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.

An overview of Parafrankia (Nod+/Fix+) and Pseudofrankia (Nod+/Fix-) interactions through genome mining and experimental modeling in co-culture and co-inoculation of Elaeagnus angustifolia.

In many frankia, the ability to nodulate host plants (Nod+) and fix nitrogen (Fix+) is a common strategy. However, some frankia within the Pseudofrankia genus lack one or two of these traits. This phenomenon has been consistently observed across various actinorhizal nodule isolates, displaying Nod- and/or Fix- phenotypes. Yet, the mechanisms supporting the colonization and persistence of these inefficient frankia within nodules, both with and without symbiotic strains (Nod+/Fix+), remain unclear. It is also uncertain whether these associations burden or benefit host plants. This study delves into the ecological interactions between Parafrankia EUN1f and Pseudofrankia inefficax EuI1c, isolated from Elaeagnus umbellata nodules. EUN1f (Nod+/Fix+) and EuI1c (Nod+/Fix-) display contrasting symbiotic traits. While the prediction suggests a competitive scenario, the absence of direct interaction evidence implies that the competitive advantage of EUN1f and EuI1c is likely contingent on contextual factors such as substrate availability and the specific nature of stressors in their respective habitats. In co-culture, EUN1f outperforms EuI1c, especially under specific conditions, driven by its nitrogenase activity. Iron-depleted conditions favor EUN1f, emphasizing iron's role in microbial competition. Both strains benefit from host root exudates in pure culture, but EUN1f dominates in co-culture, enhancing its competitive traits. Nodulation experiments show that host plant preferences align with inoculum strain abundance under nitrogen-depleted conditions, while consistently favoring EUN1f in nitrogen-supplied media. This study unveils competitive dynamics and niche exclusion between EUN1f and EuI1c, suggesting that host plant may penalize less effective strains and even all strains. These findings highlight the complex interplay between strain competition and host selective pressure, warranting further research into the underlying mechanisms shaping plant-microbe-microbe interactions in diverse ecosystems. IMPORTANCE: While Pseudofrankia strains typically lack the common traits of ability to nodulate the host plant (Nod-) and/or fix nitrogen (Fix-), they are still recovered from actinorhizal nodules. The enigmatic question of how and why these unconventional strains establish themselves within nodule tissue, thriving either alongside symbiotic strains (Nod+/Fix+) or independently, while considering potential metabolic costs to the host plant, remains a perplexing puzzle. This study endeavors to unravel the competitive dynamics between Pseudofrankia inefficax strain EuI1c (Nod+/Fix-) and Parafrankia strain EU1Nf (Nod+/Fix+) through a comprehensive exploration of genomic data and empirical modeling, conducted both in controlled laboratory settings and within the host plant environment.

Whole genome sequencing of Streptomyces antnestii sp. nov. with a potency to become an industrial strain.

Streptomyces Strain San01 is isolated from the soil of ant-nest found in the tea estate of Darjeeling, India. The morphology, biochemical, as well as the molecular characteristics, proved that San01 belonged to the genus Streptomyces. The average nucleotide identity (ANI) value between the genome sequence of the studied strain and its closest phylogenetic neighbors were very low and also could be distinguished from its closest neighbour with broad range of phenotypic data. The draft genome sequence of isolate San01 (NZ_RZYA00000000.1) was estimated to be 9.12 Mbp in size with 71.2% of GC content and it encompasses 39 biosynthetic gene clusters that emphasize the biotechnological potential of this isolate.Based on the phenotypic, genetic and genomic data, isolate San01 (=JCM 34633 = NCTC 14543) merits to be recognized as a type strain of a novel species and hereby propose the name Streptomyces antnestii sp. nov. Incidentally, this is the first report on Streptomyces genomes from Darjeeling, India.

Rhizospheric nano-remediation salvages arsenic genotoxicity: Zinc-oxide nanoparticles articulate better oxidative stress management, reduce arsenic uptake, and increase yield in Pisum sativum (L.).

