Medico legal aspects on neuromonitoring in thyroid surgery: informed consent on malpractice claims.

AIM: The purpose of this paper is to provide a forensic profile framework of neuromonitoring in thyroid surgery, regarding the information given to the patient and its classification as part of professional liability in the event of recurrent injury. METHOD: Evaluation and reflections on the required behaviour of the surgeon on providing details on the operation before the informed consent is given and to outline the possible legal implications regarding professional liability as a result of recurrent injury. In particular, if it is an obligation to inform the patient about using this method and if it is possible for the surgeon to freely choose whether to employ this method, which is still burdened by a certain percentage of error and for that reason it cannot be defined a "standard of care". RESULTS: To recognize neuromonitoring the role of standard of care in surgery of the thyroid means attribute a role of method able to avoid the surgeon to cause iatrogenic damage to the laryngeal nerve. For the foregoing reasons that is not true, determining false positives and false negatives, and this can be a double edged sword for the surgeon. CONCLUSIONS: Although the progress in the field of thyroid surgery made in the last decade, currently there is no scientific reassuring evidence to completely avoid the possibility of producing an iatrogenic lesion of the laryngeal nerve. Information given to the patient prior to surgery should respect the requirements of completeness, freedom and honesty in order to allow the patient to self-determination.

Cell tropism and molecular epidemiology of Anaplasma platys-like strains in cats.

Bacterial species of the genus Anaplasma are tick transmitted pathogens that negatively impact on animal productions and generate veterinary and public health concerns. This paper reports the identification, molecular characterization and phylogeny of novel unclassified A. platys-like strains in cats. Interestingly, these novel strains are closely related to conspecific strains recently identified in ruminants, and significantly differ from A. platys. A. platys-like strains in cats, unlike ruminants strains, show tropism for platelets. Results have implications in the diagnostic scenario of animal anaplasmosis and provide background for reconstructing the evolutionary history of species genetically related to A. platys.

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.

Intraoperative recurrent laryngeal nerve monitoring in thyroid surgery: is it really useful?

AIM: The aim of this study was to evaluate the ability of intraoperative recurrent laryngeal nerve monitoring to predict the postoperative functional outcome and the potential role of this technique in reducing the postoperative nerve palsy rate. MATERIALS AND METHODS: Between June 2007 and December 2011, 1693 consecutive patients who underwent thyroidectomy by a single surgical team were evaluated. We compared patients who have had a neuromonitoring and patients who have undergone surgery with the only visualization. Patients in which NIM was not utilized (Group A) were 942 against the others 751 (group B). RESULTS: In group A there were 28 recurrent laryngeal nerve injuries (2.97%) of which 21 were transients (2.22%) and 7 were permanents (0.74%). In group B there were 20 recurrent laryngeal nerve injuries (2.66%) of which 14 (1.86%) transients and 6 (0.8%) permanents. Differences between the two groups were not statistically significative. CONCLUSIONS: The technique of intraoperative neuromonitoring in thyroid surgery is safe and reliable in excluding postoperative recurrent laryngeal nerve palsy; it has high accuracy, specificity, sensitivity and negative predictive value. Neuromonitoring is useful to identify the recurrent laryngeal nerve and it can be a useful adjunctive technique for reassuring surgeons of the functional integrity of the nerve but it does not decrease the incidence of injuries compared with visualization alone. Its application can be particularly recommended for high-risk thyroidectomies.

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.

In situ-PCR for the detection of Mycobacterium paratuberculosis DNA in paraffin-embedded tissues.

Molecular biological techniques have permitted the rapid and sensitive detection of the Mycobacterium paratuberculosis genome in infected tissues, most commonly by polymerase chain reaction amplification of sequences in the IS900 DNA insertion sequence. The aim of this work was the detection of M. paratuberculosis DNA in ovine tissues by in situ-polymerase chain reaction, which is sensitive and localises the signal within the tissue sample. Paraffin embedded tissues from three acid-fast positive ovine guts with classical lesions of paratuberculosis, and from negative control samples were tested. A 413-bp fragment of the IS900 sequence was amplified in-situ and hybridised to an internal PCR-synthesised digoxygenin-labelled probe. The samples from sheep affected by paratuberculosis clearly showed cell-specific cytoplasmic signals in mucosal and submucosal macrophages. This technique could be useful both in the diagnosis and study of the pathogenesis of infections in which involvement of M. paratuberculosis is suspected.

Expression and antigenic characterization of recombinant Mycoplasma agalactiae P48 major surface protein.

