Epidemiology and Genetic Diversity of Grapevine Leafroll-Associated Viruses in British Columbia.

Grapevine leafroll disease (GLD) is a complex associated with one or more virus species belonging to the family Closteroviridae. The majority of viruses in this complex are vectored by one or more species of mealybugs (Pseudococcidae) and/or scale insects (Coccidae). Grape-growing regions of British Columbia (BC), including Okanagan, Similkameen, and Fraser valleys and Kamloops (BC central interior), Vancouver, and Gulf islands, were surveyed during the 2014 and 2015 growing seasons for the presence of four major grapevine leafroll-associated viruses, including Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3, and GLRaV-4. In total, 3,056 composite five-vine samples were collected from 153 Vitis vinifera and three interspecific hybrid vineyard blocks. The results showed GLRaV-3 to be the most widespread, occurring in 16.7% of the composite samples, followed by GLRaV-4 (3.9%), GLRaV-1 (3.8%), and GLRaV-2 (3.0%). Mixed infections of two or more GLRaVs were found in 4.1% of the total samples. The relative incidence of GLRaVs differed among regions and vineyard blocks of a different age. Characterization of partial CO1 region from a total of 241 insect specimens revealed the presence of Pseudococcus maritimus, Parthenolecanium corni, and other Pulvinaria sp. in BC vineyards. Spatial patterns of GLRaV-3 infected grapevines in three vineyard blocks from three different regions in the Okanagan Valley showed variable degrees of increase in disease spread ranging from 0 to 19.4% over three growing seasons. Regional differences in the relative incidence and spread of GLD underline the need for region-based management programs for BC vineyards.

Application of Next Generation Sequencing for Diagnostic Testing of Tree Fruit Viruses and Viroids.

Conventional detection of viruses and virus-like diseases of plants is accomplished using a combination of molecular, serological, and biological indexing. These are the primary tools used by plant virologists to monitor and ensure trees are free of known viral pathogens. The biological indexing assay, or bioassay, is considered to be the "gold standard" as it is the only method of the three that can detect new, uncharacterized, or poorly characterized viral disease agents. Unfortunately, this method is also the most labor intensive and can take up to three years to complete. Next generation sequencing (NGS) is a technology with rapidly expanding possibilities including potential applications for the detection of plant viruses. In this study, comparisons are made between tree fruit testing by conventional and NGS methods, to demonstrate the efficacy of NGS. A comparison of 178 infected trees, many infected with several viral pathogens, demonstrated that conventional and NGS were equally capable of detecting known viruses and viroids. Comparable results were obtained for 170 of 178 of the specimens. Of the remaining eight specimens, some discrepancies were observed between viruses detected by the two methods, representing less than 5% of the specimens. NGS was further demonstrated to be equal or superior for the detection of new or poorly characterized viruses when compared with a conventional bioassay. These results validated both the effectiveness of conventional virus testing methods and the use of NGS as an additional or alternative method for plant virus detection.

Characterization of isolates of Acanthamoeba from the nasal mucosa and cutaneous lesions of dogs.

Acanthamoeba spp. are free-living amoebae that are ubiquitously distributed in the environment and can cause encephalomyelitis in animals and humans. The factors that contribute to Acanthamoeba infections include parasite biology, genetic diversity, environmental spread, and host susceptibility. The aim of the present study was to characterize isolates of Acanthamoeba from the nasal mucosa and cutaneous lesions of dogs in order to access the occurence and pathogenicity of these organisms in this animal group. We studied 13 isolates of Acanthamoeba confirmed by polymerase chain reaction. They were sequenced, the genotype was determined, and their potential of pathogenicity was evaluated.

First observation of edge localized modes mitigation with resonant and nonresonant magnetic perturbations in ASDEX Upgrade.

