Neosporosis in a captive Parma wallaby (Macropus parma)

Infection with Neospora caninum has been diagnosed in a variety of animal species; however, reports in marsupials are rare. A captive Parma wallaby (Macropus parma) died suddenly and was subjected to necropsy examination. The main finding was necrotizing myocarditis associated with protozoan parasites. The protozoa were identified as N. caninum by use of immunohistochemistry and partial gene sequence analysis. Neospora and Toxoplasma should be considered a possible cause of disease in captive marsupials. Further work is required to determine whether marsupials are an accidental or terminal host of this protozoan in order to better understand the host-parasite relationship.     

Haematological Changes in the Tammar Wallaby (Macropus eugenii) Following Intraperitoneal Administration of Lipopolysaccharide

Intraperitoneal injection of lipopolysaccharide promoted a cellular haematological response in the peripheral blood of tammar wallabies. The changes in the treated wallabies were characterised by a decrease in the total leucocyte concentration from the baseline value at the 2 and 4h time-points and a mild increase in neutrophil concentration at 24h. Morphological changes in neutrophils including: increased numbers of neutrophils with a hyposegmented nucleus and cytoplasmic abnormalities were evident. No significant changes were observed in the fibrinogen concentration or serum electrophoretograms. As the changes in leucocyte concentration of tammar wallabies administered LPS were transient and mild, assessment of leucocyte morphology should be included in any laboratory investigations of macropodid health. Correspondence and offprint requests to: P. Clark, School of Clinical Sciences, Division of Veterinary and Biomedical Sciences, Murdoch University, South St, Murdoch, Western Australia, 6150. e-mail: pclark@murdoch.edu.au     

A longitudinal study of changes in blood leukocyte numbers in the tammar wallaby, Macropus eugenii

Changes in leukocyte numbers were monitored over a 3-year period in a small group of captive tammar wallabies, Macropus eugenii, maintained in the animal research facilities at Macquarie University (NSW, Australia). The neutrophil to lymphocyte ratio (N/L), a commonly used parameter in the assessment of health status in wildlife populations, was not useful when applied between animal populations but did reliably predict changes within individual animals and between animals within the study cohort. This study also demonstrated the importance of obtaining haematological values from animals on more than one occasion to ensure that differential cell counts from asymptomatic individuals do not unduly influence the determination of reference values.     

Ultrastructure and molecular histology of rabbit hind-limb collateral artery growth (arteriogenesis)

Previous studies in the canine heart had shown that the growth of collateral arteries occurs via proliferative enlargement of pre-existing arteriolar connections (arteriogenesis). In the present study, we investigated the ultrastructure and molecular histology of growing and remodeling collateral arteries that develop after femoral artery occlusion in rabbits as a function of time from 2 h to 240 days after occlusion. Pre-existent arteriolar collaterals had a diameter of about 50 μm. They consisted of one to two layers of smooth muscle cells (SMCs) and were morphologically indistinguishable from normal arterioles. The stages of arteriogenesis consisted of arteriolar thinning, followed by transformation of SMCs from the contractile- into the proliferative- and synthetic phenotype. Endothelial cells (ECs) and SMCs proliferated, and SMCs migrated and formed a neo-intima. Intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) showed early upregulation in ECs, which was accompanied by accumulation of blood-derived macrophages. Mitosis of ECs and SMCs started about 24 h after occlusion, whereas adhesion molecule expression and monocyte adhesion occurred as early as 12 h after occlusion, suggesting a role of monocytes in vascular cell proliferation. Treatment of rabbits with the pro-inflammatory cytokine MCP-1 increased monocyte adhesion and accelerated vascular remodeling. In vitro shear-stress experiments in cultured ECs revealed an increased phosphorylation of the focal contacts after 30 min and induction of ICAM-1 and VCAM-1 expression between 2 h and 6 h after shear onset, suggesting that shear stress may be the initiating event. We conclude that the process of arteriogenesis, which leads to the positive remodeling of an arteriole into an artery up to 12 times its original size, can be modified by modulators of inflammation. Received: 16 March 1999 / Accepted: 4 August 1999     