Pea (Pisum sativum L.), a legume, has a high nutritional content, but arsenic (As) in the agro-ecosystem poses a significant bottleneck to its yield, especially in South East Asia, by severely hampering ontogeny. The present study proposes a rhizospheric nano-remediation strategy to evade As-genotoxicity and improve crop yield using biogenic zinc-oxide nanoparticles (ZnONPs). Similar to any other source of environmental stress, As-toxicity caused rapid oxidative bursts with deterioration in morpho-physiological attributes (germination rate, shoot length, and root length decreased by 62 %, 16 %, and 14.9 % respectively in the negative control, over normal control). Reactive oxygen species (ROS) accumulation (12.8 and 9-fold increase in leaves and roots) overburdened antioxidative defense, and loss of cellular homeostasis resulted in membrane damage (82.75 % increase) and electrolyte-leakage (2.6-fold increase) in negative control. The study also reveals a significant increase in nuclear area, nuclear fragmentation, and micronuclei formation in root tip cells under As-stress, indicating severe genomic instability and increased programmed cell death (3.3-fold increase in early apoptotic cells) due to leaky plasma membrane and unrepaired DNA damage. Application of ZnONPs significantly reduced As-toxicity in peas due to its adsorption in the rhizosphere, causing diminished As-uptake and better antioxidant response. Improved phytochelatin synthesis enhanced vacuolar sequestration of arsenic, which reduced As-interference. Comparatively better flowering time (7.74-19.36 % reduction in flowering delay) with greater transcript abundance of GIGANTIA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) genes; better photosynthetic activity (1.3-1.9-fold increased chlorophyll autofluorescence); increased pollen viability; lesser genotoxicity (decreased tail DNA in comet assay) was noticed. A maximum increase of 37.5 % in pod number and seed zinc content (1.67-fold) was observed while seed arsenic content decreased under ZnONPs treatment. However, the highest dose of ZnONPs (400 mg L-1) induced NP-toxicity in pea plants under our experimental conditions, while optimum stress-alleviation was observed up to 300 mg L-1.

Phytocompounds from Phyllanthus acidus (L.) Skeels in the management of Monkeypox Virus infections.

Monkeypox is a communicable disease similar to smallpox, primarily occurring in African countries. However, recently it has spread to countries outside Africa and may arise as the next threat after COVID-pandemic. The causative organism, i.e. Monkeypox Virus (MPV) spreads from one individual to another primarily through inhalation of respiratory droplets or through contact with skin lesions of infected individuals. No known drugs are available specifically for MPV. Due to its similarity with smallpox, treatment of monkeypox is being attempted through the administration of the smallpox vaccine. Therefore, we evaluated the efficacy of the plant Phyllanthus acidus against MPV since it is traditionally used in the treatment of chickenpox and smallpox. Through functional annotation, PASS prediction and Network pharmacology analysis, the effectiveness of these chosen P. acidus-derived phytocompounds against MPV was confirmed. Target prediction of the phytocompounds identified in GC-MS analysis of the plant extract showed them to be associated with 76 human proteins. The compounds also show good binding affinity with selected viral proteins: DNA polymerase (DNApol), Putative Virulence Factor (vPVF) and Cytokine Binding Protein. Prediction of Activity Spectra for Substances (PASS) and functional annotation of the target proteins further support their antiviral nature through interaction with these proteins. The compounds were found to modulate pathways related to symptoms of viral infection and this may help in maintaining homeostasis. Our study demonstrates antiviral activity as well as the therapeutic potential of the plant against MPV infection.Communicated by Ramaswamy H. Sarma.

Neurological potency of native plants from sub-Himalayan West Bengal through reverse pharmacology.

Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and epilepsy, pose a growing global health challenge due to an aging population. These conditions share common processes, including protein accumulation, oxidative stress, and neuro-inflammation, making their treatment complex and costly. Network pharmacology, an innovative approach integrating systems biology and computational biology, offers insights into multi-target formulations and the repurposing of existing medications for neurodegenerative diseases. We shortlisted 730 bioactive compounds from 25 traditional Himalayan plants, assessed their drug-like properties using ADME criteria, and predicted their potential target proteins through reverse docking and pharmacophore mapping. Our study identified 287 compounds with high gastrointestinal absorption and good blood-brain barrier permeability. These compounds were subjected to target prediction, yielding a list of 171 potential target proteins. Functional annotation and pathway enrichment analysis highlighted their involvement in steroid hormone-related pathways, MAPK signaling, FOXO signaling, TNF signaling, VEGF signaling, and neurotrophin signaling. Importantly, one plant, Valeriana jatamansi, exhibited an association with beta-amyloid binding activity, a potential therapeutic approach for AD. From our study we could understand how these plants modulate our body to manage these diseases. However, further in vitro and in vivo validation is needed before commercial and public use of this data.

Biofilm-forming antimicrobial-resistant pathogenic Escherichia coli: A one health challenge in Northeast India.

This study aimed to investigate the prevalence of Shiga toxin-producing Escherichia coli (STEC), Enteropathogenic E. coli (EPEC), and Enterotoxigenic E. coli (ETEC) in common food animals (cattle, goats, and pigs) reared by tribal communities and smallholder farmers in Northeast India. The isolates were characterized for the presence of virulence genes, extended-spectrum beta-lactamases (ESBL) production, antimicrobial resistance, and biofilm production, and the results were statistically interpreted. In pathotyping 141 E. coli isolates, 10 (7.09%, 95% CI: 3.45%-12.66%) were identified as STEC, 2 (1.42%, 95% CI: 0.17%-5.03%) as atypical-EPEC, and 1 (0.71%, 95% CI: 0.02%-3.89%) as typical-EPEC. None of the isolates were classified as ETEC. Additionally, using the phenotypic combination disc method (ceftazidime with and without clavulanic acid), six isolates (46.1%, 95% CI: 19.22%-74.87%) were determined to be ESBL producers. Among the STEC/EPEC strains, eleven (84.6%, 95% CI: 54.55%-98.08%) and one (7.7%, 95% CI: 0.19%-36.03%) strains were capable of producing strong or moderate biofilms, respectively. PFGE analysis revealed indistinguishable patterns for certain isolates, suggesting clonal relationships. These findings highlight the potential role of food animals reared by tribal communities and smallholder farmers as reservoirs of virulent biofilm-forming E. coli pathotypes, with implications for food contamination and zoonotic infections. Therefore, monitoring these pathogens in food animals is crucial for optimizing public health through one health strategy.

Livestock, pets and humans as carriers of methicillin-resistant Staphylococcusaureus and comparative evaluation of two PCR protocols for detection.

Staphylococcus aureus are Gram positive bacteria known to acquire antibiotic resistance rapidly and pose a major challenge to clinicians worldwide. Infections by methicillin resistant Staphylococcus aureus (MRSA) are usually associated with increased mortality and prolonging of treatment. Samples (n = 706) from diverse sources (livestock, pets, animal handlers, human hospital) were collected and screened for the presence of MRSA by phenotypic and genotypic methods. The incidence of Staphylococcus aureus was greater in goats (42.00%; 28.20 - 56.80%, confidence interval [CI] 95.00%) followed by cattle (13.50%; 9.20 - 18.80%, CI 95.00%), humans (12.90%; 9.30 - 17.40%, CI 95.00%) and dogs (12.90%; 8.10 - 19.20%, CI 95.00%). Significantly higher incidence of MRSA was observed in dogs (65.00%; 40.80 - 84.60%, CI 95.00%), compared to other hosts namely cattle (48.00%; 26.50 - 64.30%, CI 95.00%), humans (35.00%; 20.20 - 52.50%, CI 95.00%) and goats (10.00%; 1.20 - 30.40%, CI 95.00%). All the S. aureus isolates were further screened for thermostable nuclease (nuc gene) by polymerase chain reaction (PCR). The incidence of nuc gene in cattle, dog, goat and human were found to be 3.30% (1.30 - 6.60%, CI 95.00%), 5.20% (2.30 - 9.90%, CI 95.00%), 28.00% (16.20 - 42.50%, CI 95.00%) and 9.10% (6.00 - 13.00%, CI 95.00%), respectively. Comparative evaluation of two PCR primers (mecA-162 and mecA-310) indicated the former one as more rational choice for detection of MRSA. Overall, the results of our study indicated possible risk of zoonotic transmission of MRSA from canines.

