Italian Isolates Research Articles

Susceptibility of European sheatfish Silurus glanis to a panel of ranaviruses

Summary The current study was undertaken in order to assess the risk that different ranaviruses might impose on European sheatfish aquaculture. As the European sheatfish virus (ESV) is a known pathogen causing losses in European sheatfish aquaculture, it was assumed that closely related exotic ranaviruses might also be able to infect European sheatfish and probably cause disease and mortality in this species. The differential susceptibility of European sheatfish (Silurus glanis) to various ranavirus isolates was assessed at two different temperatures (15°C and 25°C) in a recirculation system. Fish were infected experimentally with a panel of ranavirus isolates including ESV, European catfish virus (ECV), European catfish virus isolate 24 (ECV-24), Epizootic haematopoietic necrosis virus (EHNV), Rana esculenta virus isolate Italy 282/ I02 (REV), short-finned eel virus (SERV), Bohle iridovirus (BIV), guppy virus 6 (GV6), doctor fish virus (DFV) and Frog virus 3 (FV3). Significant mortalities were observed, as expected, in fish infected with ESV at 15°C (100%) as well as at 25°C (86/83%). Fish infected with ECV at 15°C showed no clinical signs of disease (8% mortality), whereas those fish infected at 25°C exhibited a cumulative mortality of 54%. Fatal disease was also induced by Italian isolate ECV-24 at 25°C (81%). Virus isolates ESV, ECV and ECV-24, generally the most genetically closely related viruses, were successfully isolated from dead fish by cell culture with subsequent identification by polymerase chain reaction (PCR) and sequence analysis. However, no mortality or clinical signs of disease were observed in the groups of sheatfish infected with the other ranaviruses investigated in the study, and none of those viruses were re-isolated in cell culture or identified by PCR. It was concluded that European sheatfish are susceptible to infection with ESV, ECV and ECV-24 under laboratory conditions, but not to infection with EHNV, REV, SERV, BIV, GV6, DFV or FV3. For ESV, the incubation period was shorter at 25°C compared to 15°C water temperature, but whereas all fish died after ESV infection at 15°C, some fish survived the infection at 25°C. Futhermore, the very young sheatfish were susceptible to ECV and ECV-24 at 25°C, whereas there was no significant mortality in the group of older sheatfish challenged with ECV at 15°C. Therefore, the clinical characteristics of the disease seem to depend on the age of the fish as well as on the water temperature.

Genotypic and phenotypic features of enteropathogenic Escherichia coli isolated in industrialized and developing countries

Typical EPEC are considered a leading cause of diarrhoea in developing countries, while atypical EPEC have been isolated more frequently in developed areas. The actual geographic distribution of the two EPEC subgroups is controversial, since data can be highly influenced by laboratory resources. This study aimed to compare the distribution of typical and atypical EPEC among children in developed and developing countries, and to characterize the bacterial isolates, using a unique methodological approach. A total of 1,049 E. coli were isolated from faeces of children with acute diarrhoea in Mozambique, Angola and Italy, and processed by PCR to assess the presence of a large panel of virulence genes. All isolates classified as EPEC were further characterized by evaluating adherence and capability to induce actin rearrangement on Hep-2 cells. Overall we isolated 59 EPEC, likewise distributed in the three countries, representing the 5.04%, 4.44% and 6.97% of all Mozambican, Angolan and Italian isolates, respectively. Nevertheless, the geographic distribution of the two EPEC subgroups was not homogeneous: in Italy we isolated 28 aEPEC but no tEPEC, while in Angola and Mozambique the percentage of the two subgroups was comparable. Twelve atypical EPEC were FAS positive and able to induce localized-like adherence on Hep-2 cells, but no correlation with the geographic origin of isolates was observed. Atypical EPEC are present in sub-Saharan areas in a percentage similar to that of typical strains, and are not mainly restricted to industrialized countries, as it was previously supposed.

