Characterization of fenarimol-resistant mutants of Aspergillus nidulans

The fitness of twelve fenarimol-resistant mutants of Aspergillus nidulans was tested with respect to spore germination, germ tube elongation, hyphal growth and sporulation. Half of the strains tested were isolated on triarimolcontaining media. The other strains were selected on imazalil-or cycloheximide-containing media (Van Tuyl, 1977).A number of mutant strains produced spores with unimpaired viability on synthetic medium. In other strains a reduction in spore viability was noticed. The rate of germ tube elongation of all resistant mutants was significantly lower than that of the wild-type strain. Mutant strains with a low degree of resistance always had an almost normal mycelial growth rate, whereas growth of some of the strains with a relatively high degree of resistance was significantly slower. Spore production on malt agar was highest in the wild-type strain and was found to be lower in fenarimol-resistant mutants. In most of the mutant strains tested a high degree of cross-resistance between fenarimol, imazalil and triadimefon was established; in some of them cross-resistance to these chemicals was low or even absent.Possible implications of the results described for the chance of development of resistance in phytopathogenic fungi to sterol biosynthesis-inhibiting fungicides are discussed.     

Effects of Escherichia coli heat-stable enterotoxin b on small intestinal villi in pigs, rabbits, and lambs

Culture supernates from two strains of E. coli were placed into different ligated intestinal sections (loops) of each animal. The two bacterial strains were identical except that one contained a plasmid carrying the heat-stable toxin b (STb) gene, while the other did not. Morphometric techniques were used to assess villous epithelial surface areas and mucosal volumes in both intestinal segments exposed to STb-positive (test) and to STb-negative (control) supernates. In pigs whose intestines were exposed to STb-positive supernatants for 2 hours, both villous epithelial surface area and mucosal volume were significantly smaller in test loops than in control loops (P less than 0.02). In test loops of pigs incubated for 1 hour, and in test loops of lambs incubated for 2 hours, there was a decrease in villous epithelial surface area which approached the test for significance but did not meet it (0.05 less than P less than 0.10). Rabbit test loops did not differ from rabbit control loops in either villous epithelial surface area or mucosal volume. Histological examination of the tissues from all three species revealed epithelial changes in porcine and ovine tissues only. In porcine and ovine tissues, epithelium at villous tips was seen to be cuboidal or squamous, or even to be absent. Villi with similarly altered epithelium were seen in control loops, but were seen much more frequently in test loops. These epithelial changes were seen as early as 30 minutes of incubation in pigs. Intestinal tissues from these pigs were examined by transmission electron microscopy, but no difference between test and control tissues was seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microscopic alterations in jejunal epithelium of 3-week-old pigs induced by pig-specific, mouse-negative, heat-stable Escherichia coli enterotoxin

The objective of this study was to determine whether exposure of swine jejunum to crude culture filtrates containing Escherichia coli pig-specific, mouse-negative, heat-stable enterotoxin (STb) induces structural alterations in the jejunal mucosa of pigs. Two ligated intestinal loops in each of twelve 3-week-old pigs were exposed for 2 hours to sterile E coli culture filtrates from each of the following strains: 431 (STa-producing), 1261 (STa and STb-producing), and 1790 (STb-producing); recombinant strain HB101-pRAS-1 (STb-producing); the nontoxigenic K-12 variant HB101; or trypticase soy broth. Formalin-fixed sections from these loops were examined for sloughed cells around villi, and a lesion score was determined, indicating a change in villous epithelium from columnar to cuboidal or squamous cell types or to discontinuous epithelium. Villous lengths and crypt depths also were determined. For loops exposed to culture filtrates containing STa and STb or containing only STb, lesion scores and numbers of sloughed cells were greater (P less than 0.05) and villous length was shorter (P less than 0.01) than in loops not exposed to toxin. For loops exposed to culture filtrates containing STa, lesion scores, villus lengths, and numbers of sloughed cells were not different from those of loops not exposed to toxin. Therefore, exposure of swine jejunum to STb induced structural alterations in intestinal mucosa (ie, loss of villous absorptive cells and partial atrophy of villi) that were consistent with those causing compromised absorptive capacity.     

In vitro detection of Shiga toxin using porcine alveolar macrophages.

Porcine alveolar macrophages were found to be highly susceptible to the cytolytic effects of a toxin (Shiga toxin [Stx]) produced by certain strains of Escherichia coli and sometimes associated with clinical disease in pigs and other animals. In comparison with the cells that are most commonly used for Stx detection and titration in vitro (namely, Vero cells), porcine alveolar macrophages appeared to be generally more sensitive and test results could be obtained in less time. Moreover, unlike Vero cells, porcine alveolar macrophages need not be continuously propagated to ensure immediate availability. They can simply be removed from a low-temperature repository, thawed, seeded, and shortly thereafter exposed to the sample in question. These characteristics suggest that porcine alveolar macrophages may be useful in developing a highly sensitive and timely diagnostic test for Stx.     

Development and validation of species-specific primers that provide a molecular diagnostic for virus-vector longidorid nematodes and related species in German viticulture

The nematode species Longidorus attenuatus, L. elongatus, L. macrosoma and Paralongidorus maximusare economically important pests to the viticulture industry due to their ability to vector two nepoviruses (Raspberry Ringspot Virus and Tomato Black Ring Virus) to grapevines. In Germany, these species occur in vineyard soil with other non-vector but morphologically similar longidorid species, L. helveticus, L.profundorum and L. sturhani. Species-specific primers were designed from ribosomal DNA for all seven species to facilitate taxonomic identification for non-specialists. Primers were assessed for their reliability by screening, where possible, a number of populations of each species. Furthermore, their selectivity and sensitivity were determined when challenged with closely related longidorid species and general nematode communities typical of vineyard soil. A multiplex approach using a common forward primer combined with species-specific reverse primers enabled three target nematode species to be detected in the same PCR reaction. All primers were highly specific, detecting all nematode developmental forms from disparate populations and were sufficiently sensitive to detect a single target nematode within a whole nematode community typical of a vineyard soil comprising of a range of non-target species. Given their specificity, sensitivity and reliability, these diagnostic primers should be of great benefit to both phytosanitary/quarantine services related to the viticulture industry and also as a decision management tool for growers.     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

Immunohistochemical study of endothelial nitric oxide synthase in the trigeminal ganglia of a crotaline snake Trimeresurus flavoviridis

The immunoreactivity of constitutive endothelial nitric oxide synthase (eNOS) was studied in the trigeminal ganglia (TG) of a crotaline snake, Trimeresurus flavoviridis. eNOS immunoreactivity was found in TG neurons of different sizes. The percentage of eNOS-positive TG neurons was significantly higher in the mandibular division than in the infrared-related divisions, the maxillary division and ophthalmic ganglion (p<0.001). These findings suggest that eNOS in the TG of crotaline snakes is involved in constitutive neurotransmission in the TG, and is minimally involved in processing in the infrared-sensory system.