Italian Isolates Research Articles

First evidence of bluetongue virus serotype 16 in Croatia

This study described the first report of BTV-16 in Croatia. Serological evidence occurred in cattle at the end of September and continued during October and November 2004. All positive animals were in the Dubrovnik-Neretva County, a region located in the southernmost part of Croatia. BTV-16 infection was also detected in goats and sheep. Apart from few cases reported in Greece between 1999 and 2000, BTV-16 has never been reported in the Balkanic peninsula before. The BTV strain was isolated from cattle blood samples and typed as BTV-16. When the S5 was sequenced, it showed 100% homology with the BTV-16 vaccine isolate produced by Ondersterpoort Biological Product (SA) and used in Italy during the 2004 BT vaccination campaign. On the other hand no complete homology was found when the same RNA segment sequence was compared with that of the homologous Italian field isolate. As no evidence of livestock movements from Italy was demonstrated, an eolic transmission of the infection through infected Culicoides was hypothesised. According to the local meteostations, in several occasions, during the 2004 summer months, the west–east breeze blew with a speed above 50 km/h from Italy towards the Dubrovnik County. It is concluded that the BTV-16 which infected Croatian livestock was similar to the homologous OBP vaccine isolate and it is likely that it was introduced from Italy into the Southern regions of Croatia through infected Culicoides carried by the wind.

Biochemical characterisation and antioxidant activity of mycelium of Ganoderma lucidum from Central Italy

Abstract Ganoderma lucidum species is currently popular and used in the formulation of nutraceuticals and as functional foods, but a broad biochemical characterisation of its mycelium has not yet been reported. In this study new Italian and Chinese isolates, both identified as G. lucidum, were molecularly and biochemically characterised and compared. The mycelia differ both in terms of the enzymatic activities and in protein content revealed by 2D-PAGE electrophoretograms. The ethanolic extracts were screened for their possible antioxidant activities using three different tests: chelating activity on Fe 2+ , lipoxygenase assay and DPPH free radical scavenging. Only a fraction containing the low molecular weight compounds (L) showed antioxidative properties, whereas the soluble intracellular polysaccharides fraction (P) was ineffective. The correlation between total phenol content and scavenging activity on DPPH assay was also discussed. Increased interest in the identification of natural molecules with good antioxidant properties suggests further investigations into the use of Italian G. lucidum in the formulation of nutraceuticals and functional foods.

Seroconversion against SU5 derived synthetic peptides in sheep experimentally infected with different SRLV genotypes

Synthetic peptides were generated, corresponding to SU5 domain of envelope glycoprotein of Italian SRLV isolates It-561 and It-Pi1, belonging respectively to MVV- and CAEV-like genotypes. The peptides, encompassing an N-terminal variable and a C-terminal conserved antibody-binding site, were used in an ELISA assay to analyse the sera of two groups of sheep experimentally infected with these isolates. The kinetics and specificity of the humoral response to the homologous and heterologous antigen and the affinity maturation of the sera were evaluated. Seroconversion occurred between week 3 and 8. The response to SU5 antigen was mostly type-specific. The few broadly reacting sera may reflect the production of antibodies directed to the SU5 constant antibody-binding site. All sera underwent with time avidity maturation, resulting in the appearance of high affinity antibodies. This study suggests constant monitoring of the circulating viral variants to develop a panel of diagnostic peptides representative of local genotypes.

Identification of the novel KI Polyomavirus in paranasal and lung tissues

KI is a novel polyomavirus identified in the respiratory secretions of children with acute respiratory symptoms. Whether this reflects a causal role of the virus in the human respiratory disease remains to be established. To investigate the presence of KIV in the respiratory tissue, we examined 20 fresh lung cancer specimens and surrounding normal tissue along with one paranasal and one lung biopsy from two transplanted children. KIV-VP1 gene was detected in 9/20 lung cancer patients and 2/2 transplanted patients. However, amplification of the sequence coding for the C-terminal part of the early region of KIV performed on the 11 positive cases was successful only in two malignant lung tissues, one surrounding normal tissue, and 1/2 biopsies tested. Phylogenetic analysis performed on the early region of KIV (including the four Italian isolates), BKV and JCV revealed the presence of three distinct clades. Within the KIV clade two sub-clades were observed. A sub-clade A containing the four Italian strains, and a sub-clade B comprising the Swedish and Australian isolates. Interestingly, the two Italian strains identified in normal tissue clustered together, whereas those detected in malignant tissue fell outside this cluster. In vitro studies are needed to investigate the transforming potential of KIV strains.