Small ruminant lentiviruses in Jordan: evaluation of sheep and goat serological response using recombinant and peptide antigens

Small ruminant lentiviruses infect sheep and goats worldwide, causing chronic progressive diseases and relevant economic losses. Disease eradication and prevention is mostly based on serological testing. The goal of this research was to investigate the presence of the small ruminant lentiviruses (SRLVs) in Jordan and to characterize the serological response in sheep and goat populations. A panel of sera were collected from flocks located in Northern Jordan and Jordan Valley. The samples were tested using three ELISA assays: a commercially available ELISA based on p25 recombinant protein and transmembrane peptide derived from British maedi-visna virus (MVV) EV1 strain, an ELISA based on P16-P25 recombinant protein derived from two Italian strains representative of MVV- and caprine arthritis encephalitis virus (CAEV)-like SRLVs, and an ELISA based on SU5 peptide from the same two Italian isolates. The results indicate that both MVV- and CAEV-like strains are present in Jordan and that the majority of the viruses circulating among sheep and goat populations belong to the MVV-like genotype.

Molecular epidemiology of Italian clinicalCryptococcus neoformansvar.grubiiisolates

Cryptococcus neoformans variety grubii is the major etiological agent of cryptococcal meningitis in both immunocompromised and immunocompetent patients. The current PCR-based molecular methods are not sufficient to discriminate among the different populations of this yeast. Therefore, the aim of the present study was to investigate the genotypes of the Italian clinical C. neoformans var. grubii isolates by multilocus sequence typing (MLST). A total of 53 isolates, each representative of a single case, were studied. Genotyping was performed using the ISHAM Cryptococcus MLST consensus scheme and the results were compared to the publically available global C. neoformans var. grubii MLST dataset. A total of 16 genotypes were identified; 14 were new genotypes, one was identical to sequence type (ST) ST81, which had been previously reported from Thailand, and one to ST23 already identified in Uganda, the USA and Korea. Sequence type ST61 was the most numerous, including 16 isolates. Network phylogenetic analysis showed that the Italian isolates could be divided into at least three clusters with similarities with those recovered in Africa, Asia and Americas. Distribution of the STs among the isolates could not be correlated to the hospital in which they were recovered or to the HIV status of the patients. The majority of the isolates belonged to the molecular type VNI; three belonged to the rare molecular type VNII and one to the VNB group, which until now had not been described in Europe. The results reveal that the Italian C. neoformans var. grubii population presents a distinct variability, displaying a high number of new genotypes, and probably recombines sexually.

Detection and Molecular Characterization of Pepper Mild Mottle Virus from Turkey

AbstractUsing double‐antibody sandwich–enzyme‐linked immunosorbent assay (DAS‐ELISA), pepper mild mottle virus (PMMoV) was detected in 27 pepper (Capsicum spp.) plants of 3000 tested and found to be present in Adana, Antalya, Kahramanmaraş, Mersin and Şanlıurfa, all provinces devoted to pepper production in southern Turkey. Results of reverse transcription‐polymerase chain reaction (RT‐PCR) using primers specific to RNA‐dependent RNA polymerase (RdRp) and capsid protein (CP) genes confirmed those of ELISA by amplifying all PMMoV‐infected plants. Restriction fragment length polymorphism (RFLP) and PCR assays using sequence characterized amplified region (SCAR) marker primers showed that PMMoV from Turkey overcomes L3‐gene‐mediated resistance, so pepper plantations are susceptible to PMMoV infection. Sequences of CPs showed high amino acid identities (92–99%) with their homologues in the database and, furthermore, to share a distinguished molecular print found common uniquely in pathotypes P1,2,3. The phylogenetic tree allocated the Turkish isolates in one cluster together with PMMoV pathotypes P1,2,3 of the Italian, Spanish and Israeli isolates, all reported to overcome the L3‐resistance‐breaking gene in pepper. This is the first molecular information on PMMoV isolates present in Turkey, for which this information could have guiding significance in future pepper resistance breeding in the country.

