Equus asinus papillomavirus (EaPV1) provides new insights into equine papillomavirus diversity.

We detected a novel papillomavirus (EaPV1) from healthy skin and from sun associated cutaneous lesions of an Asinara (Sardinia, Italy) white donkey reared in captivity in a wildlife recovery centre. The entire genome of EaPV1 was cloned, sequenced, and characterised. Genome is 7467 bp long, and shows some characteristic elements of horse papillomaviruses, including a small untranslated region between the early and late regions and the lack of the retinoblastoma tumour suppressor binding domain LXCXE in E7. Additionally, a typical E6 ORF is missing. EaPV1 DNA was detected in low copies in normal skin of white and grey donkeys of the Asinara Island, and does not transform rodent fibroblasts in standard transformation assays. Pairwise nucleotide alignments and phylogenetic analyses based on concatenated E1-E2-L1 amino acid sequences revealed the highest similarity with the Equine papillomavirus type 1. The discovery of EaPV1, the prototype of a novel genus and the first papillomavirus isolated in donkeys, confirms a broad diversity in Equidae papillomaviruses. Taken together, data suggest that EaPV1 is a non-malignant papillomavirus adapted to healthy skin of donkeys.

Detection and characterization of Mycoplasma spp. and Salmonella spp. in free-living European tortoises (Testudo hermanni, Testudo graeca, and Testudo marginata).

Free-living and captive chelonians might suffer from upper respiratory tract disease (URTD), a pathology primarily caused by Mycoplasma agassizii. Wild tortoises can also be an important reservoir of Salmonella spp., which are commensal in the host reptile but are potential zoonotic agents. Between July 2009 and June 2010, we screened free-living European tortoises (spur-thighed tortoises Testudo graeca, Hermann's tortoises Testudo hermanni, marginated tortoises Testudo marginata) temporarily housed in a wildlife center in Italy. We molecularly characterized 13 Mycoplasma isolates detected in all Testudo spp. studied, and three PCR-positive animals showed typical URTD clinical signs at the time of sampling. Three Salmonella enterica serotypes (Abony, Potsdam, Granlo), already related to reptile-associated human infections, were also identified. These results highlight the potential role played by wildlife recovery centers in the spread and transmission of pathogens among wild chelonians and to humans.

Identification and characterization of novel Mycoplasma spp. belonging to the hominis group from griffon vultures.

Mycoplasmas are commensals and pathogens of various avian species, and are also regularly found in birds of prey, although their significance to birds' health remains unclear. Here we describe two novel Mycoplasma isolated from the upper respiratory tract of four Eurasian griffon vultures (Gyps fulvus) housed in a wildlife recovery centre in Sardinia (Italy). By sequencing the 16S rRNA gene and the entire 16S/23S intergenic spacer region, the new strains were classified within the Mycoplasma taxonomy at the group and cluster levels, showing that the two isolates fall into the Mycoplasma synoviae and Mycoplasma hominis clusters of the hominis group, respectively. We combined molecular tools and immunoblotting methods in order to further characterize these isolates, and antigenic analyses overall confirmed the molecular findings. Different levels of pathogenicity and prevalence of these strains might have different implications for the conservation and reintroduction of vultures.

Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts.

This study was conducted to isolate, to culture, and to characterize embryonic cell lines from in vitro produced vitrified sheep blastocysts. Embryos were produced and vitrified at the expanded blastocyst stage. Ten inner cell masses arising from day 6-7 blastocysts were isolated by immunosurgery, disaggregated, and cultured onto mitomocin-C-inactivated mouse STO fibroblasts (MIF). After 5 or 6 days of culture the primary cell colonies were disaggregated, seeded in a new MIF, and cultured for 3 or 4 days to form new colonies called Passage 1. These cells were then disaggregated and cultured for other two passages. The primary cell colonies and Passage 2 colonies expressed stage specific embryonic markers SSEA-1, SSEA-3, and SSEA-4, and were alkaline phosphatase positive. In the absence of feeder layer and human leukemia inhibitory factor (LIF), these cells differentiated into variety of cell types and formed embryoid bodies. When cultured for an extended period of time, embryoid bodies differentiated into derivatives of three embryonic germ (EG) layers. These were characterized by detection of specific markers for differentiation such early mesoderm (FE-C6), embryonic myosin (F1-652), neural precursor (FORSE-1), and endoderm (anti-cytokeratin 18). To our knowledge, this is the first time that embryonic cell lines from in vitro produced and vitrified ovine blastocysts have been isolated and examined for detection of SSEA markers, and embryoid bodies have been cultured and examined for specific cell surface markers for differentiation.

