganisms in the GI tract. In addition, the comparison of the bacterial community in the foregut of kangaroos and other herbivores and the rumen might give us insight into the mechanism of fiber degradation and help us exploit approaches to improve the feed efficiency and subsequently, reduce the methane emission from herbivores.

COMPARISON OF MEDETOMIDINE-KETAMINE AND BUTORPHANOL-AZAPERONE-MEDETOMIDINE IN CAPTIVE BENNETT'S WALLABIES (MACROPUS RUFOGRISEUS).

The objective of this study was to compare a traditional partially reversible medetomidine-ketamine sedation with a more reversible butorphanol-azaperone-medetomidine combination in Bennett's wallabies ( Macropus rufogriseus ) maintained in a zoological collection. Fourteen animals were divided into two treatment groups. Individuals in group 1 received an intramuscular (i.m.) injection of butorphanol (0.54 ± 0.05 mg/kg), azaperone (0.22 ± 0.02 mg/kg), and medetomidine (0.16 ± 0.02 mg/kg). Individuals in group 2 received an i.m. injection of ketamine (5.43 ±1.16 mg/kg) with medetomidine (0.05 ± 0.014 mg/kg). For group 1, sedation was reversed with atipamezole (0.81 ± 0.069 mg/kg i.m.) and naltrexone (1.08 ± 0.09 mg/kg i.m.). For group 2, sedation was reversed with atipamezole (0.27 ± 0.056 mg/kg i.m.). There were no significant differences between the groups in mean time to induction, time spent on gas anesthesia, or time to standing after reversal was administered. Animals in both groups required supplemental gas anesthesia to facilitate intubation. No adverse reactions or effects were noted with either protocol; however, the BAM protocol did not provide sufficient sedation for handling in all animals and may not be suitable for use in this species.

SURGICAL CORRECTION OF A CERVICAL SPINAL FRACTURE IN A BENNETT'S WALLABY (MACROPUS RUFOGRISEUS).

Macropods are susceptible to trauma, and fractures of the cervical vertebrae due to collisions are relatively common. A 4-yr-old, intact male Bennett's wallaby (Macropus rufogriseus) was presented acutely nonambulatory and tetraparetic but with motor function present in all limbs. Cervical radiographs revealed a C4 vertebral fracture. Surgical stabilization was obtained through a ventral midline approach whereby the vertebral bodies were distracted and then secured with screws and polymethylmethacrylate (PMMA). Postoperative radiographs revealed restoration of the spinal canal and appropriate implant placement. The patient was discharged 4 days postoperatively and gradually recovered full neurologic function per the owner over the ensuing 10 wk. Subsequent radiographs obtained 10.5 mo postoperatively revealed a healed fracture with stable implants. The ventral spinal distraction and stabilization technique using screws and PMMA, as are used in the domestic dog, was successful in this wallaby despite its smaller vertebral size and comparable lack of soft-tissue support.

Artificial light at night desynchronizes strictly seasonal reproduction in a wild mammal.

Change in day length is an important cue for reproductive activation in seasonally breeding animals to ensure that the timing of greatest maternal investment (e.g. lactation in mammals) coincides with favourable environmental conditions (e.g. peak productivity). However, artificial light at night has the potential to interfere with the perception of such natural cues. Following a 5-year study on two populations of wild marsupial mammals exposed to different night-time levels of anthropogenic light, we show that light pollution in urban environments masks seasonal changes in ambient light cues, suppressing melatonin levels and delaying births in the tammar wallaby. These results highlight a previously unappreciated relationship linking artificial light at night with induced changes in mammalian reproductive physiology, and the potential for larger-scale impacts at the population level.

Experimental manipulation of female reproduction demonstrates its fitness costs in kangaroos.

