Field Comparison of Cyclopentanone Versus Carbon Dioxide as an Attractant for Adult Mosquitoes in Southeast Queensland, Australia.

Cyclopentanone is a saturated monoketone typically used as an intermediate in the manufacture of pharmaceuticals, biologicals, insecticides, and rubber chemicals. Recently, it has been demonstrated that cyclopentanone activates the cpA CO2 receptor neuron on the maxillary palp of mosquitoes, suggesting that it may be a viable alternative to CO2 as an attractant for mosquitoes. Furthermore, semifield experiments showed that traps baited with cyclopentanone attract Culex quinquefasciatus Say at a similar rate to those baited with CO2. We evaluated the field efficacy of cyclopentanone as an alternative to CO2 in Centers for Disease Control (CDC) light traps and counterflow geometry (CFG) traps commonly used to collect mosquitoes in surveillance programs. Three pairwise trials and four Latin square trials were conducted across three peri-urban sites, comprising two saltwater sites and one freshwater site, in southeast Queensland, Australia. In all trials, CO2-baited traps outperformed traps baited with cyclopentanone. Carbon dioxide-baited CDC traps collected significantly more total mosquitoes, Aedes vigilax (Skuse), Culex sitiens Weidemann, and Culex annulirostris Skuse, than those baited with ≥99% cyclopentanone in pairwise trials. Similarly, in almost all Latin square trials, CO2-baited CDC and CFG traps collected significantly greater numbers of total mosquitoes, Ae. vigilax, Cx. annulirostris, Culex orbostiensis Dobrotworsky, and Cx. sitiens when compared with CFG traps baited with 20% cyclopentanone. Our trials indicate that cyclopentanone is not effective as a mosquito attractant in the field and cannot be used as a simple substitute for CO2 in commonly used mosquito surveillance traps.

Emerging tropical diseases in Australia. Part 5. Hendra virus.

Hendra virus (HeV) was first isolated in 1994, from a disease outbreak involving at least 21 horses and two humans in the Brisbane suburb of Hendra, Australia. The affected horses and humans all developed a severe but unidentified respiratory disease that resulted in the deaths of one of the human cases and the deaths or putting down of 14 of the horses. The virus, isolated by culture from a horse and the kidney of the fatal human case, was initially characterised as a new member of the genus Morbillivirus in the family Paramyxoviridae. Comparative sequence analysis of part of the matrix protein gene of the virus and the discovery that the virus had an exceptionally large genome subsequently led to HeV being assigned to a new genus, Henipavirus, along with Nipah virus (a newly emergent virus in pigs). The regular outbreaks of HeV-related disease that have occurred in Australia since 1994 have all been characterised by acute respiratory and neurological manifestations, with high levels of morbidity and mortality in the affected horses and humans. The modes of transmission of HeV remain largely unknown. Although fruit bats have been identified as natural hosts of the virus, direct bat-horse, bat-human or human-human transmission has not been reported. Human infection can occur via exposure to infectious urine, saliva or nasopharyngeal fluid from horses. The treatment options and efficacy are very limited and no vaccine exists. Reports on the outbreaks of HeV in Australia are collated in this review and the available data on the biology, transmission and detection of the pathogen are summarized and discussed.

Emerging tropical diseases in Australia. Part 3. Australian bat lyssavirus.

Since its discovery in a juvenile black flying fox (Pteropus alecto) in 1996, Australian bat lyssavirus (ABLV) has become the cause of a potentially important emerging disease for health authorities in Australia, with two human deaths (one in 1996 and one in 1998) attributed to the virus in the north-eastern state of Queensland. In Australia, the virus has been isolated from all four species of flying fox found on the mainland (i.e. P. alecto, P. scapulatus, P. poliocephalus and P. conspicillatus) as well as a single species of insectivorous bat (Saccolaimus flaviventris). Australian bat lyssavirus belongs to the Lyssavirus genus and is closely related, genetically, to the type strain of Rabies virus (RABV). Clinically, patients infected with ABLV have displayed the 'classical' symptoms of rabies and a similar disease course. This similarity has led to the belief that the infection and dissemination of ABLV in the body follows the same pathways as those followed by RABV. Following the two ABLV-related deaths in Queensland, protocols based on the World Health Organization's guidelines for RABV prophylaxis were implemented and, presumably in consequence, no human infection with ABLV has been recorded since 1998. ABLV will, however, probably always have an important part to play in the health of Australians as the density of the human population in Australia and, consequently, the level of interaction between humans and flying foxes increase.