Genetic characterization of northeastern Italian population isolates in the context of broader European genetic diversity

Population genetic studies on European populations have highlighted Italy as one of genetically most diverse regions. This is possibly due to the country's complex demographic history and large variability in terrain throughout the territory. This is the reason why Italy is enriched for population isolates, Sardinia being the best-known example. As the population isolates have a great potential in disease-causing genetic variants identification, we aimed to genetically characterize a region from northeastern Italy, which is known for isolated communities. Total of 1310 samples, collected from six geographically isolated villages, were genotyped at >145 000 single-nucleotide polymorphism positions. Newly genotyped data were analyzed jointly with the available genome-wide data sets of individuals of European descent, including several population isolates. Despite the linguistic differences and geographical isolation the village populations still show the greatest genetic similarity to other Italian samples. The genetic isolation and small effective population size of the village populations is manifested by higher levels of genomic homozygosity and elevated linkage disequilibrium. These estimates become even more striking when the detected substructure is taken into account. The observed level of genetic isolation in Friuli-Venezia Giulia region is more extreme according to several measures of isolation compared with Sardinians, French Basques and northern Finns, thus proving the status of an isolate.

Comparison ofAlternariaspp. Collected in Italy from Apple withA. maliand Other AM-Toxin Producing Strains

Since 1999, a disease of apple caused by an Alternaria sp. has been affecting orchards in northern Italy resulting in necrotic spots on leaves and on fruit. Forty-four single-spored isolates were obtained from diseased plant materials to investigate the diversity of this fungus in Italy and to compare these isolates to isolates of Alternaria associated with apple disease in previous studies, including A. mali, causal agent of apple blotch. All isolates, including the reference strains, were tested for pathogenicity utilizing in vitro bioassays on detached leaf or on fruit ('Golden Delicious'). In addition, morphological characterizations were conducted describing both the three-dimensional sporulation pattern and the colony morphology of each isolate. In order to assess the genetic diversity within the Italian Alternaria population, sequence characterization of specific loci and anonymous regions (endoPG, OPA1-3, OPA2-1, and OPA10-2) and genetic fingerprinting based on amplified fragment length polymorphism and inter simple sequence repeat markers were performed. The single spore isolates exhibited differential pathogenicity, which did not correlate with the morphological groupings or to groupings defined by molecular approaches. Moreover, 10 pathogenic isolates out of the 44 single-spored tested were positive for the host-specific AM-toxin gene based upon polymerase chain reaction amplification using specific primers for the AM-toxin gene. This suggests that the production of the AM-toxin may be involved in pathogenesis by some of the Italian isolates of A. alternata from apple. However, this research also suggests that a number of different Alternaria genotypes and morphotypes may be responsible for the apple disease in Italy and that a single taxon cannot be defined as the sole causal agent.

Characterization of Citrus tristeza virus isolates recovered in Syria and Apulia (southern Italy) using different molecular tools.

Citrus tristeza virus (CTV) is the causal agent of the most important virus disease of citrus. CTV isolates differing in biological and molecular characteristics have been reported worldwide. Recently, CTV was detected in Syria in citrus groves from two Governorates (Lattakia and Tartous) and several CTV outbreaks have been reported in Apulia (southern Italy) since 2003. To molecularly characterize the CTV populations spreading in Syria and Italy, a number of isolates from each region was selected and examined by different molecular approaches including: Multiple Molecular Markers analysis (MMM), real time RT-(q)PCR, single strand conformation polymorphism (SSCP) of the major coat protein (CP) gene (P25), and sequence analysis of the CP (P25), P18, P20 and RdRp genes. SSCP analysis of CP25 yielded two distinct simple patterns among the Syrian isolates and three different patterns in the Italian isolates. Based on MMM analysis, all Syrian CTV isolates were categorized as VT-like genotype, whereas the Italian isolates reacted only with the markers specific for the T30 genotype. These findings were also confirmed by RT-qPCR and by sequencing analysis of four genomic regions. The Italian isolates had nucleotide identities which varied: from 99.5 to 99.8 for the CP gene; from 97.4% to 98.3% for the P18 gene; from 98.6% to 99.8% for the P20 and from 97.8% to 99.1% for the partial RdRp sequenced. High sequence identity was found for all genomic regions analyzed between the Syrian isolates (from 98.9% to 99.6%). These results show that the CTV populations spreading in Apulia and Syria are associated with different genotypes, indicating different potential impacts on the citrus trees in the field. Since in both areas the introduction of the virus is relatively recent, infected plants resulted to contain a single and common genotype, suggesting that CTV is spreading from the first outbreaks by aphids or local movement of autochthonous infected plant material.