First experiments with nonaxisymmetric magnetic perturbations, toroidal mode number n=2, produced by newly installed in-vessel saddle coils in the ASDEX Upgrade tokamak show significant reduction of plasma energy loss and peak divertor power load associated with type-I edge localized modes (ELMs) in high-confinement mode plasmas. ELM mitigation is observed above an edge density threshold and is obtained both with magnetic perturbations that are resonant and not resonant with the edge safety factor profile. Compared with unperturbed type-I ELMy reference plasmas, plasmas with mitigated ELMs show similar confinement, similar plasma density, and lower tungsten impurity concentration.

Potentially pathogenic Acanthamoeba in swimming pools: a survey in the southern Brazilian city of Porto Alegre.

Between May 2006 and March 2007, 65 water samples were collected from both heated and unheated swimming pools in the city of Porto Alegre, the capital of the Brazilian state of Rio Grande do Sul. The aim was to explore the problem posed by, and the pathogenic potential of, Acanthamoeba in the pools. Free-living amoebae in the samples were isolated by culture with Escherichia coli and identified from trophozoite and cyst morphology and the results of a PCR with Acanthamoeba-specific oligonucleotide primers. Potential pathogenicity was assessed in osmotolerance and thermotolerance assays. Thirteen (20%) of the water samples investigated were found positive for free-living amoebae, all identified as belonging to morphological groups II (nine isolates) or III (four isolates) of the genus Acanthamoeba. All 13 isolates were found positive in the Acanthamoeba-specific PCR, and the results of the tolerance assays indicated that five (38%) of the isolates should be considered potentially pathogenic. The results of this first study on the occurrence and distribution of Acanthamoeba in the water of swimming pools in Porto Alegre confirm the presence of potentially pathogenic types that may present a risk to human health.

Microarray immunoassay for the detection of grapevine and tree fruit viruses.

Development and application of DNA microarrays for plant disease diagnosis has to date been limited, and for antibody arrays even more so. In this work, an antibody microarray procedure was developed and its usefulness for the detection of plant viruses demonstrated. Using the conventional monoplex immunoassay ELISA technique as a benchmark, the procedure was used to detect several grapevine and tree fruit viruses. In a direct labelling approach, Arabis mosaic virus (ArMV), and Grapevine fanleaf virus (GFLV) were detected after incubating the antibody array with alkaline phosphatase-conjugated viral extract. Indirect detection using a double or triple antibody sandwich format also resulted in good reaction signals, using either a chromogenic or fluorescence dye. In a multiplex system, four grapevine viruses were detected without compromising sensitivity and specificity. Compared to ELISA, the antibody microarray system is similar with respect to sensitivity and specificity, and a high correlation (R(2), 0.759) was observed in regression analysis of virus concentration measurements. Receiver operating characteristic (ROC) curve analysis provided evidence of the good performance of the microarray system (AUC>0.8).

First Report of Colombian datura virus in Brugmansia in Canada.