Sexing of in vitro produced ovine embryos by duplex PCR.

The aim of this article was to develop a fast and easy duplex polymerase chain reaction (PCR) method, for sex determination of ovine in vitro produced embryos prior to implantation. We tested the approach with 107 samples of autosomal cells (oviductal sheep cells and male lamb fibroblasts), divided into three groups for each sex according to the number of cells employed (30, 5, 2, respectively). We then used the test on 21 embryos at blastocyst stage. On the same day the embryos were transferred in pairs into 11 recipient synchronized ewes. The PCR utilized two different sets of primers: the first pair recognized a bovine Y-chromosome-specific sequence (SRY), that showed 100% homology with the corresponding sequence of the ovine Y-chromosome and is amplified in males only. The second pair recognized the bovine 1.715 satellite DNA (SAT) which was amplified in all ovine samples but, when submitted to the GenBank database did not show homology with any of the reported ovine sequences. However, after sequencing, ovine amplification product showed 98% homology with the bovine specific satellite sequence. The autosomal samples were amplified with 85.0% efficiency and 91.2% accuracy, while amplification was successful with all 21 embryos (100% efficiency). Eight lambs were born and the sex as determined by PCR corresponded to the anatomical sex in seven (87.5% accuracy). These results confirm that this method can be applied in ovine breeding programs to manipulate sex ratio of offspring.

Comparison of different vitrification protocols on viability after transfer of ovine blastocysts in vitro produced and in vivo derived.

We compare different vitrification protocols on the pregnancy and lambing rate of in vitro produced (IVP) and in vivo derived (IVD) ovine embryos. Ovine blastocysts were produced by in vitro maturation, fertilization and culture of oocytes collected from slaughtered ewes or superovulated and inseminated animals. Embryos were cryopreserved after exposure at room temperature either for 5 min in 10% glycerol (G), then for 5 min in 10% G + 20% ethylene glycol (EG), then for 30 s in 25% G + 25% EG (glycerol group), or for 3 min in 10% EG + 10% dimethyl sulphoxide (DMSO), then for 30s in 20% EG + 20% DMSO + 0.3 M sucrose (DMSO group). One group of in vitro produced embryos was cryopreserved similarly to the DMSO group, but with 0.75 M sucrose added to the vitrification solution (DMSO 0.75 group). Glycerol group embryos were then loaded into French straws or open pulled Straws (OPS) while the DMSO group embryos were all loaded into OPS and directly plunged into liquid nitrogen. Embryos were warmed with either a one step or three step process. In the one step process, embryos were placed in 0.5 M sucrose. The three-step process was a serial dilution in 0.5, 0.25 and 0.125 M sucrose. The embryos of DMSO 0.75 group were warmed directly by plunging them into tissue culture medium-199 (TCM-199) + 20% foetal bovine serum (FBS) in the absence of sucrose (direct dilution). Following these manipulations, the embryos were transferred in pairs into synchronised recipient ewes and allowed to go to term. The pregnancy and the lambing rate within each group of IVP and IVD embryos indicated that there was no statistical difference among the vitrification protocols.

Effect of recombinant human FSH and LH on in vitro maturation of sheep oocytes; embryo development and viability.

