Emergence of Novel Chlamydia trachomatis Sequence Types among Chlamydia Patients in the Republic of Belarus.

Chlamydia trachomatis (CT) is a major cause of sexually transmitted diseases worldwide. The multilocus sequence typing (MLST) of clinical samples from random heterosexual chlamydia patients who were either asymptomatic or reported clinical manifestations of genital chlamydiosis (n = 63) in each of the seven major regions of the Republic of Belarus in 2017-2018 revealed 12 different CT sequence types (STs). We found seven known STs, ST4, ST6, ST9, ST13, ST38, ST95 and ST110, and five novel variants, namely ST271-ST275, which have not been detected elsewhere thus far. The ST4 variant was predominant (27/63, 42.9%) and detected in six out of seven regions. The two most common STs, ST9 and ST13, were regularly seen in four out of seven regions. In contrast, the remaining STs, ST6, ST38, ST95, ST110, and novel STs271-275, surfaced randomly in different parts of the country. The emergence of novel STs was registered in two regions, namely Minsk (ST271 and ST275) and Brest (ST271, ST272, ST273, and ST274). All the STs of detected CT strains were clustered into two Groups, I and III, which are characteristic of CT urogenital strains. No STs typical for Group II, specific to the LGV strains, were revealed. Our study contributes to better understanding the genetic diversity and molecular evolution of CT, one of the most important pathogens in public health worldwide.

Genetic Evidence of Multiple Introductions of Lumpy Skin Disease Virus into Saratov Region, Russia.

Lumpy skin disease virus (LSDV) is the causative agent of lumpy skin disease (LSD) that has been recently reported in the South-East and North Asian parts of the Russian Federation. During 2017-2019, there were more than 30 LSD outbreaks in Saratov Region despite active inoculation of cattle with heterologous vaccine. Importantly, the first case of the novel recombinant LSDV strain was reported here in 2017. This study aimed to determine the main clonal lineage(s) of LSDV strains circulated within Saratov Region and other regions of Russia since the first introduction of LSDV. The molecular typing and subtyping based on the coding regions of the G-protein-coupled chemokine receptor (GPCR) gene resulted in a discrimination of all outbreak-related LSDV strains into two main types, such as Type I and Type II, and subtypes Ia-d and IIa-g. Phylogenetically, eleven LSDV lineages were revealed in Russia including the five ones in Saratov Region. They were the following: (i) the Neethling wild Type Ia/2017; (ii) the recombinant Saratov IIc/2017/2019; (iii) the specific Dergachevskyi IId/2017; (iv) the Khvalynsky IIg/2018, and (v) the Haden-Type IIa lineage for the six LSDV strains detected in cattle immunized with heterologous vaccine during the last LSD outbreak in the Saratov Region, Nesterovo Village, in 2019 (Nesterovo-2019 strains). A single LSDV strain detected in Saratov Region in 2017 had the same Type Ia that was identified in 2016 in the bordered Republic of Kazakhstan. Phylogeographic analysis demonstrated three nominal clusters of LSDV types in the following Russian Federation territories: (I) the Central European part; (II) the South-East of the European part; (III) the North Asian part. Cluster I was represented by mainly Type I strains, while both Clusters 2 and 3 contained predominantly Type II strains. The Clusters I and II partially overlapped, while Cluster 3 was separate. Multiple introductions of LSDV into Saratov Region in 2017-2019 using GPCR-based molecular typing and subtyping were revealed. This scheme is a promising tool for molecular discrimination of LSDV strains derived from both vaccinated and unvaccinated against LSD cattle as well as for molecular epidemiology.

Molecular and cellular effects of food contaminants and secondary plant components and their plausible interactions at the intestinal level.

The intestinal mucosa is not simply a barrier allowing entry of compounds such as nutrients or chemicals, and restricting that of others. Intestinal cells and activities perform selective absorption, biotransformations and efflux back to the lumen. Furthermore, food substances affect both bioavailability and intestinal function. Some are able to act as transcriptional regulators and enzyme modulators. This review points out plausible interactions between food contaminants and/or natural constituents at molecular and cellular levels and focuses on the effects of classical (pesticides and veterinary drugs), environmental (heavy metals, PCBs, dioxins, etc.) and food processing generated (PAHs, heterocyclic amines, etc.) contaminants on absorption, metabolism and efflux. Special attention is given to secondary metabolites of molds (mycotoxins) and plants (polyphenols). Molecular targets are briefly described as well as regulation mechanisms. Where possible, data referred to deal with human intestinal functions in vivo, and with in vitro studies on human intestinal Caco-2 cells; however, since data related to the intestine are rather scarce, effects on molecular targets in liver are also considered. This review also points out the urgent need for fully validated high throughput in vitro tools to screen combinations of substances, at realistic intestinal concentrations. A higher priority could then be given to combinations of nutrients, xenobiotics and food contaminants, with hazardous or beneficial impacts on human health.

Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of zearalenone and deoxynivalenol.

A multianalyte lateral-flow technique using colloidal gold-labeled monoclonal antibodies was developed for the rapid simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEA). The results of this qualitative one-step test were interpreted visually. A very simple and fast sample preparation was used, and the assay procedure could be accomplished within 10 min. When applied to spiked wheat samples, the technique gave accurate and reproducible results. Cut-off levels of 1500 and 100 microg kg(-1) for DON and ZEA, respectively, were observed. The described multianalyte format can be used as a reliable, rapid and cost-effective on-site screening technique for the simultaneous determination of mycotoxins in grain samples.

Fluorescence polarisation immunoassays for strobilurin fungicides kresoxim-methyl, trifloxystrobin and picoxystrobin.

Fluorescence polarisation immunoassays (FPIAs) based on monoclonal antibodies for detection of three strobilurin fungicides - kresoxim-methyl (KM), trifloxystrobin (TF) and picoxystrobin (PC), were developed and optimised. Fluorescein-labelled derivatives of target antigens (tracers) were synthesised and purified by thin-layer chromatography. Influence of tracer structures on the assay parameters was investigated. For KM and TF, the best assay performance was achieved with the homologous pairs of reagents. For the PC assay, the heterologous tracer, i.e. fluorescein-labelled derivative of TF, was used. The developed FPIAs were applied to the determination of KM, TF and PC in red wine. Most optimal sample preparation was achieved with cross-linked poly(vinylpyrrolidone) as a sorbent. This clean-up is simple, rapid and allows determination of all three strobilurin fungicides in one sample. Detection limits of the developed FPIAs in red wine were 28, 6 and 5ng/mL for KM, TF and PC, respectively. Recovery in spiked samples averaged between 80% and 104%. Intra- and inter-assay coefficients of variance were less than 12%. The developed FPIA methods can be applied to screening of wine samples for KM, TF and PC residues without complicated cleanup.

Development of monoclonal antibodies against pirimiphos-methyl and their application to IC-ELISA.

To detect the organophosphorus (OP) pesticide pirimiphos-methyl in grain samples, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed and optimized. By the active esters method, pirimiphos-methyl hapten A was conjugated to keyhole limpet hemocyanin to be used as the immunogen for the production of monoclonal antibodies, and pirimiphos-methyl hapten B was conjugated to ovalbumin to be used as coating antigen. By using the monoclonal antibody and the coating antigen, an IC-ELISA has been developed. Under the established optimized conditions, the IC-ELISA showed an IC50 of 4.2 ng/mL with a detection limit of 0.07 ng/mL. The IC-ELISA showed negligible cross-reactivity with other OP pesticides except with pirimiphos-ethyl. Recoveries of pirimiphos-methyl from spiked grain samples ranged from 83 to 96%.

Immunochromatography using colloidal gold-antibody probe for the detection of atrazine in water samples.

An immunochromatography (ICG) strip test for rapid detection of atrazine in water samples was developed. A monoclonal antibody (MAb) specific to atrazine was produced from the cloned hybridoma cell (AT-1-M3) and used to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) and ICG strip. MAb conjugated to colloidal gold, and that was applied to the conjugate pad of the ICG strip. The visual detection limit for the ICG strip was 3 ng/mL. This test required only 10 min to get results and one step of sample to perform the assay. The results of water samples spiked with 5, 10, 20, and 50 ng/mL of atrazine by ICG strip were in good agreement with those obtained by DC-ELISA. The ICG strip was sufficiently sensitive and accurate to be useful for rapid screening of atrazine in various water samples.

Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of the organophosphorus pesticide parathion-methyl.

A fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the detection of parathion-methyl (PM) was developed and optimized. Fluorescein-labeled PM derivatives (tracers) with different structures were synthesized and purified by thin-layer chromatography. The influence of immunogen and tracer structures on the assay characteristics was investigated. PM concentration determinable by the FPIA ranged from 25 to 10000 ppb. The detection limit was 15 ppb. Methanol extracts of vegetable, fruit, and soil samples were diluted 1/10 for the analysis. Recovery in spiked samples averaged between 85 and 110%. The method developed is characterized by high specificity and reproducibility (CV ranged from 1.5 to 9.1% for interassay and from 1.8 to 14.1% for intra-assay). The FPIA method can be applied to the screening of food and environmental samples for PM residues without complicated cleanup.

Lateral-flow colloidal gold-based immunoassay for the rapid detection of deoxynivalenol with two indicator ranges.