Colombian datura virus (CDV) was first described in 1968 (3) and has since been reported in Europe (4), Japan (see 4 for additional references), and the United States (1,2). CDV is a member of the family Potyviridae with flexuous, filamentous nucleocapsids that can be transmitted by mechanical inoculation and grafting and is known to be vectored by the common aphid Myzus persicae. In the fall of 2007, five Brugmansia plants of unknown species from a Parks Board Collection in a Lower Mainland nursery, British Columbia, Canada, were found to be displaying symptoms typical of a viral infection: chlorotic flecking and mottling on leaves, leaf shrivel, and vein banding. Symptomatic leaves from these five plants were tested by ELISA (Immuno Strip Test, Agdia, Elkhart, IN) for several common viruses including Impatiens necrotic spot, Tobacco mosaic, Cucumber mosaic, and Tomato spotted wilt viruses and found to be negative for all. However, rub inoculations onto the herbaceous indicators Nicotiana occidentalis and N. benthamiana resulted in severe symptom formation including necrosis, wilting, shriveling, stunted growth, petiole and stem tip collapse, as well as collapse from the base of the plants, and plant death within 2 weeks after inoculation. A leaf dip assay of the original infected Brugmansia sample and infected N. benthamiana tissue revealed flexuous, potyvirus-like particles with the electron microscope (EM). On the basis of the Brugmansia leaf symptoms and the EM results, a possible infection with CDV was suspected. Primers CDV-3 and CDV-NIb5, specific to CDV (4), were used in a reverse transcription (RT)-PCR assay that amplified an approximate 1,600-bp fragment from the original Brugmansia sample and inoculated N. bentamiana and N. occidentalis plants. The amplified portion of the genome is the extreme 3' terminus and includes the 3' noncoding sequence, the viral coat protein gene, and part of the viral replicase gene. Fragments were cloned into pCR2.1-TOPO (Invitrogen, San Diego, CA) and two clones from each plant (total of six clones) were sequenced in both directions. Sequences of all clones were essentially identical, with only three nucleotide differences among the clones (GenBank Accession No. EU571230). BLASTn analysis revealed the highest match to several CDV isolates ranging from 98.7 to 99.5% nucleotide sequence identity. BLASTp analysis of the 451 amino acid viral polyprotein translation product gave a similarly high match with CDV isolates, with the highest match to a Hungarian isolate of CDV (GenBank Accession No. CAD26690) of 99.8% identity, or only one mismatch out of 451 amino acids. An additional group of 15 large symptomless Brugmansia plants, located approximately 6 m from the five symptomatic plants, were also tested by RT-PCR and found to be positive. These 15 plants were of a different but also unknown species of Brugmansia. In conclusion, analysis of symptomatic Brugmansia from a Canadian collection by transfer of disease to herbaceous indicators, EM, RT-PCR, and genomic sequence comparisons, are consistent with the detection and identification of the potyvirus Colombian datura virus. To our knowledge, this is the first report of this viral pathogen in Canada. References: (1) S. Adkins et al. Phytopathology (Abstr.) 95(suppl.):S2, 2005. (2) C. R. Fry et al. J. Phytopathol. 152:200, 2004. (3) R. P. Kahn and R. Bartels. Phytopathology 58:58, 1968. (4) J. Schubert et al. J. Phytopathol. 154:343, 2006.

First Report of Aster Yellow Phytoplasmas ('Candidatus Phytoplasma asteris') in Canadian Grapevines.

In North America, elm yellows, aster yellows (AY), and X-disease phytoplasmas have been detected in American grapevines (1), and recently, Bois noir was detected in Canadian vineyards from British Columbia (BC) and Ontario (ON) (2). Typical symptoms of grapevine yellows (GY) include leaf rolling and chlorosis, uneven or total lack of lignification of canes, flower abortion or berry withering, and stunting. In 2006 and 2007, independent surveys were conducted by the Canadian Food Inspection Agency (CFIA) and Agriculture and Agri-Food Canada (AAFC) to detect phytoplasmas in Canadian vineyards containing different cultivars in BC, ON, Québec (QC), Nova Scotia, New Brunswick, and Prince Edward Island. The CFIA collected and tested 651 fresh leaf samples from recently imported grapevines and older grapevines in the same or neighboring blocks displaying symptoms typical of those associated with disease caused by phytoplasmas. Many vineyards were surveyed only once. AAFC collected and tested 3,485 samples from symptomatic and asymptomatic grapevines from established vineyards in ON, BC, and QC. The same vineyards were sampled in ON and BC both years; QC vineyards were only sampled in 2007. AAFC-collected leaf samples were freeze dried and stored at -20°C before processing. CFIA samples were tested by a modified real-time PCR assay and TaqMan probe targeting the 16S ribosomal RNA gene that detects a wide range of known phytoplasmas (2). Positive samples were confirmed by conventional PCR using the phytoplasma-specific primers P1/P7 (3) and the resulting ~1,800-bp fragment was cloned and sequenced as previously described (2). DNA extracted by AAFC was amplified by nested PCR technology with universal phytoplasma specific primer pairs P1/P6 and R16R2/R16F2 (3) and the resulting 1,200-bp fragment was cloned and sequenced. Two plants, one located in ON in 2006 and the other in BC in 2007, were found to be infected with an AY-like phytoplasma by the CFIA. The phytoplasmas detected in both infected plants had a 99.9% nt sequence identity with AY phytoplasma sequences from GenBank (Accession Nos. AF222063 and AY665676, respectively), with the BC isolate also showing 100% identity to a strain of AY, ash witches'-broom phytoplasma (GenBank Accession No. AY566302). AAFC detected phytoplasma DNA in both years in a total of 17 symptomatic plants and 21 asymptomatic plants from different vine varieties in ON, BC, and QC. Positive samples were found to have a 99.0% nt sequence identity to AY subgroup 16SrI-A (GenBank Accession No. AY180956). Sequences were exchanged for confirmation of phytoplasma identity and were deposited in Genbank under Accession Nos. FJ659844 and FJ824597. Phytoplasma strains were identified for all plants in which phytoplasmas were detected. Results show that AY is present in vineyards in the provinces of ON, BC, and QC. To our knowledge, this is the first report of AY being detected in grapevines in Canada. References: (1) E. Boudon-Padieu. Bull. O I V, 79:299, 2003. (2) M. Rott et al. Plant Dis. 91:1682, 2007. (3) E. Tanne et al. Phytopathology 91:741, 2001.

