A Rapid and Novel Multiplex PCR Assay for Simultaneous Detection of Multiple Viruses Associated with Bovine Gastroenteritis.

Bovine viral diarrheal virus (BVDV) and bovine coronavirus (BCoV) are prevalent viral infections in buffalo calves that result in significant economic losses globally. However, Bovine picobirnavirus (BPBV) Group I and II has been an emerging causes of gastrointestinal infection as has been detected with mixed of BVDV as well as BCV. To combat economic losses and viral infection, a rapid and innovative multiplex-PCR assay (M-PCR) was developed to simultaneously identify BVDV, BCV, and BPBV. The assay employed three primer pairs, each specific to a particular virus. Notably, the primers for BCV and BVDV, targeting the transmembrane (M) Mebus gene and 5'UTR genes, respectively, were self-designed. To validate the assay, 300 samples of buffalo calf feces were subjected to the standardized multiplex PCR. The results demonstrated that 54 (18%) samples tested positive for multiple viruses, with 16.67% samples infected by BVDV, 0.9% by BCoV, and 0.13% by BPBV, as detected by the M-PCR assay. In summary, this developed assay is characterized by high specificity, sensitivity, throughput, and speed, enabling the simultaneous detection of the three viruses in a single reaction tube. Consequently, it holds potential for epidemiological investigations. It is worth noting that, to the best of our knowledge, this is the first reported multiplex assay for the worldwide detection of BVDV, BCoV, and BPBV. This novel assay promises to aid in the detection of mixed infections in the gastrointestinal tract.

Synergistic Effect of Zinc-Chitosan Nanoparticles and Hydroxychloroquine to Inhibit Buffalo Coronavirus.

Zinc ions can hinder the synthesis of proteins required for accomplishing several stages of the viral life cycle. The intracellular zinc concentration can be increased by using zinc ionophores which transport zinc ions into the cells and hinder viral replication. (Hydroxy)chloroquine is an example of a zinc ionophore, but both zinc and (hydroxy)chloroquine can be toxic to the host organism. The nanocarriers may serve as camouflage to evade the adverse effects of drugs, chemicals, and nanoparticles on the host. We formulated ZnO nanoparticles with flower-like morphology (ZnONFs). It was further decorated with chitosan along with hydroxychloroquine (as a zinc ionophore) (CHCZnO NPs). We have chosen the cationic polymer chitosan since it is biocompatible, biodegradable and binds easily with the cells, and enhances the transport of drugs across cell membranes. The formulation was investigated for size, shape, surface charge, and interaction of chemicals used. We evaluated the formulations for cytotoxicity, and biocompatibility in embryonated chicks and their efficacy against bovine coronavirus (BCoV) isolated from a buffalo calf, and pneumo-enteric coronaviruses isolated from a buffalo calf with promising results in comparison to ZnONFs/hydroxychloroquine alone. Furthermore, we elucidate the mechanism underlying the lysosomotropic effect of various formulations on Vero cells infected with the buffalo coronavirus.

Multiplex PCR for rapid differential diagnosis of co-prevalent species of Theileria (Theileria annulata and Theileria orientalis) in cattle.

Theileriosis is a tick-borne disease that causes enormous losses in the dairy industry. There are several species of Theileria that can infect bovines. Generally, more than one species are prevalent in any geographical area; thus, chances of co-infections are high. Differentiation of these species may not be possible by microscopic examination or serological tests. Therefore, in this study, a multiplex PCR assay was standardized and evaluated for rapid and simultaneous differential detection of two species of Theileria viz., Theileria annulata and Theileria orientalis. Species-specific primers were designed to target the merozoite piroplasm surface antigen gene (TAMS1) of T. annulata and the major piroplasm surface protein gene of T. orientalis, yielding specific amplicon of 229 bp and 466 bp, respectively. The sensitivity of multiplex PCR was 102 and 103 copies for T. annulata and T. orientalis, respectively. The simplex and multiplex PCRs were specific and showed no cross-reactivity with other hemoprotozoa for either primer. For comparative evaluation, blood samples from 216 cattle were tested by simplex and multiplex PCR for both species. Using multiplex PCR, 131 animals were found infected for theileriosis, of which 112 were infected with T. annulata, five were infected with T. orientalis, and 14 had mixed infections. This is the first report of T. orientalis from Haryana, India. Representative sequences of T. annulata (ON248941) and T. orientalis (ON248942) were submitted in GenBank. The standardized multiplex PCR assay used in this study was specific, sensitive, for the screening of field samples.