First Report of Alfalfa mosaic virus Infecting Tedera (Bituminaria bituminosa (L.) C.H. Stirton var. albomarginata and crassiuscula) in Australia.

Tedera (Bituminaria bituminosa (L.) C.H. Stirton vars albomarginata and crassiuscula) is being established as a perennial pasture legume in southwest Australia because of its drought tolerance and ability to persist well during the dry summer and autumn period. Calico (bright yellow mosaic) leaf symptoms occurred on occasional tedera plants growing in genetic evaluation plots containing spaced plants at Newdegate in 2007 and Buntine in 2010. Alfalfa mosaic virus (AlMV) infection was suspected as it often causes calico in infected plants (1,2) and infects perennial pasture legumes in local pastures (1,3). Because AlMV frequently infects Medicago sativa (alfalfa) in Australia and its seed stocks are commonly infected (1,3), M. sativa buffer rows were likely sources for spread by aphids to healthy tedera plants. When leaf samples from plants with typical calico symptoms from Newdegate (2007) and Buntine (2010) were tested by ELISA using poyclonal antisera to AlMV, Bean yellow mosaic virus (BYMV) and Cucumber mosaic virus (CMV), only AlMV was detected. When leaf samples from 864 asymptomatic spaced plants belonging to 34 tedera accessions growing at Newdegate and Mount Barker in 2010 were tested by ELISA, no AlMV, BYMV, or CMV were detected, despite presence of M. sativa buffer rows. A culture of AlMV isolate EW was maintained by serial planting of infected seed of M. polymorpha L. (burr medic) and selecting seed-infected seedlings (1,3). Ten plants each of 61 accessions from the local tedera breeding program were grown at 20°C in an insect-proof air conditioned glasshouse. They were inoculated by rubbing leaves with infective sap containing AlMV-EW or healthy sap (five plants each) using Celite abrasive. Inoculations were always done two to three times to the same plants. When both inoculated and tip leaf samples from each plant were tested by ELISA, AlMV was detected in 52 of 305 AlMV-inoculated plants belonging to 36 of 61 accessions. Inoculated leaves developed local necrotic or chlorotic spots or blotches, or symptomless infection. Systemic invasion was detected in 20 plants from 12 accessions. Koch's postulates were fulfilled in 12 plants from nine accessions (1 to 2 of 5 plants each), obvious calico symptoms developing in uninoculated leaves, and AlMV being detected in symptomatic samples by ELISA, inoculation of sap to diagnostic indicator hosts (2) and RT-PCR with AlMV CP gene primers. Direct RT-PCR products were sequenced and lodged in GenBank. When complete nucleotide CP sequences (666 nt) of two isolates from symptomatic tedera samples and two from alfalfa (Aq-JX112758, Hu-JX112759) were compared with that of AlMV-EW, those from tedera and EW were identical (JX112757) but had 99.1 to 99.2% identities to the alfalfa isolates. JX112757 had 99.4% identity with Italian tomato isolate Y09110. Systemically infected tedera foliage sometimes also developed vein clearing, mosaic, necrotic spotting, leaf deformation, leaf downcurling, or chlorosis. Later-formed leaves sometimes recovered, but plant growth was often stunted. No infection was detected in the 305 plants inoculated with healthy sap. To our knowledge, this is the first report of AlMV infecting tedera in Australia or elsewhere.

