TESTUDINID HERPESVIRUS 3: DETECTION AND MOLECULAR CHARACTERIZATION OF STRAINS IN ITALIAN TESTUDO SPP.

Gli Herpesvirales sono un gruppo di virus a DNA ontogeneticamente antico, in grado di infettare diverse specie animali inclusi i rettili. Tra le diverse famiglie di herpesvirus gli Alphaherpesvirus sono la sottofamiglia di Herpesvirales maggiormente coinvolta nelle infezioni di rettili e sono re-sponsabili di lesioni necrotizzanti di gravit\ue0 variabile a carico di diversi organi; spesso le lesioni portano a morte il soggetto sia a causa dei danni indotti direttamente dal virus che per l'insorgenza di infezioni batteriche secondarie. Tra tutte le specie di rettili le tartarughe, le lucer-tole e i serpenti sono gli animali domestici pi\uf9 comuni. I proprietari di tartarughe sono, tra le tipologie di proprietari di rettili, quelli pi\uf9 proni ad acquistare animali prelevati in natura e ad abbandonare specie alloctone sul territorio. Inoltre, sono presenti numerosi allevamenti amatoriali di queste specie che non rispettano le buone norme di allevamento e introducono animali senza effettuare regolare quarantena. La somma di questi comportamenti ha avuto molteplici conse-guenze, tra le quali la diffusione di malattie esotiche e non esotiche nelle popolazioni autoctone di cheloni. Tra tutti i virus che possono causare infezioni nelle tartarughe, gli Herpesvirus sono i maggior-mente rappresentati, con 5 gruppi in grado di infettare testuggini terrestri e acquatiche. Inoltre, sulla base di quanto riportato in letteratura, gli herpesvirus sono una delle principali cause di morte tra i cheloni. Sebbene questi agenti eziologici abbiano un ruolo di rilievo sia da un punto di vista della bioconservazione che economico, non sono presenti informazioni sulla presenza del virus in Italia e, in linea generale, pochissime informazioni sono disponibili sulle caratteristiche genetiche e sui meccanismi di interazione ospite-patogeno. Questo lavoro ha lo scopo di identificare la presenza di Testudinid herpesvirus (TeHVs) in Italia, caratterizzare il genoma del virus ed iniziare ad investigare i meccanismi di interazione ospi-te-patogeno tra TeHV3 e Testudo graeca. Per valutare la presenza dei TeHVs in Italia, abbiamo effettuato uno studio prospettico effet-tuando test ELISA e PCR su tutte le tartarughe presenti nell'Oasi WWF di Vanzago ed abbiamo fatto, inoltre, uno studio retrospettivo effettuando la PCR sui campioni d'archivio dell'Istituto di Anatomia Patologica Veterinaria dell'Universit\ue0 di Milano. Esame necroscopico ed istopatologi-co completo sono stati effettuati su tutti i soggetti deceduti spontaneamente provenienti dall'Oasi di Vanzago. Inoltre, da uno dei soggetti provenienti dall'Oasi \ue8 stato possibile effettuare l'isolamento virale. Utilizzando ceppi di TeHV3 provenienti da Italia, USA e Svizzera, \ue8 stato possibile sequenziare il genoma di TeHV3. La valutazione dell'interazione ospite-patogeno \ue8 stata fatta utilizzando una libreria fagica screenata con siero iperimmune di T. graeca, ottenuto da uno studio di trasmissione precedente. Il nostro studio ha dimostrato che tutti i campioni positivi, sia prospettici che retrospettivi, erano positivi per TeHV3, tranne un caso retrospettivo positivo per TeHV1. . Il genoma di TeHV3 \ue8 risultato essere lungo, circa, 150.080 nucleotidi, con una configurazione genomica di tipo D ed \ue8 risultato altamente colineare con il genoma dell'Human herpesvirus 1. Da un punto di vista immunologico, abbiamo individuato tre potenziali proteine responsabili della risposta immunitaria dell'ospite, TE-17, UL-15 e la gB. Sebbene si fosse inizialmente ritenuto che la gB fosse la principale proteina responsabile della risposta immunitaria, valutazioni successive hanno dimostrato che l'orientamento della sequenza che codifica per la gB nel fagemide fosse antisenso e che, quindi, non potesse essere trascritta. Per valutare il possibile ruolo antigenico di gB, il fagemide originale \ue8 stato modificato in modo tale da contenere un solo frammento codificante. Le tecniche utilizzate per modificare il fagemide sono state: restrizione enzimatica, clonaggio con PCR e direct site mutagenesis con PCR. Sebbene siano stati fatti numerosi tentativi e utilizzate metodologie differenti, a tutt'oggi, non \ue8 stato possibile ottenere colonie batteriche contenti il fagemide modificato e, quindi, non \ue8 stato possibile confermare l'ipotesi originale.Herpesvirales is an ancient group of DNA viruses that infect different animal species, including reptiles. The Alphaherpesvirus is the major subfamily of Herpesvirales involved in reptile infections, and responsible for the onset of variably severe necrotizing lesions, affecting different body parts, and frequently associated with the animal's death. The death of the animal can be caused directly by the effect of the virus, or more frequently by the onset of secondary bacterial infections. Today, the reptile species most commonly kept as household pets are tortoises, lizards, and snakes. Compared to the owners of the other two groups of reptiles, at least in Europe, pet tortoise owners frequently buy animals that are collected from the wild (especially exotic species) and are also prone to abandon these animals into the wild. Moreover, good breeding practices are frequently not applied to reptile breeding centers, which commonly introduce new animals in their enclosure without testing and/or quarantining the animals. These habits have different consequences, including spreading both exotic and non-exotic diseases on Italian soil. Among all viruses infecting chelonians, herpesviruses are the group with the highest number of members, with more than five groups affecting turtles and tortoises. International literature re-ports herpesviruses as one of the most relevant causes of death in chelonians. Despite the relevance of the disease from both a conservational and economical point of view, no information is available on the presence of these viruses in Italy, and very little is known about the genetic characteristics and the host-pathogen interaction mechanisms. This work aims to identify the presence of Testudinid herpesviruses (TeHVs) in Italy, characterize the genome of the virus, and start to understand the host-pathogen interaction mechanisms between TeHV3 and Testudo graeca. To evaluate the presence of TeHVs in Italy, we carried out a prospective study, performing the ELISA test on live animals in the WWF Vanzago's Oasis, combined with PCR on both prospective and retrospective samples collected from the archive of the anatomical pathology section of the University of Milan. Complete necropsy and histological evaluation were performed on all prospective samples after their natural death. Furthermore, viral isolation was successfully carried out on one of the Vanzago cases. Using both TeHV3 strains collected in Italy, USA, and Switzerland, complete genome sequencing was performed. Evaluation of T. graeca immune response against TeHV3 was evaluated by creating a bacteriophage expression library screened with hyperimmune tortoise sera, obtained from a previous transmission study. Our study demonstrates that from both the prospective and retrospective samples, all that were positive were TeHV3, but one retrospective sample was TeHV1 positive. TeHV3 genome sequencing demonstrated that the viral genome is at least 150,080 nucleotides long, arranged in a D-type configuration, and extensively co-linear with the human herpesvirus 1 genome. From an immunological point of view, we were able to identify three relevant TeHV3 candidate antigenic proteins, TE-17, UL-15, and gB. Although we initially supposed the gB was the most relevant antigenic protein in T. graeca immune response against TeHV3, further investigation showed that the gB sequence in the evaluated phagemid was antisense compared to the origin of replication, and could not be transcribed. To assess the possible immunological relevance of TeHV3 gB protein in boosting host immune response, we performed enzymatic restriction, PCR cloning and PCR direct site mutagenesis on the original phagemid, to obtain new structures containing only one of the three possible immunogenic proteins previously identified. Despite the numerous attempts and techniques used, we are currently not able to obtain viable bacteria to confirm our hypothesis

