Analysis of the competitiveness between a non-aflatoxigenic and an aflatoxigenic Aspergillus flavus strain on maize kernels by droplet digital PCR

Non-aflatoxigenic Aspergillus flavus strains are used as a biocontrol system on maize fields to decrease the aflatoxin biosynthesis of aflatoxigenic A. flavus strains. A. flavus strain AF36 was the first commercially available biocontrol strain and is authorized for use on maize fields by the US Environmental Protection Agency, e.g., in Texas and Arizona. A droplet digital PCR (ddPCR) assay was developed to analyze the mechanisms of competition and interaction of aflatoxigenic and non-aflatoxigenic A. flavus strains. This assay enables the parallel identification and quantification of the biocontrol strain A. flavus AF36 and the aflatoxigenic A. flavus strain MRI19. To test the assay, spores of both strains were mixed in varying ratios and were incubated on maize-based agar or maize kernels for up to 20 days. Genomic equivalent ratios (genome copy numbers) of both strains were determined by ddPCR at certain times after incubation and were compared to the spore ratios used for inoculation. The aflatoxin biosynthesis was also measured. In general, A. flavus MRI19 had higher competitiveness in the tested habitats compared to the non-aflatoxigenic strain, as indicated by higher final genomic equivalent ratios of this strain compared to the spore ratios used for inoculation. Nevertheless, A. flavus AF36 effectively controlled aflatoxin biosynthesis of A. flavus MRI19, as a clear aflatoxin inhibition was already seen by the inoculation of 10% spores of the biocontrol strain mixed with 90% spores of the aflatoxigenic strain compared to samples inoculated with only spores of the aflatoxigenic A. flavus MRI19.

Optimization of Conditions for The Multiplex PCR for Diagnostics of Horse Strangles with Subspecies Differentiation of Streptococcus Equi Subsp Equi

As a result of the work performed, the conditions for setting up multiplex PCR with electrophoretic detection for the diagnosis of horse strangles were determined, allowing the identification and differentiation of S. equi subsp. equi in one reaction. It was found that the developed PCR protocol for the detection and species differentiation of S. equi subsp. equi with electrophoretic detection in a “multiplex” format has a high specificity and does not lead to amplification of PCR products with DNA of closely related microorganisms, saprophytic microflora, and bacterial pathogens. The sensitivity of the protocols for the detection and species differentiation of S. equi subsp. equi with electrophoretic detection was assessed. Diluted DNA samples of two S. equi subspecies were used as objects of research: S. equi subsp. equi and S. equi subsp. zooepidemicus. DNA samples were diluted by two-fold dilutions, starting from a concentration of 5 ng (which corresponds to 2 million 170 thousand copies in the genomic equivalent) to 1.19 * 10-6 ng (which corresponds to 0.71 copies in the genomic equivalent). DNA detection limit for S. equi subsp. equi was 66 copies in genomic equivalent or 152 fg, DNA of S. equi subsp. zooepidemicus – 132 copies in genomic equivalent or 305 fg.

Randomized, Double-Blinded Clinical Trial for Human Norovirus Inactivation in Oysters by High Hydrostatic Pressure Processing

ABSTRACTContamination of oysters with human noroviruses (HuNoV) constitutes a human health risk and may lead to severe economic losses in the shellfish industry. There is a need to identify a technology that can inactivate HuNoV in oysters. In this study, we conducted a randomized, double-blinded clinical trial to assess the effect of high hydrostatic pressure processing (HPP) on Norwalk virus (HuNoV genogroup I.1) inactivation in virus-seeded oysters ingested by subjects. Forty-four healthy, positive-secretor adults were divided into three study phases. Subjects in each phase were randomized into control and intervention groups. Subjects received Norwalk virus (8FIIb, 1.0 × 104genomic equivalent copies) in artificially seeded oysters with or without HPP treatment (400 MPa at 25°C, 600 MPa at 6°C, or 400 MPa at 6°C for 5 min). HPP at 600 MPa, but not 400 MPa (at 6° or 25°C), completely inactivated HuNoV in seeded oysters and resulted in no HuNoV infection among these subjects, as determined by reverse transcription-PCR detection of HuNoV RNA in subjects' stool or vomitus samples. Interestingly, a white blood cell (granulocyte) shift was identified in 92% of the infected subjects and was significantly associated with infection (P= 0.0014). In summary, these data suggest that HPP is effective at inactivating HuNoV in contaminated whole oysters and suggest a potential intervention to inactivate infectious HuNoV in oysters for the commercial shellfish industry.