First Report of Bois Noir Phytoplasma in Grapevine in Canada.

During the summer and fall of 2006, a survey was done to detect European phytoplasmas of quarantine significance in Canadian vineyards. This survey was developed as one of the 2006 import requirements for grapevine nursery stock from Europe. This addresses the increased concerns regarding inadvertent phytoplasma introductions. Grapevines imported in 2006 and established grapevines were observed for symptoms typical of those associated with diseases caused by phytoplasmas on grapevine. Samples were tested from 155 grapevines. One plant, located in the lower Okanagan Valley, British Columbia, tested positive by a modified real-time PCR assay and TaqMan probe targeting the 16S region of the ribosomal RNA gene (1), which detects a wide variety of known phytoplasmas. The sample was further analyzed and found to be positive by conventional PCR with the phytoplasma-specific primers, P1/P7 (3), and Stolbur specific primers, STOL11f2/r1 (2). Additional PCR tests with primers specific to flavescence doree (FD9f/r) (2) and western X disease (P1/W INT) (3) were negative. These phytoplasmas are also known to infect grapevine. The approximate 1,800-bp fragment obtained with P1/P7 was sequenced (GenBank Accession No. EU086529) and found to have 99.7% nucleotide sequence identity to the Stolbur STOL #11 isolate (GenBank Accession No. AF248959) originally isolated from eastern Europe. This was the highest match to any available phytoplasma sequence obtained and indicates that the phytoplasma in the British Columbian sample is an isolate of bois noir, a pest of quarantine significance to Canada. Additional phylogenetic analysis using CLUSTAL W (Lasergene; DNASTAR, Madison, WI) confirmed this result. The presence and identity of the phytoplasma was confirmed from a second tissue sample that was analyzed by PCR and sequenced using the same test procedures as for the first sample, with identical results. The bois noir phytoplasma belongs to the stolbur group (16SrVII) with the principal vector being a cixiid planthopper. Stolbur phytoplasmas cause diseases in other crops, but bois noir disease is caused by a specific member of that group and is the only stolbur phytoplasma known to infect grapevines in Europe. The infected grapevine was from a lot of 1,965 plants of Grenache clone 70 on rootstock 3309 clone 143 that was imported from Europe in 2006. All plants in this importation have been destroyed. This phytoplasma has not been detected in any other grapevines in Canada. Additional import conditions requiring hot water treatment of European vines have been implemented for 2007. Further survey work for phytoplasma in grapevine will continue. References: (1) N. M. Christensen et al. Mol. Plant-Microbe Interact. 17:1175, 2004. (2) X. D. Daire et al. Eur. J. Plant Pathol. 103:507, 1997. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

Little cherry virus-2: sequence and genomic organization of an unusual member of the Closteroviridae.