Emerging tropical diseases in Australia. Part 4. Mosquitoborne diseases.

Mosquito-borne diseases continue to be a serious public-health concern in Australia. Endemic alphaviruses (including Ross River and Barmah Forest viruses) account for the majority of the arboviral notifications, while some flaviviruses (Murray Valley encephalitis, Japanese encephalitis and Kunjin viruses) cause occasional outbreaks of encephalitis. Dengue epidemics are increasing in frequency in northern Queensland, with the largest outbreak in 50 years occurring during the 2008-2009 wet season. Of great concern are the threats posed by the importation of exotic arboviruses, such as West Nile, chikungunya and Rift Valley fever viruses, the introduction of exotic vectors, and the potential range expansion of key Australian vectors. Environmental and anthropogenic influences provide additional uncertainty regarding the future impact of mosquito-borne pathogens in Australia. This review discusses the trends, threats and challenges that face the management of mosquito-borne disease in Australia. Topical mosquito-borne pathogens of biosecurity and public-health concern, and the potential impacts of environmental and global trends, are discussed. Finally, a short overview of the public-health response capability in Australia is provided.

Emerging tropical diseases in Australia. Part 1. Leptospirosis.

Human leptospirosis is a zoonotic disease of global importance that causes significant morbidity and mortality, particularly in developing nations. In this review, the history, epidemiology, transmission, clinical presentation and treatment of this disease, and its impact in Australia, are discussed. Central to this review is the delineation of diagnostic methods for the disease and the challenges that this disease presents for both the clinician and diagnostic laboratory. This information should furnish clinicians with an updated tool to help overcome a number of problems associated with the diagnosis of leptospirosis.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, inter-serovar convergence, and evidence of attenuation in Leptospira reference collections.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

First Report of Iresine viroid 1 in Celosia plumosa in the Netherlands.

In 2008, in the framework of surveying for pospiviroids, nine symptomless clones of Celosia plumosa (Voss) Burv. (Amaranthaceae) from a Dutch breeding company were tested by reverse transcription (RT)-PCR with primer sets Pospi1-RE/FW and Vid-RE/FW (4). In four samples, amplicons of 227 nt were obtained with primers Pospi1-RE/FW. Sequencing of the amplicons showed identities of more than 99% to the partial sequence of Iresine viroid 1 (IrVd-1) from Alternanthera sessilis, NCBI GenBank Accession No DQ846886 (2). Subsequently, a set of primers was designed to amplify the complete viroid genome, i.e., IrVd-FW1 5'-GCG GAA GAA ACA GGA GCT CGW CT-3' and IrVd-RE1 5'-CGC GWG GAG TTC TCC GGT CTT TA-3' - identical to nt 168 to 190 and 145 to 167 of the complete IrVd-1 sequences in the NCBI GenBank (Nos. DQ094293, DQ094294, NC_003613, and X95734). One isolate from C. plumosa was amplified with this primer pair and amplicons were cloned into the pGEM-T Easy Vector System II. Sequencing of one individual cDNA clone (GenBank Accession No. GU911350) revealed a genome size of 370 nt and 98.1% sequence identity to the IrVd-1 isolate from Vinca major, GenBank Accession No. DQ094293 (1). Hence, the viroid was identified as IrVd-1. The isolate from C. plumosa was also mechanically inoculated to 10 healthy plants of C. plumosa, chrysanthemum (Chrysanthemum × morifolium) cv. White Delianne, potato (Solanum tuberosum) cv. Nicola, and tomato (Solanum lycopersicum) cv. Moneymaker. No symptoms were observed over a 6-week period, and RT-PCR with primers Pospi1-RE/FW on bulked samples of five plants per species only identified IrVd-1 in both samples of C. plumosa. For tomato, these results confirm those of Spieker (3). Therefore, in contrast to the other pospiviroids, it seems unlikely that IrVd-1 poses a threat to potato and tomato. References: (1) X. Nie et al. Can. J. Plant Pathol. 27:592, 2005. (2) R. P. Singh et al. Plant Dis. 90:1457, 2006. (3) R. L. Spieker. J. Gen. Virol. 77:2631, 1996. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