Genetic and serological typing of European infectious haematopoietic necrosis virus (IHNV) isolates

Infectious haematopoietic necrosis virus (IHNV) causes the lethal disease infectious haematopoietic necrosis (IHN) in juvenile salmon and trout. The nucleocapsid (N) protein gene and partial glycoprotein (G) gene (nucleotides 457 to 1061) of the European isolates IT-217A, FR-32/87, DE-DF 13/98 11621, DE-DF 4/99-8/99, AU-9695338 and RU-FR1 were sequenced and compared with IHNV isolates from the North American genogroups U, M and L. In phylogenetic studies the N gene of the Italian, French, German and Austrian isolates clustered in the M genogroup, though in a different subgroup than the isolates from the USA. Analyses of the partial G gene of these European isolates clustered them in the M genogroup close to the root while the Russian isolate clustered in the U genogroup. The European isolates together with US-WRAC and US-Col-80 were also tested in an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (MAbs) against the N protein. MAbs 136-1 and 136-3 reacted equally at all concentrations with the isolates tested, indicating that these antibodies identify a common epitope. MAb 34D3 separated the M and L genogroup isolates from the U genogroup isolate. MAb 1DW14D divided the European isolates into 2 groups. MAb 1DW14D reacted more strongly with DE-DF 13/98 11621 and RU-FR1 than with IT-217A, FR-32/87, DE-DF 4/99-8/99 and AU-9695338. In the phylogenetic studies, the Italian, French, German and Austrian isolates clustered in the M genogroup, whereas in the serological studies using MAbs, the European M genogroup isolates could not be placed in the same specific group. These results indicate that genotypic and serotypic classification do not correlate.

Age-related hearing loss in four Italian genetic isolates: An epidemiological study

The objective of this study was to estimate the prevalence of hearing impairment in four genetically isolated Italian villages (Carlantino, Campora, Gioi-Cardile, and Stoccareddo), 1682 subjects were recruited from all the individuals participating in a multidisciplinary study. They underwent otoscopy and pure-tone audiometry and completed a questionnaire. The audiological data show that the percentage of impaired people increases with age and in particular becomes relevant aged over 40. For this reason we decided to compare the PTA values of individuals aged 40 or older. The PTA values of Stoccareddo and Carlantino are statistically different from PTAs of the other villages. Campora and Gioi-Cardile, both located within the Cilento National Park, have similar middle-low frequency PTA values while some differences are present at high frequencies. Using pedigrees it was possible to calculate the heritability of the trait. For Carlantino and Gioi-Cardile the percentage of the phenotype variation attributable to genetic variation is not significant, while for Campora the heritability value is 0.49 (p = 0.01) suggesting that genetic factors may have an important role.

PCR-based monitoring of recent isolates of tobacco blue mold from Europe reveals the presence of two genetically distinct phenotypes differing in fungicide sensitivity

Bioassays testing the fungicide sensitivity against metalaxyl of Peronospora tabacina isolates collected in German tobacco fields in 2005 revealed the presence of two phenotypes, resistant and sensitive. DNA fingerprints using SSR and minisatellite primers allowed separation of the samples into two groups. The differences in amplification patterns coincided with the sensitive and resistant reaction of the isolates in metalaxyl bioassays. New primers were developed which allowed PCR-based detection of P. tabacina and differentiation of the metalaxyl-sensitive and the metalaxyl-resistant phenotype, respectively. Screening of recent blue mold isolates from Germany and other European countries for metalaxyl sensitivity with leaf disk bioassays coincided completely with the PCR-based identification of the two phenotypes. In Germany, exclusively resistant isolates were found between 2002 and 2004. These still dominate. Since 2005 the co-occurrence of sensitive isolates has been shown. No similar monitoring has been done in any other European country. However, we found the resistant phenotype reaction in a French isolate of 2004 and in two out of three Italian isolates in 2007. Two isolates from Poland and Bulgaria in 2007 were sensitive to metalaxyl. For all 58 isolates tested since 2002 the metalaxyl bioassay-based resistance type and the PCR-based tests for sensitive and resistant genotype coincided. Using genotype-specific primers for future population studies may help to trace the sources of the pathogen from which yearly propagation starts.