Hepatitis C virus genotype 4d in Southern Italy: Reconstruction of its origin and spread by a phylodynamic analysis

Hepatitis C Virus (HCV) genotype 4 predominates in Middle East and Central Africa countries. Recently, it has become also prevalent in Southern European countries where it is thought to have been introduced through immigration and the movement of intravenous drug users. In Italy, the prevalence of genotype 4 is particularly high (4.5%) in Southern regions, such as Calabria, and reaches values of 8.4% in specific areas where there appears to be endemic circulation of this genotype. In the present study, the phylogeny of HCV subtype 4d isolated from 19 Italian patients in Calabria was investigated by analysing a fragment of the NS5B viral genomic region. A Bayesian coalescent-based framework was used to estimate origin and spread of the HCV 4d in this area. The mean evolutionary rate HCV 4d NS5B sequences was estimated using a dataset of sequences sampled at known times and a relaxed clock constant model that best fitted the data. By using a Bayesian coalescent method, the Italian 4d isolates collected in Calabria were found to share a common ancestor with reference 4d isolates whose origin was traced back to 1940s. The genotype 4d epidemic in Southern Italy was maintained in a steady non-expanding phase until the late 1970s after that it grew exponentially up to 1990s probably sustained by the vast increase of unsafe blood transfusions and the spread of illicit intravenous drug users.

Using forensic microsatellites to decipher the genetic structure of linguistic and geographic isolates: A survey in the eastern Italian Alps

The study of geographically and/or linguistically isolated populations could represent a potential area of interaction between population and forensic genetics. These investigations may be useful to evaluate the suitability of loci which have been selected using forensic criteria for bio-anthropological studies. At the same time, they give us an opportunity to evaluate the efficiency of forensic tools for parentage testing in groups with peculiar allele frequency profiles. Within the frame of a long-term project concerning Italian linguistic isolates, we studied 15 microsatellite loci (Identifiler kit) comprising the CODIS panel in 11 populations from the north-eastern Italian Alps (Veneto, Trentino and Friuli Venezia Giulia regions). All our analyses of inter-population differentiation highlight the genetic distinctiveness of most Alpine populations comparing them either to each other or with large and non-isolated Italian populations. Interestingly, we brought to light some aspects of population genetic structure which cannot be detected using unilinear polymorphisms. In fact, the analysis of genotypic disequilibrium between loci detected signals of population substructure when all the individuals of Alpine populations are pooled in a single group. Furthermore, despite the relatively low number of loci analyzed, genetic differentiation among Alpine populations was detected at individual level using a Bayesian method to cluster multilocus genotypes. Among the various populations studied, the four linguistic minorities (Fassa Valley, Luserna, Sappada and Sauris) showed the most pronounced diversity and signatures of a peculiar genetic ancestry. Finally, we show that database replacement may affect estimates of probability of paternity even when the local database is replaced by another based on populations which share a common genetic background but which differ in their demographic history. These findings point to the importance of considering the demographic and cultural profile of populations in forensic applications, even in a context of substantial genetic homogeneity such as that of European populations.