The complete genomic sequence of variant USA6b of Little cherry virus-2 (LChV-2), has been determined and is 15045 nucleotides in length, coding for 11 open reading frames (ORFs). The sequence shares 77.2% identity with a previously published, ca. 6 kb partial replicase sequence of LChV-2 (variant USA6a). Both LChV-2/USA6a and LChV-2/USA6b were obtained from the same tree infected with little cherry disease, and would suggest a mixed infection. LChV-2/USA6b is more closely related to the partially determined genomic sequence of a Canadian isolate of LChV-2, strain LC5 (92.9% identity). LChV-2/USA6b has an unusual genomic organization compared to other members of the Closteroviridae. The LChV-2/USA6b genome is potentially ambi-sense, with a negative sense ORF0 at the 5' terminus, from which an 18.1 kDa protein of unknown function can be expressed in vitro. The N-terminal region of the LChV-2/USA6 ORF1a translation product does not code for a papain-like protease motif. ORF1 codes for a novel motif, of unknown function, also present in isolates of the Grapevine leafroll associated virus-3, (genus Ampelovirus) as well as viruses of the family Flexiviridae. ORF3 lacks an AUG start codon, but could potentially be expressed via read-through of the ORF2 stop codon. At the 3' end, there is a re-organization of encoded genes compared with other members of the Closteroviridae including separation of the coat protein and coat protein duplicate genes by 4 other genes as found for LChV-2/LC5.

A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease.

Several recombinant clones expressing antigens from Echinococcus granulosus were isolated previously from a parasite cDNA library using cystic hydatid disease (CHD) patients' sera or rabbit hyperimmune antiserum against a lipoproteic fraction from bovine cyst fluid. Six of these antigens were expressed in Escherichia coli and the purified recombinant proteins were tested in enzyme-linked immunosorbent assay (ELISA) for specific IgG with a panel of sera from patients with surgically confirmed (n = 58) or immunologically diagnosed (n = 71) CHD. Sera from clinically normal individuals (n = 203) and sera from individuals with other helminthic infections (n = 65) were assayed for the assessment of specificity. A cut-off value was determined by receiver-operating-characteristic plots for each antigen. A recombinant antigen B subunit (AgB8/2) presented the highest sensitivity (93.1%), considering the group of sera from patients with CHD surgically confirmed, and specificity (99.5%) and is proposed as the basis for an immunodiagnostic test. The other recombinant antigens tested presented sensitivities between 58.6% and 89.7%, and three of them were considered of complementary value. In subclass-specific ELISA, different IgG isotypes showed dominance in the response for each of the recombinant antigens. There was a clear predominance of IgG4 response for all antigens tested, indicating that this would be the subclass of choice to be assessed for these recombinant proteins.

PCR-restriction fragment length polymorphism analysis (PRA) of Mycobacterium leprae from human lepromas and from a natural case of an armadillo of Corrientes, Argentina.

Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) which relies on the amplification of a 439-bp portion of the hsp65 gene present in all mycobacteria, followed by two distinct digestions (with BstEII and HaeIII) of the PCR product, offers a rapid and easy alternative that allows identification of the species without the need for specialized equipment. Wild leprosy in the nine-banded armadillo (Dasypus novemcinctus) is characterized by the presence of multiple bacilli in internal organs such as lymph nodes, spleen and liver, as well as in nerves and skin. We could observe this in 9 out of 132 animals captured in Corrientes, Argentina, an area endemic for leprosy in humans. Mycobacterium leprae were recognized in those naturally infected animals through different techniques. Three samples of extracted DNA of the mycobacteria present in the spleen, liver and popliteal lymph node of a naturally infected animal during the Experimental Program in Armadillo (PEA) and three samples of human lepromas were processed by PRA. The patterns of the six samples analyzed were identical and were characteristic of M. leprae. These studies, made for the first time in Argentina, corroborate the initial discoveries in South America made by our investigative group on the detection of armadillos naturally infected with the Hansen bacillus.

Complete nucleotide sequence of cherry necrotic rusty mottle virus.