First Report of Tomato apical stunt viroid in the Symptomless Hosts Lycianthes rantonnetii and Streptosolen jamesonii in the Netherlands.

In 2009, in the framework of surveying for pospiviroids, samples of various ornamental plants from the Netherlands were tested by reverse transcription (RT)-PCR with the primer pairs Pospi1-RE/FW and Vid-RE/FW (2). With primer pair Pospi1-RE/FW, amplicons of the expected size were obtained in two samples of symptomless plants of Lycianthes rantonnetii and Streptosolen jamesonii. Sequencing of the amplicons, which were expected to correspond with partial pospiviroid genomes, showed identities of 100 and 98% to the sequence of Tomato apical stunt viroid (TASVd), NCBI GenBank Accession No. AM777161 (3). For the amplification of the complete viroid genomes, RT-PCRs were performed with primer pair CEVd-FW/RE (1). Sequencing of these amplicons yielded sequences of 364 nt and identities to TASVd AM777161 of 100 and 98.1%, respectively. Therefore, both isolates were identified as TASVd. The sequence variant from S. jamesonii was submitted to the NCBI GenBank as No. GU911351. In addition, both isolates were mechanically inoculated to four tomato plants (Solanum lycopersicum) of cv. Moneymaker. All inoculated plants developed chlorosis and growth reduction after 4 weeks and TASVd infections were confirmed in a bulked sample by RT-PCR with primer pair CEVd-FW/RE after 6 weeks. Hence, two more ornamental host plant species have been identified that may act as symptomless sources of pospiviroid inoculum. References: (1) N. Önelge. Turk. J. Agric. For. 21:419, 1997. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (3) J. Th. J. Verhoeven et al. Plant Dis. 92:973, 2008.

Hypomagnesaemia in the first 10 days of severe leptospirosis.

Magnesium imbalance in leptospirosis has, for the most part, been neglected by the medical and leptospirosis communities. In a recent, retrospective study, serum concentrations of magnesium were followed in 15 patients with severe leptospirosis. The results revealed that 14 of the 15 patients developed hypomagnesaemia at some time during the first 10 days of their illness. In severely ill patients, such magnesium deficiency can worsen clinical outcome. Magnesium concentrations may affect a number of organ systems and mental status. Since altered mental status in leptospirosis is a poor prognostic indicator, it is suggested that serum concentrations of magnesium be monitored closely in patients with leptospirosis. Any hypomagnesaemia can then be treated promptly, in an effort to reduce the morbidity and mortality attributable to the disease.

Leptospirosis and Goodpasture's syndrome: testing the aetiological hypothesis.

Leptospiral pathogens have a world-wide distribution and cause a spectrum of disease ranging from a mild, influenza-like illness to Weil's disease, which manifests itself in multi-organ failure. Recently, Leptospira-reactive sera from 40 leptospirosis patients were investigated in an ELISA designed to detect antibodies to the human glomerular basement membrane (GBM). The aim was to determine if host-derived leptospiral immunoglobulins cross-react with proteins in the human GBM, so facilitating the development of Goodpasture's syndrome. As all 40 sera were found negative in the anti-GBM ELISA, the hypothesis that, during the immune phase of leptospirosis, patients are at risk of developing Goodpasture's syndrome was not supported. Further work is required to determine if leptospirosis is a risk factor in the development of any other pulmonary-renal syndromes that are associated with auto-immune diseases, such as Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, Behçet's disease, IgA nephropathy and systemic lupus erythematosus.

