Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for <Emphasis Type="Italic">Xiphinema diversicaudatum</Emphasis>, <Emphasis Type="Italic">X. index</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis>

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.     

RNA‐transfected CD40‐activated B cells as a vaccine strategy in canine malignancies

Introduction: Cell‐based vaccine strategies using dendritic cells as cellular adjuvant have entered phase III trials in humans and have been found to be safe, feasible, and potentially efficacious. Canine patients are generally smaller than adult human patients, which makes production of canine dendritic cell (DC) vaccines problematic, given patient size and the small number of available DC precursors. Here we describe feasibility studies of a novel cell‐based vaccine strategy which uses CD40‐activated B‐cells (CD40‐B) loaded with RNA. This strategy is based on our observations that RNA‐transfected human CD40‐B can drive anti‐tumor T cell responses. One advantage of using CD40‐B cells is the ability to expand this cell population ex vivo, allowing for the numbers of cells required for therapeutic vaccines. Methods: Twenty milliliters of blood were drawn from 6 normal dogs and 5 canine lymphoma patients. Peripheral blood mononuclear cells were separated by Ficoll centrifugation. Culture conditions for B cell activation were optimized using CD40‐ligand, canine IL‐4, and Toll‐like receptor stimulus with CpGoligodinucleotides (ODN). Cyclosporine was added to eliminate peripheral T lymphocytes. Proliferation and activation of CD40‐B cells were demonstrated by CFSE dilution of B cells quantified by flow cytometry. Gene transfer was achieved by mRNA electroporation. Results: Marked in vitro stimulation and proliferation of canine peripheral B cells were achieved with soluble trimeric CD40L, canine IL‐4, and ODN. CD40‐B cells showed dramatic upregulation of MHC class II molecules and CD21 (B‐cell activation marker). After two weeks in culture, cells were negative for CD3 and CD4. Canine CD40‐B cells were efficiently transfected with mRNA, with >60% of CD40‐B expressing green fluorescent protein after GFP mRNA electroporation. Conclusion: RNA‐transfected CD40‐B cells can be efficiently generated from normal and tumor‐bearing dogs. These results provide rationale to test tumor RNA‐transfected CD40‐B as a novel therapeutic approach to treating canine malignancies. Clinical trials in canine lymphoma have been proposed.     

Adherence of Actinobacillus pleuropneumoniae serotype 1 to swine buccal epithelial cells involves fibronectin

The swine pathogen Actinobacillus pleuropneumoniae serotype 1 was investigated for its ability to adhere to swine, rat, and human buccal epithelial cells (BEC). The highest number of bacteria adhered was to swine BEC. This binding ability was affected by heating, extreme pH, treatment with sodium dodecyl sulfate, ethylenediamine tetraacetate, or periodate, and proteolysis, suggesting that cell-surface glycoproteins participate in adherence and that adherence is based mostly on ionic interactions. Mannose and swine fibronectin may play a direct role in this interaction. Convalescent-phase serum from naturally infected pigs inhibited the adhesion. There was a correlation between bacterial pathogenicity as well as host specificity and the capacity for adherence to swine BEC. Adhesion to swine BEC provides a convenient method to study in vitro the adherence of A. pleuropneumoniae and other pathogens of the pig respiratory tract.     

Identification of Yersinia enterocolitica in Minced Meat: A Comparative Analysis of API 20E,Yersinia Identification Kit and a 16S rRNA‐based PCR Method