Pathogenicity of a QX strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the Ma5 and 4/91 serotypes

The aims of this study were firstly to evaluate the pathogenicity of an Italian isolate of the QX strain of infectious bronchitis (IB) virus using 1-day-old female specific pathogen free chicks (layer type) and 1-day-old female commercial broiler type chickens, and secondly to assess the level of protection induced in these birds by a vaccination programme including the IB Massachusetts and 4/91 serotype live attenuated vaccines. Unvaccinated birds showed clinical signs of varying severity, predominantly affecting the upper respiratory tract. Vaccinated birds appeared healthy, with the exception of a very mild conjunctivitis affecting a limited number of the broilers. Vaccination fully protected specific pathogen free birds, since no histopathological lesions were observed, nor was virus detected following challenge. In broilers, replication of the challenge virus was not prevented but was significantly reduced. This study confirms that vaccination at 1 day old and at 14 days of age using the Ma5 and 4/91 IB vaccines may be instrumental in reducing the economic impact of QX IB virus infections in layer and broiler farms.

Laboratory tests for evaluating the level of attenuation of bluetongue virus

One of the most important steps when preparing a live attenuated vaccine is the assessment of the level of attenuation in target animals. It is costly and time consuming as it requires, on each occasion, a large number of susceptible animals and contained accommodation. This study assessed the consistency of the bovine foetal aorta endothelial (BFA) cell line and newborn mice for evaluating the attenuation level of BTV4, BTV9 and BTV16 Italian field isolates. Following serial passages in BHK 21c13 or Vero cell cultures, BTV attenuated clones demonstrated a reduced replication capability in the BFA cells compared to the homologous virulent strains. Similarly, following intracerebral inoculation, the attenuated clones were completely innocuous to newborn mice contrary to the homologous virulent strains which killed all animals within 10 days. Vaccines produced with the BTV9 or BTV4 attenuated clones were safe, immunogenic and capable of preventing clinical symptoms and viraemia in sheep following challenge with homologous virulent virus. The two assays may be valuable indicators of the gradual changes occurring in the BTV population leading to virus attenuation, they can predict the safety of a BTV attenuated vaccine and, in turn, reduce the number of sheep and cattle required to assess the level of attenuation attained.

Linkage studies in familial idiopathic basal ganglia calcification: Separating the wheat from the chaff

Idiopathic Basal Ganglia Calcification (IBGC) is a neuropsychiatric condition characterized by brain calcinosis, heterogeneous motor impairment and behavioral symptoms. The IBGC1 locus was the first region linked to this phenotype in an American family, but another kindred from Spain was also reported as possibly associated with this locus. Our group excluded this locus in additional families together with an independent study of an Australian pedigree with IBGC, but without clinical symptoms. Recently, a large Italian family from a population isolate was excluded from IBGC1. However, there are unusual aspects concerning this Tyrolean family, especially if we consider that almost all the clinically affected subjects manifested symptoms and signs suggestive of a dysmorphic syndrome, associated with neuropsychiatric symptoms. Curiously, some of the clinical features in this kindred match with the autosomal dominant chromosomal instability syndrome reported in Japan. Previous studies show that the definition of an autosomal dominant pattern of inheritance is an assumption that might be considered cautiously in familial IBGC, due to the limited clinical penetrance for the brain calcifications and especially when there is no access to all the parents neuroimaging data. Families from an Italian isolate, such as Tyrol, with high inbreeding rates, are more likely to manifest recessive syndromes. Nevertheless, the current debate regarding the nosology of this heterogeneous phenotype demands the establishment of standard diagnostic criteria. The current identification of loci or mutations responsible for FIBGC might help to elucidate this intriguing neuropsychiatric condition.

Variation of hemoglobin levels in normal Italian populations from genetic isolates

Normal hemoglobin levels vary greatly according to genetic and acquired factors. As a consequence there is no general agreement on the definition of anemia in terms of hemoglobin levels. Here we compare the hemoglobin levels of subjects recruited from normal genetically isolated Italian populations whose medical history, life style habits and results of laboratory tests are available. After the exclusion of pathological samples we analyzed the hemoglobin levels of 3,849 subjects (1,661 males and 2,188 females) and evaluated the hemoglobin heritability. Normal subjects of different age groups from a northern Italian isolate have significantly higher hemoglobin levels when compared to matched subjects of southern Italian isolates. The estimated heritability of hemoglobin levels ranges from 0.34 to 0.42 in the different isolates. Our study provides a dataset of hemoglobin levels for normal subjects of different geographical origin and indicate that hemoglobin levels are substantially influenced by heritable components.

