Gut microbiota of the European Brown Hare (Lepus europaeus).

Diseases of the gastrointestinal tract due to changes in the bacterial flora have been described with increasing incidence in the European brown hare. Despite extensive demographic and phylogeographic research, little is known about the composition of its gut microbiota and how it might vary based on potential environmental or host factors. We analysed the intestinal and faecal microbiota of 3 hare populations by Illumina MiSeq 16S rRNA gene amplicon sequencing. The phyla and OTU abundance composition differed significantly between intestinal and faecal samples (PERMANOVA: P = 0.002 and P = 0.031, respectively), but in both sample types Firmicutes and Bacteroidetes dominated the microbial community composition (45.51% and 19.30% relative abundance). Intestinal samples contained an enrichment of Proteobacteria compared with faecal samples (15.71-fold change, P < 0.001). At OTU level, a significant enrichment with best BLAST hits to the Escherichia-Shigella group, Eubacterium limosum, Sphingomonas kyeonggiensis, Flintibacter butyricus and Blautia faecis were detected in intestinal samples (P < 0.05). In our statistical model, geographic location and possibly associated environmental factors had a greater impact on the microbiota composition than host factors. Population had a significant effect on the composition of abundant intestinal and faecal OTUs, and on the abundance of potential pathogenic bacteria of the family Enterobacteriaceae, regularly associated with intestinal dysbiosis in hares, in faecal samples. Our study is the first to describe the microbiota in brown hares and provides a foundation to generate hypothesis aiming to test the role of gut health in population fluctuations of the species.

Multiple strain infections and high genotypic diversity among Mycobacterium avium subsp. paratuberculosis field isolates from diseased wild and domestic ruminant species in the eastern Alpine region of Austria.

Johne's disease, or paratuberculosis, is a chronic fatal ruminant gastroenteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP) whose foodborne zoonotic potential and association with Crohn's disease are still under debate. The disease is widespread but its epidemiology and epizootiology remains elusive. Wildlife is suspected to play a major role. After a surge in MAP seroprevalence in Austrian cattle, paratuberculosis was declared a notifiable disease in Austria in 2006. At the same time a rise in MAP cases in wild ruminant populations in the Austrian province of Styria was reported. All five autochthonous ruminants were affected. Genetic analysis of isolates, yielded numerous genotypes (>15) and several multiple strain infections (15%) across host species. Identical MIRU-VNTR profiles were identified in different species and sampling locations. On the other hand varying MIRU-VNTR profiles were revealed at the same location and in conspecifics. Our data, taken together with earlier epidemiological studies on MAP and other mycobacteria, raised concerns about the organisms' ecology. Constraints regarding in vitro culture of this highly fastidious organism potentially bias our current understanding of its epidemiology. We suggest that MAP infections could be polyclonal and question the informative value of genotyping a single MAP colony derived from a single specimen for epidemiological analysis of MAP.

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.

An electrochemical impedance spectroscopy (EIS) assay measuring the calcification inhibition capacity in biological fluids.

Pathological calcification of the cardiovascular system is one of the major causes of high mortality and morbidity in dialysis patients. The inhibition of ectopic calcification relies (I) on the formation of calciprotein particles (CPPs), nanospherical complexes of calcium phosphate mineral, fetuin-A and other acidic serum proteins, and (II) on the stabilization of calcium phosphate prenucleation clusters by fetuin-A monomers. In supersaturated serum, mineral ion aggregation leads to a change in the electrical impedance. In this work, we present a method based on electrochemical impedance spectroscopy (EIS) to establish an impedance trace of mineral ion clustering in vitro. In the presence of 20 µM of serum protein fetuin-A, a prototypic calcification inhibitor, we measured a change in impedance (Δ(R)) of 195.52 ± 27.78%Ω compared to 430.41 ± 11.36%Ω in inhibitor-free samples. We also identified a CPP-formation dependency on the actual content of ions and protein in the samples under investigation. Two-step ripening of CPP was also observed. The presented method may form the basis of a simple label-free bedside or online test to be used in routine clinical practice for estimating the calcification risk in serum.