Nanotechnology: a contemporary therapeutic approach in combating infections from multidrug-resistant bacteria.

In the 20th century, the discovery of antibiotics played an essential role in the fight against infectious diseases, including meningitis, typhoid fever, pneumonia and Mycobacterium tuberculosis. The development of multidrug resistance in microflora due to improper antibiotic use created significant public health issues. Antibiotic resistance has increased at an alarming rate in the past few decades. Multidrug-resistant bacteria (superbugs) such as methicillin-resistant Staphylococcus aureus (MRSA) as well as drug-resistant tuberculosis pose serious health implications. Despite the continuous increase in resistant microbes, the discovery of novel antibiotics is constrained by the cost and complexities of discovery of drugs. The nanotechnology has given new hope in combating this problem. In the present review, recent developments in therapeutics utilizing nanotechnology for novel antimicrobial drug development are discussed. The nanoparticles of silver, gold and zinc oxide have proved to be efficient antimicrobial agents against multidrug-resistant Klebsiella, Pseudomonas, Escherichia Coli and MRSA. Using nanostructures as carriers for antimicrobial agents provides better bioavailability, less chances of sub-therapeutic drug accumulation and less drug-related toxicity. Nanophotothermal therapy using fullerene and antibody functionalized nanostructures are other strategies that can prove to be helpful.

Characterization of sulfur-oxidizing bacteria isolated from mustard (Brassica juncea L.) rhizosphere having the capability of improving sulfur and nitrogen uptake.

AIMS: The present investigation was carried out to isolate, screen and characterize potential sulfur-oxidizing bacteria (SOB) isolated from mustard field's soil. METHODS AND RESULTS: A total of 130 bacteria were isolated and after screening five maximum sulfate-producing isolates were optimized for culture conditions. The incubation time of 48 h was found optimum for all bacterial isolates and 30°C was the best temperature for the growth of SSD11, SSR1 and SSG8 whereas 35°C for SSF17. The pH 8 was found best for all four isolates except SSF17 (6 pH). Media having glucose as a carbon source and ammonium sulphate as an N-source were producing maximum sulphate. The isolates SSF17, SSR1 and SSG8 were identified as Burkholderia cepacia (accession no. MT559819), Enterobacter cloacae (accession no. MT559820) and Klebsiella oxytoca (accession no. MT372097), respectively, on the basis of morphological, biochemical and molecular characterization. The isolates were also found to increase N and S uptake efficiently in both wheat and mustard crops. CONCLUSION: This study strongly concludes that SOB isolated from the mustard field can oxidize sulfur in vitro and in vivo conditions. The three best isolates come out of the study were identified as Burkholderia, Enterobacter and Klebsiella strains. Also, inoculation of SOB increased the uptake of S and N nutrient in mustard and wheat crops and thus may be proved as an important plant growth-promoting bacteria having the biofertilization capability. SIGNIFICANCE AND IMPACT OF THE STUDY: As we know, our soil is continuously deteriorating day by day due to excessive utilization and immoderate use of chemical fertilizers. The SOB could minimize the application of chemical fertilizers thus reducing environmental deterioration by improving soil health in sustainable agricultural practices.

Molecular and Physiological Mechanisms to Mitigate Abiotic Stress Conditions in Plants.

