TRICHINELLOSIS EMERGENCY IN SARDINIA

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Detection of serum antibodies to hepatitis E virus in domestic pigs in Italy using a recombinant swine HEV capsid protein

Background: The hepatitis E virus (HEV) has been detected in both humans and animals, particularly pigs, worldwide. Several evidences, including human infection following consumption of raw contaminated meat, suggest a zoonotic transmission of HEV. In Italy, large circulation of genotype 3 HEV has been reported in swine, and recent studies have confirmed the involvement of this genotype in autochthonous human cases. Result: In this study 111 sera collected from healthy pigs in two Italian regions were tested for anti-HEV IgG antibodies. For specific HEV antibody detection in swine, we developed ELISA and Western blotting methods, using a truncated capsid (ORF2) protein lacking the first 111 amino acids of a swine HEV genotype 3 strain. The ORF2-based ELISA revealed anti-HEV antibodies in 104 out of 111 pigs compared with 102 detected with a commercial ELISA kit. A lower number of sera reacted with the recombinant ORF2 protein in a Western blotting format (81/111). Using a Latent class analysis (LCA), the estimated sensitivities for ELISA-ORF2 and ELISA-kit tests were 0.961 and 0.936, respectively, whereas specificities were 0.599 and 0.475. The estimated sensitivity of Western blotting was 0.775, and the specificity was 0.944. Conclusions: The overall results confirm the high prevalence of HEV seropositive healthy pigs in Italy. Through comparisons with a commercial ELISA test, the swine genotype 3 HEV antigen produced in this study was proven suitable to detect anti-HEV antibodies in pig sera by both ELISA and Western Blotting

Evidence of Hepatitis E Virus (HEV) infection in human and pigs in Sardinia, Italy

Introduction. The aim of this study was to determine the seroprevalence of anti-HEV antibodies in humans sera and to study HEV prevalence in swine from different Sardinian farms, testing viral HEV-RNA in bile samples. Methods. In the first six months of 2008, 532 subjects of whom 402 blood donors and 130 workers at zoonotic risk, were enrolled. Anti-HEV were determined with an enzyme linked immunosorbent assay (ELISA). In positive subjects, RNA was extracted and tested by RT-Nested-PCR. From July 2006 to March 2007, 95 bile samples were collected from randomly selected pigs. RNA was extracted from 250 ?l of bile and tested by RT-Nested-PCR. Results. The overall prevalence of anti-HEV antibodies was 4.3%; 5.0% among blood donors and 2.3% among workers at zoonotic risk, with no statistically significant differences between sex, age classes and occupation. The search for HEV-RNA in the subjects positive for antibodies, gave negative results. HEV genome was detected in 6 of the 95 swine bile samples tested. Sequences were clustered within the genotype 3 and are edited on GenBank under accession number: from FJ850960 to FJ850962 and from FJ883000 to FJ883002. Discussion. The overall prevalence of anti-HEV shows that the virus circulates without giving origin to cases of acute hepatitis. The low prevalence value found in workers at zoonotic risk do not apparently support the hypothesis of professional risk. In this study, HEV-RNA was isolated from pigs in Sardinia for the first time confirming the role of swine as HEV reservoir and the possibility of virus transmission to humans

Relationship of Late Lactation Milk Somatic Cell Count and Cathelicidin with Intramammary Infection in Small Ruminants

Late lactation is a critical moment for making mastitis management decisions, but in small ruminants the reliability of diagnostic tests is typically lower at this stage. We evaluated somatic cell counts (SCC) and cathelicidins (CATH) in late lactation sheep and goat milk for their relationship with intramammary infections (IMI), as diagnosed by bacteriological culture (BC). A total of 315 sheep and 223 goat half-udder milk samples collected in the last month of lactation were included in the study. IMI prevalence was 10.79% and 15.25%, respectively, and non-aureus staphylococci were the most common finding. Taking BC as a reference, the diagnostic performance of SCC and CATH was quite different in the two species. In sheep, receiver operating characteristic (ROC) analysis produced a higher area under the curve (AUC) value for CATH than SCC (0.9041 versus 0.8829, respectively). Accordingly, CATH demonstrated a higher specificity than SCC (82.92% versus 73.67%, respectively) at comparable sensitivity (91.18%). Therefore, CATH showed a markedly superior diagnostic performance than SCC in late lactation sheep milk. In goats, AUC was <0.67 for both parameters, and CATH was less specific than SCC (61.90% versus 65.08%) at comparable sensitivity (64.71%). Therefore, both CATH and SCC performed poorly in late lactation goats. In conclusion, sheep can be screened for mastitis at the end of lactation, while goats should preferably be tested at peak lactation. In late lactation sheep, CATH should be preferred over SCC for its higher specificity, but careful cost/benefit evaluations will have to be made