The gene encoding the P48 major surface lipoprotein of M. agalactiae has been recently characterised. Since its product plays an important role in the immune response of infected animals, in this study we analysed a recombinant P48 expressed in E. coli. Multiple point mutations were introduced by site directed mutagenesis in order to convert four tryptophan TGA codons, which are a typical feature of the mycoplasma genetic code, into the standard TGG. The mutated p48 gene was subcloned into pGex-2T and expressed in fusion with glutathione-S transferase. Following purification steps, P48 was eluted from carrier protein by thrombin digestion and used in Western blot and indirect ELISA using well-characterised sheep sera. Results demonstrate that specific antibodies against P48 are detected 3 weeks after onset of clinical disease and the recombinant P48 is a diagnostically relevant marker of M. agalactiae infection.

An accessory open reading frame (orf-x) of jaagsiekte sheep retrovirus is conserved between different virus isolates.

Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of a contagious lung tumour of sheep known as sheep pulmonary adenomatosis (syn: ovine pulmonary carcinoma, jaagsiekte). JSRV exhibits a simple genetic organization, characteristic of the type D and type B retroviruses, with the canonical retroviral sequences gag, pro, pol and env encoding the structural proteins of the virion. An additional open reading frame (orf-x), of approximately 500 bp overlapping pol, is present in the only two complete sequences of JSRV published to date. Since very little information is available on the biology of JSRV it is important to establish if orf-x is conserved between different virus isolates. In this study we analysed the orf-x region of JSRV isolates collected from the United Kingdom, Italy, Spain and South Africa. In addition we also analysed the presence of orf-x in JSRV-related endogenous sequences (enJSRVs) present in the sheep genome. Orf-x was highly conserved in all the exogenous isolates (n=10) and in most of the endogenous sequences (n=8). Thus orf-x may be an accessory gene of JSRV and haves a biological function which might be advantageous to JSRV. Phenetic analysis conducted on the complete orf-x nucleotide sequences seems to highlight the presence of three distinct groups statistically well supported by bootstrapping: i) exogenous JSRV sequence from the UK; ii) exogenous JSRV sequences from Southern Europe and iii) the exogenous South African strain plus all the endogenous sequences analyzed and collected from Australia, Italy, UK and South Africa.

P48 major surface antigen of Mycoplasma agalactiae is homologous to a malp product of Mycoplasma fermentans and belongs to a selected family of bacterial lipoproteins.

A major surface antigenic lipoprotein of Mycoplasma agalactiae, promptly recognized by the host's immune system, was characterized. The mature product, P48, showed significant similarity and shared conserved amino acid motifs with lipoproteins or predicted lipoproteins from Mycoplasma fermentans, Mycoplasma hyorhinis, relapsing fever Borrelia spp., Bacillus subtilis, and Treponema pallidum.

Genetic and antigenic characterization of caev (caprine arthritis-encephalitis virus) recombinant transmembrane protein.

The env gene fragment of an Italian strain of Caprine Arthritis Encephalitis virus (CAEV) coding for the hydrophilic region of transmembrane protein was amplified, cloned and expressed in prokaryotic system as fusion protein with glutathione-S-transferase. Sequence analysis revealed 63 to 66% amino acid homology, when compared with three ovine lentiviruses and 83% when compared with one caprine lentivirus. The recombinant transmembrane protein was efficiently expressed, purified under denaturing conditions and used as antigen in western blotting and ELISA. Sera from clinically diseased goats strongly reacted in western blotting and naturally infected animals seroconverted between 20 and 33 weeks of age. An indirect ELISA performed with this antigen showed improved sensitivity in comparison with agar gel immunodiffusion test. Our results confirm that transmembrane protein is an important immunological marker in CAEV infection and its use as antigen may enhance the validity of serological diagnosis of Caprine Arthritis Encephalitis.

Dermatophilosis in sheep: first report in Italy, experimental reproduction and evaluation of immune response.

A severe exudative-crustous and proliferous dermatitis in a 2 year old sheep caused by Dermatophilus congolensis (observed for the first time in Italy), is reported. The disease was reproduced experimentally in sheep, goats, rabbits and guinea pigs, whose skin was treated in different ways before infection. E.L.I.S.A. and Immunoblotting tests carried out in experimentally infected sheep, showed the antigenic complexity of the pathogen and the existence of cross-immunity to the protein components. Intradermoreaction tests were carried out in all animals. The development of a positive reaction only in rabbits and guinea pigs, confirmed that these animals have a cellular immunity against Dermatophilus congolensis.