Molecular characterization of the ITS-2 fragment of Paramphistomum leydeni (Trematoda: Paramphistomidae)

Paramphistomosis has shown an increased spread over the last years in Argentina, being in some regions an emerging parasitosis, which has motivated their study and identification. Even when morphological features were reported, molecular characterization appears as a complementary and reliable tool. Samples of Paramphistomum leydeni collected since 2002 from different origin, definitive hosts, natural or experimental infections and preservation method were subjected to PCR-RFLP for ITS2+ fragment, and compared to Fasciola hepatica and Notocotilidae cercarie. Frozen and ethanol fixed samples amplified fragments of 500 bp for P. leydeni and Notocotilidae samples, while F. hepatica eggs produced a 540 bp amplicon. Restriction fragments obtained from endonucleases HinfI, HhaI, BsuRI, TaqI, and TruI were identical for all P. leydeni samples, supporting the morphological classification previously performed. Four selected amplicons were sequenced and reported at GenBank, given a consensus sequence ITS-2+ of 441 bp. This first report of molecular characterization for P. leydeni, improves the current knowledge of the genus and establishes precedents for further specimen classifications.     

Occurrence of Monilinia laxa and M. fructigena after introduction of M. fructicola in peach orchards in Spain

Seventeen field surveys were done in four commercial orchards during six consecutive fruit-growing seasons from 2006 until 2011 in order to determine the current frequencies of occurrence of M. laxa, M. fructigena, and M. fructicola and their relative contributions to postharvest brown rot in peaches and nectarines in the Ebro Valley. The relative frequencies of occurrence of Monilinia spp. were determined on three sources of primary inoculum and on three sources of secondary inoculum. The major relative frequencies of Monilinia spp. were significantly recorded (P?=?0.05) from mummified fruit on the trees (approx. 42 %) and 7-day-old harvested fruit with brown rot (32 %), followed by that recovered from mummified fruit on the orchard bed (14 %), pruned branches on the orchard bed (8 %) and latent infections of immature fruit (3 %). We found that: (a) the relative frequency of M. fructicola has increased over the years to coexist on the same level as at the time M. laxa, (b) M. fructigena is no longer a cause of brown rot in harvested peaches, (c), a progressive reduction in the time of the first appearance of Monilinia airborne conidia (r?=??0.30, P?=?0.003), and the time of the first latent infection (r?=??0.44, P?=?0.0001) was detected along years after correlation analysis, and (d) these displacements are not associated with an increased incidence of brown rot disease. The M. fructicola increase was due to its significantly increased presence in 7-day-old harvested fruit with brown rot (r?=?0.73, P?=?0.0009), in latent infections of immature fruit (r?=?0.68, P?=?0.002), on pruned branches on the orchard bed (r?=?0.56, P?=?0.018), and on mummified fruit sampled on the trees (r?=?0.53, P?=?0.03). This progressive increase was accompanied by a progressive reduction in the relative frequency of occurrence of M. laxa in 7-day-old harvested fruit with brown rot (r?=??0.55, P?=?0.021) and M. fructigena on mummified fruit sampled on the trees (r?=??0.51, P?=?0.03).     

Apoptotic effects of tamoxifen on leukocytes from horse peripheral blood and bronchoalveolar lavage fluid

A reduction in inflammatory cell apoptosis is an important concept in the maintenance of inflammation and a potential target for the resolution of inflammation in many inflammatory diseases. Dysregulation of apoptosis has been implicated in a range of diseases, including tumors, neurodegenerative disorders and autoimmunity, and may also be implicated in allergic asthma. In horses, recurrent airway obstruction (RAO) is an asthma-like condition that is characterized increased survival neutrophil bronchial. Tamoxifen is a synthetic, non-steroidal, anti-estrogen agent that is widely used for treating all stages of breast cancer and has been approved for the prevention of breast cancer in high-risk women. The observed efficacy of tamoxifen has been attributed to both growth arrest and the induction of apoptosis. Therefore, the aim of our study was to evaluate the ability of tamoxifen to induce apoptosis in vitro in granulocytic cells from peripheral blood and in mononuclear cells from bronchoalveolar lavage fluid (BALF) in horses. Flow cytometry using commercial AnnexinV-FITC and propidium iodide was used to quantify early and late apoptotic leukocytes, respectively. The results showed a significant increase in early apoptosis in peripheral blood and bronchial granulocytic cells treated with tamoxifen. The rate of early apoptosis of mononuclear cells from blood and BALF when incubated with tamoxifen was significantly lower compared with granulocytic cells. We did not observe a direct effect of tamoxifen on late apoptosis in any of the in vitro assays in the cell types used here. These results indicate that the apoptotic mechanisms under these experimental conditions would affect only blood and BALF granulocytic cells, particularly in early apoptosis. Finally, further in vitro and in vivo studies are needed to better understand apoptotic mechanisms because tamoxifen could be used to treat chronic, inflammatory pathologies associated with granulocytes and allergic diseases, such as asthma or equine RAO.     

