Specificity of the carbon immunoassay (CIA) test for the diagnosis of Toxoplasma infections.

A rapid and low cost procedure, the carbon immunoassay (CIA) test, was evaluated for the diagnosis of Toxoplasma gondii infections. Using a closely related parasite (Besnoitia jellisoni) as antigen, and homologous or heterologous immune sera, it was demonstrated by light and electron microscopy that CIA is a very reliable and specific test. As it is neither expensive nor time-consuming, it can be recommended for general and routine laboratory use.     

The organophosphorous insecticide chlorpyrifos affects the neuroepithelial junction, the bioelectric parameters of the skin of the frog Caudiverbera caudiverbera, and the structure of model cell membranes

The current study examines the acute effects of the organophosphorus pesticide chlorpyrifos on a sympathetic synapse of the frog Caudiverbera caudiverbera. Nerve stimulation was followed immediately by a transient increase in the short-circuit current (SCC) and in the potential difference (PD), which consisted of a rapid and then a slow component. Chlorpyrifos concentrations from 5 μM to 1.0 mM caused a dose-dependent block of both components to a 10% of their control values, which was reversed by washout. The pesticide blockade did not affect the skin response to noradrenaline. X-ray diffraction and fluorescence spectroscopy studies on membrane models showed marked phospholipid perturbation, which favors changes in ion channel conformation and interferes with receptor proteins, thus altering noradrenergic transmission and Cl⁻ secretion in the mucous glands. The foregoing results may be interpreted as a reversible inhibition of the neuroepithelial synapse to nerve stimulation, possibly due to non-specific lip μd-protein perturbation, interference with synaptic transmission, and transient Cl⁻ channel inactivation.     

Molecular cloning,purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein

The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis.     

Phenotype and function of murine discrete Peyer's patch macrophage derived - dendritic cells

The phenotype and function of peritoneal cavity macrophage-derived dendritic cells (PEC-DC) was previously reported. In this study we have gone further in using our established culture system to generated discrete Peyer's patch dendritic cells (DPP-DC) from murine discrete Peyer's patch macrophages (DPP-M?), following stimulation with granulocyte macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) for 7 days. DPP-M? from murine small intestines were obtained by mechanical disruption of discrete Peyer's patches (DPP), followed by metrizamide density gradient centrifugation to remove Peyer's patch resident DC and debri, after which an overnight adherent step in tissue culture medium was carried out for macrophage enrichment. Characterization of the generated DPP-DC was carried out using well-established criteria of morphology, expression of membrane antigens and capacity for antigen presentation. Dendritic cells expressed DEC-205, F4/80 and CD34 at high levels, but exhibited very low CD11c levels. They were shown to present soluble protein antigen to CD3(+) spleen T cells. A comparison of the surface antigen expression in the progenitor DPP-M? population and the generated DPP-DC showed a significant decrease in MHC class II levels and a marked down regulation of the co-stimulatory molecule CD86 (B7-2). High expression of the haemopoietic progenitor marker CD34 indicates that the generated DC, possess a haemopoietic rather than myeloid origin. Taken together, these results may provide a better understanding of the complex network regulating mucosal immune responses.     

Strawberry Anthracnose: Histopathology of Colletotrichum acutatum and C. fragariae

ABSTRACT Ontogeny of the invasion process by Colletotrichum acutatum and C. fragariae was studied on petioles and stolons of the strawberry cultivar Chandler using light and electron microscopy. The invasion of host tissue by each fungal species was similar; however, each invasion event occurred more rapidly with C. fragariae than with C. acutatum. Following cuticular penetration via an appressorium, subsequent steps of invasion involved hyphal growth within the cuticle and within the cell walls of epidermal, subepidermal, and subtending cells. Both species of fungi began invasion with a brief biotrophic phase before entering an extended necrotrophic phase. Acervuli formed once the cortical tissue had been moderately disrupted and began with the development of a stroma just beneath the outer periclinal epidermal walls. Acervuli erupted through the cuticle and released conidia. Invasion of the vascular tissue typically occurred after acervulus maturation and remained minimal. Chitin distribution in walls of C. fragariae was visualized with gold-labeled wheat germ agglutinin. The outer layer of bilayered walls of conidia, germ tubes, and appressoria contained less chitin than unilayered hyphae in planta.     

Quantification of airborne spores of <Emphasis Type="Italic">Monilinia fructicola</Emphasis> in stone fruit orchards of California using real-time PCR

The fungal pathogen Monilinia fructicola causes blossom blight and fruit brown rot of stone fruits in California. In this study, spore densities in the air were monitored in six orchard/year combinations with Burkard spore traps. A real-time PCR assay was developed to efficiently quantify the dynamics of spore density in these orchards during the growing season. Different patterns of dynamics of spore density were observed in these orchards. A linear relationship between numbers of spores counted with a compound microscope and those determined with the real-time PCR assay was obtained, using the same samples of spore traps. Spore density in five of six orchard/year combinations ranged from 0.0 to 0.05 spores l⁻¹, except for that in orchard 4, which showed much higher values of spore density in the air, as well as higher values and wider range of incidences of blossom infection and fruit rot than those in the other orchards. The results demonstrated a potential method to quantitatively determine spore inoculum potential in orchards by using a real-time PCR assay.     

Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.     

Diversity of insecticide resistance mechanisms and spectrum in European populations of the codling moth, Cydia pomonella

Only a few of the registered insecticides against Cydia pomonella L. are still effective in areas where insecticide resistance has emerged in this pest. Resistance mechanisms are multiple, and their lone or cumulative effects in a single population are not completely understood. A detailed estimation of resistance spectrum is still required to define the suitable insecticides to use against a given population. The efficacy of ten insecticides was therefore investigated together with the resistance mechanisms expressed in four laboratory strains and 47 field populations of C. pomonella from five countries. Bioassays were performed using topical applications of diagnostic concentrations on diapausing larvae, and resistance mechanisms were analysed on adults emerging from control insects. All populations exhibited a reduced susceptibility to at least one insecticide when compared with the susceptible laboratory strain. Cross-resistances were observed between azinphos-methyl or phosalone and more recent compounds such as spinosad and thiacloprid. Resistances to azinphos-methyl, diflubenzuron, spinosad, tebufenozide and thiacloprid were significantly correlated with mixed-function oxidase activity, while increased glutathione-S-transferase and reduced non-specific esterase activities were correlated with resistance to azinphos-methyl and emamectin, respectively. Conversely, resistances to azinphos-methyl, tebufenozide and thiacloprid were negatively correlated with increased esterase activity. None of the observed mechanisms explained the loss of susceptibility of populations to chlorpyrifos-ethyl, and no significant correlation was detected between resistance to deltamethrin and the presence of the kdr mutation. The suitability of such non-target instars to monitor insecticide resistance in field populations is discussed.     

Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host–pathogen interaction.