A lateral-flow immunoassay using a colloidal gold-labelled monoclonal antibody was developed for the rapid detection of deoxynivalenol (DON). Different parameters, such as the amount of immunoreagents, type of the materials, composition of the blocking solution and of the detector reagent mixture, were investigated to provide the optimum assay performance. The experimental results demonstrated that such a visual test had an indicator range rather than a cut-off value. Thus, tests for DON determination with two different indicator ranges of 250-500 and 1000-2000 microg kg(-1) were designed. The method allowed detection of DON at low and high concentration levels, which could be useful for research and practical purposes. The assay applied to spiked wheat and pig feed samples demonstrated accurate and reproducible results. The applicability of the developed lateral-flow test was also confirmed under real field conditions. The test strips prepared in Belgium were sent to Mexico, where they were used for the screening of DON contamination in different bread wheat entries from Fusarium Head Blight inoculated plots. The results were compared with those obtained by ELISA and LC-MS/MS. A poor correlation between ELISA and LC-MS/MS was observed. Visual results of the dipstick tests were in a good agreement with the results of the LC-MS/MS method. Coupled with a simple and fast sample preparation, this qualitative one-step test based on the visual evaluation of results did not require any equipment. Results could be obtained within 10 min. The described assay format can be used as a simple, rapid, cost-effective and robust on-site screening tool for mycotoxin contamination in different agricultural commodities.

Investigation of several parameters influencing signal generation in flow-through membrane-based enzyme immunoassay.

Rapid-response analytical tests that can be performed at the point of sampling are based on a visual detection system. The influence of different factors on the signal generation in a membrane-based enzyme immunoassay was investigated. The research was applied to a flow-through immunoassay for the detection of ochratoxin A (OTA). This assay format is a very convenient, simple and fast qualitative screening tool. Conjugates of OTA with horseradish peroxidase (HRP) and alkaline phosphatase (AP) were used as enzyme tracers. A new conjugate OTA-AP has been synthesized in our laboratory and its performance in the assay was compared with that of OTA-HRP. Different substrate systems for HRP and AP were compared. Several reagents, including polymers and surfactants, were tested for their possible effect on signal generation with the use of OTA-HRP conjugate. Polymers such as poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG) 6000 exerted a favourable effect on signal amplification, whereas surfactants negatively affected assay performance. The highest signal amplification (30-70% compared to the standard assay procedure) was achieved using 0.5% PVA in tetramethylbenzidine (TMB) Colorburst substrate solution and phosphate-buffered saline (PBS) for the washing step. It allowed more reliable visual estimation of the results from OTA-HRP assay. Exclusion of the detergent (Tween 20) from the washing solution exerted a favourable effect on assay performance using both enzyme tracers. The assay using OTA-HRP was more susceptible to matrix interferences than the assay with OTA-AP. Signal development in the matrix was better for the OTA-AP assay and visual estimation of the results was easier to perform in this case. For the analysis of spiked wheat samples, OTA-AP conjugate gave a more sensitive, stable and reproducible assay with a cut-off level of 4 microg kg(-1) for OTA. The application of the new OTA-AP conjugate resulted in improved assay performance for the food samples.

Direct competitive ELISA based on a monoclonal antibody for detection of aflatoxin B1. Stabilization of ELISA kit components and application to grain samples.

A direct competitive enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody has been developed and optimized for detection of aflatoxin B1 (AFB1), and an ELISA kit has been designed. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for aflatoxin monitoring. AFB1 concentrations determinable by ELISA ranged from 0.1 to 10 microg L(-1). The IC50 value was 0.62 microg L(-1). Recovery from spiked rice samples averaged between 94 and 113%. The effect of different reagents on the stability of HRP-AFB1 conjugate solution was studied. The performance of a stabilized enzyme tracer in ELISA was determined and compared with that of a freshly prepared control solution of HRP-AFB(1) conjugate. The results showed that stabilizing media containing 0.02% BSA, 0.1% Kathon CG, and 0.05 mol L(-1) calcium chloride in 0.05 mol L(-1) Tris-HCl buffer (pH 7.2) maintained the activity of HRP-AFB1 at a dilution of 1:1000 for a period of at least 12 months at room temperature whereas the reference conjugate solution without the additives lost its activity within a few days. Several additives were tested for their stabilizing effect on a monoclonal antibody (MAb) immobilized on the surface of polystyrene microtitre plates. It was shown that immobilized MAb, treated with post-coating solutions containing PVA, BSA, and combinations of these substances with trehalose, retained its activity for at least 4 months at 4 degrees C, whereas the untreated MAb-coated plate lost its activity within 2 days.