Epidemiological surveillance for <i>Trichinella britovi</i> infection in free-ranging pigs of Sardinia

The aim of the present work was to investigate on Trichinella sp. infection in free-ranging pigs of the Orgosolo municipality

RUOLO DEL LABORATORIO DI BIOLOGIA MOLECOLARE NELLE CONTAMINAZIONI IN AMBITO OSPEDALIERO DI PSEUDOMONAS AERUGINOSA

Obiettivi: Lo scopo del lavoro è stato valutare la presenza di P.aeruginosa (Pa) e le mutazioni responsabili della produzione di alginato in campioni provenienti da diversi riuniti odontoiatrici quali modello di possibili infezioni nosocomiali. In particolare il gene mucA codifica per una proteina coinvolta nella produzione di alginato in Pa, le mutazioni presenti nel promotore del gene o lungo la parte amino-terminale della proteina, modulano un’iper-espressione di alginato, conferendo al biofilm batterico una barriera pressoché impermeabile agli antimicrobici. Questo aspetto deve essere considerato durante l’utilizzo di microbicidi ossidanti quali H2O2, in grado di determinare mutazioni cromosomiche in Pa, inoltre i perossidi rappresentano i disinfettanti d’elezione nei riuniti, rendendo questi presidi ad alto rischio per contaminazioni di ceppi di Pa farmaco resistenti. Materiali e Metodi: In 90 campioni prelevati su 20 riuniti odontoiatrici la presenza/titolo di Pa, e il profilo nucleotidico di mucA sono stati valutati attraverso PCR real time e Sequenziamento capillare (ABI 310) . Inoltre è stata valutata la massa del biofilm totale calcolando i genomi batterici con PCR quantitativa, amplificando una regione conservata per i batteri del gene rrs, la calibrazione è stata eseguita utilizzando ceppi di Pa a titolo noto e con profilo allelico conosciuto per il gene mucA. Risultati: I risultati hanno evidenziato una contaminazione da Pa nell’ 8% dei riuniti esaminati. Il 20% dei ceppi presentavano mutazioni missense nella regione codificante del gene (GGG/GCG in posizione 63). Conclusioni: Il presente lavoro può rappresentare un metodo utile nello screening per la prevenzione di infezioni nosocomiali sostenute da Pseudomonas spp. 1. Szmolka A, Libisch B, Paszti J, et al. Virulence and antimicrobial resistance determinants of human pathogenic and commensal strains of Pseudomonas aeruginosa. Acta Microbiol Immunol Hung 2009;56:399-402. 2. Hay ID, Gatland K, Campisano A, et al. Impact of alginate overproduction on attachment and biofilm architecture of a supermucoid Pseudomonas aeruginosa strain. Appl Environ Microbiol 2009;75:6022-5

USEFULNESS OF AN MULTIPLEX REALTIME PCR FOR DETECTION OF DIFFERENT CAPRINE ARTHITIS VIRUS GENOTYPES

Introduction: A serological survey carried out between 2005 and 2008 by the “Istituto Zooprofilattico Sperimentale” of Sardinia; showed that CAEV infection is spread throughout the regional territory with a prevalence of positive farms around 80% and a seropositivity of more than 70% of tested animals. The CAE is a serious threat to goat-sheep heritage and is likely to compromise an economy that is a fundamental source of income for breeders, especially in those areas considered "agronomically" difficult. It can be inferred therefore that the disease will become more important in the coming years than it has been in the past. Currently, there are no vaccines for the control of the infection, thus the only prophylaxis that can be implemented is hygienic- sanitary. Prevention measures are based on the removal of healthy infected heads. In this context, laboratory testing for CAEV-positive samples plays a crucial role in the entire diagnostic procedure. The aim of the work was to evaluate a molecular system based on real-time PCR amplification on relatively gag gene sequences in the SRLV, as already indicated by several authors [1,2]. Methods: A total of 50 blood samples from farms in southern Sardinia with a clinical diagnosis for CAEV infections have been anlyzed and proviral DNA was obtained from buffy-coat by GeneProof Pathogen Kit (Brno-Czech Republic), 2 μl of DNA extract was used in a real-time PCR reaction by using the kit SYBR Premix (TaKara-Clontech ®) with a LightCycler II Roche® apparatus following the manufacture istructions [3]. The PCR target region was identified by multiple sequence alignment program Geneious Biomatters Ltd., on gag gene sequences available in the DNA data Bank (GenBank). Melting curve analysis showed two different peaks after real time PCR reaction. Positive samples showed a Tm peak ranged from 82 °C to 88 °C, while the negative sample was identified for a unique peak positioned from 73 °C to 77 °C, Figure 1. These samples resulted 90% CAEV positives and the analysis of the melting curve revealed several multiple-infection (different CAEV variants in the same subject), in this case the most detected sequences are referenced to genotypes B3 and E (subtype Volterra, Fonni, Roccaverano, Seui) GenBank accession n. JF502417, JF502416, EU293537, GQ381130 Conclusions: The presented method/approach could be suitable for a rapid and complete laboratory diagnosis of CAEV infection

