Isolation and identification of mycobacteria from captive reptiles

The occurrence of Mycobacterium species in clinically healthy pet reptiles was studied in Italy during the period 2004-2006. The feces samples of 223 animals were examined bacteriologically. Thirty-seven strains were isolated, in particular from 13/18 (72.2%) ophidians, 13/134 (9.7%) saurians and 11/71 (15.5%) chelonians. The isolates were classified, after HPLC analysis of bromophenacyl esters of cell wall mycolic acids, as Mycobacterium fortuitum (14 strains, 37.8%), Mycobacterium fortuitum-like (17, 45.9%), Mycobacterium peregrinum (4, 10.8%), and Mycobacterium chelonae (1, 2.7%). M. fortuitum was isolated from seven pythons, five saurians and two turtles; M. fortuitum-like from six saurians, six pythons and five turtles; M. peregrinum from four turtles; M. chelonae from one lizard. One isolate from an Iguana iguana could not be identified by HPLC analysis showing a previously unreported profile. Comparative 16S rDNA sequencing showed a low similarity with Mycobacterium triviale (97.2%) and Mycobacterium confluentis (97.1%). On the basis of such data the unidentified bacterium turned out to belong to a not yet described Mycobacterium species.     

Distribution of ghrelin-producing cells in the gastrointestinal tract of pigs at different ages

Ghrelin is involved in many biological processes, ranging from appetite regulation and the release of growth hormone to the regulation of gastrointestinal motility and secretion processes. Ghrelin expression is not homogenously distributed throughout the gastrointestinal tract; expression is species-specific and can also depend on the animal age. This study was performed to investigate ghrelin immunolocalization in the gastrointestinal tract of pigs at different ages: 1 day (birth), 28 days (weaning), 2 months, 4 months, and 7 months (pre-puberty). Tissue samples were collected along the entire gastrointestinal tract and were examined by immunohistochemistry and double-immunofluorescence. Histometry was performed by counting the number of endocrine ghrelin immunopositive cells in the gastrointestinal mucosa. Ghrelin was found to be present along the swine alimentary canal from the stomach to the caecum. In all regions of the alimentary canal of the animals studied, ghrelin-immunoreactive (IR) cells co-localized with chromogranin-A and were therefore identified as endocrine cells. In the gastric fundus, ghrelin-immunoreactivity was partially detected in co-localization with H-K-adenosine triphosphatase and pepsinogen. Ghrelin-IR endocrine cells were abundant in the oxyntic mucosa but less present in the small intestine and rare in the large intestine. The cell density of the ghrelin-IR endocrine cells was lowest in the oxyntic mucosa of 1-day-old pigs. We can conclude that gastric ghrelin expression is not related merely to age but could also potentially be influenced by food intake.     

Development of Real-Time PCR for in wood-detection of Ceratocystis platani, the agent of canker stain of Platanus spp.

Ceratocystis platani is a quarantine fungal pathogen agent of canker stain, a destructive disease affecting Platanus. Despite its diagnosis being critical for disease control, there is still no effective diagnostic tool as all known mycological and biological detection assays are problematical. In this study we developed highly effective Real-Time PCR methods based on the use of an intercalating dye, EvaGreen, and on a Taqman probe. We designed primers and probe on the internal transcribed spacer (ITS) region of rDNA, and used them for the amplification and detection of a 95?bp C. platani amplicon. Inference of standard curves revealed that both Real-Time procedures have similar and high values of amplification efficiency when applied to a range of templates, e.g. genomic fungal DNA and DNA extracted from diseased wood. The methods were sensitive with a detection limit of 10?fg???l^?1? C. platani genomic DNA. They were specific as they did not yield any detection signal when applied to non-target fungal taxa colonizers of Platanus wood. Reliability was demonstrated through the positive detection of a collection of C. platani isolates and of wood samples collected from naturally infected trees. Robustness was positively verified through detection on artificially infected trees, which were tested at different times after death, up to 27?months. Generating a standard curve with a target-amplicon-containing plasmid enabled an absolute quantification and a comparison between the discoloured wood of resistant and susceptible genotypes. The importance of the methods for studies on pathogen epidemiology and host resistance is also discussed.     