Immunocytochemical and ultrastructural characterization of somatolactin cells from the gilthead sea bream ( Sparus aurata L., Teleostei): an ontogenic study (from newly hatched to adults)

For the first time, somatolactin (SL) cells have been ultrastructurally identified and characterized during the ontogeny of gilthead sea bream, Sparus aurata, using specimens ranging in age from hatching to 15 months. The SL cells were identified by an immunogold method using anti-cod SL serum. The SL-immunoreactivity was mostly located on the secretory granules of the cells, although some vesicles of variable size and shape with a medium electron-dense content, and some irregular secretory granules and polymorphic or very irregular masses that can arise from the fusion of several secretory granules, also presented immunogold labeling. In adults, the SL cells were mainly found in the pars intermedia, where they were organized in discontinuous cell cords lying against the neurohypophysis or surrounding the neurohypophyseal branches. Some SL cells, however, appeared isolated or in small groups in the pars intermedia, in the proximal pars distalis and, rarely, in the rostral pars distalis. The SL cells were variable in shape, with processes directed towards the neurohypophysis or blood vessels, or intermingling among other adenohypophyseal cells. The secretory granules were mostly round, although some were oval, bilobate or pear-shaped, with a homogeneous, very electron-dense content and a narrow, dense or clear, halo. Different SL cell populations can be distinguished according to secretory granule size. Our findings indicate that SL is stored in the secretory granules and released by exocytosis. SL cells showing involutive features were only found in adults. SL cells can be ultrastructurally identified in one-day-old larvae although similar characteristics to those found in adults can be positively identified only after 4 days. Secretory granules increased in number, size and heterogeneity during development. Synaptic-like structures between axon terminals of the neurohypophysis and the SL cells were found in larvae from one-day-old onwards. In juveniles of 118 days of age, two different populations of secretory granules (immunogold-labeled and non-immunogold-labeled) can be found in the same or different SL cells, findings that suggest the existence of two different molecular forms of SL at this age. There was a clear increase in the complexity of the pituitary gland and in the heterogeneity of the SL cells during development, the latter observation probably reflecting different functional cell stages or production of SL molecules. Accepted: 22 January 2001     

Development of the penis and clitoris in the tammar wallaby, Macropus eugenii

 The development of the phallus from the indifferent stage to sexual dimorphism has not been described in any marsupial. This study describes the morphological and histological changes occurring in the development of the phallus of the tammar wallaby. The development of the penis and clitoris in the tammar closely follow the most widely accepted model for the development of the same organs in eutherian mammals. The urogenital plate that is present in both sexes at birth hollows out to form a urogenital groove at approximately 70 days postpartum (p.p.). There is then greater growth of the phallus in males than in females, which results in sexual dimorphism in length approximately 100 days p.p. In males, the urogenital groove secondarily closes over at this time and fuses in the midline and by 128 days p.p. the penile urethra is fully formed. In females, the groove remains open. The clitoris changes little morphologically from the time of formation of the urogenital groove until adulthood. The pattern of development of the penis in the tammar is similar to that seen in eutherian mammals. There is strong evidence that penis development is androgen-dependent in the tammar, yet unusually it becomes sexually dimorphic at a time when androgen content of the developing testis is low. Accepted: 10 November 1998     

Ultrastructural study of the sinus venosus in embryos of the dogfish ( Scyliorhinus canicula )