Phylogenetic analysis of human coronavirus NL63 circulating in Italy

Background Five known human coronaviruses infect the human respiratory tract: HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63 and HCoV-HKU1. Objectives To evaluate the prevalence of HCoV-NL63 in hospitalized adult patients and to perform molecular characterization of Italian strains. Study Design HCoV-NL63 was sought by RT-PCR in 510 consecutive lower respiratory tract (LRT) samples, collected from 433 Central-Southern Italy patients over a 1-year period. Phylogenetic analysis was performed by partial sequencing of S and ORF1a. Additional S sequences from Northern Italy were included in the phylogenetic trees. Results HCoV-NL63 was detected in 10 patients (2.0%) with symptomatic respiratory diseases, mainly during winter. Phylogenetic analysis indicated a certain degree of heterogeneity in Italian isolates. The ORF1a gene clustering in phylogenetic trees did not match with that of the S gene. Conclusions As observed by others, HCoV-NL63 is often associated with another virus. Phylogenetic characterization of HCoV-NL63 circulating in Italy indicates that this virus circulates as a mixture of variant strains, as observed in other countries.

THE SMALL GENOMIC SEGMENT OF TOMATO SPOTTED WILT VIRUS ISOLATE BR20 IS NECESSARY BUT NOT SUFFICIENT TO INDUCE LETHAL NECROSIS IN NICOTIANA BENTHAMIANA AND LOCAL NECROTIC LESIONS IN NICOTIANA TABACUM cv. WHITE BURLEY

SUMMARY A consistently different phenotype was observed in Nicotiana benthamiana and Nicotiana tabacum cv. White Burley plants infected by the Brazilian Tomato spotted wilt virus (TSWV) isolate Br20 and the two Italian isolates p272 and p105/43.14. Based on the phenotype of experimentally obtained reassortants carrying the small (S) genomic segment of p272 or p105/43.14 and the medium (M) and large (L) segments of Br20, we concluded that the small segment of Br20 is required for eliciting lethal necrosis in N. benthamiana and local necrotic lesions in N. tabacum. To evaluate whether the Br20 nucleocapsid (N) or the non structural (NSs) protein encoded by the S segment were able to cause the distinctive symptoms, either alone or in combination, the three allelic variants (Br20, p272 and p105/43.14) of each gene were transiently expressed in planta through agroinfiltration. All the alleles of the N and NSs proteins accumulated abundantly in infiltrated N. benthamiana and N. tabacum leaves. No specific necrotic reaction was observed in any of the single or combined agro-infiltrations of the Br20 alleles, suggesting that the S-encoded proteins of isolate Br20 are not sufficient to elicit the specific symptomatic response and that the co-involvement of other regions of the genome might be necessary.

Assessment of efficacy of a bivalent BTV-2 and BTV-4 inactivated vaccine by vaccination and challenge in cattle

The efficacy of a bivalent inactivated vaccine against bluetongue virus (BTV) serotypes 2 (BTV-2) and 4 (BTV-4) was evaluated in cattle by general and local examination, serological follow-up, and challenge. Thirty-two 4-month-old calves were randomly allocated into 2 groups of 16 animals each. One group was vaccinated subcutaneously (s/c) with two injections of bivalent inactivated vaccine at a 28-day interval, and the second group was left unvaccinated and used as control. Sixty-five days after first vaccination, 8 vaccinated and 8 unvaccinated calves were s/c challenged with 1 mL of 6.2 Log 10 TCID 50/mL of an Italian field isolate of BTV serotype 2, while the remaining 8 vaccinated and 8 unvaccinated animals were challenged by 1 mL of 6.2 Log 10 TCID 50/mL of an Italian field isolate of BTV serotype 4. Three additional calves were included in the study and used as sentinels to confirm that no BTV was circulating locally. At the time of the challenge, only one vaccinated animal did not have neutralizing antibodies against BTV-4, while the remaining 15 showed titres of at least 1:10 for either BTV-2 or BTV-4. However, the BTV-2 component of the inactivated vaccine elicited a stronger immune response in terms of both the number of virus neutralization (VN) positive animals and antibody titres. After challenge, no animal showed signs of disease. Similarly, none of the vaccinated animals developed detectable viraemia while bluetongue virus serotype 2 and 4 titres were detected in the circulating blood of all unvaccinated animals, commencing on day 3 post-challenge and lasting 16 days. It is concluded that administration of the bivalent BTV-2 and BTV-4 inactivated vaccine resulted in a complete prevention of detectable viraemia in all calves when challenged with high doses of BTV-2 or BTV-4.

