A WIDESPREAD NOVEL GAMMAHERPESVIRUS IN APPARENTLY HEALTHY WILD QUOKKAS (SETONIX BRACHYURUS): A THREATENED AND ENDEMIC WALLABY OF WESTERN AUSTRALIA.

Five genetically distinct macropodid marsupial herpesviruses have been reported [Macropodid alphaherpesviruses 1 and 2 (MaHV-1 and -2); Macropodid herpesviruses 3 to 5 (MaHV-3 to -5)]. MaHV-2 was originally isolated from an outbreak of fatal disease in captive quokkas (Setonix brachyurus) that were in contact with other macropodid species. This warranted a survey of the presence of herpesviruses in this threatened and endemic Western Australian (WA) wallaby. Blood samples from 142 apparently healthy quokkas were tested for exposure to MaHV-1 and -2 by serology. Of these 142, 121 [Rottnest Island (RI), n = 93; mainland WA, n = 28] were tested for herpesvirus infection by polymerase chain reaction (PCR). Antibodies to MaHV-1 and -2 were detected in one individual [prevalence, 0.7%; 95% confidence interval (CI), 0.1%-3.2%] from the mainland and none from RI. However, a novel gammaherpesvirus [designated Macropodid herpesvirus 6 (MaHV-6)] was detected by PCR in the blood of 13 of 121 individuals (11%; 95% CI, 6.2-17.2). Infection with MaHV-6 was significantly more prevalent on the mainland (7/28; i.e., 25%) compared with RI (6/93; i.e., 6.45%; difference in sample proportions, 95% CI, 6%-32%; P = 0.015). There was no association (P > 0.05) between infection with MaHV-6 and differences in hematology, blood chemistry, peripheral blood cell morphologies, or on clinical status. There was a significant association between infection with MaHV-6 and the presence of Theileria spp. in blood [odds ratio (OR) = 11.0; 95% CI, 2.31-52.3; P = 0.001] and yeast in the nasal lining (OR = 7.0; 95% CI, 1.54-31.8; P = 0.021), suggesting that quokkas may be more susceptible to infection with these microorganisms if also infected with MaHV-6. MaHV-6 infection may be a catalyst for vulnerability to disease with other infectious agents and may pose a significant threat to other macropods. These findings have implications for in situ and ex situ management programs of quokkas.

ANTIBODY RESPONSE TO EPSILON TOXIN OF CLOSTRIDIUM PERFRINGENS IN CAPTIVE ADULT SPRINGBOK (ANTIDORCAS MARSUPIALIS), IMPALA (AEPYCEROS MELAMPUS), ALPACA (VICUGNA PACOS), AND RED-NECKED WALLABY (MACROPUS RUFOGRISEUS) OVER A YEAR.

Enterotoxemia is an important issue in various zoological taxa. In this study, serologic responses over a 1-yr period after vaccination with a multivalent clostridial vaccine were evaluated in 10 adult springboks (Antidorcas marsupialis), 12 impalas (Aepyceros melampus), seven alpacas (Vicugna pacos), and five red-necked wallabies (Macropus rufogriseus). Antibody production to the Clostridium perfringens type D epsilon toxin component of the vaccine was measured using an indirect enzyme-linked immunosorbent assay and determined as the percentage of inhibition (% inhib). Initial % inhib was (0.01-18.9)%. All animals received initial vaccination with a booster vaccine 4 weeks apart. Serum samples were collected at T0 (nonvaccinated), 15, 30, 60, 180, and 360 days postvaccination (dpv) for analysis. The vaccine induced a high antibody response that peaked at 15, 30, and 60 dpv in springboks, 30 and 60 dpv in impalas (P < 0.01), and 60 dpv in alpacas and wallabies (P < 0.01). The booster vaccine was followed by a high antibody response, which slowly decreased with time. The antibody response was significantly higher at 360 dpv than at T0 in wallabies and alpacas (P < 0.01). In impalas and springboks, it appeared that a booster every 6 mo might be required to maintain an antibody response above baseline (P < 0.01). Because no challenge studies were performed, it is unknown whether the measured humoral immune responses would have been protective. Further research is warranted to investigate protective effects of antibodies to inoculation challenge in nondomestic species.