 An ultrastructural study of the development of the sinus venosus has been carried out on seven embryos of the dogfish (Scyliorhinus canicula L.) between 10.5 and 69 mm of total length (TL). The sinus venosus appears at the end of the looping process of the cardiac tube, namely in the 10.5 mm embryo, when the heart reaches its adult tetracameral S-form. The endocardium of the smallest embryo is constituted of a single layer of cubic cells. In larger embryo, these cells progressively acquire a squamous appearance as well as electron-dense cytoplasmic inclusions. The subendocardium is progressively populated by ganglion cells, Schwann cells and bundles of amyelinic fibers that can first be recognised in the embryo of 34 mm TL. Some subendocardial mesenchymal cells located in earlier embryos close to the entrance of the ducts of Cuvier might be their ectomesenchymal progenitors. The myocardium is initially constituted of a single layer of cubic cells. In the embryos of 19, 27 and 34 mm TL, the myocardium becomes multilayered, and the myocardiocytes develop myofibrils randomly arranged throughout the sarcoplasm. In later embryos, the myocardiocytes are innervated and arranged in oval bundles surrounded by a basal lamina. The epicardium covers the sinus venosus by the retrograde migration of the epithelium already established around the atrioventricular groove and, in a lesser degree, by the adhesion of mesothelial cells that are floating free in the pericardial cavity. This process has finished in the embryo of 34 mm TL. The differentiation of the sinus venosus (including the endocardial and myocardial differentiation as well as the epicardial covering) progresses in an anteroventral-posterodorsal direction. Accepted: 12 June 1998     

Haematological response to naturally occurring disease in the western quoll (Dasyurus geoffroyi)

Haematological data from clinically ill western quolls was retrospectively analysed. A lymphocytosis was the most commonly encountered haematological abnormality, observed in 17/23 animals. In contrast, a neutrophilia was uncommon, present in only 1/23 cases. Two animals exhibited a polycythaemia, four animals exhibited a mild anaemia and one animal a severe anaemia. The last exhibited a marked regenerative response with 12% polychromatophilic erythrocytes.     

Some Haematological Values of Farmed Fallow Deer (>Dama dama)

Haematological examinations have been carried out on 62 healthy farmed male and female fallow deer after they had been shot during a routine cull. Comparisons were made between the sexes, between young (up to 2 years) and older (>2 years) animals, and between age groups according to sex. The following parameters (means and SDs) were examined on all samples: erythrocyte count, haemoglobin concentration, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, total leucocyte count, and numbers of neutrophils, eosinophils, basophils, lymphocytes, band neutrophils and monocytes. Only minor differences in red cell and white cell parameters between different groups of animals were found. However, significant differences in mean cell volume, mean cell haemoglobin concentration, eosinophils and band neutrophils were discovered in different age and sex groups. The values provide useful information for diagnostic purposes in fallow deer. Correspondence and offprint requests to: G. Vengušt, Institute for Breeding and Health Care of Wild Animals, Fishes and Bees, University of Ljubljana, Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia. e-mail: vengusgo@vf.uni-lj.si     

Reproduction in male swamp wallabies (Wallabia bicolor): puberty and the effects of season

This study describes pubertal changes in testes and epididymides and seasonal changes in the adult male reproductive organs and plasma androgen concentrations of the swamp wallaby (Wallabia bicolor). Pre-pubescent males had testes with solid seminiferous cords and spermatogenesis only to the stage of gonocytes. Their epididymides had empty lumina along their entire length. The testes of three males undergoing puberty had some lumen formation and mitotic activity. Their epididymides were similar in appearance to those of adult males but were entirely devoid of any cells within the lumen of the duct. Three other pubescent males showed full lumen formation in the testes and spermatogenesis up to the elongating spermatid stage. Their epididymides were similar in appearance to those of adult males but with no spermatozoa in the duct. However, cells of testicular origin were found in the lumen of the duct in all regions suggesting that testicular fluids and immature germ cells shed into the rete testes flow through the seminiferous tubules into the epididymis before the release of mature testicular spermatozoa. The weights of testes and epididymides of adult males showed no change throughout the year but prostate weight and plasma androgen concentrations varied significantly with season, with maximums in spring and summer and minimums in winter. The volume fraction of Leydig cells and seminiferous tubules was significantly lower in winter than in summer; but, despite this, maturing spermatozoa were found in the testes throughout the year. Females in the area conceived year-round, suggesting that seasonal changes in the male reproductive tract did not prevent at least some males from breeding throughout the year.     