Intraspecies Transmission of BASE Induces Clinical Dullness and Amyotrophic Changes

The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrPTSE), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrPTSE type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle.

Fusarium Wilt of Gerbera Caused by a Fusarium sp. in Brazil.

In 2006, gerbera (Gerbera jamesonii) plants, cvs. Basic, Xena, and Olimpia grown for cut flower production in two greenhouse farms in the region of Guarapuava, Paraná, Brazil, exhibited symptoms of a wilt disease. Affected plants (approximately 20, 60, and 50% on cvs. Basic, Xena, and Olimpia, respectively) were stunted and developed yellow leaves unilaterally with initially brown and eventually black streaks in the vascular system. The first symptoms occurred 2 months after transplanting during the hottest period of the summer with an average air temperature of 27°C. Vascular streaks in the yellow leaves were continuous with a brown discoloration in the vascular system of the crown and upper taproot. Occasionally, the leaves of affected plants turned red. A Fusarium sp. was consistently and readily isolated onto a Fusarium-selective medium from symptomatic vascular tissue sampled from the crown of infected plants. Colonies were identified as Fusarium oxysporum on the basis of colony and conidia morphology (1) after subculturing on potato dextrose agar. Since F. oxysporum f. sp. chrysanthemi has been previously reported on Chrysanthemum morifolium, Argyranthemum frutescens, and gerbera (4), pathogenicity tests were carried out by using one monoconidial isolate obtained from wilted plants and one Italian isolate (F. oxysporum f. sp. chrysanthemi MASS 6). The isolates of F. oxysporum were grown in casein hydrolysate in shake culture (90 rpm) for 10 days at 25°C with 12 h of fluorescent light per day. Healthy rooted plants of 30-, 20-, and 45-day-old C. morifolium (cv. Captiva), A. frutescens (cv. Stella 2000), and gerbera (cvs. Jaska, Dalma, and Excellence), respectively, were inoculated by separately dipping roots into a conidial suspension (5 × 107 conidia/ml) of the two isolates of F. oxysporum. Plants were transplanted (one plant per pot) into pots (3.5 liter vol). Noninoculated plants served as control treatments. Plants (15 per treatment) were grown in a glasshouse at an average day temperature of 32°C and night temperature of 23°C (minimum 21°C and maximum 43°C). Wilt symptoms and discoloration of the vascular system in roots, crown, and petioles developed within 29 days on C. morifolium, 26 days on A. frutescens, and 14 days on gerbera. Noninoculated plants remained healthy. F. oxysporum was consistently reisolated from infected plants. The pathogenicity test was carried out twice. Gerbera wilt caused by F. oxysporum f. sp. chrysanthemi was recently reported in Italy (2) and Spain (3). Currently, the wilt of gerbera in the area of Paraná is limited to two farms. To our knowledge, this is the first report of the disease in Brazil as well as in South America.

First report of Olive latent virus 2 in wild castor bean (Ricinus communis) in Italy