Weak Universality Induced by Q=±2e Charges at the Deconfinement Transition of a (2+1)-Dimensional U(1) Lattice Gauge Theory.

Matter-free lattice gauge theories (LGTs) provide an ideal setting to understand confinement to deconfinement transitions at finite temperatures, which is typically due to the spontaneous breakdown (at large temperatures) of the center symmetry associated with the gauge group. Close to the transition, the relevant degrees of freedom (Polyakov loop) transform under these center symmetries, and the effective theory depends on only the Polyakov loop and its fluctuations. As shown first by Svetitsky and Yaffe, and subsequently verified numerically, for the U(1) LGT in (2+1) dimensions, the transition is in the 2D XY universality class, while for the Z_{2} LGT, it is in the 2D Ising universality class. We extend this classic scenario by adding higher charged matter fields and show that the critical exponents γ and ν can change continuously as a coupling is varied, while their ratio is fixed to the 2D Ising value. While such weak universality is well known for spin models, we demonstrate this for LGTs for the first time. Using an efficient cluster algorithm, we show that the finite temperature phase transition of the U(1) quantum link LGT in the spin S=1/2 representation is in the 2D XY universality class, as expected. On the addition of Q=±2e charges distributed thermally, we demonstrate the occurrence of weak universality.

Development of a novel visual isothermal amplification assay for rapid detection of Brucella spp.

Brucellosis is an economically important livestock disease worldwide besides having a noteworthy impact on human health. In this study, a rapid, simple, and ultra-sensitive nuclei-acid diagnostic technique was developed for the detection of brucellosis harnessing saltatory rolling circle amplification (SRCA). The diagnostic method was developed using World Organization for Animal Health (WOAH) approved primers targeting the bcsp31 gene of the Brucella genome. The assay can be accomplished within 90 min at a temperature of 65 °C without the requirement of sophisticated instrumentation. The result interpretation can be done with the naked eye with the aid of SYBR green dye. The developed technique displayed 100% specificity by amplifying only 10 reference and field strains of Brucella spp. and there was no cross-reactivity with the other tested pathogens. The lower limit of detections of SRCA and end-point PCR assays were 9.7 fg/µL (2.7 genome copies of Brucella) and 970 fg/µL, respectively. Thus, the developed SRCA assay was found to be 100× more sensitive than the end-point PCR assay. To the best of our knowledge, our study is the first one to develop an SRCA-based assay for the detection of brucellosis and it can be a diagnostic tool for resource-constrained laboratories and veterinary hospitals.

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.

Neem and Turmeric in the management of Covid Associated Mucormycosis (CAM) derived through network pharmacology.

Mucormycosis or 'Black Fungus' has been known to target immunocompromised individuals even before the emergence of COVID-19. Nevertheless, the present circumstances provide the best opening for Covid Associated Mucormycosis (CAM), as the global pandemic is engulfing a large part of human population making them immunocompromised. This drastic increase in Mucormycosis infections has to be addressed as early as possible. There is a growing tendency of relying upon herbal drugs that have minimal side effects and does not compromise our immune system. Recently, the concept of network pharmacology has grabbed the attention of modern science, especially advanced medical sciences. This is a new discipline that can use computational power to systematically catalogue the molecular interactions between botanical formulations and the human body. In this study, Neem and Turmeric was considered as the target plants and an attempt was made to reveal various aspects through which phytocompounds derived from them may effectively manage CAM menace. We have taken a step-by-step approach for identifying the target proteins and ligands associated with Mucormycosis treatment. Functional network analysis and Molecular docking approaches were applied to validate our findings. Quercetin derived from both Neem and Turmeric was found to be one of the main phytocompounds working against Mucormycosis. Along with that, Caffeic acid, Curcumin, Kaempferol, Tetrahydrocurcumin and Myricetin also play a pivotal role in fighting against Black-Fungus. A thorough analysis of our result suggested a triple-front attack on the fungal pathogens and the approaches are necrosis inhibition, iron chelation and immuno-boosting.Communicated by Ramaswamy H. Sarma.