Agriculture production faces many abiotic stresses, mainly drought, salinity, low and high temperature. These abiotic stresses inhibit plants' genetic potential, which is the cause of huge reduction in crop productivity, decrease potent yields for important crop plants by more than 50% and imbalance agriculture's sustainability. They lead to changes in the physio-morphological, molecular, and biochemical nature of the plants and change plants' regular metabolism, which makes them a leading cause of losses in crop productivity. These changes in plant systems also help to mitigate abiotic stress conditions. To initiate the signal during stress conditions, sensor molecules of the plant perceive the stress signal from the outside and commence a signaling cascade to send a message and stimulate nuclear transcription factors to provoke specific gene expression. To mitigate the abiotic stress, plants contain several methods of avoidance, adaption, and acclimation. In addition to these, to manage stress conditions, plants possess several tolerance mechanisms which involve ion transporters, osmoprotectants, proteins, and other factors associated with transcriptional control, and signaling cascades are stimulated to offset abiotic stress-associated biochemical and molecular changes. Plant growth and survival depends on the ability to respond to the stress stimulus, produce the signal, and start suitable biochemical and physiological changes. Various important factors, such as the biochemical, physiological, and molecular mechanisms of plants, including the use of microbiomes and nanotechnology to combat abiotic stresses, are highlighted in this article.

Metal/metal oxide nanoparticles: Toxicity concerns associated with their physical state and remediation for biomedical applications.

Metal/metal oxide nanoparticles show promise for various applications, including diagnosis, treatment, theranostics, sensors, cosmetics, etc. Their altered chemical, optical, magnetic, and structural properties have differential toxicity profiles. Depending upon their physical state, these NPs can also change their properties due to alteration in pH, interaction with proteins, lipids, blood cells, and genetic material. Metallic nanomaterials (comprised of a single metal element) tend to be relatively stable and do not readily undergo dissolution. Contrarily, metal oxide and metal alloy-based nanomaterials tend to exhibit a lower degree of stability and are more susceptible to dissolution and ion release when introduced to a biological milieu, leading to reactive oxygen species production and oxidative stress to cells. Since NPs have considerable mobility in various biological tissues, the investigation related to their adverse effects is a critical issue and required to be appropriately addressed before their biomedical applications. Short and long-term toxicity assessment of metal/metal oxide nanoparticles or their nano-formulations is of paramount importance to ensure the global biome's safety; otherwise, to face a fiasco. This article provides a comprehensive introspection regarding the effects of metal/metal oxides' physical state, their surface properties, the possible mechanism of actions along with the potential future strategy for remediation of their toxic effects.

The Importance of Nanomedicine in Prophylactic and Theranostic Intervention of Bacterial Zoonoses and Reverse Zoonoses in the Era of Microbial Resistance.

Emergence of multidrug resistance (MDR), extensively drug resistance (XDR) and pandrug resistance (PDR) strains of bacteria in communicable diseases of zoonotic and reverse zoonotic importance is the major hurdle of one health concept. Increasing level of resistance against antibiotics among bacterial population throughout the world, slow pace of new antibacterial drug discovery and enhanced pace of resistance development by pathogenic bacteria possess major challenges for human and animal health as well as life in future. Alternative management strategy in terms of improved prophylactic vaccine; early, easy and effective diagnostics and therapeutic drugs against those resistant bacteria is the need of the hour. In this context nanomedicine can fit into the multifaceted demands as an effective prophylactic and theranostic alternative to control the communicable diseases in a cost effective manner in the era of microbial resistance. The current review is focused towards delineating the application of nanomaterials as vaccine or drug delivery system, diagnostics and directly acting antimicrobial therapeutic agents in combating the important zoonotic and reverse zoonotic bacterial diseases in recent scenario along with their potential benefits, limitations and future prospects to formulate successful eradication strategies.

Development of Multiwalled Carbon Nanotube-Based Immunobiosensor for the Detection of Bluetongue Virus.

Bluetongue (BT) disease is a noncontagious disease of domestic and wild ruminants (mainly sheep, cattle, deer) caused by the bluetongue virus (BTV) which is an orbivirus of the Reoviridae family and transmitted by vector Culicoides biting midges. It is a reportable disease of considerable socioeconomic concern and of major importance for the international trade of animals and animal products. Conventional diagnostic methods, such as virus propagation and isolation, immunoassays and also various molecular methods have been developed for the detection of the BTV. Here, we present a novel, rapid and pen-side test for the detection of BTV using multiwalled carbon nanotube (MWCNTs) based immunosensor. Though it is not reported yet. The MWCNTs were prepared, characterized and functionalized with carboxyl group. Viral antibodies were conjugated successfully with functionalized MWCNTs and coated on screen printed carbon electrode (SPCE). These SPCE were evaluated by using electrochemical sensor with an antigen specific to BTV antibodies, resulted in the self-assembled layer of antigen-antibody on the surface of SPCE. The approach described in the present study is a prototype for the development of simple and economic diagnostic tool which will provide the routine screening of BT disease at the door of farmers, thereby increasing the income of farmers by decreasing the cost of diagnosis.