Light and electron microscopic examination of endothelial cells from bovine retinal vessels in long-term cultures

The aim of the present work was to examine and compare the ultrastructure of bovine retinal endothelial cells (BRECs) in vitro during several passages in a medium selective for endothelial cells. The identity of the endothelial cells was confirmed immunohistochemically, up to the tenth passage. Changes in their ultrastructure in comparison to endothelial cells in vivo occurred at the onset of culturing and not progressively with repeated passages. The cultured BRECs show high metabolic activity in all passages. While retaining their identity as endothelial cells, they modify their lipid metabolism, so that lipids are stored. This change in lipid metabolism was induced by the medium. Received: 19 May 1999 / Accepted: 4 August 1999     

Salivary epithelial cells in the peripheral blood films of two yellow-footed rock wallabies ( Petrogale xanthopus )

Epithelial cells were observed in the blood films of two yellow-footed rock wallabies (Petrogale xanthopus). These cells originated from the parotid salivary gland which overlies the cranial jugular vein in macropodids. Inadvertent sampling of the parotid salivary gland may occur when performing venepuncture of the jugular vein in macropodids.     

Theoretical Analysis of Drug Treatment in Haematological Disease Using Lanchester (Osipov) Linear Law

The Lanchester linear law <$>{\rm dx\over dt}=-Dxy<$><$>/{\rm dy\over dt}=-Axy<$> was used theoretically to analyse the interaction between drugs and cancer cells. The simulations were conducted with respect to different killing rates, different initial amounts of drug, different initial numbers of cancer cells, different half-lives of drugs in vivo, different production rates of survived cancer cells and different dose regimens. The results do not support the division of a dose of drug either homogeneously or heterogeneously.     

Osmotic Fragility and Erythrocyte Size in Iguana iguana (Sauria – Iguanidae) in Captivity

The erythrocyte size and osmotic fragility were studied in blood samples from adult (n= 40) and juvenile (n= 40) specimens of Iguana iguana. In fresh preparations the erythrocytes were large, oval cells. The largest diameters were 17.06 ± 2.5 mm (juvenile) and 16.20 ± 1.25 mm (adults), and the smallest diameters were 8.23 ± 1.87 mm (juvenile) and 9.00 ± 1.00 mm (adults). In fixed and stained preparations, the largest erythrocyte diameters were 15.28 ± 3.3 mm (juvenile) and 15.51 ± 1.3 mm (adults), and the smallest were 7.82 ± 0.65 mm (juvenile) and 7.85 ± 0.8 mm (adults). The haematocrit value for both juvenile and adult specimens was 27 ± 2%; the red blood cell counts were 1.3 ± 0.43×10¹²/l (juvenile) and 1.2 ± 0.35×10¹²/l (adults). Although no significant differences were observed in the cumulative osmotic curves, the derivative curve of adult specimens indicates the presence of at least two erythrocyte populations with osmotic fragilities at about 70 and 60 mm NaCI, representing 27% and 73% of the total cells, respectively. In samples from juvenile specimens, a major peak at about 70 mm NaCI was observed, which represented 85% of the total cell population. The difference in osmotic resistance between these erythrocyte subpopulations is correlated with their respective geometrical parameters, and compared to that of erythrocytes from other vertebrates.     