Evaluation of cytological diagnostic accuracy for canine splenic neoplasms : an investigation in 78 cases using STARD guidelines

Cytology represents a useful diagnostic tool in the preliminary clinical approach to canine splenic lesions, and may prevent unnecessary splenectomy. However, few studies have evaluated diagnostic accuracy of cytology in the diagnosis of canine splenic neoplasms. The aim of this study was to determine overall accuracy, sensitivity, specificity, positive and negative predictive values (i.e. diagnostic accuracy indexes) of cytology for canine splenic neoplasms following Standards for the Reporting of Diagnostic Accuracy Studies (STARD) guidelines. A consecutive series of canine splenic cytological samples was retrospectively retrieved from the database of the Diagnostic Pathology Service of the Department of Veterinary Medicine (DIMEVET\u2014University of Milan). Histopathology was set as the diagnostic reference standard. Cytological cases were enrolled when slides were available for review and when the same lesion was submitted for histopathology. Seventy-eight (78) lesions were included in the study. By histopathology, 56 were neoplastic and 22 were non-neoplastic. Cytology had an overall accuracy of 73.08% (95% C.I. 61.84%-82.50%), sensitivity of 64.29% (95% C.I. 50.36%-76.64%), specificity of 95.45% (95% C.I. 77.16%-99.88%), and positive and negative predictive values of 97.3% (95% C.I. 84.01%-99.60%) and 51.22% (95% C.I. 42.21%-60.15%), respectively. Low sensitivity and negative predictive value were balanced by very high specificity and positive predictive value. When positive for neoplasia, cytology represents a useful diagnostic tool to rule in splenic neoplasia, prompting surgery independently from other diagnostic tests. Conversely, a negative cytological result requires additional investigations to confirm the dog to be disease free