In-silico studies on wild orange (Citrus macroptera Mont.) compounds against COVID-19 pro-inflammation targets.

One-fifth of COVID-19 patients suffer a severe course of COVID-19 (SARS-CoV-2) infection; however, the specific causes remain unclear. Despite numerous papers that have been flooded in different scientific journals clear clinical picture of COVID-19 aftermath persists to remain fuzzy. The survivors of severe COVID-19infection having defeated the virus are just the starting of an uncharted recovery path. Currently, there is no drug available that is safe to consume to combat this pandemic. However, researchers still struggling to find specific therapeutic solutions. The present study employed an in silico approach to assessing the inhibitory potential of the phytochemicals obtained from GC-MS analysis of Citrus macroptera against inflammatory proteins like COX-2, NMDAR and VCAM-1 which remains in a hyperactive state even after a patient is fully cured of this deadly mRNA virus. An extensive molecular docking investigation of the phyto-compounds at the active binding pockets of the inflammatory proteins revealed the promising inhibitory potential of the phytochemicals. Reasonable physicochemical attributes of the compounds following Lipinski's rule of five, VEBER and PAINS analysis further established them as potential therapeutic candidates against aforesaid inflammatory proteins. MM-GBSA binding free energy estimation revealed that Limonene was the most promising candidate displaying the highest binding efficacy with the concerned VCAM-1 protein included in the present analysis. An interesting finding is the phytochemicals exhibited better binding energy scores with the concerned COX-2, VCAM-1 and NMDA receptor proteins than the conventional drugs that are specifically targeted against them. Our in silico results suggest that all the natural phyto-compounds derived from C. macroptera could be employed in Post covid inflammation complexities after appropriate pre-clinical and clinical trials for further scientific validation.Communicated by Ramaswamy H. Sarma.

Detection of the peptidyl epitope for vaccine development against MPV.

Background: Monkeypox Virus (MPV) is the cause of zoonotic disease characterized by skin-eruption with pus cell formation and lymphadenopathy. This virus belongs to the Orthopoxvirus genus with DNA as its genetic material. Previously, this infection was reported from Africa and occasionally from USA and UK. However, recently there is a sudden surge of infection in non-epidemic countries and a new strain of MPVhas been discovered. Therefore it is important to revisit the phylogeny of MPV with the addition of new strains. Recently WHO also stressed the need of developing vaccines for new strains. In this scenario we have two objectives for this study -first, to reveal the exact phylogenetic position of the 2022 strain and second, to identify specific peptides which may be used for vaccine development in the future. Methods: The phylogenetic analysis was done with the help of Bayesian phylogeny. The dN/dS calculation was performed based on DNA polymerase genes of selected MPV strains. The peptidyl-epitope was searched in MPV2022/2 SLO strain with the help of several algorithms implemented in Allergen FP v.1.0, NetMHCII 2.3, MHCpred and Toxin Pred. The structure prediction of the proteins and peptides was performed through Hpepdock. The quality of the structures was validated through the Ramachandran plot. The molecular dynamics and simulation were performed through Gromacs software. The interaction between peptide and protein was assessed through Ligplot software. Results: The phylogenetic analysis revealed that the considered 2022 MPVstrains were close to the USA strains. The evolutionary analysis showed the volatile nature of the genome. Moreover, 9-mer peptide sequence was identified as an epitope for vaccine development. Conclusions: The emergence of more virulent strains in near future may not be ruled out. Immunocompromised patients are more susceptible to this virus hence sub-unit vaccine is a better choice than a recombinant or attenuated vaccine against monkeypox. We have identified a small stretch of specific peptide which may be used for developing a subunit vaccine against this virus.

Salt Stress Tolerance in Casuarina glauca: Insights from the Branchlets Transcriptome.

