Robust and sensitive monoclonal enzyme-linked immunosorbent assay for the herbicide molinate

This paper reports on the generation of monoclonal antibodies and the development of a new enzyme-linked immunosorbent assay (ELISA) for the detection of molinate (S-ethyl hexahydroazepine-1-carbothioate). Hybridoma cells were generated using spleen and lymph node cells from a mouse immunized with S-2-carboxyethyl hexahydroazepine-1-carbothioate conjugated to keyhole limpet hemocyanin. After screening with a competitive ELISA, two monoclonal antibodies, mAbs 16C11 and 14D7, with IC(50) values of 82 +/- 2 and 173 +/- 8 ng/mL, respectively, were selected. MAb 16C11 can detect molinate concentrations of 1 ng/mL with no cross-reactivity to any other thiocarbamate pesticides; however, it was susceptible to the presence of organic solvents and to variation in buffer ionic strength. MAb 14D7 tolerated concentrations up to 5% of propylene glycol and 12.5% of acetonitrile in the assay buffer. The sensitivity of mAb 14D7 was further improved by decreasing the amount of coating antigen in the ELISA; the final inhibition assay showed an IC(50) of 69.2 +/- 1.4 ng/mL. In summary, mAb14D7 provided a more sensitive and robust assay, as compared with previous polyclonal antibody-based assays, with the additional advantage of being based upon a consistent and unlimited source of a defined reagent.     

Incidence of ‘Candidatus Liberibacter europaeus’ and phytoplasmas in Cacopsylla species (Hemiptera: Psyllidae) and their host/shelter plants

Psyllids, as vectors of phloem-restricted plant pathogens, are serious agricultural pests. Fruit tree phytoplasmas are transmitted by different Cacopsylla spp., while other psyllids are known vectors of liberibacters. Recently, the bacterium ??Candidatus Liberibacter europaeus?? was found in pear trees and in Cacopsylla pyri (Linnaeus), the vector of ??Ca. Phytoplasma pyri??. This new species does not cause symptoms in plants and is probably a symbiont rather than a pathogen. Based on these findings and the assumption that ??Ca. Liberibacter europaeus?? is widespread, we studied its distribution in the genus Cacopsylla and in the respective host and shelter plants (where psyllids aestivate and overwinter), as well as its possible co-presence with ??Ca. Phytoplasma?? spp. We tested 14 Cacopsylla species and 11 plant species from northwestern Italy, Hungary and Israel, characterized by warm oceanic, temperate continental and warm Mediterranean climatic conditions, respectively. ??Ca. Liberibacter europaeus?? was common within the Cacopsylla genus, being present in nine of the 14 species screened as well as in most host plants, whereas none of the shelter plants tested positive for this bacterium. Altogether, these findings indicate the presence of ??Ca. Liberibacter europaeus?? in continental zones, whereas it does not seem to be widespread in the Mediterranean region. Furthermore, lack of specific symptoms in all infected plants confirms an endophytic relationship with this bacterium, while its abundance in insects suggests a beneficial role for the host. Co-infections with phytoplasmas, observed in insects and plants, require further study to evaluate the possible interactions between them.     

Phytoestrogen content of beverages, nuts, seeds, and oils

Phytoestrogens are secondary plant metabolites that have received increasing attention for their bioactivity, in particular due to their structural and functional similarity to 17beta-estradiol. Although urinary and plasma phytoestrogens can be used as biomarkers for dietary intake, this is often not possible in large epidemiological studies or in the assessment of general exposure in free-living individuals. Accurate information about dietary phytoestrogens is therefore important, but there are very limited data concerning food contents. In this study was analyzed a comprehensive selection of tea, coffee, alcoholic beverages, nuts, seeds, and oils for their phytoestrogen content using a newly developed sensitive method based on LC-MS incorporating (13)C 3-labeled standards. Phytoestrogens were detected in all foods analyzed, although the contents in gin and bitter (beer) were below the limit of quantification (1.5 microg/100 g). Lignans were the main type of phytoestrogens detected. Tea and coffee contained up to 20 microg/100 g phytoestrogens and beer (except bitter) contained up to 71 microg/100 g, mainly lignans. As these beverages are commonly consumed, they are a main source of dietary lignans. The results published here will contribute to databases of dietary phytoestrogen content and allow a more accurate determination of phytoestrogen exposure in free-living individuals.     

