Spanish Strains Research Articles

Genotype distribution of clinical isolates of Trichosporon asahii based on sequencing of intergenic spacer 1

The sequence polymorphisms of intergenic transcribed spacer and the antifungal susceptibility profile of 18 Trichosporon asahii isolates from Spain, Argentina, and Brazil together with 43 intergenic transcribed spacer 1 sequences deposited in the GenBank were analyzed. Six genotypes were detected instead of 5 genotypes described previously. Genotype 1 was the most common found comprising 57.3% of all strains, followed by genotype 3 (14.7%) and genotype 5 (13.1%). Spanish strains had members in all genotypes except 2, whereas South American isolates were grouped with genotypes 1, 3, and 6. Our results indicate that all genotypes are present in at least 2 countries suggesting a worldwide distribution. On the other hand, genotype 6 was not previously described but was only composed of 2 South American strains isolated from a subcutaneous abscess and skin. All isolates showed amphotericin B MICs ≥2 mg/L supporting the in vitro resistance of this specie to this antifungal. Three isolates from South America showed high MICs to all antifungals analyzed. The true epidemiologic usefulness of classifying T. asahii in genotypes should be ascertaining analyzing a high number of isolates from many countries.

Yessotoxins profile in strains of Protoceratium reticulatum from Spain and USA

Seven strains of Protoceratium reticulatum isolated from Spain and the USA were cultured in the laboratory. Yessotoxins (YTXs) quantification and toxin profile determination were performed by LC–FLD and LC–MS/MS. The four Spanish strains were found to produce YTX and known YTX analogs, however, YTX was not detected in any of the three USA strains. Among the strains that produced YTXs, toxin production ranged between 2.9 and 28.6 pg/cell. The YTX profile was substantially different between strains, in three out of the four Spanish strains YTX was the main toxin and in the fourth homoYTX was the prominent toxin. This work demonstrates that YTX is not always the main toxin in P. reticulatum and a high variability in YTX amounts and profile found in other locations is confirmed.

Analysis of world strains of Anaplasma marginale using major surface protein 1a repeat sequences

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host–pathogen and tick–pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains and phylogenetic studies. MSP1a is involved in the adhesion and transmission of A. marginale by ticks and varies among geographic strains in the number and sequence of amino-terminal tandem repeats. The aim of this study was to characterize the genetic diversity of A. marginale strains collected from countries in North and South America, Europe, Asia, Africa and Australia, inclusive of all continents. In this study, we characterized 131 strains of A. marginale using 79 MSP1a repeat sequences. These results corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The phylogenetic analyses of MSP1a repeat sequences did not result in clusters according to the geographic origin of A. marginale strains but provided phylogeographic information. Seventy-eight percent of the MSP1a repeat sequences were present in strains from a single geographic region. Strong (≥80%) support was found for clusters containing sequences from Italian, Spanish, Chinese, Argentinean and South American strains. The phylogenetic analyses of MSP1a repeat sequences suggested tick–pathogen co-evolution and provided evidence of multiple introductions of A. marginale strains from various geographic locations worldwide. These results contribute to the understanding of the genetic diversity and evolution of A. marginale and tick–pathogen interactions.

Phenotypic and Genetic Diversity in Strains of Common Blight Bacteria (Xanthomonas campestrispv.phaseoliandX. campestrispv.phaseolivar.fuscans) in a Secondary Center of Diversity of the Common Bean Host Suggests Multiple Introduction Events

