Cohabitation of a <Emphasis Type="Italic">Brucella melitensis</Emphasis> infected Alpine ibex (<Emphasis Type="Italic">Capra ibex</Emphasis>) with domestic small ruminants in an enclosure in Gran Paradiso National Park,in Western Italian Alps

After the first report of Brucella melitensis infection from a 7-year-old alpine ibex (Capra ibex) buck living in Gran Paradiso National Park (GPNP), further studies demonstrated the presence of the infection in ibex and chamois. Considering that livestock herds keep on sharing pastures with more than 3,500 ibex and 9,000 chamois in the park, our aim was to demonstrate under controlled conditions the possibility of Brucella infection passing from wild ruminants to livestock. A 7-year-old male alpine ibex with clinical signs of brucellosis and serologically positive was released in a 5,000 m² enclosure together with five goats and two sheep rams. Due to poor condition, ibex was suppressed at day 40, domestic ruminants stayed into the enclosure potentially contaminated by ibex for further 38 days. During this period, we had monitored our animals taking blood from domestic ruminants every 15 days and tested the serum to Rose Bengal agglutination test and Complement Fixation test. Domestic animals tested negative at serology at all sampling time and at isolation, while B. melitensis biovar 3 was isolated from ibex tissues. Our data show that transmission of infection from ibex to livestock is not easy. After 40 days of strict cohabitation and 38 days of permanence in an area where an infected ibex lives, no one of the domestic animals contracted infection. In spite of the limitation of our field trial, we have demonstrated that long direct and indirect contact between alpine ibex and domestic animals will not easily lead to an infection of the latter. Further investigations are needed to confirm our results and evaluate the effective risk of B. melitensis transmission from alpine ibex to livestock.     

Techniques for noninvasive genetic monitoring of Alpine Ibex <Emphasis Type="Italic">Capra ibex</Emphasis>

Noninvasive sampling is of increasing importance for the molecular genetic monitoring of wild animal populations, although reduced quality and quantity of such samples’ DNA can affect genetic data and their subsequent interpretation. Consequently, we performed a pilot study to establish a feasible approach for the genetic investigation of free-ranging Alpine ibex Capra ibex Linnaeus, 1758 populations. Establishing an ibex-specific PCR-RFLP based on Cytochrome b gene differences allowed the discrimination of noninvasive ibex samples from those of other sympatric ungulates. In addition, we established a quantitative PCR for ibex samples. The quantification of 35 faecal samples clearly exhibited a strong variability of DNA contents among samples and individuals. Furthermore, we performed threefold genotyping experiments on six microsatellite loci to determine the extent of genotyping errors in reference to blood samples of the respective individuals. The analyses exhibited a strong dependence of erroneous microsatellite genotypes on the starting amount of template DNA. Variability in reliability was observed between individual loci, resulting in a mandatory high DNA concentration necessary for consistent genotyping. This study serves as basis for further ibex research and we propose the application of DNA quantification of faecal samples to focus genotyping efforts solely on suitable samples.     

Spatial behaviour of adult male Alpine ibex<Emphasis Type="Italic">Capra ibex ibex</Emphasis> in the Gran Paradiso National Park,Italy

During a two year preliminary study, the spatial organization of a group of male Alpine ibexCapra ibex ibex Linnaeus, 1758 was examined in the Gran Paradiso National Park, Western Italian Alps, Italy. From December 1995 to January 1998 we measured annual, seasonal home range and home range during the rut, plus altitudinal migration of 13 radio-collared adult Alpine ibex. The small annual home range size showed a traditional use of space, confirmed by the high overlapping values between home ranges of consecutive years: the ibex used the same places from year to year. This was also true during periods of rut. Home ranges closely overlapped in consecutive ruts, while their size changed from winter to winter. Snow cover limited the movements of the ibex; winter and spring home ranges were smaller than those in summer and autumn. Mean vertical movement patterns were similar in the two years, showing the highest values in summer and the lowest in spring. Space use was never proportional to availability for each altitudinal range.     