Pepper chat fruit viroid: biological and molecular properties of a proposed new species of the genus Pospiviroid.

In autumn 2006, a new disease was observed in a glasshouse-grown crop of sweet pepper (Capsicum annuum L.) in the Netherlands. Fruit size of the infected plants was reduced up to 50%, and plant growth was also slightly reduced. Here we show that the disease is caused by a previously non-described viroid. The pepper viroid is transmitted by both mechanical inoculation and pepper seeds and, when inoculated experimentally, it infects several solanaceous plant species inducing vein necrosis and reduced fruit and tuber size in tomato and potato, respectively. The viroid RNA genome consists of 348 nucleotides and, with minor modifications, it has the central conserved and the terminal conserved regions characteristic of members of the genus Pospiviroid. Classification of the pepper viroid within the genus Pospiviroid is further supported by the presence and structure of hairpins I and II, the presence of internal and external RY motifs, and phylogenetic analyses. The primary structure of the pepper viroid only showed a maximum of 66% nucleotide sequence identity with other viroids, which is far below the main species demarcation limit of 90%. According to its biological and molecular properties, we propose to assign the pepper viroid to a new species within the genus Pospiviroid, and to name this new species Pepper chat fruit viroid.

First Report of Tomato yellow leaf curl virus in Tomato in the Netherlands.

Tomato yellow leaf curl virus (TYLCV) is an economically important virus with tomato (Solanum lycopersicum L.) as its main host. The virus is widely distributed in subtropical areas and is transmitted by the tobacco whitefly (Bemisia tabaci) in a persistent manner. TYLCV has a quarantine status (IIAII) in the European Union (EU directive 2000/29/EC). It was not previously recorded in the Netherlands. In September 2007, symptoms were observed in tomato crops in a few greenhouses located in close proximity from each other in the western part of the Netherlands. Infected plants showed TYLCV-like symptoms, i.e., stunting, leaf curl, and marginal and interveinal chlorosis. Similar symptoms were evoked after grafting symptomatic tips onto healthy tomato seedlings, whereas no viruses were transmitted by mechanical inoculation to herbaceous test plants. Extracted DNA from symptomatic leaves was used in PCR with two sets of primers for universal detection of begomoviruses (1,2). Analysis of the overlapping amplified products revealed the highest identity to isolate TYLCV-Alm (GenBank Accession No. AJ489258) from Almeria, Spain. To amplify the remaining 60% of the virus genome, three additional primer sets were designed: TYLCV965F 5'-GGCAGCCAAGTACGAGAACC-3' and TYLCV1736R 5'-CCACTATCTTCCTCTGCAATCC-3'; TYLCV1598F 5'-TACTTGCGAACAGTGGCTCG-3' and TYLCV2282R 5'-TCCAAATCGATGGCAGATCAG-3'; TYLCV2229F 5'-ATGCGTCGTTGGCAGATTG-3' and TYLCV68R 5'-CAGTGACGTCTGTGGAACCCT-3'. Analysis of the five overlapping PCR products of one isolate revealed a total virus genome of 2,781 nucleotides. The complete sequence of the Netherlands Isolate (GenBank Accession No. FJ439569) showed 99.3% nucleotide identity to isolate TYLCV-Alm (AJ489258), and therefore, the virus was identified as TYLCV-Alm. After the initial identification, a survey was conducted in all tomato crops in a surrounding area of approximately 40 km2. TYLCV was found in 19 of 27 cultivations. The identity of one isolate per cultivation was confirmed by sequence analysis of the products obtained with the Wyatt and Brown primers (2) occasionally in combination with the Deng primers (with 99.1 to 100% and 99.2 to 100% nucleotide identity to the Netherlands isolate [FJ439569], respectively) (1). As many as 25 symptomatic plants were recorded per greenhouse. A subsequent survey of 34 randomly selected tomato growers in other areas of the country revealed no further infections. Results of the sequence analyses and surveys suggested that the outbreak resulted from a single introduction of the virus, whereas the insect vector B. tabaci accounted for local spread. Measures taken to eliminate the virus included the removal and subsequent destruction of infected tomato plants as well as eradication of B. tabaci. No TYLCV infections were found during surveys in 2008, and therefore, it is believed that the virus was eradicated effectively. References: (1) D. Deng et al. Ann. Appl. Biol. 125:327, 1994. (2) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