ABSTRACT Common bacterial blight (CBB) disease of the common bean (Phaseolus vulgaris) is caused by Xanthomonas campestris pv. phaseoli and the brown-pigmented variant X. campestris pv. phaseoli var. fuscans. CBB first was described in Castilla y León County, Spain, in 1940, and is now a major constraint on common bean production. In this secondary center of diversity of the common bean, large-seeded Andean cultivars predominate, although medium-seeded Middle American cultivars also are grown. Xanthomonad-like bacteria associated with CBB in Castilla y León were characterized on the basis of carbohydrate metabolism, brown pigment production, genetic analyses (repetitive-element polymerase chain reaction [rep-PCR] and random amplified polymorphic DNA [RAPD]) and pathogenicity on cultivars representing the two common bean gene pools (Andean and Middle American). X. campestris pv. phaseoli was more prevalent (80%) than X. campestris pv. phaseoli var. fuscans (20%). Patterns of carbohydrate metabolism of Spanish CBB bacteria were similar to those of known strains; and only X. campestris pv. phaseoli var. fuscans strains utilized mannitol as a sole carbon source. rep-PCR and RAPD analyses revealed relatively little genetic diversity among Spanish X. campestris pv. phaseoli strains, and these strains were placed together with New World strains into a large cluster. Similar to other New World strains, representative Spanish X. campestris pv. phaseoli strains were highly pathogenic on bean cultivars of both gene pools, showing no gene pool specialization such as that found in certain East African strains. Genetic analyses and pathogenicity tests confirmed and extended previous results, indicating that these East African strains represent distinct xanthomonads that independently evolved to be pathogenic on common bean. X. campestris pv. phaseoli var. fuscans strains were more closely related and genetically distinct from X. campestris pv. phaseoli strains. However, two distinct clusters of X. campestris pv. phaseoli var. fuscans strains were identified, one having the most New World strains and the other having the most African strains. Spanish strains were placed in both clusters, but all strains tested were highly pathogenic on bean cultivars of both gene pools. Together, our results are consistent with multiple introductions of CBB bacteria into Spain. These findings are discussed in terms of breeding for CBB resistance and the overall understanding of the genetic diversity and evolution of CBB bacteria.

Detection of hepatitis E virus in liver, mesenteric lymph node, serum, bile and faeces of naturally infected pigs affected by different pathological conditions

The objective of the present study was to detect hepatitis E virus (HEV) in different samples from naturally infected pigs and to characterise genetically the detected strains. Serum, bile, liver, lymph nodes and faeces of 69 animals from 1 week to 4 months of age with different pathological conditions were collected. Reverse transcriptase-polymerase chain reaction (RT-PCR) to detect HEV and histopathology of tissues was conducted. Positive RT-PCR samples were sequenced and phylogenetically analysed. HEV was detected in at least one sample in 26 out of 69 animals (37.7%). Bile was the most frequently positive sample, followed by mesenteric lymph nodes, liver, faeces and serum. HEV was detected in pigs of 1 ( n = 7), 2 ( n = 8) and 3 ( n = 11) months of age. A total of 22 of 69 (31.9%) pigs had mild to moderate hepatitis and 15 of them were HEV RT-PCR positive in at least one of the tested samples.The highest sensitivity of viral detection was achieved using samples that cannot be obtained from live pigs, such as liver, mesenteric lymph node and bile. Phylogenetic analyses confirmed that all Spanish swine HEV strains detected belonged to genotype III. Therefore, genotype III strains are present in a relative high proportion of pigs between 1 and 3 months of age. Through this study, it cannot be ruled out if concomitant infections may influence the distribution of HEV in infected pigs.

Robotized Thermal and Chlorophyll Fluorescence Imaging of Pepper Mild Mottle Virus Infection in Nicotiana benthamiana