Traceability of plant contribution in olive oil by amplified fragment length polymorphisms

Application of DNA molecular markers to traceability of foods is thought to bring new benefit to consumer's protection. Even in a complex matrix such as olive oil, DNA could be traced with PCR markers such as the amplified fragment length polymorphisms (AFLPs). In this work, fluorescent AFLPs were optimized for the characterization of olive oil DNA, to obtain highly reproducible, high-quality fingerprints, testing different parameters: the concentrations of dNTPs and labeled primer, the kind of Taq DNA polymerase and thermal cycler, and the quantity of DNA employed. It was found that correspondence of fingerprinting by comparing results in oils and in plants was close to 70% and that the DNA extraction from olive oil was the limiting step for the reliability of AFLP profiles, due to the complex matrix analyzed.     

A radiographic study of breed‐specific vertebral heart score and vertebral left atrial size in Chihuahuas

Although echocardiography is the gold standard for the diagnosis of cardio‐structural disease, thoracic radiography is a rapid, cost‐effective, and widely accessible method for evaluating cardiac size in dogs. The vertebral heart score (VHS) and the vertebral left atrial size (VLAS) are established as objective measures of cardiomegaly on thoracic radiographs. However, several studies have shown significant variations in the VHS among different breeds. The Chihuahua is predisposed to both congenital and acquired cardiac diseases. The aim of this prospective, single‐center, cross sectional study was thus to evaluate the VHS and the VLAS in healthy adult Chihuahua dogs. A total of 30 Chihuahuas were included. The VHS values in our sample population of Chihuahuas were 10.0 ± 0.6 (95% range, 8.9‐11.0). This was significantly greater than the canine reference value of 9.7 ± 0.5 established by Buchanan and Bücheler (P = .002). The VLAS of Chihuahuas in our study was 1.8 ± 0.2 (95% range, 1.3‐2.1). This was significantly lower than the values previously reported by Malcolm et al (2.07 ± 0.25; P = .0004). The VHS and the VLAS were not influenced by sex, body weight, short or long hair, and body condition score in normal Chihuahuas. Our results indicated that breed‐specific reference values for radiographic VHS and VLAS are needed. In Chihuahuas, the values found in this study can be used as a normal reference in order to help avoid overinterpretation of cardiomegaly in these dogs.     

Environmental DNA reveals the temporal and spatial extent of spawning migrations of European shad in a highly fragmented river basin

1. Artificial barriers on lowland rivers impede the spawning migrations of anadromous fishes, preventing access to historical spawning areas. In the cryptic European shads Alosa alosa and Alosa fallax (‘shad’ hereafter), this has resulted in population declines across their range. Conservation programmes aim to facilitate the passage of migrators over these barriers and so require baseline information on the spatial and temporal extent of current migrations.
2. Here, a shad-specific environmental DNA (eDNA) assay was used to quantify the spatial extent of shad spawning migrations in the River Severn basin, western England. This basin is characterized by the presence of multiple barriers in the lower catchment. In 2017, the eDNA assay was piloted in the River Teme, an important shad spawning tributary, and then applied in 2018 and 2019 across the lower Severn basin.
3. In all years, shad DNA was detected between mid-May and mid-June, with the maximum spatial extent of shad distribution being in early June when shad eDNA was detected upstream of weirs that were generally considered as impassable. In 2018, this included the detection of shad above the most upstream weir on the main River Severn that required individual fish to have passed six weirs.
4. Although barriers inhibit the spawning migrations of shad, this eDNA assay showed that some highly vagile individuals might be able to ascend these barriers and migrate considerable distances upstream. This suggests that efforts to increase the permeability of these barriers could result in relatively high numbers of migrating shad reaching upstream spawning areas. These results demonstrate that this eDNA assay could also be used across their range, to further quantify the spatial extent of their spawning, including in highly fragmented rivers and those where shad are believed to spawn only occasionally and are rarely observed.