G- and C-banded karyotype of the markhor<Emphasis Type="Italic">Capra falconeri heptneri</Emphasis>, and comparison of its banding patterns with the Alpine ibex<Emphasis Type="Italic">Capra ibex</Emphasis> and cattle<Emphasis Type="Italic">Bos taurus</Emphasis>

Here we present the first data on chromosome banding forCapra falconeri heptneri (Zalkin, 1945) (Bovidae: Caprinae), a critically endangered subspecies of the markhor, and compare its G- and C-banding patterns with those of the congeneric Alpine ibexC. ibex Linnaeus, 1758 and the evolutionarily more distant cattleBos taurus Linnaeus, 1758. The two goat species have identical karyotypes whereasB. taurus, which has the same diploid number (2n = 60) and autosomal fundamental number (aFN) differs in the morphology of two pairs of autosomes (9 and 14) and of the X chromosome, as well as in the amount of C heterochromatin. Although the study supports the earlier idea of karyotype homogeneity within the genusCapra, new comparative cytogenetic data for unstudied yet congeneric and other related species are necessary for our understanding of the pattern of chromosome evolution within the subfamily Caprinae and, more broadly, the family Bovidae.     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

Management of a caseous lymphadenitis outbreak in a new Iberian ibex (<Emphasis Type="Italic">Capra pyrenaica</Emphasis>) stock reservoir

Background

In 2010, an Iberian ibex (Capra pyrenaica hispanica) stock reservoir was established for conservation purposes in north-eastern Spain. Eighteen ibexes were captured in the wild and housed in a 17 hectare enclosure. Once in captivity, a caseous lymphadenitis (CLA) outbreak occurred and ibex handlings were carried out at six-month intervals between 2010 and 2013 to perform health examinations and sampling. Treatment with a bacterin-based autovaccine and penicillin G benzatine was added during the third and subsequent handlings, when infection by Corynebacterium pseudotuberculosis was confirmed. Changes in lesion score, serum anti-C. pseudotuberculosis antibodies and haematological parameters were analyzed to assess captivity effects, disease emergence and treatment efficacy. Serum acute phase proteins (APP) Haptoglobin (Hp), Amyloid A (SAA) and Acid Soluble Glycoprotein (ASG) concentrations were also determined to evaluate their usefulness as indicators of clinical status.Once in captivity, 12 out of 14 ibexes (85.7%) seroconverted, preceding the emergence of clinical signs; moreover, TP, WBC, eosinophil and platelet cell counts increased while monocyte and basophil cell counts decreased. After treatment, casualties and fistulas disappeared and both packed cell volume (PCV) and haemoglobin concentration significantly increased. Hp, SAA and ASG values were under the limit of detection or showed no significant differences.

Conclusions

A role for captivity in contagion rate is suggested by the increase in antibody levels against C. pseudotuberculosis and the emergence of clinical signs. Although boosted by captivity, this is the first report of an outbreak of caseous lymphadenitis displaying high morbidity and mortality in wild ungulates. Treatment consisting of both vaccination and antibiotic therapy seemed to prevent mortality and alleviate disease severity, but was not reflected in the humoural response. Haematology and APP were not useful indicators in our study, perhaps due to the sampling frequency. Presumably endemic and irrelevant in the wild, this common disease of domestic small ruminants is complicating conservation efforts for the Iberian ibex in north-eastern Spain.

     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

Sonication assisted <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation enhances the transformation efficiency in flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A sonication-assisted, Agrobacterium-mediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and cotyledons excised from about 10-day-old flax seedlings grown in vitro were placed into a 10 mM MgSO₄ solution, and inoculated with an A. tumefaciens vector bearing the mgfp5-ER gene driven by the CaMV 35S promoter. The explants were subjected to pulses of ultrasound delivered by a sonicator apparatus (35 kHz) for 0–150 s and co-cultivated for 2 h at 27°C. The dried hypocotyls and cotyledons were grown on a selective MS medium to promote shoot regeneration. An electron microscopic study showed that the sonication treatment resulted in thousands of microwounds on and below the surface of the explants. A stereo microscope Leica MZ 12 equipped with a GFP adaptor was used to assess the infection and transformation of plant tissues in real time. After only 48 h and for at least 30 days after bacteria elimination, signs of transgene expression could be seen as a bright fluorescence. Our results show that treatment with ultrasound facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends on the duration of the treatment and the frequency used. SAAT could be a promising tool for enhancing transformation efficiency in flax.     