Femoral bone perfusion through the nutrient foramen during growth and locomotor development of western grey kangaroos (Macropus fuliginosus).

The nutrient artery passes through the nutrient foramen on the shaft of the femur and supplies more than half of the total blood flow to the bone. Assuming that the size of the nutrient foramen correlates with the size of the nutrient artery, an index of blood flow rate (Qi) can be calculated from nutrient foramen dimensions. Interspecific Qi is proportional to locomotor activity levels in adult mammals, birds and reptiles. However, no studies have yet estimated intraspecific Qi to test for the effects of growth and locomotor development on bone blood flow requirements. In this study, we used micro-CT and medical CT scanning to measure femoral dimensions and foramen radius to calculate femoral Qi during the in-pouch and post-pouch life stages of western grey kangaroos (Macropus fuliginosus) weighing 5.7 g to 70.5 kg and representing a 12,350-fold range in body mass. A biphasic scaling relationship between Qi and body mass was observed (breakpoint at ca. 1-5 kg body mass right before permanent pouch exit), with a steep exponent of 0.96±0.09 (95% CI) during the in-pouch life stage and a statistically independent exponent of -0.59±0.90 during the post-pouch life stage. In-pouch joeys showed Qi values that were 50-100 times higher than those of adult diprotodont marsupials of the same body mass, but gradually converged with them as post-pouch adults. Bone modelling during growth appears to be the main determinant of femoral bone blood flow during in-pouch development, whereas bone remodelling for micro-fracture repair due to locomotion gradually becomes the main determinant when kangaroos leave the pouch and become more active.

Evaluation of medetomidine-alfaxalone and medetomidine-ketamine in semi-free ranging Bennett's wallabies (Macropus rufogriseus).

Twenty-six adult semi-free-ranging Bennett's wallabies were anesthetized. Animals in group MA received medetomidine 0.1 mg/kg and alfaxalone 4 mg/kg i.m. in a 5-ml dart, whereas those in group MK received medetomidine 0.1 mg/kg and ketamine 5 mg/kg i.m. in a 3-ml dart. Dosages were based on estimated body weights. The wallabies were allowed to recover spontaneously or, if still nonresponsive at the end of the procedure, were given atipamezole 0.5 mg/kg (half the dose via i.m. and the other half via i.v.). Heart rate and respiratory rate were monitored at 5-min intervals, temperature at 10-min intervals, and two arterial blood samples were taken for blood gas analysis. Statistical analysis was performed by using analysis of variance (P < 0.05). The use of 5-ml darts in group MA compared with 3-ml darts in group MK could potentially increase the risk of iatrogenic trauma and should be considered. Induction and maintenance of anesthesia were satisfactory in both groups. There were no significant differences between the groups in mean time to first effect, recumbency, and approach, or to time to sternal recumbency and standing after reversal with atipamezole. Although bradycardia was present in both groups, no statistical differences were calculated for respiratory rate and heart rate, whereas the mean cloacal temperature was significantly lower in group MA (P = 0.01). Mixed acid-base disturbances occurred in both groups. All but one animal in group MK needed atipamezole at the end of the procedure. No adverse effects were observed after recovery.

The effect of pregnant and oestrous females on male testosterone and behaviour in the tammar wallaby.

Tammar wallaby females (Macropus eugenii) are seasonally breeding marsupials with a post-partum oestrus after a highly synchronised birth period when testosterone concentrations rise in males. Chemical communication appears to be important for mating, as males show checking behaviour, sniffing the urogenital opening (UGO) and the pouch of females. This study investigates whether the presence of pregnant and oestrous females directly influences testosterone in males and if oestrous odours or secretion from the pouch or UGO are attractive. Concentrations of plasma testosterone were measured in males housed with pregnant and oestrous females during two consecutive cycles in the breeding season, and an artificially induced cycle in the non-breeding season. Males were also tested for their interest in swabs taken from the urogenital opening (UGO) or pouch of oestrous females. Testosterone increased sharply in males in the presence of pregnant and oestrous females during all cycles in both seasons, but there was no change when males were exposed to non-cycling females in lactational or seasonal diapause. Males had no preference for either oestrous or non-oestrous samples taken from the pouch or from the UGO from oestrous females. This study confirms that the increase in plasma testosterone in tammar males can be induced through the presence of pregnant and oestrous females, regardless of season and that the increase began when the females were in late-pregnancy. This confirms that the male's reproductive state is dependent on a signal from females and is not blocked through seasonal effects.