After infecting a susceptible host, pathogens spread throughout the plant. Depending on pathogen type and strain, the severity of symptoms varies greatly. In the case of pepper mild mottle virus (PMMoV) infection in Nicotiana benthamiana, newly developing leaves display visual symptoms (symptomatic leaves). In this study, two PMMoV strains were used, the Spanish strain (PMMoV-S) being more virulent than the Italian strain (PMMoV-I). Plants infected with PMMoV-I could recover from the virus-induced symptoms. Leaves that were fully developed at the start of PMMoV infection remained symptomless. In these asymptomatic leaves, an increase in temperature, initiating from the tissue adjacent to the main veins, was observed 7 d before the Chl fluorescence pattern changed. Virus immunolocalization on tissue prints matched well with the concomitant pattern of Chl fluorescence increase. The temperature increase, associated with the veins, was shown to be related to stomatal closure. Upon PMMoV-I infection, the appearance of thermal and Chl fluorescence symptoms as well as virus accumulation were delayed by 3 d compared with PMMoV-S-induced symptoms. The temperature increase of whole symptomatic leaves was also correlated with a decrease in stomatal aperture. In contrast to the persistent increase in symptomatic leaf temperature observed during PMMoV-S infection, the temperature of symptomatic leaves of PMMoV-I-infected plants decreased gradually during recovery. We propose that the earliest temperature increase is caused by a systemic plant response to the virus infection, involving the control of water loss. In conclusion, thermography has potential as an early reporter of an ongoing compatible infection process.

Genomes of Helicobacter pylori from native Peruvians suggest admixture of ancestral and modern lineages and reveal a western type cag-pathogenicity island

BackgroundHelicobacter pylori is presumed to be co-evolved with its human host and is a highly diverse gastric pathogen at genetic levels. Ancient origins of H. pylori in the New World are still debatable. It is not clear how different waves of human migrations in South America contributed to the evolution of strain diversity of H. pylori. The objective of our 'phylogeographic' study was to gain fresh insights into these issues through mapping genetic origins of H. pylori of native Peruvians (of Amerindian ancestry) and their genomic comparison with isolates from Spain, and Japan.ResultsFor this purpose, we attempted to dissect genetic identity of strains by fluorescent amplified fragment length polymorphism (FAFLP) analysis, multilocus sequence typing (MLST) of the 7 housekeeping genes (atpA, efp, ureI, ppa, mutY, trpC, yphC) and the sequence analyses of the babB adhesin and oipA genes. The whole cag pathogenicity-island (cagPAI) from these strains was analyzed using PCR and the geographic type of cagA phosphorylation motif EPIYA was determined by gene sequencing. We observed that while European genotype (hp-Europe) predominates in native Peruvian strains, approximately 20% of these strains represent a sub-population with an Amerindian ancestry (hsp-Amerind). All of these strains however, irrespective of their ancestral affiliation harbored a complete, 'western' type cagPAI and the motifs surrounding it. This indicates a possible acquisition of cagPAI by the hsp-Amerind strains from the European strains, during decades of co-colonization.ConclusionOur observations suggest presence of ancestral H. pylori (hsp-Amerind) in Peruvian Amerindians which possibly managed to survive and compete against the Spanish strains that arrived to the New World about 500 years ago. We suggest that this might have happened after native Peruvian H. pylori strains acquired cagPAI sequences, either by new acquisition in cag-negative strains or by recombination in cag positive Amerindian strains.

