Development of a novel sensitive single-tube nested PCR assay for the detection of African swine fever virus.

African swine fever (ASF) is a highly fatal and contagious viral disease caused by African swine fever virus (ASFV). It has caused significant economic losses to the swine industry and poses a serious threat to food security worldwide. Diagnostic tests with high sensitivity are essential for the effective management of ASF. Here, we describe a single-tube nested PCR (STN-PCR) assay for the detection of ASFV in which two consecutive amplification steps are carried out within a single tube. Two pairs of primers (outer and inner) were designed to target the p72 gene of ASFV. The primer concentrations, annealing temperatures, and number of amplification cycles were optimized to ensure the consecutive utilization of outer and inner primer pairs during amplification while minimizing the likelihood of amplicon contamination. In comparison with two conventional endpoint PCR assays (one of which is recommended by the World Organization for Animal Health), the newly developed STN-PCR assay demonstrated a 100-fold improvement in the limit of detection (LOD), detecting 100 copies of ASFV genomic DNA, whereas the endpoint PCR assays could detect no fewer than 10,000 copies. The clinical performance of the STN-PCR assay was validated using 95 tissue samples suspected of being positive for ASFV, and the assay showed 100% specificity. A Cohen's kappa value of 0.91 indicated perfect agreement between the assays. This new STN-PCR assay is a potentially valuable tool that will facilitate the control of ASF.

Carrageenans as biostimulants and bio-elicitors: plant growth and defense responses.

In the context of climate change, the need to ensure food security and safety has taken center stage. Chemical fertilizers and pesticides are traditionally used to achieve higher plant productivity and improved plant protection from biotic stresses. However, the widespread use of fertilizers and pesticides has led to significant risks to human health and the environment, which are further compounded by the emissions of greenhouse gases during fertilizer and pesticide production and application, contributing to global warming and climate change. The naturally occurring sulfated linear polysaccharides obtained from edible red seaweeds (Rhodophyta), carrageenans, could offer climate-friendly substitutes for these inputs due to their bi-functional activities. Carrageenans and their derivatives, known as oligo-carrageenans, facilitate plant growth through a multitude of metabolic courses, including chlorophyll metabolism, carbon fixation, photosynthesis, protein synthesis, secondary metabolite generation, and detoxification of reactive oxygen species. In parallel, these compounds suppress pathogens by their direct antimicrobial activities and/or improve plant resilience against pathogens by modulating biochemical changes via salicylate (SA) and/or jasmonate (JA) and ethylene (ET) signaling pathways, resulting in increased production of secondary metabolites, defense-related proteins, and antioxidants. The present review summarizes the usage of carrageenans for increasing plant development and defense responses to pathogenic challenges under climate change. In addition, the current state of knowledge regarding molecular mechanisms and metabolic alterations in plants during carrageenan-stimulated plant growth and plant disease defense responses has been discussed. This evaluation will highlight the potential use of these new biostimulants in increasing agricultural productivity under climate change.

First Seroepidemiological Investigation of Hepatitis E Virus Infection in Backyard Pigs from Northeastern India: Prevalence and Associated Risk Factors.

Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis globally, with zoonotic potential, and pigs are considered the major reservoir. To determine the seroprevalence of HEV infection in pigs reared in backyard conditions in the northeastern region of India, blood samples were collected from 400 pigs from five northeastern states (80 samples from each state) and tested for IgG antibodies against HEV using an ELISA assay. Questionnaires on farm characteristics and management practices were completed, and risk factors associated with HEV were studied using univariate and multivariate analysis. The apparent seroprevalence of HEV infection was 51% (46.1-55.9, 95% CI), with a true prevalence of 52.98% (47.22-58.75, 95% CI). The risk factors significantly associated with higher HEV seropositivity were as follows: lack of disinfection (OR 4.65), feeding swill (restaurant and bakery waste) (OR 2.55), failure to follow the all-in-all-out production system (OR 3.47), and medium holding size (OR 9.83), which refers to mixed rearing of younger and older age groups. This study demonstrates that HEV is widespread among pigs reared in northeastern India. The risk factor analysis conducted in this study provides valuable insights into the prevalence of HEV in the region.