Haematology and blood chemistry in free-ranging quokkas (Setonix brachyurus): Reference intervals and assessing the effects of site, sampling time, and infectious agents.

Quokkas (Setonix brachyurus) are small macropodid marsupials from Western Australia, which are identified as of conservation concern. Studies on their blood analytes exist but involve small sample sizes and are associated with very little information concerning the health of the animals. Blood was collected from free-ranging quokkas from Rottnest Island (n = 113) and mainland (n = 37) Western Australia, between September 2010 and December 2011, to establish haematology and blood chemistry reference intervals. Differences in haematology and blood chemistry between sites (Rottnest Island v mainland) were significant for haematology (HMT, p = 0.003), blood chemistry (BLC, p = 0.001) and peripheral blood cell morphology (PBCM, p = 0.001). Except for alkaline phosphatase, all blood chemistry analytes were higher in mainland animals. There were also differences with time of year in HMT (p = 0.001), BLC (p = 0.001) and PBCM (p = 0.001) for Rottnest Island quokkas. A small sample of captive animals (n = 8) were opportunistically sampled for plasma concentrations of vitamin E and were found to be deficient compared with wild-caught animals. Fifty-eight of the 150 quokkas were also tested for the presence of Salmonella, microfilariae, Macropodid herpesvirus-6, Theileria spp., Babesia spp., trypanosomes, Cryptococcus spp. and other saprophytic fungi. All eight infectious agents were detected in this study. Infectious agents were detected in 24 of these 58 quokkas (41%), with more than one infectious agent detected for all 24 individuals. Salmonella were detected concurrently with microfilariae in 8 of these 24 quokkas, and this mixed infection was associated with lower values across all haematological analytes, with Salmonella having the greater involvement in the decreased haematological values (p < 0.05). There was no evidence for an effect of sex on HMT, BLC and PBCM. Our data provide important haematological and blood chemistry reference intervals for free-ranging quokkas. We applied novel methods of analyses to HMT and BLC that can be used more broadly, aiding identification of potential disease in wildlife.

The pharmacokinetics of single dose intramuscular amoxicillin trihydrate in tammar wallabies (Macropus eugenii).

Five tammar wallabies (Macropus eugenii) were injected intramuscularly with 10 mg/kg amoxicillin trihydrate. Serial blood samples were collected through to 26 hr postinjection. Plasma amoxicillin concentrations were measured using high-performance liquid chromatography. Pharmacokinetic parameters were estimated using noncompartmental analysis. The terminal half-life (1.77 +/- 0.40 hr) was comparable to that previously reported in domestic small ruminants. Without intravenous kinetic data, it is unclear whether the terminal phase is elimination- or absorption-dependent; both scenarios have been reported in domestic species. Plasma concentrations of amoxicillin remained above a reported minimum inhibitory concentration (MIC) breakpoint for staphylococci and streptococci for at least 8 hr; the MIC breakpoint for enterobacteria and enterococci was never attained.

Hematology and serum biochemistry of the brush-tailed rock-wallaby (Petrogale penicillata).

In Australia the brush-tailed rock-wallaby (Petrogale penicillata) is the subject of a national recovery plan, and several sites have been selected for reintroductions. Condition of wild populations and individual animals can be monitored using hematologic and serum biochemistry analytes, and hematologic variables have been correlated with postrelease survival in other species. Prior to such monitoring, reference values for blood variables are required, but these data have not been available for the brush-tailed rock-wallaby. During four trapping periods from November 2004 to August 2005, 116 blood samples were collected from 44 brush-tailed rock-wallabies in a wild colony in southeast Queensland. Some variables varied with sex, age, method of restraint, lactation demands, and trapping period. After partitioning, when required, reference ranges for hematology and serum biochemistry variables were established. This study provides the most comprehensive serum biochemistry reference range for any macropodid marsupial yet published.