Climate change and the accelerated rate of population growth are imposing a progressive degradation of natural ecosystems worldwide. In this context, the use of pioneer trees represents a powerful approach to reverse the situation. Among others, N2-fixing actinorhizal trees constitute important elements of plant communities and have been successfully used in land reclamation at a global scale. In this study, we have analyzed the transcriptome of the photosynthetic organs of Casuarina glauca (branchlets) to unravel the molecular mechanisms underlying salt stress tolerance. For that, C. glauca plants supplied either with chemical nitrogen (KNO3+) or nodulated by Frankia (NOD+) were exposed to a gradient of salt concentrations (200, 400, and 600 mM NaCl) and RNA-Seq was performed. An average of ca. 25 million clean reads was obtained for each group of plants, corresponding to 86,202 unigenes. The patterns of differentially expressed genes (DEGs) clearly separate two groups: (i) control- and 200 mM NaCl-treated plants, and (ii) 400 and 600 mM NaCl-treated plants. Additionally, although the number of total transcripts was relatively high in both plant groups, the percentage of significant DEGs was very low, ranging from 6 (200 mM NaCl/NOD+) to 314 (600 mM NaCl/KNO3+), mostly involving down-regulation. The vast majority of up-regulated genes was related to regulatory processes, reinforcing the hypothesis that some ecotypes of C. glauca have a strong stress-responsive system with an extensive set of constitutive defense mechanisms, complemented by a tight mechanism of transcriptional and post-transcriptional regulation. The results suggest that the robustness of the stress response system in C. glauca is regulated by a limited number of genes that tightly regulate detoxification and protein/enzyme stability, highlighting the complexity of the molecular interactions leading to salinity tolerance in this species.

Prevalence, toxinotyping, antimicrobial susceptibility and biofilm-forming ability of Clostridium perfringens isolated from free-living rodents and shrews.

BACKGROUND AND OBJECTIVES: Clostridium perfringens (C. perfringens), is a spore-forming and toxin-producing pathogenic Gram-positive rod-shaped bacterium with immense public health/zoonotic concern. Rodents are well-known reservoirs and vectors for a large number of zoonoses and strong links have been recognized between synanthropic rodents and foodborne disease outbreaks throughout the world. To date, no study has been conducted for studying the prevalence of C. perfringens in rodents and shrews. In this study, we investigated faecal samples from free-living rodents and shrews trapped in Meghalaya, a North-eastern hill state of India for the presence of virulent and antimicrobial-resistant C. perfringens. METHODS: A total of 122 animals comprising six species of rodents and one species of shrews were trapped: Mus musculus (n = 15), Mus booduga (n = 7), Rattus rattus (n = 9), Rattus norvegicus (n = 3), Bandicota indica (n = 30), Bandicota bengalensis (n = 32) and Suncus murinus (n = 26). The faecal swabs were collected and processed for the isolation of C. perfringens. Toxinotyping was done using PCR. Antimicrobial susceptibility testing and biofilm forming ability testing were done using Kirby Bauer disc diffusion method and crystal violet assay. RESULTS: C. perfringens was isolated from 27 of the 122 faecal swabs (22.1%), from six species of rodents and shrews. Five of the host species were rodents, Bandicota bengalensis (25%), Bandicota indica (16.7%), Rattus norvegicus (33.3%), Mus musculus (13.3%), Mus booduga (42.8%) and Suncus murinus (shrew) (29.6%). The common toxinotype was type A (59.2%) followed by Type A with beta2 toxin (33.3%), Type C (3.7%) and Type C with beta2 toxin (3.7%). None of the isolates harboured cpe, etx, iap, and NetB genes and therefore none was typed as either B, D, E, F, or G. Nine isolates (33.3%) turned out to be multi-drug resistant (MDR), displaying resistance to three or more categories of antibiotics tested. Twenty-three out of twenty-seven isolates (85.2%) were forming biofilms. CONCLUSION: Globally, this is the first study to report the prevalence of C. perfringens and its virulence profile and antimicrobial resistance in free-living rodents and shrews. The rodents and shrews can potentially contaminate the food and environment and can infect humans and livestock with multi-drug resistant/virulent Type A and Type C C. perfringens.

Development and evaluation of a novel polymerase spiral reaction based testing technique for same-day visual detection of Campylobacter coli in pork.