First Report of Solanum jasminoides Infected by Citrus exocortis viroid in Germany and the Netherlands and Tomato apical stunt viroid in Belgium and Germany.

Recent identifications of Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd) in Solanum jasminoides (3,4) prompted the testing of this plant species for infections with other pospiviroids. From autumn of 2006 to spring of 2007, samples from symptomless plants of S. jasminoides were collected in Belgium (3 samples ranging from 75 to 150 plants), Germany (3 samples ranging from 1 to 200 plants), and the Netherlands (3 samples ranging from 2 to 200 plants). Samples were tested for pospiviroids by reverse transcription (RT)-PCR assays using the Pospi1-FW/RE and Vid-FW/RE (2) and PSTV-Nb-FW (5'-ggatccccggggaaacctgga-3')/RE (5'-ggatccctgaagcgctcctcc-3') primer sets. Each set amplifies several but not all pospiviroids. The first and last primer sets amplified PCR products from six samples. The full-length genomes of all six isolates were amplified using primer pairs CEVd-FW1/RE1 (1) and CEVd-FW2 (5'-gtgctcacctgaccctgcagg-3')/RE2 (5'-accacaggaacctcaagaaag-3'), which are fully complementary to both Citrus exocortis viroid (CEVd) and Tomato apical stunt viroid (TASVd). Sequence analysis of the PCR products identified CEVd from two samples each from Germany and the Netherlands and TASVd from one sample each from Germany and Belgium (plants were imported from Israel). Although the sequences of the different CEVd isolates from S. jasminoides were not identical, all exhibited more than 95% identity with a CEVd isolate from Vicia faba (GenBank Accession No. EF494687). Both TASVd sequences were identical and showed 99.2% identity to a TASVd isolate from tomato (GenBank Accession No. AY 062121). Two nucleotide sequences of CEVd were submitted to the NCBI GenBank (Accession Nos. EU094207 and EU094208). The two other CEVd sequences and the TASVd sequence were submitted to the EMBL Nucleotide Sequence Database as Accession Nos. AM774356, AM774357, and AM777161. In addition to identification from S. jasminoides by sequence analysis, TASVd infection in the S. jasminoides sample from Germany and CEVd in one sample from the Netherlands was confirmed by mechanical inoculation to tomato followed by RT-PCR using the two CEVd-FW/RE primer pairs and analysis of the sequenced PCR product. Infection by CEVd and TASVd was also confirmed in the German samples by Northern hybridization and TASVd was confirmed in the Belgian sample by return-polyacrylamide gel electrophoresis. To our knowledge, these are the first reports of CEVd and TASVd in S. jasminoides. The viroids do not reduce the quality of S. jasminoides plants; however, the infected plants may act as infection sources for other crops. References: (1) N. Önelge. Turk. J. Agric. For. 21:419, 1997. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (3) J. Th. J. Verhoeven et al. Plant Dis. 90:1359, 2006. (4) J. Th. J. Verhoeven et al. Plant Pathol. 57:399, 2008.

First Report of Potato spindle tuber viroid in Tomato in Belgium.