A study of the distribution of total, free, short-chain acyl and long-Chain acyl carnitine in whole blood and plasma of arabian sand gazelles (Gazella subgutturosa marica)

This study was conducted to investigate the presence of total carnitine, free carnitine, short-chain acyl carnitine, long-chain acyl carnitine, acyl carnitine levels and the ratio of acyl carnitine to free carnitine in erythrocytes, whole blood and plasma of Arabian sand gazelles (Gazella subgutturosa marica). Results showed erythrocyte levels of carnitine significantly higher compared to either whole blood or plasma of Arabian sand gazelles (p<0.001). Moreover, the relative distribution of acyl carnitine was higher in erythrocytes compared to most species (p<0.001), however, it was similar to the Arabian camel (p<0.001). The ratio of acyl carnitine to free carnitine was significantly higher in erythrocytes, whole blood and plasma compared to most species (p<0.001). The observed discrepancies in carnitine levels among species and different tissues is well known, however, the reasons for these discrepancies are obscure. The higher carnitine content and a higher acyl carnitine to free carnitine ratio in blood of Arabian sand gazelles may suggest that these animals possess an adaptive mechanism which may be found in other desert animal species. This is the first report describing deer carnitine distributions.     

Evaluation of the Cell-Dyn 3500 Haematology Analyser for Bovine Blood

A complete blood count provides critical information for disease diagnosis as well as prophylaxis in herd health programmes. During the last 30 years, automated haematology analysers have increasingly replaced microscopic methods. Because most haematology analysers are made for human blood, adjustments are required for the examination of blood from domestic animal species. A total of 399 blood samples, collected from cows immediately prior to parturition to 5 days post partum, were analysed using the blood analyser Cell-Dyn 3500 (Abbott Diagnostica, Delkenheim, Germany). Results of erythrocyte, platelet and leucocyte counts as well as haematocrit were compared with results obtained using reference methods. The two parameters, carry-over and within-batch precision, were within the tolerated ranges. High correlations were calculated for erythrocytes (r=0.859), leucocytes (r=0.960), haematocrit (r=0.968) neutrophils (r=0.973), eosinophils (r=0.876) and lymphocytes (r=0.802). Moderate correlations were calculated for platelets (r=0.706) and monocytes (r=0.578) whereas no meaningful correlation could be established for basophilic granulocytes. Correspondence and offprint requests to: Dr Ulrich Bleul, Klinik für Fortpflanzungskunde, Universit?t Zürich, Winterthurerstrasse 260, 8057 Zürich, Switzerland.     

Development of the digestive tract of sea bass (Dicentrarchus labrax L). Light and electron microscopic studies

The developing gut of sea bass was studied by light and electron microscopy, four phases being established. Phase I, from hatching to the opening of the mouth, was a lecitotrophic period, in which the gut appeared as a straight undifferentiated tube lined by a simple epithelium that became stratified in the most caudal region. The epithelial cells increased in length towards the caudal zone, as did the number and height of the apical microvilli and the magnitude of the lamellar structures in their basal region. Cilia were more numerous in the caudal region than in the rest of the gut. Signs of lipid but not of protein absorption were found in the epithelial cells at this phase. Phase II, from the opening of the mouth to the complete resorption of the yolk sac, was a lecitoexotrophic period in which an esophagus, a gastric region, an intestine and a rectum, the last two separated by a valve, were present. During this phase the differentiation of the gut started at the esophagus and the rectum. In the esophagus, the epithelium became stratified and goblet cells containing acid mucosubstances, including sulphomucins, appeared. In the epithelial cells of the rectum, supranuclear vacuoles and an incipient endocytotic apparatus that seemed to be involved in the absorption and digestion of proteins were found. In both regions the mucosa was folded. Phase III, from the complete resorption of the yolk sac to the appearance of the first gastric glands, initiated the exclusively exotrophic period. During this phase the intestine formed the mucosa folds, while the first pyloric caeca and the epithelial cells acquired the ultrastructural features of mature absorptive cells with many lipid inclusions. Goblet cells containing neutral mucosubstances appeared and increased in number in both the intestine and the rectum. Neutral mucosubstances were also present in the cells lining the gastric region. During phase IV, from the appearance of the first gastric glands onwards, the intestinal absorptive surface increased with the formation of new pyloric caeca and two intestinal loops. The stomach acquired its definitive anatomy and histology with the development of the caecal and pyloric regions alongside differentiated gastric glands. The glandular cells had the ultrastructural features of the cells that secrete both pepsinogen and hydrochloride acid in the adult teleost stomach. Accepted: 7 February 2001     