First record of Pseudomonas syringae pv. viburni in the UK

Various Pseudomonas syringae pathovars cause leaf spots and cankers of hardy nursery stock species in the UK, including P.s. pv. syringae on lilac, P.s. pv. berberidis on Berberis spp. and P.s. pv. philadelphi on Philadelphus spp. (Roberts, 1997). Leaf spots on some other hosts are attributed to P.s. pv. syringae or to P. syringae pv. undetermined. Occasionally Central Science Laboratory has received samples of Viburnum spp. with watersoaked leaf spots from which P. syringae has been isolated; one was from Viburnum tinus in 1986. The isolate, a typical LOPAT group 1 member of P. syringae (Lelliott & Stead, 1987), was placed in the National Collection of Plant Pathogenic Bacteria (NCPPB) as NCPPB 3450 and was listed under P.s. pv. viburni with a ‘b’ ranking, denoting authenticity not verified. More recently, plants of Viburnum sargentii with typical bacterial leaf spots were received from a Lincolnshire nursery and from which LOPAT 1 strains were isolated. Host tests confirmed pathogenicity on Viburnum sargentii but it was weaker on Viburnum opulus var. Sterile. Two isolates from V. sargentii on the same nursery, taken at different times (CSL 4205 and 4206), were acceded to the NCPPB as NCPPB 4265 and NCPPB 4266, respectively. Fatty acid profiles obtained were typical of P. syringae. REP-PCR based on primers from the repetitive enterobacterial consensus sequence and BOX-PCR, based on primers from the BOX A subunit, were carried out (Stead et al., 2000). Fingerprints were compared with those of reference strains of all validly named P. syringae pathovars held in the NCPPB, including P.s. pv. viburni (NCPPB 1921, the only authentic strain). upgma dendrograms of both assays showed that NCPPB 3450 did not have the profile of P.s. pv. viburni but was very similar to that of P.s. pv. maculicola and P.s. pv. tomato. However, NCPPB 4265 and NCPPB 4266 had profiles very similar to that of the P.s. pv. viburni type strain and were different from all other reference strains. Bradbury (1986) lists the known distribution as USA and Spain, with the Spanish strains coming from a rot of tobacco. This is the first authentic record of P.s. pv. viburni in the UK. The precise taxonomic position of NCPPB 3450 remains uncertain. This work was funded by DEFRA Plant Health Division Project PH0154.

Different European-type vaccines against porcine reproductive and respiratory syndrome virus have different immunological properties and confer different protection to pigs

Immunization of piglets with two different European-type modified live vaccines against porcine reproductive and respiratory syndrome (PRRS) virus produced different outcomes. After vaccination, pigs became viremic (42 days), neutralizing antibodies did not develop, and frequencies of virus-specific gamma-interferon-secreting cells (IFN-γ-SC) were low. Levels of interleukin-10 (IL-10) produced by peripheral blood mononuclear cells (PBMC) seemed to inversely correlate with interferon-gamma responses. After a challenge with a virulent Spanish strain, one vaccine (V3) protected piglets against viremia while the other (V1) did not. The vaccine V3 induced the highest IFN-γ-SC frequencies. IL-2, IL-4 or transforming growth factor-beta responses were not detected at any time for neither of the vaccines. In contrast, haptoglobin rose in sera of viremic pigs after the challenge. These results indicated a strong involvement of IFN-γ, and maybe IL-10, in the development of immunity against PRRS virus.

Genetic Characterization of Anaplasma (Ehrlichia) platys in Dogs in Spain

This paper reports the first genetic characterization of Anaplasma (Ehrlichia) platys in Spain from a naturally infected dog. The dog presented clinical signs compatible with canine ehrlichiosis. After DNA extraction and PCR amplification, 16S rRNA gene and citrate synthase gene ( gltA) of this agent were amplified. The GenBank accession number for the nucleotide sequence of the 16S rRNA gene of this strain is AY530806. The A. platys strains registered in France and Japan showed the highest similarity to the 16S rRNA gene sequence obtained from the Spanish strain. In the amplification of the gltA gene, a 1443 bp fragment was obtained, and three nucleotide differences were detected in comparison with other strains sequences. These data confirm the presence of A. platys in a dog showing clinical signs compatible with ehrlichiosis in Spain.

Antigenic and molecular characterization of isolates of the Italy 02 infectious bronchitis virus genotype

As part of an epidemiological surveillance of infectious bronchitis virus (IBV) in Spain, four Spanish field isolates showed high S1 spike sequence similarities with an IBV sequence from the GenBank database named Italy 02. Given that little was known about this new emergent IBV strain we have characterized the four isolates by sequencing the entire S1 part of the spike protein gene and have compared them with many reference IBV serotypes. In addition, cross-virus neutralization assays were conducted with the main IBV serotypes present in Europe. The four Spanish field strains and the Italy 02 S1 sequence from the NCBI database were established as a new genotype that showed maximum amino acid identities with the 4/91 serotype (81.7% to 83.7%), the D274 group that included D207, D274 and D3896 strains (79.8% to 81.7%), and the B1648 serotype (79.3% to 80%). Furthermore, on the basis of these results, it was demonstrated that the Italy 02 genotype had been circulating in Spain since as early as 1997. Based on the average ratio of synonymous:non-synonymous (dS/dN) amino acid substitutions within Italy 02 sequences, no positive selection pressures were related with changes observed in the S1 gene. Moreover, phylogenetic analysis of the S1 gene suggested that the Italy 02 genotype has undergone a recombination event. Virus neutralization assays demonstrated that little antigenic relatedness (less than 35%) exists between Italy 02 and some of the reference IBV serotypes, and indicated that Italy 02 is likely to be a new serotype.