Reference intervals for hematologic and biochemical constituents and protein electrophoretic fractions in captive common buzzards (Buteo buteo)

BACKGROUND: Increasing interest in wildlife care leads to the need for new tools to evaluate animal health. Laboratory investigations require reference intervals against which to compare the results obtained. For common buzzards, only a few studies have been performed to establish hematologic and biochemical reference intervals. OBJECTIVES: The aim of this work was to develop reference values for routine hematologic and biochemical constituents and protein electrophoretic fractions and evaluate possible seasonal differences in values for healthy common buzzards. METHODS: Heparinized blood samples were collected from 23 captive, clinically healthy common buzzards between February 2001 and June 2003. A CBC, routine biochemical analysis, and protein electrophoresis were performed. Data distribution was assessed and results from birds sampled in spring, summer, and winter were compared. Results from alternative methods for hemoglobin (Hgb; estimated as HCT / 3 vs spectrophotometry), total protein (biuret vs refractometry), and albumin (bromcresol green vs electrophoresis) concentrations also were compared. RESULTS: Reference intervals were calculated as 10-90th percentiles. In spring and summer, total WBC and heterophil counts, and urea, total protein, prealbumin, and beta- and gamma-globulins concentrations were significantly different from winter values. Results obtained by alternative methods for Hgb, total protein, and albumin concentrations were significantly different from those obtained by standard methods, although estimated and spectrophotometric Hgb values were significantly correlated. CONCLUSIONS: The reference values obtained in this study for hematologic and plasma biochemical constituents and their seasonal variation in healthy, captive common buzzards will be useful in the clinical evaluation of these birds in rehabilitation settings.     

GIS-models work well, but are not enough: Habitat preferences of Lanius collurio at multiple levels and conservation implications

Species conservation largely depends on knowledge of habitat needs of target species. GIS-models are increasingly used to assess habitat preferences and distribution of target species, but their accuracy is constrained by availability of digital data layers. We developed a two-steps approach aiming at showing pros and cons of landscape (GIS)- and site-level habitat models, identifying key habitat factors for conservation of a threatened bird species, the red-backed shrike Lanius collurio. A spatially explicit GIS-model was generated using landscape variables, and a second model at site level was developed using fine-scale variables measured on the ground. The GIS-based model was then extrapolated to the entire region to obtain a map of distribution of suitable habitats. Positive associations between shrike occurrence and both hedgerow length and partial shrub cover were detected at both scales. Shrikes were also positively associated with grassland cover at landscape level and with partial cover of untilled herbaceous vegetation at the finer scale, and negatively affected by lucerne cover. The GIS-model led to an affordable map of predicted habitat suitability which should help conservationists to focus on different local priorities, but was unable to identify effects of untilled and lucerne cover. Site-level model gave fine details for habitat management, but its application elsewhere requires ground-measurements of factors. Combining the multiscale models could indicate more urgent actions at large scales (e.g. maintaining suitable habitats, or improving connectivity among isolated patches) and draw a detailed figure of the most suitable habitat for the species. Shrike occurrence was associated with a higher number of shrub and tree species: the indicator value of the species should ensure general benefits for biodiversity from dedicated management.     

Cannabinoid receptors are widely expressed in goldfish: molecular cloning of a CB2-like receptor and evaluation of CB1 and CB2 mRNA expression profiles in different organs