During August of 2006, a sample of a tomato plant (Solanum lycopersicum, formerly Lycopersicum esculentum) from a greenhouse in Belgium was received for diagnosis. The plant showed severe growth reduction and the young leaves were chlorotic and distorted. In the greenhouse, the disease had been spreading slowly along the row. These observations suggested the presence of a viroid infection, and reverse transcriptase (RT)-PCR with two sets of universal pospiviroid primers (Pospi1-RE/FW and Vid-FW/RE; 3) yielded amplicons of the expected size (approximately 196 and 360 bp). Sequence analysis of the larger PCR product revealed that the genome was 358 nt and 100% identical to two isolates of Potato spindle tuber viroid (PSTVd) previously submitted to the NCBI GenBank (Accession Nos. AJ583449 from the United Kingdom and AY962324 from Australia). A pathogen associated with the symptomatic tomato plants was therefore identified as PSTVd. Tracing the origin of the infection revealed the following information: during November of 2005, 8-day-old tomato seedlings raised from seed by a Dutch nursery were transferred to a small part of the greenhouse of the Belgian grower; 7 to 8 weeks later, the plants were transplanted to their final destination; during May of 2006, the grower first observed growth reduction in a single plant; several weeks later, similar symptoms were observed in two more plants in the same row close to the first symptomatic plant; and by September, there were approximately 20 symptomatic tomato plants, all located in two adjacent rows. The viroid outbreak was fully eradicated by destroying all tomato plants in the affected rows as well as in two adjacent rows at both sides. The absence of further infections was confirmed by testing approximately 1,200 tomato plants in pooled samples for PSTVd by RT-PCR (2) and real-time RT-PCR (1). The origin and the method of introduction and spread of the viroid remain unclear. References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) R. A. Mumford et al. Plant Pathol. 53:242, 2004. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

First Report of Tomato chlorotic dwarf viroid in Petunia hybrida from the United States of America.

In November 2005, 13 accessions of Petunia hybrida from the United States of America entered the post-entry quarantine station of the Plant Protection Service in the Netherlands. The plants were inspected and tested for quarantine organisms according to Directives 95/44 and 97/46 of the European Union. No virus and viroid symptoms were observed in the imported plants or in mechanically inoculated plants of Chenopodium quinoa, Nicotiana benthamiana, and N. occidentalis-P1 (3). Testing for pospiviroids by return-polyacrylamide gel electrophoresis (1) and reverse transcriptase-PCR with universal pospiviroid primers Pospi1-RE/FW (2) indicated the presence of pospiviroids in 3 and 11 P. hybrida accessions, respectively. The 196-bp amplicons of six accessions were sequenced. Sequence analysis showed the highest identity for all amplicons to both isolates of Tomato chlorotic dwarf viroid (TCDVd) in NCBI GenBank, Accession Nos. AF162131and AY372399, from Canada and the United States, respectively. Additional RT-PCRs with the Pospi1-RE/FW primers in opposite order and the semi-universal pospiviroid primers Vid-RE/FW (2) for one isolate, followed by sequence analysis, confirmed the identity as TCDVd. The isolate consisted of 359 nucleotides (GenBank Accession No. DQ859013) and showed sequence identities of 98.6 and 96.1% to the Canadian and American tomato isolates of this viroid, respectively. The next highest sequence identity was 90.0% to two accessions of Potato spindle tuber viroid (GenBank Accession Nos. AJ593449 and AY962324). On the basis of these results, the viroid from P. hybrida was identified as TCDVd. To our knowledge, this is the first report of TCDVd in this plant species. Reference: (1) J. W. Roenhorst et al. EPPO Bull. 30:453, 2000. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (3) J. Th. J. Verhoeven and J. W. Roenhorst. EPPO Bull. 33:305, 2003.

First Report of Potato virus M and Chrysanthemum stunt viroid in Solanum jasminoides.