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous cAMP-dependent protein kinase

 Single Ca²⁺-activated K⁺ channels of human erythrocytes were studied with the patch-clamp technique, to identify the mechanisms of their modulation by phosphorylation. In the cell-attached configuration, the openings of these channels were infrequent, as expected by the low cell Ca²⁺ content. After patch excision, the activity increased to levels determined by the Ca²⁺ concentration (0.5–10 μM) in the bath solution, then the channel activity ran down within a few minutes, to reach values of open probability lower than 0.10. The perfusion of the patch with MgATP increased the channel activity, with delayed and variable effects. Furthermore, the application of a mixture of cAMP (1 mM), MgATP (1 mM) and theophylline (1 mM) to the cytoplasmic side of excised patches led to dramatic enhancement of channel activity, which appeared within 20–120 s and decayed in tens of seconds after wash-out. The activation of the channel by the mixture was reversibly blocked by PKI_5–24, a peptide inhibitor specific to cAMP-dependent protein kinase (PKA). The level of activation promoted by cAMP and ATP was dependent on the Ca²⁺ concentration in the bathing solution. These results provide direct evidence that an endogenous PKA modulates the calcium sensitivity of Ca²⁺-activated K⁺ channels of human erythrocytes. Received: 19 February 1998 / Received after revision: 14 April 1998 / Accepted: 20 April 1998     

Haematological characteristics of wild quokka (Setonix brachyurus)

Blood was collected from quokkas (Setonix brachyurus), from a mainland and an island population, and analysed as part of an assessment of animal health. Mean corpuscular haemoglobin concentration was the only analyte that was significantly different between the mainland and island populations. Quokkas from both the wild populations had lesser erythrocytic values than the captive animals. The wild quokkas also possessed three types of haemoparasites that were not recognised in the captive animals. This study illustrates the differences in the haematological characteristics between wild and captive animals.     

Experimental proof of contamination of blood components by (1→3)-β-D-glucan caused by filtration with cellulose filters in the manufacturing process

 The level of (1→3)-β-D-glucan in blood is a diagnostic index of fungal infection because it is released from the fungal cell wall. However, high levels of plasma (1→3)-β-D-glucan in patients administered blood components may give false positive results. High levels of (1→3)-β-D-glucan have been detected in blood components. We suspected that (1→3)-β-D-glucan from cellulose filters had been eluted into blood components by filtration in the manufacturing process. To investigate the contamination of blood components by (1→3)-β-D-glucan from cellulose filters, in vitro experiments were performed by using six cellulose filters and a nylon filter. Human serum albumin (HSA) solution (100 ml) was flowed through each filter after rinsing with 100 ml of distilled water, and (1→3)-β-D-glucan in each fraction was determined by Fungitec G test MK. The concentration of (1→3)-β-D-glucan eluted from cellulose filters in 100-ml distilled water fractions ranged from 6 to 207 pg/ml, and that of HSA fractions ranged from 33 to 20,784 pg/ml. These data showed that remarkably higher (1→3)-β-D-glucan levels were detected in HSA fractions flowed through cellulose filters in spite of advance rinsing with 100 ml of distilled water. In the case of a nylon filter, (1→3)-β-D-glucan was not eluted in either fraction. These results indicate that (1→3)-β-D-glucan contamination in blood components is caused by filtration with cellulose filters in the manufacturing process. Received: March 11, 2002 / Accepted: January 23, 2003 Acknowledgments We are grateful to Makoto Miyazaki and Hiroyuki Kanazawa of Seikagaku Co. for providing Fungitec G test MK and information on the determination of (1→3)-β-D-glucan. We are also grateful to Cuno, Seitz-filter-Werke, Millipore, Pall, and Baxter for providing materials. Correspondence to:K. Nagasawa