The objective of this work was to study the effect of a preparation of human recombinant gonadotrophins (r-FSH and r-LH) on the in vitro maturation (IVM) and development of sheep oocytes. In addition, the viability of fresh and vitrified blastocysts obtained after transfer was tested. Oocytes collected from slaughtered animals were divided into five different maturation groups. All groups were matured in a medium containing TCM199 with 4 mg/ml BSA, 100 microM cysteamine and 1 microg/ml estradiol-17beta. Each group was also treated with one of the following: 0.1 UI/ml r-FSH (r-FSH group), 0.1UI/ml r-LH (r-LH group), 0.1 UI/ml r-FSH and 0.1 UI/ml r-LH (r-FSH/r-LH group), 5 microg/ml FSH and 5 microg/ml LH hypophysial gonadotrophins (h-G group) as a control, or no gonadotrophins (no-G group). After in vitro fertilization with fresh ram semen, presumptive zygotes were cultured in vitro for 6-7 days and a total of 109 blastocysts were then transferred in pairs into synchronized ewes. To determine the viability of embryos after vitrification, 36 blastocysts from the r-FSH/r-LH group and 30 from the h-G group were vitrified in 10% ethylene glycol (EG) and 10% dimethylsulphoxide (DMSO) for 5min, followed by 20% EG, 20% DMSO and 0.5M Sucrose (S) for <45 s. They were loaded into open pulled straws (OPS) and plunged into LN(2). After warming, the blastocysts were transferred in pairs into synchronized ewes. The highest maturation rate was reached in the r-FSH/r-LH group (91.9%). However, no statistical difference was found when this group was compared with the h-G group (84.0%). Likewise, the cleavage rate of the r-FSH/r-LH group (81.4%) was not significantly different from that of the h-G group (82.3%). The cleavage rates of all other groups, however, were significantly lower than the r-FSH/r-LH and h-G groups. The blastocyst rate was highest in the h-G group (53.6%), and it was statistically higher than in the r-FSH/r-LH group (41.5%). The blastocyst rate was very similar between groups r-FSH and r-FSH/r-LH (42.0 and 41.5%, respectively). The lowest lambing rate (31.8%) was in the no-G group. The highest lambing rate was achieved in the r-FSH/r-LH group (66.6%). The vitrified embryos of h-G and r-FSH/r-LH groups had a very similar lambing rate (16.6% and 19.4%). In conclusion, these data provide support for the hypothesis that sheep oocytes respond to human recombinant gonadotrophins used for in vitro embryo production.

[Our experience in the treatment of superior vena cava syndrome]. / La nostra esperienza nel trattamento della sindrome della vena cava superiore.

In clinical practice SVC syndrome is an important problem, given both the nature of the disease and its fast lethal evolution. Therapy must be instituted as soon as possible because the chances of a positive result are directly related to the staging of the primary illness. Surgery, chemotherapy and high energy therapy can be used. From the literature, although controversial, the superiority of surgical therapy is clear; particularly if up-to-date vascular reconstruction techniques are employed. From March 1980 to March 1988 8 cases of SVC syndrome were observed in which the aetiology was as follows: Hodgkin's disease (2 cases); secondary catheter thrombosis (1 cases); lung carcinoma (5 cases). The 2 cases of Hodgkin's disease were treated by chemotherapy; the secondary thrombosis by open thrombectomy. In the other 5 cases an innominate vein right appendage by-pass was used (3 PTFE, 2 pericardial grafts). The results were encouraging: complete, long-term remission was observed in the Hodgkin and thrombectomy patients. A PTFE graft thrombosis occurred in 2 cases but in the other cases the by-pass is functioning well at a mean 13 months follow-up.

[Popliteal artery entrapment syndrome in our experience]. / La sindrome da intrappolamento dell'arteria poplitea nella nostra esperienza.

Six cases of popliteal entrapment syndrome are presented with emphasis on the diagnostic difficulties related to this disease in its initial functional phase. The utility of Doppler ultrasonography associated with dynamic angiography is underlined. Normally surgical treatment of the disease is problem-free. The important determining factor seems to be medial gemellus hypertrophy. In this case the procedure of choice is thought to be vascular reconstruction associated with the disinsertion of this muscle followed by its reimplantation in a lower and medial position on the semi-membranous tendon muscle in order to avoid any secondary arterial compression.