SISTEMA INTEGRATO RT-PCR SEQUENZIAMENTO PER LA DIAGNOSI DI LABORATORIO PER INFEZIONE DA VIRUS CAEV

SISTEMA INTEGRATO RT-PCR SEQUENZIAMENTO P023 PER LA DIAGNOSI DI LABORATORIO PER INFEZIONE DA VIRUS CAEV M.C. Carassino1, D. Corda1, S. Fais2, G. Orrù2, M. Liciardi1 1 Istituto Zooprofilattico Sperimentale della Sardegna, Struttura Complessa di Cagliari 2 Servizio di Biologia Molecolare (MBS) Azienda Ospedaliera Universitaria, Cagliari Introduzione: Il virus dell'artite-encefalite caprina (CAEV) è un retrovirus, appartenente al genere Lentivirus. I lentivirus dei piccoli ruminanti (SRLV) sono un gruppo di virus eterogenei dal punto di vista genetico, antigenico e biologico. Gli SRLV comprendono ad oggi almeno 5 genotipi, indicati con le lettere da A a E; in Italia sono presenti almeno 3 genotipi A(MV-like), B ed E. L'ELISA è il metodo diagnostico più diffuso in quanto consente di avere risultati rapidi e poco costosi. Tuttavia il problema della sieroconversione ritardata da parte dei soggetti infetti rende tali risultati poco significativi nelle prime fasi dell'infezione. Scopo di questo lavoro è quello di mettere a punto una modalità diagnostica molecolare, sensibile e specifica che individui i genotipi virali B ed E, in particolare i ceppi Fonni, Volterra, Seui e Roccaverano [1,2]. Materiali e metodi: Il target per la PCR è stato identificato nella regione del genoma LTR-GAG in una zona con alta percentuale di omologia disegnando una coppia di primers degenerati in grado di identificare i 4 ceppi, amplificando un frammento da 180 a 230 paia di basi. Gli esperimenti sono stati condotti su campioni di sangue intero di capre, provenienti da allevamenti del sud-Sardegna, e su un controllo positivo di coltura cellulare di cellule sinoviali di capra, di cui si è proceduto all’estrazione degli acidi nucleici con il kit “Dneasy Blood and Tissue” (Qiagen). Gli estratti sono stati quindi sottoposti ad una reazione di retrotrascrizione- amplificazione usando il kit Invitrogen One-step RT-PCR. Gli amplificati sono stati quindi analizzati tramite corsa elettroforetica su gel d'agarosio al 2%. I campioni presentanti una banda di amplificazione purificati con Qiaquick PCR Purification Kit (Qiagen), sono stati sottoposti alla reazione di Sanger per il sequenziamento, utilizzando il kit “Cycle sequencing kit BigDye Terminator V 1.1” (AB AppliedBiosystems). Il sequenziamento è stato eseguito utilizzando i sequenziatori semi-automatici ABI PRISM 310 (Applied Biosystems) e ABI PRISM 3500 GENETIC ANALIZER (Applied Byosistem). Le sequenze ottenute sono state analizzate utilizzando il programma ChromasPro 2.31 (Technelysium Pty. Ltd.) e il software online ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalo/) Risultati e conclusioni: Sono stati processati un totale di 50 campioni di estratti da sangue intero, 40 dei quali risultati positivi alla PCR, mostrando un segnale di amplificazione tra le 180 bp e le 220 bp. Il 50% dei campioni positivi sono stati sottoposti a sequenziamento e dopo analisi, con il software online BLAST (http://ww.ncbi.nlm.nih.gov/BLAST), è risultato che: 12 campioni hanno dato riscontro per quanto riguarda il virus Caev ( tabella 1) e 8 campioni non hanno portato esito conclusivo. Possiamo quindi affermare che i primers disegnati sono in grado di riconoscere la regione LTR-GAG dei vari genotipi del virus CAEV rimanendo tuttavia necessario per ora come esame di conferma il sequenziamento capillare