Monkeypox zoonotic associations: insights from laboratory evaluation of animals associated with the multi-state US outbreak.

At the onset of the 2003 US monkeypox outbreak, virologic data were unavailable regarding which animal species were involved with virus importation and/or subsequent transmission to humans and whether there was a risk for establishment of zoonotic monkeypox in North America. Similarly, it was unclear which specimens would be best for virus testing. Monkeypox DNA was detected in at least 33 animals, and virus was cultured from 22. Virus-positive animals included three African species associated with the importation event (giant pouched rats, Cricetomys spp.; rope squirrels, Funisciuris sp.; and dormice, Graphiuris sp.). Virologic evidence from North American prairie dogs (Cynomys sp.) was concordant with their suspected roles as vectors for human monkeypox. Multiple tissues were found suitable for DNA detection and/or virus isolation. These data extend the potential host range for monkeypox virus infection and supports concern regarding the potential for establishment in novel reservoir species and ecosystems.

Culture and stimulation of tammar wallaby lymphocytes.

We describe the culture and stimulation of lymphocytes from the model marsupial, the tammar wallaby (Macropus eugenii). We also describe the capacity of tammar wallaby lymphocytes isolated from blood, spleen and lymph nodes to produce soluble immunomodulatory factors. Culture conditions were optimized for mitogen-driven stimulation using the plant lectin phytohaemagglutinin (PHA). Products secreted by stimulated cells were harvested and crudely fractionated before they were added back to freshly isolated lymphocytes. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay, both stimulatory and inhibitory bioactive factors were detected in serum-free supernatants harvested from mitogen-treated peripheral blood mononuclear cells. This paper describes the capacity of leukocytes of the tammar wallaby to respond to mitogenic stimulation and to produce soluble, low-molecular-weight bioactive molecules that possess cytokine-like activity.

Plasma endotoxin activity in Eastern grey kangaroos (Macropus giganteus) with lumpy jaw disease.

Progressive pyogranulomatous osteomyelitis involving the mandible or maxilla of captive macropods, referred to as "Lumpy jaw disease (LJD)", is one of the most significant causes of illness and death in captive macropods. The aim of the present study was to evaluate the relationship between the severity of LJD and plasma endotoxin activity in kangaroos. Plasma samples obtained from moderate (n=24) and severe LJD (n=12), and healthy kangaroos (n=46), were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. Plasma endotoxin activity was measured using the Limulus amebocyte lysate (LAL)-kinetic turbidimetric (KT) assay. Plasma endotoxin activity was higher in kangaroos with severe LJD (0.199 ± 0.157 EU/ml) than in those with moderate LJD (0.051 ± 0.012 EU/ml, P<0.001) and healthy controls (0.057 ± 0.028 EU/ml, P<0.001). Our results suggest that the severity of LJD in captive macropods may be related to the plasma endotoxin activity.

Effects of temperature and duration of sample storage on the haematological characteristics of western grey kangaroos (Macropus fuliginosus).

OBJECTIVE: To investigate the effects of storage duration and temperature on haematological analyses performed on blood from the western grey kangaroo (Macropus fuliginosis). METHOD: Blood samples from five western grey kangaroos were stored at 4 degrees C, 24 degrees C and 36 degrees C. Each sample was analysed haematologically over a 5-day period. RESULTS: The blood samples maintained optimal stability at 4 degrees C. At this temperature the haematological values remained essentially unchanged for the duration of the study, while samples stored at 36 degrees C and 24 degrees C showed significant changes in some haematological measures by 12 h and 48 h, respectively. Disturbances in leukocyte morphology were evident, to varying degrees, in all samples. CONCLUSIONS: Blood samples from macropodids should be tested within 48 h of collection if stored at a room temperature of about 24 degrees C. Where testing is to be delayed for more than 48 h, samples should be refrigerated as soon as possible. Exposure of samples to heat in excess of 24 degrees C should be avoided at all times.

On the clinical chemistry of the Bennett's wallaby (Macropus rufogriseus rufogriseus).

