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BackgroundCytauxzoonosis is an emerging and life-threatening tick-borne feline disease caused by haemoprotozoan parasites of the genus Cytauxzoon. Information regarding epidemiological and clinical presentation of infections by species other than Cytauxzoon felis is scant. A case of Cytauxzoon sp. infection is described in a 2-year-old mixed breed male domestic cat from Portugal, presenting a history of acute lethargy, anorexia and pyrexia.ResultsComplete blood count revealed a severe anaemia, leucocytosis and thrombocytopenia. A pleural effusion was noticed on thoracic radiograph, and marked splenomegaly and free abdominal fluid were visualized by ultrasound. A molecular screening for the detection of causative agents of infectious anaemia was performed, and a positive result for Piroplasmorida was obtained. DNA sequencing of a 743 bp amplicon of the 18S rRNA gene (GenBank accession no. KU710344) revealed 99.9 % identity with Cytauxzoon manul.ConclusionsThis is the first report of Cytauxzoon sp. (clustering together with C. manul) in a felid from Portugal. Clinical manifestations along with molecular analysis suggest the hypothesis that domestic cats might be infected with and serve as a reservoir host for C. manul.

This study describes an efficient in vitro method using colchicinetreated triploid seedlings of Napiergrass and Pearl millet hybrids to produce hexaploid hybrids and their subsequent identification with flow cytometry. It also describes chromosome count and stomatal morphology and their use to ploidy analysis. Four-hundred and eighty triploid seeds, representing 12 different hybrids were sterilized and transferred to MS media to induce chromosome doubling. Surviving plants were analysed by flow cytometry. From six triploid (control) and hexaploid plants, the stomata sizes and frequency were analysed. Chromosome count was performed only in the plants identified as hexaploid. Seventeen plants were identified as hexaploid by flow cytometry analysis. Further confirmation of the hexaploid condition was performed with stomatal morphology (stomatal frequency reduction and stomatal length increase) and chromosome count (2n = 6x = 42). Chromosome doubling has numerous applications in Pennisetum breeding. It can be used to restore the fertility of interspecific hybrids and to improve seed size.The genus Pennisetum is one of the important genera of the family Poaceae. Pennisetum purpureum Schum. (Napiergrass) and Pennisetum glaucum (L.) R.Br. (Pearl millet) are important species widely used as forage. Pennisetum glaucum is a diploid species with 2n = 2x = 14 chromosomes and P. purpureum is tetraploid with 2n = 4x = 28 chromosomes (Martel et al. 2004). Napiergrass has been successfully crossed with Pearl millet to produce high quality and high yielding perennial interspecific forage hybrids (Hanna 1981). However, the sterility of this hybrid due to its triploid condition (2n = 3x = 21 chromosomes) has been pointed as a difficulty for its use in breeding programmes. The fertility of this hybrid can be restored with chromosome doubling. Colchicine is an alkaloid widely used for chromosome doubling and for the induction of polyploidy in plants (Pasakinskiene´2000). The technique of exposing explants to colchicine in vitro has been used in a number of cases (Kadota and Niimi 2002). Chromosome counts and stomatal morphology have been used routinely for polyploidy screening. However, these methods make the analysis of a great number of plants difficult and time-consuming. Flow cytometry represents a technology gain and has been increasingly used for highthroughput ploidy screening (Roy et al. 2001). This study describes an efficient in vitro method using colchicine-treated triploid seedlings of Napiergrass and Pearl millet hybrids to produce hexaploid hybrids and their subsequent identification with flow cytometry. It also describes chromosome count and stomatal morphology and their use for ploidy analysis. Plant materials and in vitro induction of chromosome doublingFour-hundred and eighty triploid seeds, representing 12 different hybrids (40 per hybrid), were sterilized and transferred to MS media to induce seedling development. The treatments (hybrids) have been arranged in a completely random design with four repetitions. Each...

Like other eukaryotes, the nuclear genome of plants consists of DNA with a small proportion of low-copy DNA (genes and regulatory sequences) and very abundant DNA sequence motifs that are repeated thousands up to millions of times in the genomes including transposable elements (TEs) and satellite DNA. Retrotransposons, one class of TEs, are sequences that amplify via an RNA intermediate and reinsert into the genome, are often the major fraction of a genome. Here, we put research on retrotransposons into the larger context of plant repetitive DNA and genome behaviour, showing features of genome evolution in a grass genus, Brachiaria, in relation to other plant species. We show the contrasting amplification of different retroelement fractions across the genome with characteristics for various families and domains. The genus Brachiaria includes both diploid and polyploid species, with similar chromosome types and chromosome basic numbers x = 6, 7, 8 and 9. The polyploids reproduce asexually and are apomictic, but there are also sexual species. Cytogenetic studies and flow cytometry indicate a large variation in DNA content (C-value), chromosome sizes and genome organization. In order to evaluate the role of transposable elements in the genome and karyotype organization of species of Brachiaria, we searched for sequences similar to conserved regions of TEs in RNAseq reads library produced in Brachiaria decumbens. Of the 9649 TE-like contigs, 4454 corresponded to LTR-retrotransposons, and of these, 79.5 % were similar to members of the gypsy superfamily. Sequences of conserved protein domains of gypsy were used to design primers for producing the probes. The probes were used in FISH against chromosomes of accesses of B. decumbens, Brachiaria brizantha, Brachiaria ruziziensis and Brachiaria humidicola. Probes showed hybridization signals predominantly in proximal regions, especially those for retrotransposons of the clades CRM and Athila, while elements of Del and Tat exhibited dispersed signals, in addition to those proximal signals. These results show that the proximal region of Brachiaria chromosomes is a hotspot for retrotransposon insertion, particularly for the gypsy family. The combination of high-throughput sequencing and a chromosome-centric cytogenetic approach allows the abundance, organization and nature of transposable elements to be characterized in unprecedented detail. By their amplification and dispersal, retrotransposons can affect gene expression; they can lead to rapid diversification of chromosomes between species and, hence, are useful for studies of genome evolution and speciation in the Brachiaria genus. Centromeric regions can be identified and mapped, and retrotransposon markers can also assisting breeders in the developing and exploiting interspecific hybrids.

PCR is the best method for the detection of enteric viruses present at low concentrations in environmental samples. However, some organic and inorganic compounds present in these samples can interfere in the reaction. Many of these substances are cytotoxic, too. The ZP60S filter membranes used in addition to fluorpentane treatment are quite efficient for virus concentration and simultaneous elimination of cytotoxicity from environmental samples. In this study, both procedures were used to promote the elimination of reverse transcriptase PCR (RT-PCR) inhibitors from sewage and sewage-polluted creek water. Samples were subjected separately to each of the following procedures: filtration through electropositive filter membranes (ZP60S), organic extraction with Vertrel XF, and filtration through ZP60S followed by organic extraction. Afterwards, aliquots were experimentally inoculated with rotavirus SA-11 RNA and subjected to RT-seminested PCR for amplification of the VP7 gene. Results showed that the ZP60S membranes efficiently eliminated the RT-PCR inhibitors from water samples. The sample processing method was also applied to 31 in natura sewage and creek water samples for detection of naturally occurring rotavirus. A duplex seminested PCR was used for the quick detection of couples of the four rotavirus genotypes (G1 to G4). Eight samples (25.8%) were positive, and rotavirus sequences were not detected in 23 (74.2%). Results were confirmed by direct immunoperoxidase method. In summary, the use of electropositive filter membrane is appropriate for the elimination of substances that can interfere with RT-PCR, obviating additional sample purification methods.