The isolation and identification of Yersinia enterocolitica from minced meat on CIN agar medium is still one of the major problems in food microbiology because of the low selectivity of cefsulodin–irgasan–novobiocin (CIN) agar. A total of 198 minced meat samples were collected from commercial establishments (butcher shops and supermarkets) in seven German cities in order to investigate the sensitivity and specificity of three identification techniques suitable for the differentiation of Y. enterocolitica within the rich background flora on CIN agar plates. As expected isolation of Y. enterocolitica from minced meat on CIN agar medium after 72 h enrichment in peptone, sorbitol and bile salts (PSB) broth was difficult because all plates were abundantly covered with numerous ‘typical’Yersinia‐like colonies of bull's eye appearance as well as with atypical colonies. Based on the phenotype of the colonies it was possible to detect colonies showing Yersinia‐like growth on CIN agar in 52 samples (26%). For identification of Y. enterocolitica the API 20E system (bioMerieux, Nürtingen, Germany), the Yersinia identification kit (Merlin, Bornheim‐Hersel, Germany) and a 16S rRNA based PCR assay were compared. Only in one sample (0.5%) a Y. enterocolitica strain was detected by all methods. Of the three identification systems tested for routine laboratory diagnostics the API 20E system was found to be the most suitable tool to identify Y. enterocolitica colonies within the rich background flora from minced meat samples on CIN agar plates.     

Antiviral Effect of Saccharomyces cerevisiaeβ‐glucan to Swine Influenza Virus by Increased Production of Interferon‐γ and Nitric Oxide

The aim of these experiments was to investigate the potential antiviral effect of Saccharomyces cerevisiaeβ‐glucan on the pneumonia induced by swine influenza virus (SIV). Forty colostrum‐deprived 5‐day‐old piglets were randomly divided into four groups of 10. The 20 pigs in groups 1 and 2 were administered Saccharomyces cerevisiaeβ‐glucan orally (50 mg/day/pig; En‐Bio Technology Co., Ltd) for 3 days before SIV infection and those in groups 3 and 4 were given culture medium/diluent alone. Groups 1 and 3 were inoculated intranasally with 3 ml of tissue culture fluid containing 2 × 10⁶ tissue culture infective doses 50% (TCID₅₀)/ml of SIV and those in groups 2 and 4 were exposed in the same manner to uninfected cell culture supernatant. The microscopic lung lesions induced by SIV infection (group 1 pigs) were significantly more severe than those induced by infection in animals pre‐administered β‐glucan (group 3) (P < 0.05). Significantly more SIV nucleic acid was detected in the lungs of pigs experimentally infected with SIV only (group 1) at 5, 7 and 10 days post‐inoculation (dpi) compared with lungs from pigs pre‐administered β‐glucan and infected with SIV (group 3) (P < 0.05). The concentrations of interferon‐γ (IFN‐γ) and nitric oxide (NO) in bronchoalveolar lavage fluid from pigs pre‐administered β‐glucan and infected with SIV (group 3) were significantly higher than for any other group at 7 and 10 dpi for IFN‐γ, and at 5, 7 and 10 dpi for NO (P < 0.05). Saccharomyces cerevisiaeβ‐glucan reduced the pulmonary lesion score and viral replication rate in SIV‐infected pigs. These findings support the potential application of β‐glucan as prophylactic/treatment agent in influenza virus infection.     

Significance of β2‐Toxigenic Clostridium perfringens Infections in Animals and Their Predisposing Factors – A Review

The novel β2‐toxin of Clostridium perfringens has recently been described as the cause of enteric diseases in animals. The biological activity of β2‐toxin is similar to that of the β1‐toxin with a possibly weaker cytotoxic activity. However, the production of β2‐toxin in vitro is not seen in all β2‐toxin‐gene (cpb2)‐positive C. perfringens strains, and to deduce a clinical importance solely from the detection of cpb2 is difficult. Detection of cpb2‐positive C. perfringens from various animal species with and without enteric diseases demonstrates the wide distribution of cpb2 in nature, and the presence of cpb2 gene is therefore not considered a risk by itself. Predisposing factors like low trypsin activity in the intestinal tract, antibiotic and/or antiphlogistic treatment or changes in diet can result in the selection of β2‐toxigenic C. perfringens which may lead to enteritis or enterotoxaemia.     