Cholate Conjugated Polymeric Amphiphiles as Efficient Artificial Ionophores

A family of amphiphilic copolymers containing hydrophobic cholate pendants has been prepared by copolymerization of cholic acid-based monomer 2-(methacryloxy)-ethyl cholate (MAECA) with polyethylene glycol methyl ether methacrylate (PEGMA). The polymers differ for the content of MAECA that increases from 0 to 35%. The copolymers partition within liposomes and display potent ionophoric activity forming large pores in the membrane and allowing the leakage of small inorganic ions (H+, Na+) and of large polar organic molecules (calcein). Their activity is strictly correlated to the content of cholic acid subunits, increasing as the fraction of cholate moiety increases

Chromogenic in situ hybridization for the detection of lambda and kappa immunoglobulin light chains as a potential auxiliary diagnostic technique in canine plasmacytomas

The heterogeneous morphologic features of canine plasmacytomas (PCTs) can make their differentiation from other round cell tumors challenging. Immunohistochemistry (IHC) for lambda (\u3bb) and kappa (\u43a) immunoglobulin (Ig) light chains is often equivocal because of high background staining. The chromogenic in situ hybridization (CISH) technique for light chains has shown higher sensitivity compared to IHC in human plasma cell tumors. Therefore, we aimed to validate automated CISH for light chains in canine tissues and to evaluate its diagnostic potential in canine PCTs, in conjunction with routinely used IHC markers. CISH for light chains demonstrated a clear signal in plasma cell populations of canine control tissues (lymph nodes, lymphoplasmacytic inflammation) showing a polyclonal pattern with a prevalence of \u3bb-producing cells. CISH detected monotypic light chain expression in 33 of 53 (62%) PCTs, 31 expressing \u3bb and 2 expressing \u43a. CISH was more sensitive than IHC for \u3bb light chain (58% vs. 47%, respectively) and more easily interpretable given the absence of confounding background staining. The absence of CISH staining for both \u3bb and \u43a in a considerable subset of tumors may be the result of lower light chain production by neoplastic cells. Multiple myeloma oncogene 1 (MUM1) was expressed by all but 2 PCTs (96%), which showed \u3bb expression by CISH and IHC. The identification of poorly differentiated canine PCTs requires the assessment of a panel of IHC markers, with the potential support of CISH for Ig light chains

Sex-related differences in cerebrospinal fluid plasma-derived proteins of neurological patients

none11Background and aims: Cerebrospinal fluid (CSF) protein content presents a sexual dimor- phism in humans. We investigated sex-related differences in CSF IgG levels and in the quantification of intrathecal IgG synthesis (IIS). Methods: CSF, serum albumin and IgG were measured in 1519 neurological patients and both linear and hyperbolic formulas were used for the quantification of IIS. CSF-restricted oligoclonal IgG bands (OCBs) were used as “gold standard”. Results: The linear IgG Index showed a weak agreement with OCBs in males and females (k = 0.559, k = 0.587, respectively), while the hyperbolic Reiber’s formulas had a moderate agreement with OCBs in females (k = 0.635) and a weak agreement in males (k = 0.565). Higher CSF albumin and IgG levels were found in men than in women in the whole population and in subjects without IIS after adjusting for age and for serum concentrations of albumin and IgG, respectively (Quade statistics, p < 0.000001). CSF and serum albumin and IgG levels positively correlated to age in both sexes. CSF total protein content did not correlate with CSF leukocyte numbers but was higher in patients with marked pleocytosis. Conclusions: In neurological patients, men have higher levels of CSF serum-derived proteins, such as albumin and IgG.openCastellazzi, Massimiliano; Ferri, Caterina; Alfiero, Sarah; Lombardo, Ilenia; Laudisi, Michele; Tecilla, Ginevra; Boni, Michela; Pizzicotti, Stefano; Fainardi, Enrico; Bellini, Tiziana; Pugliatti, MauraCastellazzi, Massimiliano; Ferri, Caterina; Alfiero, Sarah; Lombardo, Ilenia; Laudisi, Michele; Tecilla, Ginevra; Boni, Michela; Pizzicotti, Stefano; Fainardi, Enrico; Bellini, Tiziana; Pugliatti, Maur