Vancomycin-resistantEnterococcus faeciumClone in Swine, Europe

To the Editor: The use of antimicrobial agents for growth promotion (AGP) in food-producing animals has been extensively debated because of the risk of establishing a reservoir of antimicrobial resistance genes or antimicrobial-resistant organisms of potential relevance for human health. This concern has motivated the progressive ban of the use of different AGP in the European Union, which began in 1997 with avoparcin and will end in 2006 (1). Worldwide trade of living animals for food production or breeding and of meat products enables multidrug-resistant pathogens to spread across national borders. Intercontinental dissemination of antimicrobial-resistant bacteria associated with food animals has been described for particular clones such as Salmonella enterica Typhimurium DT104 or Escherichia coli O157:H7 and for transferable genetic elements such as the genomic island SG1 or the streptococcal plasmid pRE25 (2). Vancomycin-resistant enterococci (VRE) in European farms were initially associated with the intensive use of avoparcin; however, the persistence of VRE in food animal environments after years of avoparcin withdrawal indicates that coselection by further antimicrobial or other agents, increased fitness of strains, and mobile genetic elements cannot be ruled out (1–3). A specific clone was recently detected among vancomycin-resistant E. faecium (VREF) isolated from different swine farms in Denmark and Switzerland and from a healthy Danish woman without antimicrobial drug exposure who ate pork, chicken, and beef (4,5). Since Portugal and Spain maintain commercial trade of food-producing swine (living or meat products) between them and with other European countries, including Denmark (http://www.dgv.min-agricultura.pt/dgv.nsf), we investigated a possible relationship among VREF swine fecal isolates from Portugal and Spain and compared these isolates with the Swiss/Danish clone. We studied 3 VREF from a Figueira da Foz slaughterhouse in central Portugal (1997–1998) and 3 VREF isolates from 3 Spanish slaughterhouses in Valencia, Lugo, and Murcia in eastern, northern, and southern Spain, respectively (1998–2000). These isolates were recovered in the course of previous surveillance studies (C. Novais/I. Herrero, unpub data). Antimicrobial susceptibility was tested for 13 antimicrobial agents by using the agar dilution method (6). Clonal relationships were analyzed by pulsed-field gel electrophoresis (PFGE) and characterization of pur-K alleles by amplification and further sequencing (6,7; http://efaecium.mlst.net). Species identification, genes coding for antimicrobial resistance genes or for putative virulence traits, and the backbone structure of Tn1546 were analyzed by polymerase chain reaction followed by sequencing when necessary (6,8). Broth and filter mating were performed by using E. faecium GE1 as recipient strain (6). Following criteria published elsewhere (6), the VREF isolates studied were considered a single clone (0–4 bands difference by PFGE). Some vancomycin-susceptible E. faecium swine isolates (VSEF) from Spain and Switzerland showed an SmaI-PFGE pattern closely related to that of VREF isolates (data not shown [4]; ). Representative VREF of each country harbored the allele 9 of the housekeeping gene purK, previously found among E. faecium isolates from swine and healthy persons (7). All VREF isolates were resistant to glycopeptides (vanA), erythromycin [erm(B)], and tetracycline. Two Spanish isolates were also highly resistant to streptomycin and kanamycin [aph(3´)-IIIa] (Table). All VREF isolates tested carried a Tn1546 type D, previously found in isolates from food-producing animals (8). This element showed alterations in orf1 and a G-T point mutation in the position 8234 at vanX. Transfer of vancomycin resistance was detected for the Swiss (4), Spanish, and Portuguese isolates and was associated with erythromycin resistance in all cases. Tetracycline resistance was also transferable in the Spanish strains. No virulence traits were detected. Table Features of vancomycin-resistant Enterococcus faecium swine isolates from European countries* We describe the simultaneous occurrence of a VREF strain among swine in 4 distant European countries for at least a 4-year period. Tn1546 type D has been largely described in European swine isolates, which indicates stability of this particular type among the high diversity of Tn1546 described to date (8). The finding of a group of genetically closely related strains, which include both VSEF and VREF isolates and which harbor a particular purK allele previously associated with E. faecium swine strains, might mirror wide dissemination of a host-specific clone more prone than others to acquire and spread different antimicrobial resistance, as reported for human clinical E. faecium isolates (9). Since enterococci from swine are able to colonize in the human gut (5,7) and isolates harboring purK-9 can be recovered from hospitalized patients with severe infections (10), specific swine enterococcal strains might represent a risk for antimicrobial resistance spread in the clinical setting. Further analyses need to be performed to understand the role of international animal movements, animal feed, and colonized farmers in the spread of this particular strain and to assess whether this clone shows an increased fitness in the porcine intestine when compared to other E. faecium strains.

