Plant R2R3-MYB transcription factors (TFs) have been suggested to play crucial roles in the response to diverse abiotic and biotic stress factors but there is little molecular evidence of this role in soybean plants. In this work, we identified and functionally characterized an R2R3-MYB TF, namely, GsMYB15, from the wild soybean ED059. Protein and promoter sequence analysis indicated that GsMYB15 is a typical R2R3-MYB TF and contains multiple stress-related cis-elements in the promoter region. GsMYB15 is located in the nucleus and exhibits transcriptional activation activity. QPCR assays suggested that the expression of GsMYB15 could be induced by NaCl, insect attacks and defense-related hormones (MeJA and SA). Furthermore, GsMYB15 exhibited highest expression in pods compared to other tissues. Functional analysis of GsMYB15 demonstrated that overexpression of GsMYB15 could increase salt tolerance and enhance the resistance to H. armigera larvae in transgenic Arabidopsis plants. Moreover, overexpression of GsMYB15 also affected the expression levels of salt stress- and defense-related genes in the transgenic plants. Feeding with transgenic Arabidopsis plant leaves could significantly suppress the expression levels of immunity-related genes in H. armigera larvae. Overexpression of GsMYB15 also increased mesophyll cell levels in transgenic plants. Taken together, these results provide evidence that GsMYB15 is a positive regulator of salt stress tolerance and insect resistance in transformed Arabidopsis plants.

Equine piroplasmosis (EP) is a parasitic disease caused by Theileria equi (T. equi), Babesia caballi (B. caballi) and Theileria haneyi (T. haneyi). This disease is considered to be reportable by the World Organization for Animal Health (WOAH). Real-time quantitative PCR (qPCR) is regarded as a straightforward, rapid and sensitive diagnostic method to detect pathogens. However, qPCR has not been employed in the various epidemiological investigations of T. haneyi. In this study, we developed a new qPCR method to detect T. haneyi based on the chr1sco (chromosome 1 single-copy open reading frame (ORF)) gene, which has no detectable orthologs in T. equi or B. caballi. A TaqMan MGB probe was used in the development of the qPCR assay. A plasmid containing the chr1sco gene was constructed and used to establish the standard curves. The novel qPCR technique demonstrated great specificity for detecting additional frequent equine infectious pathogens and sensitivity for detecting diluted standard plasmids. This qPCR was further validated by comparison with an optimized nested PCR (nPCR) assay in the analysis of 96 clinical samples. The agreement between the nPCR assay and the established qPCR assay was 85.42%. The newly established method could contribute to the accurate diagnosis of T. haneyi infections in horses.

Theileria equi (T. equi) and Babesia caballi (B. caballi) are the causative pathogens of Equine piroplasmosis (EP), a disease that has brought huge economic losses and great restrictions to the global equine industry. Rapid and accurate diagnostic methods are critical for the effective monitoring of the disease. In this study, we developed novel competitive ELISA methods and western blot assays based on the EMA1 or Bc48 proteins to detect antibodies against T. equi or B. caballi, respectively. In the novel cELISA, horseradish peroxidase (HRP)-labeled monoclonal antibodies are used in place of enzyme-conjugated secondary antibodies, in order to speed up the entire procedure. These methods have high sensitivity and no cross-reactivity with antibodies against other equine diseases. In the newly developed western blot assays, we optimized the dilution of T. equi or B. caballi positive serum samples to 1:200. Compared with the commercially available kit, both the novel cELISA assay and the western blot assay showed high coincidence rates in detecting antibodies against T. equi and B. caballi. Taken together, the novel cELISA and the western blot assays for detecting antibodies against T. equi or B. caballi have the potential to rapidly test for T. equi or B. caballi and to contribute to the surveillance and control of this disease.

Streptococcus equi subspecies equi (S. equi) is the causative pathogen of strangles in horses, donkeys, and other equine animals. Strangles has spread globally and causes significant losses to the horse industry. In response to the urgent need for effective disease control, this study introduces a novel nucleic acid diagnostic method known as a real-time recombinase-assisted amplification (RAA) assay, developed based on the eqbE gene, for the rapid detection of S. equi nucleic acid. The real-time RAA method employs specifically designed probes and primers targeting the eqbE gene, enhancing the overall specificity and sensitivity of the detection. After efficiency optimization, this real-time RAA method can detect 10 or more copies of nucleic acid within 20 min. The method demonstrates high specificity for S. equi and does not cross-react with other clinically relevant pathogens. Real-time RAA diagnostic performance was evaluated using 98 nasal swab samples collected from horses and compared with the real-time PCR detection method. Results revealed that 64 and 65 samples tested positive for S. equi using real-time RAA and real-time PCR, respectively. The overall agreement between the two assays was 96.94% (95/98), with a kappa value of 0.931 (p < 0.001). Further linear regression analysis indicated a significant correlation in the detection results between the two methods (R² = 0.9012, p < 0.0001), suggesting that the real-time RAA assay exhibits a detection performance comparable to that of real-time PCR. In conclusion, the real-time RAA assay developed here serves as a highly specific and reliable diagnostic tool for the detection of S. equi in equine samples, offering a potential alternative to real-time PCR methods. In conclusion, the real-time RAA nucleic acid diagnostic method, based on the eqbE gene, offers rapid and accurate diagnosis of S. equi, with the added advantage of minimal equipment requirements, thus contributing to the efficient detection of strangles in horses.

Rotavirus A (RVA) causes severe diarrhea in donkey foals, but rapid quantitative antigen assays for vaccine development and manufacturing control are lacking. We developed a VP6-based antigen-capture ELISA (acELISA) for donkey-origin RVA using rabbit anti-RVA polyclonal IgG as the capture antibody and horseradish peroxidase-conjugated mouse anti-VP6 monoclonal antibody 3F6 as the detector antibody. Assay conditions were optimized, and performance was evaluated through standard-curve analysis, specificity and repeatability testing, Western blot (WB) comparison, median tissue culture infectious dose (TCID₅₀), reverse transcription quantitative polymerase chain reaction (RT-qPCR), ultrafiltration membrane screening, and pilot-scale tangential flow filtration (TFF) monitoring. The optimized acELISA detected VP6 at 1 ng/mL and showed a linear range of 1–25 ng/mL, with no cross-reactivity with eight common equine pathogens, and intra- and inter-assay coefficients of variation below 10%. The acELISA estimates were consistent with Western blot densitometry. In process applications, the assay supported the selection of a 100 kDa membrane and monitored pilot-scale TFF, yielding 75.69% VP6 recovery and an 18-fold increase in antigen purity. This acELISA provides a specific and practical in-process tool for quantitative antigen monitoring in donkey rotavirus vaccine production and is intended to complement, rather than replace, infectivity- and genome-based assays.

Strangles, a highly contagious disease caused by Streptococcus equi subspecies equi (S.equi), significantly impacts horse populations worldwide, with Iceland as the only exception. This disease poses serious threats to equine health and results in considerable economic losses. Consequently, the accurate, sensitive, and rapid detection of S.equi from clinical samples is essential for early warning and effective disease management. This study introduces a novel detection method that integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technologies. We specifically designed RAA primers and CRISPR RNA to target the eqbE gene of S.equi, and we have carefully optimized the reaction systems for this purpose. The newly established visual diagnostic method has shown to be highly effective, demonstrating 97.14% specificity and 100% sensitivity, with the capability to detect as few as 5.6×10⁰ copies of the target. This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of S.equi. This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of S.equi, which enables visual point-of-care diagnosis of Strangles.