In 2005, a plant of the ornamental crop Solanum jasminoides from the Netherlands was submitted for testing on viruses and viroids because of its intended use for propagation. Sap from this plant was mechanically inoculated to the test plant species Chenopodium quinoa, Nicotiana benthamiana, N. hesperis-67A, and N. occidentalis-P1 (3). N. hesperis-67A showed chlorotic local lesions and rugosity followed by vein necrosis, N. occidentalis-P1 showed necrotic local lesions and systemic leaf distortion, and the two other test plant species remained symptomless. Potato virus M (PVM) was identified by double antibody sandwich enzyme-linked immunosorbent assay using leaves from S. jasminoides and N. hesperis-67A. The plant of S. jasminoides was also tested for the presence of viroids by reverse transcriptase-polymerase chain reaction (RT-PCR) with universal pospiviroid primers Pospi1-RE/FW (2). This reaction yielded an amplicon of the expected size of 198 bp. The sequence showed 100% identity to an isolate of Chrysanthemum stunt viroid (CSVd; NCBI GenBank Accession No. AF394453). Subsequently, the complete sequence of our viroid isolate (GenBank Accession No. DQ406591) was determined from the amplicon obtained after RT-PCR using specific primers for the detection of CSVd (1). The viroid isolate from S. jasminoides consisted of 354 nucleotides and showed the highest identity (98.6%) to a chrysanthemum isolate of CSVd (GenBank Accession No. AB055974). Therefore, the viroid was identified as CSVd. To our knowledge, this is the first report of PVM and CSVd in S. jasminoides. Reference: (1) R. Hooftman et al. Acta Hortic. 432:120, 1996. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (3) J. Th. J. Verhoeven and J. W. Roenhorst, EPPO Bull. 33:305, 2003.

First Report of Tomato apical stunt viroid in Tomato in Tunisia.

In May 2005, the Plant Protection Service in the Netherlands received two tomato (Lycopersicon esculentum) plant specimens for diagnosis from a protected crop production facility of 2.5 ha near Kebili in Tunisia. Growth of the plants was reduced and leaves were chlorotic and brittle. Ripening of the fruits was delayed and their storage life was reduced from 3 weeks to 1 week. The grower reported that initially only 5% of plants showed symptoms; however, the number of symptomatic plants increased quickly to 100% as a result of increasing temperatures in the production facility. Test plant species Chenopodium quinoa, Datura stramonium, Nicotiana glutinosa, N. hesperis-67A, N. occidentalis-P1, and L. esculentum 'Money-maker' were mechanically inoculated with sap from the affected plants. Symptoms including chlorosis and stunting were observed only on L. esculentum. Reverse transcriptase-polymerase chain reaction (RT-PCR) with universal pospiviroid primers Pospi1-RE/FW (2) yielded amplicons of the expected size (196 bp) for each of the two samples. One of these amplicons was sequenced and showed the highest identity to the four isolates of Tomato apical stunt viroid (TASVd) in the NCBI Gen-Bank. Subsequently, the complete sequence of the Tunisian isolate (Gen-Bank Accession No. DQ144506) was determined by sequencing the am-plicon obtained after RT-PCR using primers developed for the detection of Citrus exocortis viroid (CEVd) (1). The isolate consisted of 363 nucleotides and showed the highest sequence identity (96.7%) to tomato isolates of TASVd from Indonesia and Israel (GenBank Accession Nos. X06390 and AY062121, respectively), 92.6% to a tomato isolate from the Ivory Coast (GenBank Accession No. K00818), and 87.7% to an isolate from Solanum pseudocapsicum (GenBank Accession No. X95293). The next highest sequence identity was 81.5% to an isolate of CEVd (GenBank Accession No. X53716). On the basis of these results, the viroid was identified as TASVd. To our knowledge, this is the first report of TASVd in Tunisia. Reference: (1) N. Önelge. Turkish J. Agric. For. 21:419, 1997. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

Prenatal diagnosis of the fragile X syndrome: loss of mutation owing to a double recombinant or gene conversion event at the FMR1 locus.