Novel sensitive isothermal-based diagnostic technique for the detection of African swine fever virus.

A rapid, simple, and sensitive diagnostic technique for the detection of African swine fever virus (ASFV) nucleic acid was developed for testing clinical samples in the field or resource-constrained settings. In the current study, the saltatory rolling-circle amplification (SRCA) technique was used for the first time to detect ASFV. The technique was developed using World Organization for Animal Health (WOAH)-approved primers targeting the p72 gene of the ASFV genome. The assay can be performed within 90 minutes at an isothermal temperature of 58°C without a requirement for sophisticated instrumentation. The results can be interpreted by examination with the naked eye with the aid of SYBR Green dye. This assay exhibited 100% specificity, producing amplicons only from ASFV-positive samples, and there was no cross-reactivity with other pathogenic viruses and bacteria of pigs that were tested. The lower limits of detection of SRCA, endpoint PCR, and real-time PCR assays were 48.4 copies/µL, 4.84 × 103 copies/µL, and 4.84 × 103 copies/µL, respectively. Thus, the newly developed SRCA assay was found to be 100 times more sensitive than endpoint and real-time PCR assays. Clinical tissue samples obtained from ASFV-infected domestic pigs and other clinical samples collected during 2020-22 from animals with suspected ASFV infection were tested using the SRCA assay, and a 100% accuracy rate, negative predictive value, and positive predictive value were demonstrated. The results indicate that the SRCA assay is a simple yet sensitive method for the detection of ASFV that may improve the diagnostic capacity of field laboratories, especially during outbreaks. This novel diagnostic technique is completely compliant with the World Health Organization's "ASSURED" criteria advocated for disease diagnosis, as it is affordable, specific, sensitive, user-friendly, rapid and robust, equipment-free, and deliverable. Therefore, this SRCA assay may be preferable to other complex molecular techniques for diagnosing African swine fever.

Ethanol Treatment Enhances Physiological and Biochemical Responses to Mitigate Saline Toxicity in Soybean.

Soil salinity, a major environmental concern, significantly reduces plant growth and production all around the world. Finding solutions to reduce the salinity impacts on plants is critical for global food security. In recent years, the priming of plants with organic chemicals has shown to be a viable approach for the alleviation of salinity effects in plants. The current study examined the effects of exogenous ethanol in triggering salinity acclimatization responses in soybean by investigating growth responses, and numerous physiological and biochemical features. Foliar ethanol application to saline water-treated soybean plants resulted in an enhancement of biomass, leaf area, photosynthetic pigment contents, net photosynthetic rate, shoot relative water content, water use efficiency, and K+ and Mg2+ contents, leading to improved growth performance under salinity. Salt stress significantly enhanced the contents of reactive oxygen species (ROS), malondialdehyde, and electrolyte leakage in the leaves, suggesting salt-induced oxidative stress and membrane damage in soybean plants. In contrast, ethanol treatment of salt-treated soybean plants boosted ROS-detoxification mechanisms by enhancing the activities of antioxidant enzymes, including peroxidase, ascorbate peroxidase, catalase, and glutathione S-transferase. Ethanol application also augmented the levels of proline and total free amino acids in salt-exposed plants, implying a role of ethanol in maintaining osmotic adjustment in response to salt stress. Notably, exogenous ethanol decreased Na+ uptake while increasing K+ and Mg2+ uptake and their partitioning to leaves and roots in salt-stressed plants. Overall, our findings reveal the protective roles of ethanol against salinity in soybean and suggest that the use of this cost-effective and easily accessible ethanol in salinity mitigation could be an effective approach to increase soybean production in salt-affected areas.