A Novel Amperometric Genosensor for Rapid Detection of Canine Parvovirus in Feces.

A highly sensitive novel amperometric genosensor has been developed for rapid detection of canine parvovirus (CPV) DNA in fecal swabs of naturally infected dogs. The genosensor is based on a single stranded 5°-thiolated (SH) DNA probe complementary to VP1/VP2 gene of canine parvo vaccine strain, immobilized covalently on a polycrystalline gold (Au) electrode. The genosensor has been characterized by scanning electron microscopy (SEM), Fourier Transform Infra-Red Spectroscopy (FTIR), cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectra (EIS). The ssDNA-SH/Au electrode was hybridized with single stranded target DNA (ss T-DNA) in the sample. This hybridization was detected by reduction in current, generated by interaction of methylene blue (MB) with free guanine of ssDNA. The current response of genosensor was determined by CV, DPV and EIS. The sensor detected single stranded genomic DNA (ss g-DNA) isolated from vaccine strain of CPV in the range, 1.0-12.0 ng/µl at 25 °C for 10 min. Subsequently, the genobiosensor was applied for detection of CPV viral DNA in fecal swabs of naturally infected dogs. The limit of detection (LOD) of the sensor was 1.0 ng/µl of fecal viral DNA. To the best of our knowledge, this is the first report on development and application of amperometric biosensor for rapid, sensitive, specific point of care detection of viral DNA of CPV in feces.

An Insight into Nanomedicinal Approaches to Combat Viral Zoonoses.

BACKGROUND: Emerging viral zoonotic diseases are one of the major obstacles to secure the "One Health" concept under the current scenario. Current prophylactic, diagnostic and therapeutic approaches often associated with certain limitations and thus proved to be insufficient for customizing rapid and efficient combating strategy against the highly transmissible pathogenic infectious agents leading to the disastrous socio-economic outcome. Moreover, most of the viral zoonoses originate from the wildlife and poor knowledge about the global virome database renders it difficult to predict future outbreaks. Thus, alternative management strategy in terms of improved prophylactic vaccines and their delivery systems; rapid and efficient diagnostics and effective targeted therapeutics are the need of the hour. METHODS: Structured literature search has been performed with specific keywords in bibliographic databases for the accumulation of information regarding current nanomedicine interventions along with standard books for basic virology inputs. RESULTS: Multi-arrayed applications of nanomedicine have proved to be an effective alternative in all the aspects regarding the prevention, diagnosis, and control of zoonotic viral diseases. The current review is focused to outline the applications of nanomaterials as anti-viral vaccines or vaccine/drug delivery systems, diagnostics and directly acting therapeutic agents in combating the important zoonotic viral diseases in the recent scenario along with their potential benefits, challenges and prospects to design successful control strategies. CONCLUSION: This review provides significant introspection towards the multi-arrayed applications of nanomedicine to combat several important zoonotic viral diseases.

Application of Polymeric Nano-Materials in Management of Inflammatory Bowel Disease.

Inflammatory Bowel Disease (IBD) is an umbrella term used to describe disorders that involve Crohn's disease (CD), ulcerative colitis (UC) and pouchitis. The disease occurrence is more prevalent in the working group population which not only hampers the well being of an individual but also has negative economical impact on society. The current drug regime used therapy is very costly owing to the chronic nature of the disease leading to several side effects. The condition gets more aggravated due to the lower concentration of drug at the desired site. Therefore, in the present scenario, a therapy is needed which can maximize efficacy, adhere to quality of life, minimize toxicity and doses, be helpful in maintaining and stimulating physical growth of mucosa with minimum disease complications. In this aspect, nanotechnology intervention is one promising field as it can act as a carrier to reduce toxicity, doses and frequency which in turn help in faster recovery. Moreover, nanomedicine and nanodiagnostic techniques will further open a new window for treatment in understanding pathogenesis along with better diagnosis which is poorly understood till now. Therefore the present review is more focused on recent advancements in IBD in the application of nanotechnology.