The following parameters were determined in blood serum of apparently healthy Bennett's wallabies (Macropus rufogriseus) using the Hitachi 917 (Roche Diagnostics, Mannheim, Germany) and/or the Vettest 8008 (IDEXX-GmbH, Woerrstadt, Germany): alkaline phosphatase, alanine aminotransferase, ammonia, alpha-amylase, aspartate aminotransferase, Ca, Cl, cholesterol, cholinesterase, creatine kinase, creatinine, gammaglutamyltransferase, glucose, iron, lactate dehydrogenase, magnesium, phosphate, potassium, protein, sodium, total bilirubin, triglyceride, and urea. The results for cholesterol, glucose, total protein, triglyceride and for the enzymes alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, gamma-glutamyltransferase and lactate dehydrogenase differed significantly between both methods (P < 0.05). There is a negative correlation between the age of the Bennett's wallabies and the activity of the alkaline phosphatase. Five protein fractions could be separated on cellulose acetate electrophoresis. The mean concentrations of fructosamine and beta-hydroxybutyrate were 447.3 micromol/L and 0.27 mmol/L, respectively. The estimated vitamin A intake had no influence on the vitamin A concentration in serum. The serum vitamin E concentration was in general low and vitamin E was below the detection limit of 0.82 micromol/L in 29 out of 42 serum samples. The use of these analytes is discussed concerning the knowledge about the physiology, nutrition and diseases of macropods.

Prothrombin time and activated partial thromboplastin time using a point-of-care analyser (Abaxis VSpro®) in Bennett's wallabies (Macropus rufogriseus).

There are few reports of coagulation times in marsupial species. Blood samples collected from 14 Bennett's wallabies (Macropus rufogriseus) under anaesthesia during routine health assessments were analysed for prothrombin time (PT) and activated partial thromboplastin time (aPTT) using a point-of-care analyser (POC) (Abaxis VSPro®). The wallabies had an aPTT mean of 78.09 s and median of 78.1 s. The PT for all wallabies was greater than 35 s, exceeding the longest time measured on the POC. Although PT was significantly longer, aPTT was similar to the manufacturer's domestic canine reference range.

Plasma endotoxin activity in kangaroos with oral necrobacillosis (lumpy jaw disease) using an automated handheld testing system.

The aim of the present study was to evaluate the reliability and effectiveness of directly determining endotoxin activity in plasma samples from kangaroos with lumpy jaw disease (LJD, n=15) and healthy controls (n=12). Prior to the present study, the ability of the commercially available automated handheld portable test system (PTS(TM)) to detect endotoxin activity in kangaroo plasma was compared with that of the traditional LAL-kinetic turbidimetric (KT) assay. Plasma samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTS(TM) was not significantly different from that of the traditional LAL-based assay. The data obtained using PTS(TM) correlated with those using KT (r(2)=0.963, P<0.001). These findings indicated that the PTS(TM) is applicable as a simplified system to assess endotoxin activity in macropods. In the present study, we demonstrated the diagnostic value of plasma endotoxin activity in kangaroos with systemic inflammation caused by oral necrobacillosis and identified plasma endotoxin activity as a sensitive marker of systemic inflammation in kangaroos with LJD. Based on ROC curves, we proposed a diagnostic cut-off point for endotoxin activity of >0.22 EU/ml for the identification of LJD. Our results indicate that the assessment of plasma endotoxin activity is a promising diagnostic tool for determining the outcome of LJD in captive macropods.

Effects of nutritional manipulation on body composition in the developing marsupial, Macropus eugenii.

When 60-day-old tammar wallaby pouch young (Macropus eugenii) are fostered to mothers at 120 days of lactation, their growth, developmental rate and maturation of their GH/IGF axes are markedly accelerated. To determine the effect of fostering on energy intake, body composition and fat accretion, we first measured total body fat and lean mass in these young. Next, we mimicked the triglyceride oleic and palmitic acid composition of 120-day milk by supplementing 60 day young with these fatty acids and comparing their growth with that of growth accelerated young. There was no difference in the weight or growth axis maturation of supplemented young but there was significantly more body fat in these and in the growth-accelerated fostered young than in controls. We conclude that the accelerated growth and GH/IGF axis maturation observed previously in fostered young is most likely due to increased milk consumption and earlier access to specific nutrients.