Validation of a short-term memory test for the recognition of people and faces.

Memorising and processing faces is a short-term memory dependent task of utmost importance in the security domain, in which constant and high performance is a must. Especially in access or passport control-related tasks, the timely identification of performance decrements is essential, margins of error are narrow and inadequate performance may have grave consequences. However, conventional short-term memory tests frequently use abstract settings with little relevance to working situations. They may thus be unable to capture task-specific decrements. The aim of the study was to devise and validate a new test, better reflecting job specifics and employing appropriate stimuli. After 1.5 s (short) or 4.5 s (long) presentation, a set of seven portraits of faces had to be memorised for comparison with two control stimuli. Stimulus appearance followed 2 s (first item) and 8 s (second item) after set presentation. Twenty eight subjects (12 male, 16 female) were tested at seven different times of day, 3 h apart. Recognition rates were above 60% even for the least favourable condition. Recognition was significantly better in the 'long' condition (+10%) and for the first item (+18%). Recognition time showed significant differences (10%) between items. Minor effects of learning were found for response latencies only. Based on occupationally relevant metrics, the test displayed internal and external validity, consistency and suitability for further use in test/retest scenarios. In public security, especially where access to restricted areas is monitored, margins of error are narrow and operator performance must remain high and level. Appropriate schedules for personnel, based on valid test results, are required. However, task-specific data and performance tests, permitting the description of task specific decrements, are not available. Commonly used tests may be unsuitable due to undue abstraction and insufficient reference to real-world conditions. Thus, tests are required that account for task-specific conditions and neurophysiological characteristics.

Detection of Cryptosporidium spp., Entamoeba spp. and Monocercomonas spp. in the gastrointestinal tract of snakes by in-situ hybridization.

This report describes the development of a diagnostic method for protozoal infections of the gastrointestinal tract of captive snakes, based on chromogenic in-situ hybridization with probes designed for the detection of 18S rRNA genes from Cryptosporidium spp., Entamoeba spp., Entamoeba invadens and Monocercomonas spp. The specificity of the probes was confirmed with the help of parasitic cultures and gene sequence analysis. The probes gave clear positive signals. Of 182 snakes examined, seven were positive with the Cryptosporidium probe, 13 with the Entamoeba probe (of which nine were also positive with the E. invadens probe), and 34 with the Monocercomonas probe.

Structural dynamics of a colloidal protein-mineral complex bestowing on calcium phosphate a high solubility in biological fluids.

The concentration of mineral solutes in mammalian blood is considerably higher than that predicted by their solubility product. The plasma protein fetuin-A inhibits calcium phosphate deposition by forming colloidal calciprotein particles (CPPs). In this article the authors present a detailed small angle neutron scattering study including contrast variation analysis providing detailed quantitative information on the three-dimensional topology of the CPPs and on their morphogenesis. In detail the authors found the following: (i) A two stage growth process showing spontaneously formed primary particles with a size of about 500 A diameter that subsequently transformed to 1000 A sized particles which were stable for at least 24 h. (ii) A particular shielding topology was observed for the second CPP state, namely, that a densely packed fetuin-A monolayer covers a mineral core and thereby prevents further crystal growth. (iii) Transmission electron microscopy analysis of in vitro synthesized second state CPPs revealed striking similarities to material retrieved from a human peritonitis patient. This latter finding underscores the importance of short- and long-term stabilizations of CPPs by fetuin-A to enable clearing of mineral debris in the body.

[Aortoduodenal fistula as a cause of gastrointestinal bleeding--difficulties in endoscopic diagnosis]. / Aortoduodenale Fistel als Ursache der gastrointestinalen Blutung - eine endoskopisch schwierige Differenzialdiagnose.