Molecular evidence for hybridization between invasive <Emphasis Type="Italic">Solidago canadensis</Emphasis> and native <Emphasis Type="Italic">S</Emphasis>. <Emphasis Type="Italic">virgaurea</Emphasis>

Hybridization between alien and native species is biologically very important and could lead to genetic erosion of native taxa. Solidago × niederederi was discovered over a century ago in Austria and described by Khek as a natural hybrid between the alien (nowadays regarded also as invasive) S. canadensis and native S. virgaurea. Although interspecific hybridization in the genus Solidago is considered to be relatively common, hybrid nature of S. × niederederi has not been independently proven using molecular tools, to date. Because proper identification of the parentage for the hybrid Solidago individuals solely based on morphological features can be misleading, in this paper we report an additive polymorphism pattern expressed in the ITS sequences obtained from individuals representing S. × niederederi, and confirm the previous hypothesis that the parental species of this hybrid are S. canadensis and S. virgaurea. Additionally, based on variability at the cpDNA rpl32-trnL locus, we showed that in natural populations hybridization occurs in both directions.     

Pedogamy in <Emphasis Type="Italic">Neidium</Emphasis> (<Emphasis Type="Italic">Bacillariophyceae</Emphasis>)

Single (unpaired) vegetative cells of freshwater pennate diatom Neidium cf. ampliatum differentiated into gametangia and produced a single zygote (auxospore) via a pedogamic process. The gametic nuclei fused after auxospore expansion had begun. The auxospore expanded in parallel to the apical axis of the gametangium.     

Biochemical and molecular characterization of <Emphasis Type="Italic">Cronobacter</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> (formerly <Emphasis Type="Italic">Enterobacter sakazakii</Emphasis>) isolated from foods

The aim of this study was to identify and characterize Cronobacter spp. isolated from a range of foods. A total of 71 Cronobacter strains were isolated from 602 foods in our laboratory. The highest contamination was observed in foods of plant origin, e.g. spices, teas, chocolate, nuts, pastries and vegetables. On the basis of genus and species identification performed using genus-specific PCR, 16S rRNA sequencing and AFLP genotyping, most of the strains belonged to Cronobacter sakazakii. Biochemical profiling by the tests included in API 20E, complemented with relevant additional tests, classified the strains into 13 biogroups. AFLP genotyping facilitated discrimination of six main groups at the 70% similarity level and strain grouping correlated clearly with species identification. Our results indicate that molecular typing by AFLP may be applied as a useful tool not only for direct comparison of Cronobacter isolates, providing traceability, but also for the reliable species classification. Moreover, tracing of these bacteria in a wider variety of foods should be important to enhance the knowledge of their transmission.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Protoplast isolation and culture for somatic hybridization of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> and <Emphasis Type="Italic">L</Emphasis>. <Emphasis Type="Italic">subcarnosus</Emphasis>

A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 10⁵ protoplasts g⁻¹ fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks) and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids. However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration.     

Anti-amoebic properties of a Malaysian marine sponge <Emphasis Type="Italic">Aaptos</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> on <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis>

Crude methanol extracts of a marine sponge, Aaptos aaptos, collected from three different localities namely Kapas, Perhentian and Redang Islands, Terengganu, Malaysia, were tested in vitro on a pathogenic Acanthamoeba castellanii (IMR isolate) to examine their anti-amoebic potential. The examination of anti-Acanthamoebic activity of the extracts was conducted in 24 well plates for 72 h at 30 °C. All extracts possessed anti-amoebic activity with their IC₅₀ values ranging from 0.615 to 0.876 mg/mL. The effect of the methanol extracts on the surface morphology of A. castellanii was analysed under scanning electron microscopy. The ability of the extracts to disrupt the amoeba cell membrane was indicated by extensive cell’s blebbing, changes in the surface morphology, reduced in cell size and with cystic appearance of extract-treated Acanthamoeba. Number of acanthapodia and food cup was also reduced in this Acanthamoeba. Morphological criteria of apoptosis in Acanthamoeba following treatment with the sponge’s extracts was determined by acridine orange-propidium iodide staining and observed by fluorescence microscopy. By this technique, apoptotic and necrotic cells can be visualized and quantified. The genotoxic potential of the methanol extracts was performed by the alkaline comet assay. All methanol extracts used were significantly induced DNA damage compared to untreated Acanthamoeba by having high percentage of scores 1, 2, and 3 of the DNA damage. Results from cytotoxicity and genotoxicity studies carried out in the present study suggest that all methanol extracts of A. aaptos have anti-amoebic properties against A. castellanii.