The use of a multidisciplinary approach for the characterization of a diploid parthenogenetic Artemia population from Torre Colimena (Apulia, Italy)

A parthenogenetic Artemia population from Torre Colimena, southern Italy, originally reported in 1998, is characterized by a multidisciplinary approach including cyst and naupliar biometry, morphometry of adults, scanning electron microscopy (SEM) of brood pouch, cytogenetics and 16S rRNA PCR-RFLP analysis. We confirmed parthenogenetic status, inferred ploidy level and determined phenotypic and molecular relationships of this population through comparisons with other asexual Artemia strains as well as bisexual species found (A. salina) or introduced (A. franciscana) in the Mediterranean basin. Cyst and naupliar sizes for Torre Colimena are among the smallest recorded for asexual Artemia while the opposite is true for chorion thickness. Discriminant analysis of adult body measurements shows increased differentiation (89.5% for the first four out of the taselve functions produced) from tetraploid parthenogenetic strains and bisexual species (A. salina and A. franciscana). Scanning electron micrographs of brood pouch reveal the characteristic morphology of asexual strains, while chromosome observations of instar-I nauplii unequivocally establish diploid>. Restriction patterns give evidence that the Torre Colimena population shares an identical set of mitochondrial DNA (mtDNA) polymorphisms with a diploid Spanish parthenogenetic strain and it is well differentiated from other tetraploid as well as bisexual auto- and allochthonous strains (A. salina and A. franciscana) from the Mediterranean. The present study may serve as a reference methodological framework for multidisciplinary characterization and biodizjersiy assessments in the genus Artemia.

Antimicrobial profiles of periodontal pathogens isolated from periodontitis patients in the Netherlands and Spain

Antimicrobial resistance of periodontal pathogens towards currently used antibiotics in periodontics has been investigated in a previous study. Microbial resistance in the periodontal microflora was more frequently observed in Spanish patients in comparison with Dutch patients. The aim of the present study was to compare antimicrobial susceptibility profiles of five periodontal bacteria isolated from periodontitis patients in Spain and in The Netherlands. Subgingival plaque samples from adult patients with periodontitis were collected and cultured on selective and non-selective plates. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Micromonas micros were isolated and used for minimal inhibitory concentration tests using the Epsilometer (E-test) technique. Eight different antibiotics were tested on all bacterial isolates. MIC50 and MIC90 values for each antibiotic and each species were determined and the percentage of resistant strains was calculated. Significantly higher MIC values were noted in Spanish strains of F. nucleatum for penicillin, ciprofloxacin, of P. intermedia for penicillin, amoxicillin and tetracycline, of M. micros for tetracycline, amoxicillin and azithromycin, and of P. gingivalis for tetracycline and ciprofloxacin. Based on breakpoint concentrations, a higher number of resistant strains in Spain were found in F. nucleatum for penicillin, amoxicillin and metronidazole, in Prevotella intermedia for tetracycline and amoxicillin, and in A. actinomycetemcomitans for amoxicillin and azithromycin. Resistance of P. gingivalis strains was not observed for any of the antibiotics tested both in Spain and The Netherlands. Differences exist in the susceptibility profiles of periodontal pathogens isolated from periodontitis patients in Spain and in The Netherlands. This implicates that antibiotic susceptibility testing is necessary to determine efficacy of antimicrobial agents. Also, clinical studies with antibiotics should take these differences into account. The information from the present study indicates that it may not be possible to develop uniform protocols for usage of antibiotics in the treatment of severe periodontitis in the European Union.