We report on three patients with severe gastrointestinal bleeding arising from aortoenteric fistula. Two patients presented with a secondary aortoduodenal fistula. In the first case bleeding occurred 8 months after aortobifemoral graft implantation. In the second patient aortobiliacal graft implantation was performed 22 years before. In the third case the aortoenteric fistula was primary and was caused by an abdominal aortic aneurysm without prior vascular intervention. In the first case diagnosis was made by urgent endoscopy visualizing ongoing bleeding from the duodenal fistula. In the two other patients urgent endoscopy and CT as well could not demonstrate the bleeding source. Aortoenteric fistula was diagnosed endoscopically during severe rebleeding some hours later. Two patients underwent surgery with implantation of an axillobifemoral bypass; the third patient declined further intervention and died. The course shows that aortoduodenal fistula can present with severe but intermittent gastrointestinal bleeding making the diagnosis in the non-bleeding interval difficult. In patients with severe gastrointestinal bleeding and a history of aortic disease (aneurysm, prior aortic graft repair or stenting) an aortoduodenal fistula should be suspected and the indication for surgical intervention should be considered early in spite of negative results of endoscopy and CT.

Toxoplasmosis in Pallas' cats (Otocolobus manul) raised in captivity.

Manuls or Pallas' cats (Felis manul, syn. Otocolobus manul) are endangered wild cats from Central Asia kept and bred in many zoos. Despite good breeding success young cats frequently die from acute toxoplasmosis. From 1998 to 2002, a breeding pair in the Schönbrunn Zoo in Vienna, Austria, gave birth to 24 kittens; 58 % of kittens died between the 2nd and the 14th week of life, mostly due to acute toxoplasmosis. The epidemiology of toxoplasmosis in Pallas' cats was examined and a control strategy to protect the kittens from fatal toxoplasmosis was developed. One 12-week-old kitten from a litter of 6 born in 2001 died of generalized toxoplasmosis. This kitten had shed T. gondii oocysts that were bioassayed in mice. Toxoplasma gondii was isolated in tissue culture inoculated with tissues of these mice. The surviving animals were immediately treated with clindamycin for 16 weeks; they acquired a natural infection and seroconverted by the end of this time without clinical signs.

Spontaneous uterine fibroleiomyoma in a captive cheetah.

Information on uterine neoplasia in felids and more so in non-domestic felids is sparse. In non-domestic felids, this may be due to the small sample size. A uterine fibroleiomyoma is described in a 17-year-old captive cheetah. The multicentric nodular tumour masses were situated in the myometrium, were well demarcated, non-encapsulated and did not show infiltrative growth. Between the neoplastic cells, numerous of varying width, and irregularly braided bundles and whorls of collagen and reticular fibres were demonstrated. Immunohistochemical examinations revealed positive reactions for both desmin, the marker for smooth and skeletal muscle cells, and vimentin, the marker for fibrocyte-derived cells.

Immunohistochemical study of retinol-binding protein in livers of polar bears (Thalarctos maritimus).

Liver tumors of unknown cause have frequently been described in polar bears. Concurrent decrease of vitamin A levels and chronic liver disease are associated with hepatic carcinogenesis in humans. More than 90% of the body's vitamin A is stored in the liver, where it is bound to an intracellular retinol-binding protein (RBP). Therefore, in this retrospective study, RBP was assessed by immunohistochemistry in liver sections of 11 polar bears. Two of these polar bears had hepatocellular carcinoma, four showed other chronic liver changes, and five had normal livers. In normal livers, the cytoplasm stained diffusely positive with intensely staining cytoplasmic granules. RBP staining was evaluated and the abundance of diffuse cytoplasmic staining and intracytoplasmic large granules was determined. All cases with pathologic liver changes had markedly decreased staining intensities for RBP compared with normal livers. The findings of this study suggest that in polar bears, as in humans, vitamin A metabolism may play a role in hepatic carcinogenesis.

Hyoid apparatus and pharynx in the lion (Panthera leo), jaguar (Panthera onca), tiger (Panthera tigris), cheetah (Acinonyxjubatus) and domestic cat (Felis silvestris f. catus).