The fragile X syndrome, an X linked mental retardation syndrome, is caused by an expanded CGG repeat in the first exon of the FMR1 gene. In patients with an expanded repeat the FMR1 promoter is methylated and, consequently, the gene is silenced and no FMR1 protein (FMRP) is produced, thus leading to the clinical phenotype. Here we describe a prenatal diagnosis performed in a female from a fragile X family carrying a large premutation. In chorionic villus DNA of the male fetus the normal maternal CGG allele and a normal pattern on Southern blot analysis were found in combination with the FRAXAC2 and DXS297 allele of the maternal at risk haplotype. A second chorionic villus sampling was performed giving identical results on DNA analysis and, in addition, expression of FMRP was shown by immunohistochemistry. We concluded that the male fetus was not affected with the fragile X syndrome. Subsequent detailed haplotype analysis showed a complex recombination pattern resembling either gene conversion or a double crossover within a 20 kb genomic region.

Variable FMR1 gene methylation of large expansions leads to variable phenotype in three males from one fragile X family.

The fragile X syndrome is caused by an expanded CGG repeat (> 200 units, full mutation) at the 5' end of the FMR1 gene, which is associated with methylation of a CpG island upstream of the FMR1 gene and down regulation of the transcription. We describe three related males with full mutations in the FMR1 gene, as defined by size, but with different percentages of unmethylated alleles (+/-90%, 35%, and 15%, respectively) as studied in leucocytes. Normal mental status was observed in the male who showed 90% lack of methylation, whereas his two cousins were retarded. The mentally normal male did show some minor facial features of the fragile X syndrome; the FMR protein was detectable in 75% of his leucocytes. In all three cases, the proportion of unmethylated FMR1 genes corresponded to the percentage of leucocytes showing FMR1 protein production. Our results indicated a direct relationship between methylation and the ability to produce FMR protein. These cases will be discussed in relation to the phenotypic effects of incompletely methylated full mutations in the FMR1 gene as observed by others.

Comparison of performance of four instruments in evaluating the effects of salmeterol on asthma quality of life.

Quality of life measures are increasingly used as important efficacy endpoints in studies of drugs for asthma. The purpose of this study was to assess both the sensitivity to change and the construct validity of four different quality of life instruments in patients with asthma. In a double-blind, parallel group study, 120 moderate asthma patients, aged between 18-70 yrs, received either inhaled salmeterol 50 micrograms b.i.d. or inhaled salbutamol 400 micrograms b.i.d. In addition to respiratory outcomes, quality of life was measured at a 6 weeks follow-up using: 1) Asthma Quality of Life Questionnaire (AQLQ); 2) Living With Asthma Questionnaire (LWAQ); 3) Sickness Impact Profile (SIP); 4) Rating Scale (RS); and Standard Gamble (SG) utilities. Salmeterol led to significant improvements over salbutamol on virtually all clinical outcomes. Although all the quality of life instruments showed the same trend in favour of salmeterol, only the AQLQ and RS utilities showed significantly greater improvement on salmeterol than on salbutamol. Except for the AQLQ, the correlation between change in lung function and change in quality of life was generally low. Whereas, the AQLQ correlated well with the patient's overall assessment of efficacy (r = 0.64), the LWAQ, SIP and utilities failed to show such a correlation. The AQLQ showed the best correlation with symptom scores. The cross-sectional correlation between the AQLQ and the LWAQ was 0.73, whereas the longitudinal correlation was only 0.29. The SG generally showed poor correlation with other measures, including the RS. In conclusion, patients given salmeterol showed a greater improvement in quality of life compared to patients given salbutamol. Of the disease-specific questionnaires the Asthma Quality of Life Questionnaire was found to be more responsive to change than the Living With Asthma Questionnaire and showed greater validity. Of the generic instruments, the rating scale utilities were most responsive. The Standard Gamble showed poor correlation with other measures.