Alteration of Liver Enzymes Is a Feature of the Myh9-Related Disease Syndrome

Background MYH9-related disease (MYH9-RD) is a rare autosomal dominant genetic syndrome characterized by congenital thrombocytopenia associated with the risk of developing progressive nephropathy, sensorineural deafness, and presenile cataract. During the collection of a large case-series of patients with MYH9-RD we noticed several cases with unexplained elevation of liver enzymes. Our aim was to evaluate if the alteration of liver tests is a feature of the MYH9-RD and to define its clinical significance.Methods and FindingsData concerning liver tests, prospectively recorded in the Italian Registry for MYH9-RD, were collected and compared with those of three control populations: patients with autoimmune thrombocytopenia, patients with inherited thrombocytopenias other than MYH9-RD, and the participants to a large epidemiologic survey in an Italian geographic isolate. Thirty-eight of 75 evaluable MYH9-RD patients (50.7%) showed an elevation of ALT and/or AST, and 17 of 63 (27.0%) an increase of GGT. The increases ranged from 1.9±0.7 to 2.7±1.6 fold the upper normal limit. The prevalence of liver test alterations was significantly higher in MYH9-RD patients than in each of the control populations, with odds ratios ranging from 8.2 (95% CIs 2.2–44.8) to 24.7 (14.8–40.8). Clinical follow-up and more detailed liver studies of a subset of patients, including ultrasound liver scan, liver elastography and liver biopsy in one case, did not show any significant structural damage or evolution towards liver insufficiency.ConclusionsElevation of liver enzymes is a frequent and previously unrecognized feature of the MYH9-RD syndrome; however, this defect does not appear to have poor prognostic value.

An endopolygalacturonase gene of Diaporthe helianthi

In an attempt to define virulence determinants of Diaporthe helianthi, the causative agent of sunflower stem canker, the gene named Dhpg , coding for an endopolygalacturonase, was investigated in the highly virulent strain 8/96 isolated from a diseased plant in France. The 1130 bp coding sequence, in addition to 1473 bp from the upstream region, were cloned, sequenced and analyzed. Dhpg was most closely related to polygalacturonase genes from phytopathogenic fungi, such as Ophiostoma ulmi , O . novo-ulmi , and Gibberella zeae . A Southern blot RFLP analysis proved that Dhpg was represented in single-copy in the genome of the isolate 8/96. Analyses carried out both in vitro on liquid cultures and in vivo on host infected tissues provided evidence of the constitutive expression of the Dhpg transcript under all tested conditions. Moreover, an European collection of D. helianthi isolates was screened for the presence of Dhpg homologues by PCR, revealing the same single band in all French and Yugoslavian isolates, while one Romanian and all Italian isolates displayed a variable pattern. This genetic variability related to the different geographic origin of D. helianthi is consistent with data previously reported for different loci on the same set of isolates. This is the first report of a polygalacturonase gene in D. helianthi .

Characterization of Fusarium verticillioides strains isolated from maize in Italy: Fumonisin production, pathogenicity and genetic variability

Fusarium verticillioides (teleomorph Gibberella moniliformis) is the main fungal agent of ear and kernel rot of maize (Zea mays L.) worldwide, including Italy. F.verticillioides is a highly toxigenic species since it is able to produce the carcinogenic mycotoxins fumonisins. In this study, 25 F. verticillioides strains, isolated from maize in different regions of Italy were analyzed for their ability to produce fumonisins, their pathogenicity and their genetic variability. A further referenced strain of G. moniliformis isolated from maize in USA was also used as outgroup. The fumonisins B1, B2, and B3 were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Pathogenicity tests were carried out by symptom observation and determination of growth parameters after inoculation of maize seeds, seedlings and wounded detached leaves. Total genomic DNA was used for Amplified Fragment Length Polymorphism (AFLP) analysis. About 20% of the analyzed strains were unable to produce fumonisins in in vitro experiments on inoculated maize flour, while, among fumonisin producers, a great variability was observed, with values ranging from 1 to 115 mg kg−1. The different analyzed strains showed a wide range of pathogenicity in terms of effect on seed germination, seedling development and of symptoms produced on detached leaves, which were not correlated with the different in vitro fumonisin production. AFLP analysis indicated the presence of genetic diversity not only between the Italian strains and the American reference but also among the Italian isolates.

First Record of Alfalfa mosaic virus in Teucrium fruticans in Italy.