Structures of the hyoid apparatus, the pharynx and their topographical positions in the lion, tiger, jaguar, cheetah and domestic cat were described in order to determine morphological differences between species or subfamilies of the Felidae. In the lion, tiger and jaguar (species of the subfamily Pantherinae) the Epihyoideum is an elastic ligament lying between the lateral pharyngeal muscles and the Musculus (M.) thyroglossus rather than a bony element like in the cheetah or the domestic cat. The M. thyroglossus was only present in the species of the Pantherinae studied. In the lion and the jaguar the Thyrohyoideum and the thyroid cartilage are connected by an elastic ligament, whereas in the tiger there is a synovial articulation. In adult individuals of the lion, tiger and jaguar the ventral end of the tympanohyal cartilage is rotated and therefore the ventral end of the attached Stylohyoideum lies caudal to the Tympanohyoideum and the cranial base. In newborn jaguars the Apparatus hyoideus shows a similar topographical position as in adult cheetahs or domestic cats. In adult Pantherinae, the Basihyoideum and the attached larynx occupy a descended position: they are situated near the cranial thoracic aperture, the pharyngeal wall and the soft palate are caudally elongated accordingly. In the Pantherinae examined the caudal end of the soft palate lies dorsal to the glottis. Differences in these morphological features between the subfamilies of the Felidae have an influence on specific structural characters of their vocalizations.

The influence of amino acids on the biomineralization of hydroxyapatite in gelatin.

The effects of pH on the calcium phosphate phase, of Tris and of amino acids, such as aspartic acid, glutamic acid, and serine on hydroxyapatite formation and morphology, were studied in double diffusion experiments. In this system, hydroxyapatite was only formed when the pH was around 7.4 or higher for the duration of the reaction. A decrease in pH resulted in the transformation of hydroxyapatite to octacalcium phosphate. Amino acids and Tris or the buffering capacity of Tris have an effect on the morphology of the synthetic hydroxyapatite. The presence of the additive results in spheres consisting of needles, blades or plates depending on the reaction system.

Recruitment to Golgi membranes of ADP-ribosylation factor 1 is mediated by the cytoplasmic domain of p23.

Binding to Golgi membranes of ADP ribosylation factor 1 (ARF1) is the first event in the initiation of COPI coat assembly. Based on binding studies, a proteinaceous receptor has been proposed to be critical for this process. We now report that p23, a member of the p24 family of Golgi-resident transmembrane proteins, is involved in ARF1 binding to membranes. Using a cross-link approach based on a photolabile peptide corresponding to the cytoplasmic domain of p23, the GDP form of ARF1 (ARF1-GDP) is shown to interact with p23 whereas ARF1-GTP has no detectable affinity to p23. The p23 binding is shown to localize specifically to a 22 amino acid C-terminal fragment of ARF1. While a monomeric form of a non-photolabile p23 peptide does not significantly inhibit formation of the cross-link product, the corresponding dimeric form does compete efficiently for this interaction. Consistently, the dimeric p23 peptide strongly inhibits ARF1 binding to native Golgi membranes suggesting that an oligomeric form of p23 acts as a receptor for ARF1 before nucleotide exchange takes place.

Residues of ochratoxin A in pet foods, canine and feline kidneys.

The occurrence of ochratoxin A (OTA) in canned (26 samples) as well as dry pet foods (17 samples) for cats and dogs was investigated. In addition, 26 feline kidney samples with or without kidney alterations were surveyed for OTA-residues. The separation and detection of OTA was carried out by an isocratic high-performance liquid chromatography system based on reversed phase with fluorescence detection. After homogenization and extraction steps, immuno-affinity columns were applied for sample clean up. OTA could be detected in 47% (n=40) of the pet food samples. Those found positive contained generally low amounts of OTA (0.1-0.8 microg/kg original substance). Higher levels were only detected in two pet food samples (3.2 and 13.1 microg/kg toxin, respectively). Low concentrations of ochratoxin A could also be found iIn tissue of cat kidneys, with 16 of the analysed kidneys (n=26) being positive. The concentration levels were between 0.35 and 1.5 microg/kg OTA in tissue. No relation between pathological findings and ochratoxin levels in feline kidneys could be assessed.