Insect growth regulators as potential insecticides to control olive fruit fly (Bactrocera oleae Rossi): insect toxicity bioassays and molecular docking approach

BACKGROUND: Olive fruit fly, Bactrocera oleae (Rossi), is a key pest in olive orchards, causing serious economic damage. To date, the pest has already developed resistance to the insecticides commonly applied to control it. Thus, in searching for new products for an accurate resistance management programme, targeting the ecdysone receptor (EcR) might provide alternative compounds for use in such programmes. RESULTS: Residual contact and oral exposure in the laboratory of B. oleae adults to the dibenzoylhydrazine‐based compounds methoxyfenozide, tebufenozide and RH‐5849 showed different results. Methoxyfenozide and tebufenozide did not provoke any negative effects on the adults, but RH‐5849 killed 98‐100% of the treated insects 15 days after treatment. The ligand‐binding domain (LBD) of the EcR of B. oleae (BoEcR‐LBD) was sequenced, and a homology protein model was constructed. Owing to a restricted extent of the ligand‐binding cavity of the BoEcR‐LBD, docking experiments with the three tested insecticides showed a severe steric clash in the case of methoxyfenozide and tebufenozide, while this was not the case with RH‐5849. CONCLUSION: IGR molecules similar to the RH‐5849 molecule, and different from methoxyfenozide and tebufenozide, might have potential in controlling this pest. Copyright © 2012 Society of Chemical Industry     

Molecular insights into fungicide resistance in sensitive and resistant Penicillium digitatum strains infecting citrus

The continuous use of chemical fungicides on citrus postharvest has led to the development of resistant strains against the fungicides in use, representing a considerable threat because the control systems are no longer effective. Evaluation of the sensitivity of 75 Penicillium digitatum strains to seven different fungicides revealed the presence of a significant number of TBZ- (84%) and IMZ-resistant (77%) strains, i.e., those fungicides most used in citrus postharvest. Molecular characterization of different P. digitatum genes involved in fungicide resistance was carried out. All P. digitatum genes were selected based on particular mechanisms of resistance due to fungicide target or mode of action. TBZ-resistance was characterized by a unique point mutation in the β-tubulin gene sequence corresponding to amino acid 200, confirming previous work on this subject. Moderate to low resistance to strobilurins did not reveal any mutation in the cytochrome b gene. DMI-resistance was evaluated by examining the CYP51 gene and four different ABC transporters PMR1, PMR3, PMR4 and PMR5. The CYP51 gene did not exhibit any mutation relating to DMI-resistance, but a five tandem repeat sequence previously described was found in the CYP51 promoter in 3 of the 75 isolates examined, whereas DMI-sensitive isolates and the other DMI-resistant isolates of P. digitatum had only one tandem repeat. Of all the ABC transporters studied, only PMR1 and PMR5 appear to be involved in fungicide resistance and several mutations were found in the promoter and the coding region for PMR5 in resistant strains compared to sensitive ones. In all cases, the resistance mechanism was consistent in both orchard or packing-house isolates and no differences conferred by either origin or fungicide pressure were observed.Consequently, since different processes have been described that confer fungicide resistance to the same compounds, such as DMIs, the hypothesis that multiple mechanisms could be acting simultaneously gains strength.     

Quantification of phenolic compounds in olive oil mill wastewater by artificial neural network/laccase biosensor

In this paper is considered a new computerized approach to the determination of concentrations of phenolic compounds (caffeic acid and catechol). An integrated artificial neural network (ANN)/laccase biosensor is designed. The data collected (current signals) from amperometric detection of the laccase biosensor were transferred into an ANN trained computer for modeling and prediction of output. Such an integrated ANN/laccase biosensor system is capable of the prediction of caffeic acid and catechol concentrations of olive oil mill wastewater, based on the created models and patterns, without any previous knowledge of this phenomenon. The predicted results using the ANN were compared with the amperometric detection of phenolic compounds obtained at a laccase biosensor in olive oil wastewater of the 2004-2005 harvest season. The difference between the real and the predicted values was <0.5%. biosensor; olive oil mill wastewater; chemical analysis; phenolic compounds.     