Wild castor bean plants (Ricinus communis), showing bright yellow mosaic leaf symptoms were collected in Vibo Valentia (Calabria region, southern Italy) during spring and summer of 2005 and 2006. Symptoms consisted of yellow speckled leaves with significant mottled areas; some leaves also contained an arabesque line pattern. Electron microscope observations of sap extracts from leaves with symptoms showed semi-spherical to bacilliform particles consistent with Alfamovirus and Oleavirus. Using a range of herbaceous plants and mechanical inoculation, a virus was isolated from castor bean; symptoms, in particular in Nicotiana benthamiana, N. clevelandii, N. occidentalis, N. glutinosa, Chenopodium quinoa, Phaseolus vulgaris cv. La Victorie and Gomphrena globosa, were comparable with those previously described for a castor bean isolate of Olive latent virus 2 (Grieco et al., 2002). The virus was identified serologically by particle decoration with an OLV2 antiserum, raised against an Italian OLV2 isolate (fig. 2; Grieco et al., 1992), and by sequencing the movement gene (MP) and the coat protein gene (CP), as described previously (Grieco et al., 2002). The nucleotide sequence of a 1696 bp amplicon, encompassing the MP and CP genes, was determined (GenBank Acc. AM600639). BLAST analysis showed 97% nucleotide identity with an OLV2/CB castor bean isolate from Greece (GenBank Acc. AJ439450) and 93% with the type isolate (GenBank Acc. X76993). Deduced amino acid sequences identities for MP protein were 98% with OLV2/CB and 94% with the type isolate and 97% and 96% for the CP protein respectively. In both proteins, amino acid substitutions were mostly concentrated in the N terminus region. Most aspects of the eco-biology of OLV2 are still unclear (e.g. vector transmission, isolate variability, natural host range). OLV2 was previously detected in wild castor bean in Greece (Vovlas et al., 2002). This new finding of a disease caused by another OLV2 isolate in the same host might suggest a potential role of castor bean in the epidemiology of OLV2, by acting as a natural virus reservoir. The authors would like to thank Ms Daniela Sorrentino for her excellent technical assistance.

Pelargonium chlorotic ring pattern virus: first report in Spain

A recent survey of symptomless ornamental geraniums ( Pelargonium zonale , P. peltatum and P. grandiflorum ) in Spain (2000–2003) revealed viral infections in 52·4% of the tested plants (Alonso & Borja, 2005). Most infected plants contained tombus-like viruses such as Pelargonium line pattern virus (PLPV), either alone or in combination with other viral pathogens. Several little-studied PLPV-related viruses have been reported in Pelargonium spp. but their distribution and importance to geranium growers is unknown since they are not usually included in phytosanitary programmes. One of these viruses, Pelargonium chlorotic ring pattern virus (PCRPV), was recently sequenced (GenBank Acc. No. AY038069) and found to be related to PLPV in nucleotide sequence and genome organization (Kinard & Jordan, 2002; Castano & Hernandez, 2005). To assess the occurrence of PCRPV in Spain 5 samples from the previous survey and 3 gathered in 2006 were tested. Total RNA was extracted using Plant RNA Reagent (Invitrogen) and reverse transcribed with Superscript II-RT (Invitrogen) and primer CH149 (5 ′ -CCGATTCTCGAGCTGCCAATC-3 ′ ), complementary to positions 1543–1523 of PCRPV. PCR used primers CH149 and CH148 (5 ′ -CGGTTTGAGCACTCTGCGTATC-3 ′ ), homologous to positions 1277–1298 of PCRPV. An amplicon of the expected size (267 bp comprising a region of the viral polymerase gene) was generated in five samples from different geographical origins (Zaragoza, Zamora, Aranjuez and Cordoba) and collecting times (2000, 2003 and 2006). The amplicons derived from two separate samples were sequenced (GenBank Acc. Nos. EF635383 and EF635384) and showed 95% nucleotide and 99–100% amino acid sequence identity with the PCRPV reference sequence. An Italian isolate of PCRPV was recovered from P. zonale with vein banding and leaf distortion (Lisa et al ., 1996; Kinard & Jordan, 2002); however from our results, PCRPV infection can also be asymptomatic. This is the first report of PCRPV in Spain.

Erm (B)-Carrying Elements in Tetracycline-Resistant Pneumococci and Correspondence between Tn 1545 and Tn 6003