Phylogenetic analysis of the L and HN gene of ophidian paramyxoviruses. Brief report.

Two reptilian paramyxoviruses, isolated from a neotropical rattlesnake (neotropical virus, NTV, ATCC VR-1408) and a bush viper (bush viper virus, BVV, ATCC VR- 1409), respectively, were analysed to determine their taxonomic position among other reptilian paramyxoviruses investigated previously by Ahne et al.. A 679 bp long region of the hemagglutinin-neuraminidase (HN) gene and a 627 bp long region of the large (L) gene were reverse transcribed, amplified by polymerase chain reaction (PCR), and sequenced. The deduced amino acid sequences were compared to mammalian paramyxoviruses belonging to the genera Respirovirus and Rubulavirus. The deduced amino acid sequences revealed 58.9 to 62% homology for the partial L protein and 41% to 47.1% homology for the partial HN protein. For phylogenetic analyses, a 518 bp L gene and a 352 bp HN gene fragment were used, both generating similar trees consisting of two distinct main groups, and some intermediate isolates. BVV clustered within group "b" while NTV clustered together with the intermediate ophidian paramyxovirus isolate Crot2-OH90.

Systemic inhibition of spontaneous calcification by the serum protein alpha 2-HS glycoprotein/fetuin.

The extracellular fluid is a metastable system with regard to calcium and phosphate ions. Active inhibitors of calcification must be present in serum to prevent the spontaneous formation of Ca2+.Pi solid phases which could otherwise precipitate to cause renal calcinosis and block small blood vessels. alpha 2-HS glycoproteins/fetuins, AHSGs, are ideal candidates for this function. AHSGs are ubiquitous and highly abundant in serum; they bind calcium and efficiently prevent de novo formation of apatitic mineral. Normocalcemic AHSG-deficient mice develop sporadic perivascular calcification. Hypercalcemia induced by dietary means or by hormone treatment results in lethal calcinosis in Ahsg-/-mice. A mineral binding structure is proposed for domain D1 of AHSG suggesting that the proposed EF-hand motif for calcium binding does not exist in AHSG. Unlike serum albumin, AHSG does not preferentially bind ionic Ca2+, but rather in the form of apatitic microcrystals.

Occurrence and structure of epipharyngeal pouches in bears (Ursidae).

The infrequent mention of epipharyngeal pouches occurring in some species of bears indicates the scarcity of morphological and functional knowledge about these structures. In order to provide precise morphological data on the structure of these remarkable formations and to verify their taxonomic utility, the pharyngeal regions of 1 spectacled bear and 3 brown bears were examined. All these individuals possessed epipharyngeal pouches, which are tubular, blind-ending outpouchings of the caudodorsal pharyngeal wall equipped with respiratory epithelium and a thick layer of elastic fibres. While the spectacled bear and Ursus arctos syriacus possessed a single pouch on the caudodorsal wall of the nasopharynx, in Ursus arctos and Ursus arctos beringianus 2 unequally sized pouches were present. Two additional sacs of smaller size, representing outpouchings of the lateral pharyngeal wall, occurred in the spectacled bear. These findings prove epipharyngeal pouches to be constant and unique morphological features of the family Ursidae, the anatomical features suggesting involvement in the respiratory system most probably in important aspects of ursid phonation. This is the first description of epipharyngeal pouches in the spectacled bear.

Individual biodosimetry at the natural radiation background level.

The present note describes new advancements to a valued tool in retrospective biodosimetry that lower the threshold of detection of radiation dose to 29 mGy. The advanced method is based on the electron paramagnetic resonance measurements of stable radiation-induced radicals in tooth enamel. Earlier this method had been used only for reconstruction of high radiation doses obtained accidentally. New opportunity of reconstruction of doses lower than 100 mGy opens a new realm of possibilities for assessing the health effects of ionizing radiation at the natural radiation background level.