A virus closely related to cherry green ring mottle virus (CGRMV) was isolated from a tree displaying typical symptoms of cherry necrotic rusty mottle disease. We have named this virus cherry necrotic rusty mottle virus (CNRMV) and report here its complete genomic sequence as determined from overlapping cloned cDNAs. CNRMV has a genome of 8,432 nucleotides excluding the 3' poly(A) sequence and codes for 7 significant open reading frames (ORFs). Five of these ORFs are conserved among all fovea-, allexi-, potex- and carlaviruses and code for a methyltransferase/helicase/polymerase polypeptide, the triple gene block movement proteins and the coat protein. Two further ORFs, ORFs 2a and 5a, are nested completely within ORFs 2 and 5, respectively. The putative translation products from these ORFs display sequence similarity with putative translation products from two similarly nested ORFs present in the CGRMV genome. The function of these two ORFs is unknown, nor are they conserved among other related viruses.

Detection and Partial Characterization of a Second Closterovirus Associated with Little Cherry Disease, Little cherry virus-2.

ABSTRACT Little cherry disease (LChD) is a complex and serious viral disease of cherry. Although originally described almost 70 years ago, there has been little progress in identifying the causal agent of the disease due to the difficulty in obtaining purified virus from infected trees. This problem was partially overcome in 1997 when the compete sequence of a closterovirus associated with LChD, Little cherry virus (LChV), was published. This virus could be associated with some, but not all, trees with LChD, indicating that another virus was also involved. We report here the partial characterization of a second closterovirus associated with LChD, Little cherry virus-2 (LChV-2), and in order to differentiate the two LChD-associated viruses, we refer to LChV as Little cherry virus-1 (LChV-1). LChV-2 is a new closterovirus with molecular similarities to Grapevine leafroll-associated virus-1 (GLRaV-1) and GLRaV-3 but only distantly related to LChV-1. Based on limited sequence comparisons, LChV-2 is the same virus previously identified in association with LChD in Canada. In reverse transcription-polymerase chain reaction detection assays using specific oligonucleotide primers to either LChV-1 or LChV-2, 27 of 28 isolates of LChD tested positive to one or both of these viruses originating from Europe and North America. These results would further confirm the association of LChV-2 with LChD. One isolate, however, tested negative to both LChV-1 and LChV-2, indicating that while this report brings us a step closer to understanding LChD, further work is required to confirm the causal agents of LChD.

Growth and form of planetary seedlings: results from a microgravity aggregation experiment.

The outcome of the first stage of planetary formation, which is characterized by ballistic agglomeration of preplanetary dust grains due to Brownian motion in the free molecular flow regime of the solar nebula, is still somewhat speculative. We performed a microgravity experiment flown onboard the space shuttle in which we simulated, for the first time, the onset of free preplanetary dust accumulation and revealed the structures and growth rates of the first dust agglomerates in the young solar system. We find that a thermally aggregating swarm of dust particles evolves very rapidly and forms unexpected open-structured agglomerates.

Comparative analysis of two different subunits of antigen B from Echinococcus granulosus: gene sequences, expression in Escherichia coli and serological evaluation.

Two different Echinococcus granulosus antigen B subunits (AgB8/1 and AgB8/2) were characterized and the structure of the genes encoding these two proteins were compared. DNA sequences were expressed in Escherichia coli and the antigens' diagnostic value was then assessed. The genomic sequence of AgB8/1 has a 92 bp intron in the position corresponding to amino acid 16; the AgB8/2 genomic sequence presents a 68 bp intron in the position corresponding to amino acid 20. Both introns are located between the putative N-terminal hydrophobic sequence and the secreted peptide. A comparison between the AgB8/1 and AgB8/2 nucleotide sequences showed a 53.5% identity among exons and a 50% identity between introns. According to the molecular diversity analysis, the elapsed time since both genes shared a common ancestor would be around 4.2x10(7) years. When the native AgB and the two recombinant antigens (rAgB8/1 and rAgB8/2) were tested in an anti-IgG ELISA, the sensitivity of the native antigen B was 77.41% and its specificity was 81.9%, while rAgB8/1 showed 54.84% of sensitivity and 80.17% of specificity and rAg138/2 had an 83.87% sensitivity and a 98.28% specificity. Statistical analysis confirms that rAgB8/2 has a better performance than rAgB8/1 and native AgB in ELISA.