Cannabinoids, the bioactive constituents of Cannabis sativa, and endocannabinoids, among which the most important are anandamide and 2-arachidonoylglycerol, control various biological processes by binding to specific G protein-coupled receptors, namely CB1 and CB2 cannabinoid receptors. While a vast amount of information on the mammalian endocannabinoid system does exist, few data have been reported on bony fish. In the goldfish, Carassius auratus, the CB1 receptor has been cloned and its distribution has been analyzed in the retina, brain and gonads, while CB2 had not yet been isolated. In the present paper, we cloned the goldfish CB2 receptor and show that it presents a quite high degree of amino acid identity with zebrafish Danio rerio CB2A and CB2B receptors, while the percentage of identity is lower with the puffer fish Fugu rubripes CB2, as also confirmed by the phylogenetic analysis. The sequence identity becomes much lower when comparing the goldfish and the mammalian CB2 sequences; as for other species, goldfish CB2 and CB1 amino acid sequences share moderate levels of identity. Western-blotting analysis shows the CB2 receptor as two major bands of about 53 and 40 kDa and other faint bands with apparent molecular masses around 70, 57 and 55 kDa. Since the distribution of a receptor could give information on its physiological role, we evaluated and compared CB1 and CB2 mRNA expression in different goldfish organs by means of qReal-Time PCR. Our results show that both CB1 and CB2 receptors are widely expressed in the goldfish, displaying some tissue specificities, thus opening the way for further functional studies on bony fish and other nonmammalian vertebrates.     

Preliminary Structure-Activity Relationship on Theonellasterol, a New Chemotype of FXR Antagonist, from the Marine Sponge Theonella swinhoei

Using theonellasterol as a novel FXR antagonist hit, we prepared a series of semi-synthetic derivatives in order to gain insight into the structural requirements for exhibiting antagonistic activity. These derivatives are characterized by modification at the exocyclic carbon-carbon double bond at C-4 and at the hydroxyl group at C-3 and were prepared from theonellasterol using simple reactions. Pharmacological investigation showed that the introduction of a hydroxyl group at C-4 as well as the oxidation at C-3 with or without concomitant modification at the exomethylene functionality preserve the ability of theonellasterol to inhibit FXR transactivation caused by CDCA. Docking analysis showed that the placement of these molecules in the FXR-LBD is well stabilized when on ring A functional groups, able to form hydrogen bonds and π interactions, are present.     

Isolation and Assessment of the in Vitro Anti-Tumor Activity of Smenothiazole A and B,Chlorinated Thiazole-Containing Peptide/Polyketides from the Caribbean Sponge,Smenospongia aurea

The study of the secondary metabolites contained in the organic extract of Caribbean sponge Smenospongia aurea led to the isolation of smenothiazole A (3) and B (4), hybrid peptide/polyketide compounds. Assays performed using four solid tumor cell lines showed that smenothiazoles exert a potent cytotoxic activity at nanomolar levels, with selectivity over ovarian cancer cells and a pro-apoptotic mechanism.     

SNP discovery and gene annotation in the surf clam Mesodesma donacium

The main objective of this research was to identify single‐nucleotide polymorphisms (SNPs) from an Expressed Sequences Tags (EST) data set generated by 454 pyrosequencing in the soft clam Mesodesma donacium. A total of 180 159 ESTs were yielded from a M. donacium cDNA library. De novo assembly was performed using stringent calling parameters, producing 10 178 contigs and 41 765 singletons. Here, a total of 2594 SNPs were discovered related to 613 consensus sequences, achieving a frequency of 1 SNPs per 260 bp. SNP variants showed that A/G, A/T and C/T were the most abundant among the identified polymorphisms. We validated a total of 12 SNPs loci by HRMA for annotated genes such as heat shock protein‐70 and the translation elongation factor 1‐alpha. The Gene Ontology analysis regarding molecular function level revealed that sequences with SNPs were mainly classified to protein and nucleotide binding, as well hydrolase activity, ion binding and oxidoreductase activity. Further, biological processes like cellular and metabolic process, biogenesis, localization and biological regulation were highly annotated. The most expressed genes were related to the mitochondrial electron transport chain, senescence‐associated protein, ubiquitin and actin. Interestingly, some relevant genes related to immune response and biomineralization showed a high abundance, such as tumor necrosis factor (TNF)‐alpha‐receptor‐like protein, serine protease inhibitor, heat shock protein, aragonite‐binding protein and ferritin. This study contributes to relevant genes associated with functional polymorphisms and gives an overview for future genetic investigations.