Überleben von Ralstonia solanacearum Biovar 2 (Rasse 3), dem Erreger der Schleimkrankheit der Kartoffel, in Boden und auf verschiedenen Materialien (Mikrokosmos)

Microcosm studies were carried out to test the survival of Ralstonia solanacearum biovar 2 (race 3) in soil at the permanent wilting point (wp) water content and at field capacity (fc) water content and on various material. Soils were placed at permanent ?5°C, 4°C, 15°C and 20°C and weekly fluctuating ?10/0/+10°C and the material at 5, 15 °C, 20°C with relative humidity (rh) uncontrolled or at constant 10% or 90%. In soil, survival was clearly dependent on temperature independent of water content. At 20°C Ralstonia solanacearum could be reisolated up to 364 days, at 15°C up to 290 days, at 4°C up to 209 days and at fluctuating temperatures (?10/0/+10°C) only up to 18 days. The lower the temperature, the more the population declined. At 15°C and 20°C appr. 10⁷ cfu/g soil were detected after 100 days, whereas at ?5°C only 10² cfu/g soil were detected after only 18 days. The pathogen was longer detectable in sandy-clay loam than in lighter sandy soil. It could be longer reisolated at wilting point and the populations did not decline as rapidly as at field capacity. Ralstonia solanacearum could best survive on material surfaces like rubber, plastic and varnished metal with maximum survival of 40 days at 5°C and 10% rh. In general there is a low risk of Ralstonia solanacearum overwintering under European climatic conditions when the fields are cleared of plant debris and the soil is frozen. Contamined material surfaces pose the risk of pathogen transmission to healthy tubers.     

The potential for the Rapid Screening of Potato Cultivars (Solanum tuberosum) for Resistance to Powdery Scab (Spongospora subterranea) using a Laboratory Bioassay

Powdery scab of potato, once established in a field, is difficult to control because of the longevity of the resting spores (cystosori) of the causal organism, Spongospora subterranea f.sp. subterranea. Host resistance is likely to be the most efficient in a long-term control strategy for preventing build-up of field inoculum and spread of the disease. Resistance screening of potato cultivars is mostly done in laborious field trials where disease development is likely to be unpredictable. A bioassay with potato tissue cultured plantlets and cystosori as inoculum is described and was tested for its potential to screen potato cultivars at an early stage for their relative susceptibility to powdery scab by comparing the lab results with field data. With cystosori inoculum of Swiss origin, the laboratory test showed clear differences between the potato cultivars in the severity of zoosporangial root infection which correlated better with ranked tuber infection data, compared to root galling. There are apparent differences in the relative trends in susceptibility between roots and tubers of five selected cultivars when using naturally infested soil instead of prepared cystosori as inoculum in the lab bioassay. Furthermore, differences in the severity of zoosporangial root infection of two selected cultivars were found when cystosori from different countries where used as inoculum. A possible host genotype × pathogen interaction is discussed. The bioassay has the potential to screen and select for resistant material at an early breeding stage thus making field trials not unnecessary but more economical. It will allow the use of a standard set of pathogen collections and facilitate testing for inoculum virulence in infested soils.     

Resistance to Botrytis cinerea in Solanum lycopersicoides is Dominant in Hybrids with Tomato, and Involves induced Hyphal Death

Botrytis cinerea causes gray mold disease and affects hundreds of plant species, including tomato (Lycopersicon esculentum). The wild nightshade, Solanum lycopersicoides, is cross compatible with tomato and is more resistant to B. cinerea, thus representing a potential source for crop improvement. Tests involving droplet inoculation of detached leaves and spray inoculation of entire seedlings demonstrated that resistance to B. cinerea varies among S. lycopersicoides accessions, with S. lycopersicoides LA2951 being the most resistant accession tested. Expression of resistance in the intergeneric hybrid (L. esculentum cv. 'VF36' × S. lycopersicoides LA2951) suggested that resistance is at least partially dominant in tomato. A green fluorescent protein-tagged B. cinerea strain was used for confocal microscopic comparison of infection in leaves of S. lycopersicoides and tomato. Even though S. lycopersicoides supported spore germination, there was evidence for hyphal lysis and death 3 days after inoculation, at a time when lesions were expanding on susceptible tomato plants. The reduced frequency of B. cinerea lesion spread on S. lycopersicoides explains why this fungus produced fewer spores in this wild nightshade than in tomato.