Evaluation of preservative distribution in thermally modified European aspen and birch boards using computed tomography and scanning electron microscopy

The aim of this experiment was to impregnate thermally modified wood using an easy and cost-effective method. Industrially processed thermally modified European aspen (Populus tremula L.) and birch (Betula pubescens Ehrh.) were collected and secondarily treated at the laboratory scale with the preservatives tung oil, pine tar and Elit Träskydd (Beckers) using a simple and effective method. Preservative uptake and distribution in sample boards were evaluated using computed tomography (CT) and scanning electron microscopy (SEM) techniques. Preservative uptake and treatability in terms of void volume filled were found the highest in Beckers and the lowest in tung oil-treated samples. Thermally modified samples had lower treatability than their counterpart control samples. More structural changes after thermal modification, especially in birch, significantly reduced the preservative uptake and distribution. The differences of preservatives uptake near the end grain were high and then decreased near the mid position of the samples length as compared with similar type of wood sample. Non-destructive evaluation by CT scanning provided a very useful method to locate the preservative gradients throughout the sample length. SEM analysis enabled the visualization of the preservative deposits in wood cells at the microstructural level.     

Evaluation of an in-house TgSAG1 (P30) IgG ELISA for diagnosis of naturally acquired Toxoplasma gondii infection in pigs

Toxoplasma gondii is an apicomplexan protozoan parasite which is able to infect a large variety of warm-blooded animals. Raw or undercooked pork has been regarded as an important source of infection for humans. The aim of this study was to evaluate an in-house enzyme-linked immunosorbent assay to diagnose natural T. gondii infection in swine using native affinity chromatography-purified T. gondii surface protein-1 (TgSAG1-ELISA) as antigen, comparing its performance to that of indirect fluorescent antibody test (IFAT) and immunoblotting (IB). To obtain a panel of sera showing the evolution of the antibody response in the time course 12 pigs were experimentally inoculated intravenously (iv) with tachyzoites of the T. gondii strains RH (clonal type I), ME49 (clonal type II) and NED (clonal type III) and serologically monitored for a period of 11 weeks. Both IFAT and ELISA showed a similar time course of antibody response to T. gondii; but by IFAT this response was characterized by rapidly rising titers with peaks at two weeks post inoculation (wpi), while the ELISA indices increased slowly and reached a maximum in most animals at five wpi. Three-hundred randomly selected sera from a total of 602 pigs of different ages derived from outdoor and indoor farms from Argentina were analyzed. Serum samples testing either positive or negative by both IFAT and IB were considered as "relative standards of comparison" (RSC). Sensitivity and specificity of TgSAG1-ELISA were obtained by a Receiver Operating Characteristics (ROC) analysis and statistical agreement among serological tests was evaluated. Antibodies to T. gondii were detected in 160 of 300 sera (53.3%) by IB, in 133 of 300 (44.3%) by IFAT and in 123 of 300 sera (41%) by TgSAG1-ELISA. One hundred and eleven sera tested positive and 118 sera tested negative by both IFAT and IB (RSC); 103 of 111 positive RSC sera tested positive by TgSAG1-ELISA, and 116 of 118 negative RSC sera tested negative by TgSAG1-ELISA. Agreement observed between RSC and TgSAG1-ELISA was almost perfect (κ=0.9124, p≥0.05) and between IFAT and IB was moderate (κ=0.53, p≥0.05). Relative sensitivity and specificity of the TgSAG1-ELISA using a cut-off index of 0.204 were of 92.8% and 98.3%, respectively. ROC analysis revealed that TgSAG1-ELISA was highly accurate (AUC=0.983) relative to the RSC. According to the results in this study, the ELISA based on affinity purified T. gondii surface antigen TgSAG1 was useful for the specific and sensitive detection of antibodies to this protozoan parasite in naturally infected pigs.     

Effect of ivermectin treatment on anti-hypodermin C titers of Asturiana cattle naturally infected with Hypoderma lineatum

The collagenase (hypodermin C) from soluble crude extracts of Hypoderma lineatum 1st-instar larvae was purified by reverse-phase HPLC and used in a new indirect ELISA test. This pure protein had several advantages over the use of crude larval extracts allowing a much better discrimination between infested and non-infested cattle. The anti-hypodermin C titers of 19 Asturiana cattle were estimated over the course of a natural H. lineatum infestation cycle, in which the effect of ivermectin treatment was also investigated. The results showed differences in the onset and ending of the infestation with respect to those described for other European countries. The ivermectin treatment proved to be very effective and treated animals had relatively low anticollagenase titers.