The developed polymerase spiral reaction-based technique specifically amplified the ceuE gene of C. coli and involved a three-step centrifugation method for DNA extraction. PSR, real-time and end-point PCR were able to detect 62 fg, 620 fg and 6.2 pg C. coli DNA/tube, respectively. PSR detection limits for artificially contaminated pork samples without enrichment, with 12 h enrichment and after 24 h enrichment were 1000 CFU/g, 100 CFU/g, and 10 CFU/g samples, respectively which were ten times better than real-time PCR. The detection performance of PSR (with 12 h enrichment) was also compared to culture (ISO10272-1:2017) method using 75 naturally-contaminated samples, which revealed the sensitivity, specificity, PPV, NPV and accuracy of 100% (95%CI, 73.2%-100%), 98.4% (95%CI, 90%-99.9%), 93.3% (95%CI, 66%-99.6%), 100% (95%CI, 92.5%-100%) and 98.7% (95%CI, 92.8%-99.9%), respectively. The advantage and novelty of this assay are its equipment-free nature, dye-based interpretation by the naked eye, and the requirement of one enzyme and one primer pair. This assay could be a better alternative to other molecular methods and may help in reducing the possible troubles (e.g., gastroenteritis, hospitalization, or death) of belated detection of C. coli in food products. This is the primary report applying the PSR for C. coli detection.

Pulsed-field gel electrophoresis fingerprinting of Listeria monocytogenes isolates recovered from foods of animal origin and fishes in North-Eastern India.

L isteria monocytogenes is a pathogen of great concern to the food industry. The present study was aimed to explore the clonal relationships amongst L. monocytogenes strains isolated from foods of animal origin (milk, beef, chevon (goat meat), pork and chicken) and fish. Forty-seven L. monocytogenes strains were characterized by pulsed-field gel electrophoresis (PFGE). The PFGE analysis using ApaI and AscI enzymes revealed 37 pulsotypes, with Simpson's discriminatory index of 0.987. This study demonstrated the presence of a few similar L. monocytogenes pulsotypes in different foods of animal origin in different places and years of isolation and this indicates that some L. monocytogenes subtypes may be ubiquitous which are acclimatizing and persisting in different foods of animal origin. This also emphasizes the importance of cross-contamination in local wet markets. Thus, the understanding of genetic diversity will contribute to the development of rational and workable strategies to control this important zoonotic infection.

Computational profiling of natural compounds as promising inhibitors against the spike proteins of SARS-CoV-2 wild-type and the variants of concern, viral cell-entry process, and cytokine storm in COVID-19.

The continuous spread and evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the rapid surge in infection cases in the coronavirus disease 2019 (COVID-19) evoke a dire need for effective therapeutics. In this study, we explored the inhibitory potential of a library of 605 phytocompounds, selected from Indian medicinal plants with reported antiviral and anti-inflammatory activities, against the receptor-binding domain of spike proteins of the SARS-CoV-2 wild-type and the variants of concern, including variants B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Our approach was based on extensive molecular docking, assessment of drug-likeness, and robust molecular dynamics simulations. We also identified promising inhibitory candidates against the host (human) proteins associated with SARS-CoV-2 spike activation and attachment, namely, ACE2 receptor, proteases TMPRSS2 and CTSL, and the endocytic regulator AAK1. In addition, we screened promising inhibitory compounds against the human proinflammatory cytokines- IL-6, IL-1ß, TNF-α, and IFN-γ, that are associated with the adverse cytokine storm in COVID-19 patients. Our analysis returned an encouraging list of promising inhibitory candidates that includes: abietatriene against the spike proteins of the SARS-CoV-2 wild-type and the variants of concern; taraxerol against the human ACE2, CTSL and TNF-α; ß-amyrin against the human TMPRSS2; cynaroside against the human AAK1 and IL-1ß; and friedelin against the human IL-6 and IFN-γ. Our findings provide substantial evidence for the inhibitory potential of these compounds and encourage further in vitro and in vivo studies to validate their use as safe and effective therapeutics against COVID-19.