Canine smooth muscle tumors: A clinicopathological study

Canine smooth muscle tumors (SMTs) commonly develop in the alimentary and female genital tracts and less frequently in soft tissue. The definition of histological criteria of malignancy is less detailed for SMTs in dogs than in humans. This study evaluated the clinicopathologic features of canine SMTs and compared the veterinary and human medical criteria of malignancy. A total of 105 canine SMTs were evaluated histologically and classified according to both veterinary and human criteria. The Ki67 labeling index was assessed in all SMTs. Estrogen receptor (ER) and progesterone receptor (PR) expression was evaluated for soft tissue SMTs. Follow-up data were available in 25 cases. SMTs were diagnosed in the female genital tract (42%), alimentary tract (22%), and soft tissue (20%). Soft tissue SMTs frequently arose in the perigenital area, pelvic cavity, and retroperitoneum. A subset of soft tissue SMTs expressed ER and/or PR, resembling the gynecologic type of soft tissue SMT in humans. SMTs were less frequently malignant when assessed with human criteria than with veterinary criteria, better reflecting their benign behavior, especially in the genital tract where human criteria tolerate a higher mitotic count for leiomyoma. Decreased differentiation was correlated with increased proliferation, necrosis, and reduced desmin expression. Mitotic count, Ki67 labeling index, and necrosis were correlated with metastases and tumor-related death. Further prognostic studies are warranted to confirm the better performance of the human criteria when assessing SMT malignancy, especially genital cases, to confirm their usefulness in ER/PR-expressing soft tissue SMTs, and to better define the most useful prognostic parameters for canine SMTs

Role of Lipophilicity in the Activity of Hexameric Cyclic Peptoid Ion Carriers

Two families of hexameric cyclic peptoids decorated with linear N-alkyl and alternated N-alkyl/N-benzyl side chains (2 a\u2013d and 3 a\u2013c, respectively) were designed and synthesized in order to correlate their logP values (from 2.55 to 6.83) to their ionophoric activities. The present contribution confirms the general ability of hexameric cyclic peptoids to behave as efficient cation carriers, corroborates their preference for Na+ ion, among the tested alkali metals, and suggests a Na+/H+ antiport transport mechanism (rate limited by the transport of the proton) for these new ionophores. Our observations indicate that in order to attain an efficient ionophoric activity, a narrow range of liphophilicity is required (4<5). Moreover, to gain information on the solid state stucture of ionophoric cyclic peptoids with linear N-side chains, X-ray crystallographic studies were performed on exemplar compound 2 a. Crystal structure of compound 2 a confirms the tendency of ionophoric cyclopeptoids with linear N-side chains to form layered assemblies

Synthesis and complexing properties of cyclic benzylopeptoids-a new family of extended macrocyclic peptoids

An efficient protocol for the solid-phase synthesis of six members of a new class of extended macrocyclic peptoids (based on ortho-, meta- and para-N-(methoxyethyl)aminomethyl phenylacetyl units) is described. Theoretical (DFT) and experimental (NMR) studies on the free and Na+-complexed cyclic trimers (3\u20135) and tetramers (6\u20138) demonstrate that annulation of the rigidified peptoids can generate new hosts with the ability to sequestrate one or two sodium cations with the affinities and stoichiometries defined by the macrocycle morphology. Ion transport studies have been also performed in order to better appreciate the factors promoting transmembrane cation translocation

Neoplastic diseases in the domestic ferret (Mustela putorius furo) in Italy : classification and tissue distribution of 856 cases (2000-2010)

Background: The aim of this study was to describe the prevalence and tissue distribution of neoplasms in Italian ferrets, compared to the epidemiological data previously reported in USA and Japan. Methods: Signalment and diagnoses of pathological submissions received between 2000 and 2010 were searched; cases with the diagnosis of neoplasm were selected and original sections reviewed to confirm the diagnosis. Results: Nine-hundred and ten samples were retrieved, 690 of which included at least one tumour for a total of 856 tumours. Ferrets with multiple neoplasms were 134 (19.4%). Median age was 5years, and F/M ratio was 0.99. Endocrine neoplasms were the most common. Other frequent tumours were cutaneous mast cell tumours, sebaceous tumours, and lymphomas. Cutaneous squamous cell carcinomas (SCC) were consistently associated with sebaceous tumours. Twenty-four abdominal spindle cell tumours with an undefined origin were observed. Lymphomas and islet cell tumours had a lower incidence compared with previous extra-European studies. Discussion: Epidemiological information on ferret tumours derives from extra-European countries, mostly USA and Japan. In these countries similar distributions with minor discrepancies have been reported. Compared to previous reports, adrenal tumours were more frequent than pancreatic islet cell neoplasms, and a higher number of mesenchymal neoplasms arising from the adrenal capsule was noted. An unusual association between SCC and sebaceous gland neoplasms and a high number of intrabdominal spindle cell neoplasms with unclear primary origin were noted and grants further investigation. Conclusions: The tissue distribution of tumours recorded in this study paralleled previous findings in ferrets from USA and Japan. Some differences have been noted in the frequency of lymphoma, adrenal mesenchymal tumours and cutaneous tumours. Some tumours that are among the most common in other species seem to be uncommon in ferrets and are characterized by distinctive predilection sites