Range of phytoplasma concentrations in various plant hosts as determined by competitive polymerase chain reaction.

ABSTRACT For competitive polymerase chain reaction (PCR), an internal standard DNA template was developed that consisted of a highly conserved, internally deleted 16S rDNA fragment of an aster yellows phytoplasma. The internal standard was calibrated using a quantified culture of Acholeplasma laidlawii. Serial dilutions of the internal standard and fixed amounts of target templates from infected plants were coamplified with the same primers, and the products obtained were quantified using an enzyme-linked immunosorbent assay procedure. Analysis of the data revealed that the phytoplasma concentration in the plants examined differed by a factor of about 4 x 10(6). Phytoplasma concentrations of 2.2 x 10(8) to 1.5 x 10(9) cells per g of tissue were identified in periwinkles infected with various phytoplasmas. High to moderate concentrations were detected in Malus domestica (apple) genotypes infected with the apple proliferation phytoplasma, Alnus glutinosa (alder) genotypes infected with the alder yellows phytoplasma, and most aster yellows-infected Populus (poplar) genotypes examined. Very low phytoplasma concentrations, ranging from 370 to 34,000 cells per g of tissue, were identified in proliferation-diseased apple trees on resistant rootstocks 4551 and 4608, yellows-diseased Quercus robur (oak) trees, and Carpinus betulus (hornbeam) trees. Such low concentrations, which corresponded to about 4 to 340 cells in the reaction mixture, could only be detected and quantified by nested PCR.

Incidence of classical swine fever (CSF) in wild boar in a densely populated area indicating CSF virus persistence as a mechanism for virus perpetuation.

A virological survey was carried out to establish the distribution of classical swine fever (CSF) virus among wild boar in the Federal State of Brandenburg, Germany. Organ materials and blood samples were collected from 11,670 wild boar shot or found dead during the period March 1995 to December 1997. In total 211 (1.8%) wild boar were positive for CSF virus or antigen. The incidence of CSF-positive animals decreased continuously from 4.6% at the beginning of the epidemic in 1995 to 0.7% in 1997. The highest incidence of positive animals (22%) was found in wild-boar piglets younger than 3 months of age in 1995. The findings were indicative for the decisive role which young wild boar play in the epidemiology of CSF. Following intrauterine transfer some of the wild-boar piglets were probably persistently infected with CSF virus as experienced experimentally. Such piglets can be held responsible for CSF virus perpetuation within the wild-boar population. No CSF virus was isolated from adult wild boar weighing more than 75 kg. During 3 years of monitoring a sufficient number of susceptible wild boar, in particular young animals, was available to maintain the infection chain in that area. It was concluded that persistently infected piglets and the high population density of wild boar in the Brandenburg region offered optimal conditions for the establishment of an CSF epidemic.

[Heterakis infection in Numida meleagris (Numididae)]. / Heterakiosis en Numida meleagris (Aves: Numididae).

A case of caecum nematodiasis is described in a guinea fowl (Numida melagris) from the Municipal Zoo, Presidencia Roque Saenz Peña (Chaco) Argentina. Nematodes obtained from the caecum were observed in optic microscopy. According to their morphometric characteristics and location in the definitive host, were identified as belonging to the family Heterakidae, species Heterakis gallinarum, (Schrank, 1788) Maden, 1949.

[Acuariosis in Numida meleagris (Aves: Numididae)]. / Acuariosis en Numida meleagris (Aves: Numididae).

A case of gastric nematodiasis is described in a gineafowl (Numida meleagris) from the Municipal Zoo, Presidencia Roque Saenz Peña (Chaco) Argentina. Nematodes obtained from the glandular stomach were observed in optic microscopy. According to their morphometric characteristics and location in the definitive host, were identified as belonging to the family Acuariidae, subfamily Acuariinae, species Dispharynx nasuta Rudolphi, 1819.