This study investigated the genetic organization of erm(B)-carrying transposons of Streptococcus pneumoniae and their distribution in tetracycline-resistant clinical isolates. By comparatively analyzing reference pneumococci carrying erm(B)/tet(M) transposon Tn1545, Tn6003, Tn6002, or Tn3872, we demonstrated a substantial correspondence between Tn1545 and Tn6003, which have the same resistance gene combination [tet(M) (tetracycline), erm(B) (erythromycin), and aphA-3 (kanamycin)]; share the macrolide-aminoglycoside-streptothricin element, containing a second erm(B); and only differ by a ca. 1.2-kb insertion (containing a putative IS1239 insertion sequence) detected in Tn1545 from S. pneumoniae reference strain BM4200. These results enabled elucidation of the structure of Tn1545, the first erm(B)-carrying transposon described in S. pneumoniae. A collection of 83 erythromycin- and tetracycline-resistant clinical pneumococci, representative of recent Italian isolates carrying erm(B) as the sole erythromycin resistance gene, was used to investigate the distribution of the different transposons. All 83 organisms were positive for tet(M) and bore an erm(B)/tet(M) transposon that could be characterized by using a specific set of primer pairs; Tn3872 was detected in 18 isolates, Tn6002 in 59 isolates, and Tn6003 in 6 (the sole kanamycin-resistant) isolates. The genetic organization of transposon Tn1545, with its specific insertion, was not detected in any of the isolates tested. The erm(B)-carrying elements of tetracycline-resistant pneumococci substantially corresponded to those [bearing a silent tet(M) gene] recently detected in tetracycline-susceptible pneumococci. Overall, in erm(B)-positive pneumococci, Tn6003 was the least common erm(B)-carrying Tn916-related element and Tn6002 the most common.

First Report of a γ 3-Proteobacterium Associated with Diseased Strawberries in Italy.

During the fall seasons of 2005 and 2006, diseased strawberry plants (Fragaria × ananassa Duch.) were observed in nurseries and production fields in Ferrara, Forli-Cesena, and Ravenna provinces (Emilia-Romagna region, northern Italy). Symptoms consisted of a conspicuous plant stunting with a poor root system. Older leaves rolled upward and displayed a marked premature purplish discoloration, while young leaves were cupped, chlorotic, generally reduced in size, and had shortened petioles. This strawberry disorder was similar to "marginal chlorosis", an infectious disease occurring in France that can be induced by two different phloem-limited uncultured bacteria: the γ 3-proteobacterium 'Candidatus Phlomobacter fragariae' and the stolbur phytoplasma (16SrXII-A). In strawberry production fields, 'Ca. P. fragariae' is reported as being the prevalent agent of this disease (1). Sixty-seven diseased plants were collected from production fields and nurseries for testing for 'Ca. P. fragariae'. Leaf samples were analyzed by 4',6-diamidine-2-phenylindole staining and PCR. Forty samples showed fluorescent DNA in the phloem, whereas no fluorescence was observed in symptomless strawberries. When tested by PCR with primers Fra4/Fra5, which amplify a 550-bp fragment of the 16S rDNA region of 'Ca. P. fragariae' (1), 13 of 36 strawberries from production fields and 1 of 31 nursery plants gave a positive reaction. On the other hand, 21 samples from nurseries and 5 from production fields tested positive for stolbur phytoplasma (3). No amplification was obtained with DNA from symptomless or healthy strawberry plants. Sequencing Fra4/Fra5 amplicons from three samples (GenBank Accession Nos. DQ362916-DQ362918) showed a 98.1 to 98.6% and a 98.3 to 98.8% identity with the published sequences of the French isolate "LG2001" (GenBank Accession No. AM110766) and the Japanese isolate J-B (GenBank Accession No. AB246669) of 'Ca. P. fragariae', respectively. Higher homology (99.2 to 99.8%) was found with another bacterium-like organism (BLO) of the γ 3-proteobacteria subgroup (GenBank Accession No. AY057392) associated with the syndrome "basses richesses" of sugar beet (SBR). Furthermore, PCR assays performed with primers Pfr1/Pfr4, specific for spoT gene of 'Ca. P. fragariae', did not show any amplification with DNA from the 14 diseased strawberry plants tested. This is in agreement with the SBR BLO identification (2). To better characterize the Italian isolates, the full-length 16S rDNA gene was analyzed with primers fd1/Fra4 and Fra5/rp1, which amplify the 5' and 3' region of 16S rDNA gene of the proteobacteria, respectively (2). PCR products from eight isolates were sequenced, and the 16S rDNA sequences obtained (GenBank Accession Nos. DQ538372-DQ538379) showed a 96.4 to 97.3% identity with the known 'Ca. P. fragariae' isolates, while a higher homology (99.4 to 99.9%) was again found with the SBR BLO. To our knowledge, this is the first report of a γ 3-proteobacterium affecting strawberry in Italy. In the genome region analyzed, our isolates are more similar to the SBR BLO than to 'Ca. P. fragariae'. Further work is in progress to investigate incidence, geographical distribution, epidemiology, and host range of this pathogen in Italy.