The Teucrium genus (Lamiaceae family) contains ~300 species of evergreen and deciduous shrubs with some species widely used as ornamental plants in rock gardens. During the springs of 2010 and 2011, some plants of Teucrium fruticans L., also known as "tree germander", growing singly in pots in a Ligurian nursery (Savona Province, northern Italy), were noted for a bright yellow calico mosaic on the leaves (~1% of ~2,000 plants inspected exhibited symptoms). Preliminary electron microscope observations of leaf-dips showed semispherical to bacilliform particles, consistent with Alfamovirus and Oleavirus, in preparations obtained only from leaves of symptomatic plants. Three symptomatic and two asymptomatic plants were checked for Cucumber mosaic virus or Alfalfa mosaic virus (AMV) in protein A sandwich (PAS)-ELISA with commercial kits (Bioreba, Reinach, Switzerland) and Olive latent virus 2 (OLV2) by immunodecoration of virus particles with an OLV2 antiserum produced against an Italian OLV2 isolate. Symptomatic plants were positive only to AMV and all asymptomatic plants were negative to all viruses checked. The virus was successfully transmitted mechanically to Chenopodium amaranticolor and Ocimum basilicum that reacted, as expected for infections caused by AMV (1), with a chlorotic local lesion followed by mosaic and bright yellow mosaic, respectively. The disease was transmitted also by grafting an infected scion on healthy T. fruticans. Symptoms appeared after ~3 weeks in one plant of six grafted. AMV infection in a symptomatic grafted plant was verified by PAS-ELISA, confirming that bright yellow mosaic symptoms observed in T. fruticans were induced by an isolate of AMV. Immunocapture reverse transcription (IC-RT)-PCR assay, following the protocol described by Wetzel et al. (4), was performed on leaf extracts from one symptomatic plant using a polyclonal serum raised against a French isolate of AMV, provided by H. Lot (INRA, Station de Pathologie Végétale, Avignon, France). Specific AMV primer pair was used in the RT-PCR reactions (2). A DNA fragment of ~750 bp, covering the entire coat protein gene (CP), was obtained after IC-RT-PCR. The amplicon was gel purified with the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI), cloned into pGEMT-easy vector (Promega) and two independent clones sequenced on both strands at MWG Biotech (Ebersberg, Germany). The consensus sequence was submitted to EMBL (No. FR854391). Pairwise comparison of the AMV-T. fruticans isolate CP sequence (named Tef-1) with those of AMV reference isolates revealed the maximum (98.0 to 97.3%) nucleotide identities with isolates belonging to subgroup I, 95.5 to 94.0% identities with subgroup IIA isolates, and 95.6% identity with the subgroup IIB isolate Tec-1 (3). Among subgroup I isolates, Tef-1 had the maximum CP nucleotide identity with the CP gene belonging to an AMV isolate identified in 2010 in Lavandula stoechas in the same geographic area, suggesting a common origin for these two viral isolates. Overall results clearly indicate that an AMV isolate was the causal agent of the calico-type mosaic observed in T. fruticans. To our knowledge, this is the first report of T. fruticans as a natural host of AMV.

Amino acid diversity of antigenic sites of Iranian type O foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is a major cause of endemic outbreaks in livestock in Iran. In this study, clinical field samples of foot-and-mouth disease virus were collected from an outbreak in Khorasan Razavi Province during April and August of 2010, and subjected to indirect sandwich ELISA and RT-PCR. The viral serotype circulating during the period was confirmed to be type O. The virus was then genetically characterized for its complete P1 genomic sequences to compare with nine corresponding nucleotide sequences of representative foot-and-mouth disease viruses (FMDVs) registered in the GenBank. The P1-coding region was 2208 nucleotides in length with 736 encoded amino acid residues. Phylogenetic analysis revealed two major lineages of A (with three additional clusters) and B. Iranian field isolate was grouped within cluster I, most closely related to Pakistani strains PAK/39/2008 and PAK/29/2008 sharing 98.37 and 98.1% amino acid identity, respectively, demonstrating the close epidemiological links between countries in the region. In contrast, our isolate showed low amino acid identity with Italian isolate O-2-Brescia (93.48%) and Argentinean isolate O1 Caseros (93.75%). Based on multiple sequence alignments, comparison of sequences showed that the characteristic amino acid mutations were found in the VP1, VP2 and VP3 proteins of isolated virus. This article is the first to report on the complete P1 genomic characterization of type O FMDV circulating in Iran