Genetic variation between Spanish and American versions of sweet corn inbred lines

AbstractConservation of maize inbred lines in different stations causes variability among strains. The objective of this research was to determine agronomic and molecular differences in American sweet corn inbreds maintained in Spain. American and Spanish strains of five sweet corn inbred lines were characterized by using 34 RAPD primers that produced 168 consistent bands. Strains of four of these inbreds were crossed in a diallel design, and hybrids were evaluated in four environments in northwestern Spain. The RAPD characterization showed no differences between strains for two inbreds, while divergence between strains was largest for the inbred 15125. Most primers did not reveal any variability between pairs of strains, while some primers produced variations at high rates. Differences in agronomic performance among Spanish and American strains were most important for P51, followed by P39, while strains 1453 or 15125 were not significantly different. Molecular differences between strains were not related to agronomic performance. Residual heterozygosity or outcrossing cannot explain these results. Lack of adaptation could have caused stress‐induced mutagenesis. Natural selection could have eliminated unfavourable selective mutations, but neutral mutations can be found at the molecular level and favourable mutations could have been selected at the agronomic level.

Identification, incidence and characterization of Fusarium proliferatumon ornamental palms in Spain

During a survey conducted from 1998 to 2002, Fusarium proliferatum was found associated with young and adult palms belonging to the genera Chamaerops, Phoenix, Trachycarpus and Washingtonia showing symptoms of wilt and dieback. The fertility and toxicological profile of 36 strains representing different locations and hosts were studied. All of them except two, which were infertile, belonged to mating population D. Both mating types (MATD-1 and MATD-2) were isolated from the same host species, showing a high potential of genetic recombination in the field. Additionally, eight strains were fertile once crossed as female. Toxin analysis showed differences in the ability of strains to produce fumonisin B1, moniliformin, beauvericin, fusaric acid and fusaproliferin. Only 17 of them produced all the toxins analyzed. Pathogenicity tests were conducted on Phoenix dactylifera and P. canariensis using nine F. proliferatum Spanish strains and two reference strains from Saudi Arabia. Eight months after inoculation all strains caused disease, with palms showing lesions on the bases of leaves and development of wilt symptoms similar to those originally observed in affected plants. This is the first report on the occurrence of F. proliferatum on P. dactylifera in Spain and also the first report of this pathogen on C. humilis, P. canariensis, P. reclinata, T. fortunei, W. filifera and W. robusta.

Erwinia amylovora strains from outbreaks of fire blight in Spain: phenotypic characteristics

Summary One hundred and thirty strains of Erwinia amylovora recovered from Spanish foci of fire blight from 1995 to 2000 were characterised and compared to reference strains from different sources and origins. Their rapid identification was performed by double antibody sandwich indirect (DASI) ELISA, using specific monoclonal antibodies against E. amylovora, and molecular confirmation by PCR using primers specific to the native plasmid pEA29. The Spanish strains of E. amylovora grew on different general and selective media producing typical colonies, except one of them that was deficient in levan production, whereas none of them grew on minimal agar medium with copper sulphate and low content of asparagine. All of them were susceptible to tetracycline, streptomycin, kasugamycin and oxolinic acid. Biochemical characterisation of selected strains by API 20E system revealed a great homogeneity, with 80% of the Spanish strains showing one of the two majority API 20E profiles described for E. amylovora, and the remaining strains showing minor differences. Pathogenicity on pear fruits and hypersensitivity reaction was confirmed, but a delayed reaction was observed for two Spanish strains. This is the first characterisation of a large collection of Spanish strains of E. amylovora.