When resources are scarce, female mammals should face a trade-off between lactation and other life-history traits such as growth, survival and subsequent reproduction. Kangaroos are ideal to test predictions about reproductive costs because they may simultaneously lactate and carry a young, and have indeterminate growth and a long breeding season. An earlier study in three of our five study populations prevented female eastern grey kangaroos (Macropus giganteus) from reproducing during one reproductive season by either inserting contraceptive implants or removing very small pouch young. We explored how individual and environmental variables affect the costs of reproduction over time, combining this experimental reduction of reproductive effort with multi-year monitoring of 270 marked females. Experimental manipulation should control for individual heterogeneity, revealing the costs of reproduction and their likely sources. We also examined the fitness consequences of reproductive effort and offspring sex among unmanipulated individuals to test whether sex allocation strategies affected trade-offs. Costs of reproduction included longer inter-birth intervals and lower probability of producing a young that survived to 7 months in the subsequent reproductive event. Weaning success, however, did not differ significantly between manipulated and control females. By reducing reproductive effort, manipulation appeared to increase individual condition and subsequent reproductive success. Effects of offspring sex upon subsequent reproductive success varied according to year and study population. Mothers of sons were generally more likely to have a young that survived to 7 months, compared to mothers of daughters. The fitness costs of reproduction arise from constraints in both acquisition and allocation of resources. To meet these costs, females delay subsequent parturition and may manipulate offspring sex. Reproductive tactics thus vary according to the amount of resource available to each individual, promoting a wide range in reproductive performance within and among individuals and populations.

Empirical tests of harvest-induced body-size evolution along a geographic gradient in Australian macropods.

Life-history theory predicts the progressive dwarfing of animal populations that are subjected to chronic mortality stress, but the evolutionary impact of harvesting terrestrial herbivores has seldom been tested. In Australia, marsupials of the genus Macropus (kangaroos and wallabies) are subjected to size-selective commercial harvesting. Mathematical modelling suggests that harvest quotas (c. 10-20% of population estimates annually) could be driving body-size evolution in these species. We tested this hypothesis for three harvested macropod species with continental-scale distributions. To do so, we measured more than 2000 macropod skulls sourced from wildlife collections spanning the last 130 years. We analysed these data using spatial Bayesian models that controlled for the age and sex of specimens as well as environmental drivers and island effects. We found no evidence for the hypothesized decline in body size for any species; rather, models that fit trend terms supported minor body size increases over time. This apparently counterintuitive result is consistent with reduced mortality due to a depauperate predator guild and increased primary productivity of grassland vegetation following European settlement in Australia. Spatial patterns in macropod body size supported the heat dissipation limit and productivity hypotheses proposed to explain geographic body-size variation (i.e. skull size increased with decreasing summer maximum temperature and increasing rainfall, respectively). There is no empirical evidence that size-selective harvesting has driven the evolution of smaller body size in Australian macropods. Bayesian models are appropriate for investigating the long-term impact of human harvesting because they can impute missing data, fit nonlinear growth models and account for non-random spatial sampling inherent in wildlife collections.

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.

Sarcoptic Mange in a Tasmanian Devil (Sarcophilus harrisii) and Bennett's Wallaby (Notamacropus rufogriseus).

Sarcoptes scabiei mites and skin lesions consistent with severe sarcoptic mange were identified in a Tasmanian devil (Sarcophilus harrisii) and Bennett's wallaby (Notamacropus rufogriseus) from Tasmania, Australia. The devil and wallaby both had severe hyperkeratotic skin lesions. All stages of mite development were identified in the devil, suggesting parasite reproduction on the host. The devil was also affected by devil facial tumor disease and several other parasites. This expands the global host range of species susceptible to this panzootic mange disease.

Absence of Coxiella burnetii in kangaroo ticks (Amblyomma triguttatum) from a high seroprevalence population of eastern grey kangaroos.

Q fever, caused by Coxiella burnetii, is an important zoonotic and public health concern worldwide. Kangaroos are thought to be a likely wildlife reservoir for C. burnetii in Australia and the kangaroo tick (Amblyomma triguttatum) has often been considered a vector. In this descriptive study of ticks collected from a population of eastern grey kangaroos (Macropus giganteus) with a high serological (84 %) and molecular (65 %) prevalence of C. burnetii in northern New South Wales, a total of 72 A. triguttatum ticks were tested by PCRs targeting the IS1111, htpAB, and com1 genes of the Coxiella genome. Despite the remarkably high prevalence of coxiellosis in the host population, none of the ticks were positive for Coxiella. This finding suggests that the kangaroo tick may not play a significant role in the transmission dynamics of C. burnetii in this particular host population.