Molecular characterization of Italian Candida parapsilosis isolates reveals the cryptic presence of the newly described species Candida orthopsilosis in blood cultures from newborns

The authors report the molecular characterization of Candida parapsilosis isolates recovered from the blood and venous central catheter tips of patients admitted to different care units of the Polyclinic Hospital, University of Messina, Italy. Among 97 presumed C. parapsilosis isolates examined, 94 were identified as C. parapsilosis sensu stricto and the remaining 3 isolates were found to belong to the cryptic species Candida orthopsilosis which was recovered only from blood cultures of neonates (<30 days old) born prematurely. No C. metapsilosis was found in this study. This study emphasizes the role of C. parapsilosis as an important nosocomial pathogen, and it also describes, for the first time, the occurrence of C. orthopsilosis in newborns.

Genetic characterization of Pyrenophora graminea isolates and the reactions of some barley cultivars to leaf stripe disease under greenhouse conditions

The genetic diversity of Pyrenophora graminea, the causal agent of barley leaf stripe disease, was characterized by restriction fragment length polymorphism (RFLP) of the rDNA internal transcribed spacer region and intersimple sequence repeat (ISSR) analysis. A total of 45 P. graminea isolates recovered from different geographic origins and barley cultivars or genotypes produced identical RFLP banding patterns with HinfI, MspI, Hin6I and BsuRI restriction enzymes. ISSR analysis indicated that there was little genetic variability among the isolates. A dendrogram based on Jaccard's coefficient separated the isolates into 4 clusters with 12% dissimilarity. In the dendrogram, the isolates collected from different geographic regions in Turkey were clustered separately from the Italian isolates. These results indicated that the Turkish isolates of P. graminea were genetically homogeneous and may be derived from a single gene pool. In addition, 13 isolates were tested for pathogenic variability on 48 barley cultivars. The cultivars Durusu, Balkan 96 (Igri), Çumra 2001 and Anadolu 98 were found to be resistant to all leaf stripe isolates.

Resistance to classical scrapie in experimentally challenged goats carrying mutation K222 of the prion protein gene

Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP). Breeding programs have been implemented to increase scrapie resistance in sheep populations; though desirable, a similar approach has not yet been applied in goats. European studies have now suggested that several polymorphisms can modulate scrapie susceptibility in goats: in particular, PRNP variant K222 has been associated with resistance in case-control studies in Italy, France and Greece. In this study we investigated the resistance conferred by this variant using a natural Italian goat scrapie isolate to intracerebrally challenge five goats carrying genotype Q/Q 222 (wild type) and five goats carrying genotype Q/K 222. By the end of the study, all five Q/Q 222 goats had died of scrapie after a mean incubation period of 19 months; one of the five Q/K 222 goats died after 24 months, while the other four were alive and apparently healthy up to the end of the study at 4.5 years post-challenge. All five of these animals were found to be scrapie negative. Statistical analysis showed that the probability of survival of the Q/K 222 goats versus the Q/Q 222 goats was significantly higher (p = 0.002). Our study shows that PRNP gene mutation K222 is strongly associated with resistance to classical scrapie also in experimental conditions, making it a potentially positive target for selection in the frame of breeding programs for resistance to classical scrapie in goats.

Influence of age, sex and ethnicity on platelet count in five Italian geographic isolates: mild thrombocytopenia may be physiological

Two population-based studies recently identified a high prevalence of unexplained thrombocytopenia, especially in men and older people (Biino et al , 2011; Santimone et al , 2011). Moreover, these studies reported a wide variability in the prevalence of thrombocytopenia in different villages. Further investigation concluded that the development of thrombocytopenia derived from the interaction of three factors: genetic factors, which set the ‘basic’ platelet count, gender-related factors, which result in lower platelet count in men and age-related factors, which are responsible for a progressive decrease in the platelet count over a life-time (Biino et al , 2011).