Organic Polymer and Metal Nano-particle Based Composites for Improvement of the Analytical Performance of Electrochemical Biosensors.

Metal nanoparticles (NPs) are described in the nanoscale and made from either pure metals or their compounds such as oxides. Metallic NPs have certain indistinct functional groups due to which these can bind with any type of ligand, antibody and drugs. Organic polymers, which conduct electricity, are called conducting polymers (intrinsically conducting polymers). They behave like semiconductors by exhibiting metallic conductivity. Process-ability is the major advantage of conducting polymers. Nanocomposite is a novel material having nano-fillers scattered in a matrix with morphology and interfacial characteristics of nano-composites including their individual property that influence their characteristics. Conducting polymers and NP composites can enhance the rate of electron transport between the current collector material (electrode) and the electrolyte; therefore they have been employed in the construction of improved electrochemical sensors such as amperometric, catalytic and potentiodynamic affinity sensors.

Nano-antimicrobials: A New Paradigm for Combating Mycobacterial Resistance.

BACKGROUND: Mycobacterium group contains several pathogenic bacteria including M. tuberculosis where the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) is alarming for human and animal health around the world. The condition has further aggravated due to the speed of discovery of the newer drugs has been outpaced by the rate of resistance developed in microorganisms, thus requiring alternative combat strategies. For this purpose, nano-antimicrobials have emerged as a potential option. OBJECTIVE: The current review is focused on providing a detailed account of nanocarriers like liposome, micelles, dendrimers, solid lipid NPs, niosomes, polymeric nanoparticles, nano-suspensions, nano-emulsion, mesoporous silica and alginate-based drug delivery systems along with the recent updates on developments regarding nanoparticle-based therapeutics, vaccines and diagnostic methods developed or under pipeline with their potential benefits and limitations to combat mycobacterial diseases for their successful eradication from the world in future. RESULTS: Distinct morphology and the underlying mechanism of pathogenesis and resistance development in this group of organisms urge improved and novel methods for the early and efficient diagnosis, treatment and vaccination to eradicate the disease. Recent developments in nanotechnology have the potential to meet both the aspects: nano-materials are proven components of several efficient targeted drug delivery systems and the typical physicochemical properties of several nano-formulations have shown to possess distinct bacteriocidal properties. Along with the therapeutic aspects, nano-vaccines and theranostic applications of nano-formulations have grown in popularity in recent times as an effective alternative means to combat different microbial superbugs. CONCLUSION: Nanomedicine holds a bright prospect to perform a key role in global tuberculosis elimination program.

Salicylic acid induces amelioration of chromium toxicity and affects antioxidant enzyme activity in Sorghum bicolor L.

AIM: Chromium (Cr(VI)) would inflict serious morphological, metabolic, and physiological anomalies in plants ranging from chlorosis of shoot to lipid peroxidation and protein degradation. Cr(VI) toxicity is often associated with oxidative stress, caused by the excessive formation of reactive oxygen species (ROS). In response, plants are equipped with a repertoire of mechanisms to counteract heavy metal (HM) toxicity. Salicylic acid (SA) plays a key role in the signal transduction pathways of various stress responses, demonstrating the protective effect of SA against abiotic stress factors. So, the present investigation was carried out to study the amelioration of pernicious effects of different concentration of Cr(VI) (0.0, 2.0, and 4.0 mg Cr(VI) kg-1 soil in the form of potassium dichromate) by treatments of salicylic acid solution viz. pretreatment and foliar spray via antioxidative enzymes and their metabolites. RESULTS: With different treatments of salicylic acid solution, the reinstatement from ill effects of Cr(VI) toxicity was contemplated but the most conspicuous effect was observed when salicylic acid solution was supplied through the foliar spray (0.50 mM). This was accompanied with an increase in ascorbate peroxidase activity and hydrogen peroxide content and decrease in peroxidase activity and ascorbic acid content. SIGNIFICANCE OF THE STUDY: This study suggests that salicylic acid when applied through pre-treatment of seeds or through a foliar spray can be used to ameliorate the toxic effects of chromium (VI). Salicylic acid has the great potential for reducing the toxicity of heavy metals without negatively impacting the growth of the plants.