Purification, properties, partial sequence and evolutionary relationships of marsupial erythrocyte carbonic anhydrase.

Carbonic anhydrase (EC 4.2.1.1) has been purified from the erythrocytes of the tammar wallaby (Macropus eugenii desmarest). The enzyme was separated into four zones of activity. The three major individual forms were isolated as discrete entities. Comparison of substrate specificity, specific activities, kinetic constants and inhibition characteristics indicated that these heteromorphs represented minor post-translational modifications of a single gene product of carbonic anhydrase II type. Double-immunodiffusion and peptide mapping confirmed this proposition. The marsupial enzyme exists as a monomer with a molecular weight of about 29 000 containing one atom of zinc per mole which is much more tightly bound to the enzyme than it is in either human carbonic anhydrase I or II. The wallaby enzyme was, like human carbonic anhydrase II, partially inactivated by p-hydroxymercuribenzoate under conditions not inhibitory for human carbonic anhydrase I. The partial sequence of 51 residues of cyanogen-bromide peptides was sufficiently homologous to allow unambiguous overlap with the sequence of both human carbonic anhydrase I and II isozymes as well as with the recently published sequence of an apparent type I-like enzyme from the turtle. It is clear that the single wallaby erythrocyte carbonic anhydrase belongs to the class of separately evolving type II isozymes which have previously been defined only for placental mammals.

Plasma cholinesterase activity of rats, western grey kangaroos, alpacas, sheep, cattle, and horses.

INTRODUCTION: Plasma cholinesterase activity levels of various species may be of interest to toxicologists or pathologists working with chemicals that interfere with the activity of plasma cholinesterase. METHODS: We used a pH titration method to measure the plasma cholinesterase activity of six mammalian species. RESULTS: Plasma cholinesterase activity varied up to 50-fold between species: sheep (88 ± 45 nM acetylcholine degraded per ml of test plasma per minute), cattle (94 ± 35), western grey kangaroos (126 ± 92), alpaca (364 ± 70), rats (390 ± 118) and horses (4539 ± 721). DISCUSSION: We present a simple, effective technique for the assay of plasma cholinesterase activity levels from a range of species. Although labour-intensive, it requires only basic laboratory equipment.

Immunology of pouch young marsupials. I. Levels of immunoglobulin transferrin and albumin in the blood and milk of euros and wallaroos (hill kangaroos: Macropus robustus, marsupialia).

The concentration of total protein, albumin, transferrin, and immunoglobulin G of adult serum, pouch young serum, milk whey and colostrum has been estimated in three species of kangaroos, Macropus robustus, Macropus rufus (= Megaleia rufa) and Macropus giganteus. No study of this kind has previously been published for any marsupial species. The three individual proteins were antigenically identical in all four kinds of fluid. Colostrum and milk whey are relatively enriched in transferrin but have low levels of immunoglobulin G. Serum concentrations of total protein, albumin and transferrin rise steadily throughout pouch life and attain adult values when the young finally leaves the pouch. Serum concentrations of immunoglobulin G are very low for the first 90-100 days of pouch life, being approximately half of those in milk whey for this period. After this the level rises rapidly and also reaches adult values when the young leaves the pouch. We suggest that in the first 90-100 days the pouch young is largely protected humorally by passive immunity acquired from the mother, and after this it increasingly makes its own responses.

Difference in concentration of progesterone in plasma between pregnant and non-pregnant quokkas (Setonix brachyurus).

The plasma progesterone concentrations during pregnancy and the oestrous cycle of the quokka were measured daily after each had been initiated by the removal of pouch young. Progesterone levels ranged from 0.6 ng/ml in the early stages of the oestrous cycle to about 2.5 ng/ml at the peak of the luteal phase. There was no significant difference between pregnant and non-pregnant states before the removal of the pouch young nor in the latter half of the cycle. However, the plasma progesterone concentration on days 3--4 after removal of the pouch young was significantly greater in pregnant animals when compared with non-pregnant animals at the same stage and also when compared with the levels before removal of young. This early peak in the concentration of progesterone in peripheral plasma is discussed in relation to the development of the previously dormant blastocyst.