First Record of the Rare Species Aeromonas culicicola from a Drinking Water Supply

We describe the recovery of the rare species Aeromonas culicicola, so far known only in mosquitoes in India, from a drinking water supply in Spain. Typing, using enterobacterial repetitive intergenic consensus-PCR, revealed that the 27 new isolates belonged to 3 very closely related strains. These strains were genetically identified by 16S rRNA gene sequencing. Spanish strains differed from the mosquito strains in three nucleotide positions. The AHCYTOEN gene was present in these water strains, which may have a public health significance.

Novel 180- and 480-Base-Pair Insertions in African and African-American Strains of Helicobacter pylori

Helicobacter pylori is a genetically diverse bacterial species that chronically infects human stomachs and sometimes causes severe gastroduodenal disease. Studies of polymorphic DNA sequences can suggest geographic origins of individual strains. Here, we describe a 180-bp insertion (ins180), which is just after the translation stop of a gene of unknown function, near the promoter of jhp0152-jhp0151 two-component signal transduction genes in strain J99, and absent from this site in strain 26695. This ins180 insertion was found in 9 of 9 Gambian (West African), 9 of 20 (45%) South African, and 9 of 40 (23%) Spanish strains but in only 2 of 20 (10%) North American strains and none of 20 Lithuanian, 20 Indian, and 20 Japanese strains. Four South African isolates that lacked ins180 and that belonged to an unusual outlier group contained a 480-bp insertion at this site (ins480), whereas none of 181 other strains screened contained ins480. In further tests 56% (10 of 18) of strains from African Americans but only 17% (3 of 18) of strains from Caucasian Americans carried ins180 (P < 0.05). Thus, the H. pylori strains of modern African Americans seem to retain traces of African roots, despite the multiple generations since their ancestors were taken from West Africa. Fragmentary ins180-like sequences were found at numerous sites in H. pylori genomes, always between genes. Such sequences might affect regulation of transcription and could facilitate genome rearrangement by homologous recombination. Apparent differences between African-American and Caucasian-American H. pylori gene pools may bear on our understanding of H. pylori transmission and disease outcome.

Nucleotide correlation between HAV isolates from human patients and environmental samples

Hepatitis A disease is endemic in Albania even though records from the Medical Authority show outbreaks involving only limited numbers of people. In the city of Lac, 200 people became ill following an outbreak of hepatitis A started on November 2002. The age distribution of the cases shows a peak in the age group 5-9 (43.5%) followed by the age group 10-14 (28%) and the age group 1-4 (18%). No cases were recorded in the age group 0-1. Drinking water and sewage samples were collected, using electropositive cartridges, during the outbreak. Rotavirus was identified by RT-PCR in two out of five drinking water samples; however, all the samples tested negative for HAV and astrovirus. Rotavirus was also identified in three out of five sewage samples from which four were HAV positive. HAV-RNA was identified in 28 (62.2%) out of 45 IgM anti-HAV positive sera collected during the outbreak. Genotype IA was expressed from all the amplified samples. Sequence analysis of the overlapping VP1-2A region shows 97-99% homology with three Italian strains IT-SCH-00, IT-ZAM-01, and IT-CAP-00 and one Spanish strain (Sa 30/06/95), whereas the phylogenetic tree built from the 168 bp sequence of the VP1-2A shows four clusters, one including 24 out of 28 RT-PCR positive sera. Considering the deduced amino acid sequence, only one substitution was identified and reported previously for genotype IB.