Nanotherapeutics: An insight into healthcare and multi-dimensional applications in medical sector of the modern world.

In recent years nanotechnology has revolutionized the healthcare strategies and envisioned to have a tremendous impact to offer better health facilities. In this context, medical nanotechnology involves design, fabrication, regulation, and application of therapeutic drugs and devices having a size in nano-range (1-100 nm). Owing to the revolutionary implications in drug delivery and gene therapy, nanotherapeutics has gained increasing research interest in the current medical sector of the modern world. The areas which anticipate benefits from nano-based drug delivery systems are cancer, diabetes, infectious diseases, neurodegenerative diseases, blood disorders and orthopedic problems. The development of nanotherapeutics with multi-functionalities has considerable potential to fill the lacunae existing in the present therapeutic domain. Nanomedicines in the field of cancer management have enhanced permeability and retention of drugs thereby effectively targeting the affected tissues. Polymeric conjugates of asparaginase, polymeric micelles of paclitaxel have been recmended for various types of cancer treatment .The advancement of nano therapeutics and diagnostics can provide the improved effectiveness of the drug with less or no toxicity concerns. Similarly, diagnostic imaging is having potential future applications with newer imaging elements at nano level. The newly emerging field of nanorobotics can provide new directions in the field of healthcare. In this article, an attempt has been made to highlight the novel nanotherapeutic potentialities of polymeric nanoparticles, nanoemulsion, solid lipid nanoparticle, nanostructured lipid carriers, dendrimers, nanocapsules and nanosponges based approaches. The useful applications of these nano-medicines in the field of cancer, nutrition, and health have been discussed in details. Regulatory and safety concerns along with the commercial status of nanosystems have also been presented. In summary, a successful translation of emerging nanotherapeutics into commercial products may lead to an expansion of biomedical science. Towards the end of the review, future perspectives of this important field have been introduced briefly.

Virus-Host Interactions: New Insights and Advances in Drug Development Against Viral Pathogens.

BACKGROUND: Viruses are the most devastating pathogens of almost all life forms including humans and animals. Viruses can replicate very fast and may affect any metabolic and physiological function of the host cell. Therefore, it has been a challenge to develop a universal and common treatment against viral pathogens, in contrast to bacterial pathogens. Virus-host interaction is a complex phenomenon and often is virus- and host cell-specific. Exciting new insights into the molecular pathogenesis and host-virus interactions have been gained over the past few decades. These advances have enabled researchers to design better antiviral drugs. METHODS: The literature related to various aspects of virus-host interactions: new insights and advances in drug development was collected from several scientific research related databases such as Science Direct, Google Scholar, Scopus, PubMed, AGRICOLA, and Medline, etc. Total number of 319 research papers was used to compile the information regarding drug development against viral pathogens. RESULTS: Clinical adequacy of antiviral drugs and their bioavailability are important parameters for effective treatment of viral infections. The problems associated with effective delivery of a drug in a safe and desired quantity have led to the search for (and design of) better drug delivery systems. In recent past, several new antiviral drugs have been developed, which have high therapeutic effectiveness against life-threatening viral diseases such as HIV, hepatitis B virus, herpes virus, dengue virus and influenza virus infections. The majority of recent advances in antiviral drug discovery were possible due to the developments in allied fields such as in vitro virus cultivation technology, molecular biology of viral-genome-encoded enzymes, complete-genome-sequence-based studies of viruses and identification of suitable targets for antiviral drugs in viral genomes. Recently, several novel drug delivery approaches including small interfering RNAs (siRNAs) have emerged to aid antiviral therapy. CONCLUSION: The present review is aimed at providing an update on research and development efforts being made to create effective antiviral chemotherapeutic agents and approaches to their delivery to appropriate targeted cells or tissues.