Sandip Kumar Khurana

Incidence of Burkholderia mallei infection among indigenous equines in India

Burkholderia mallei is the causative agent of glanders which is a highly contagious and fatal disease of equines. Considering the nature and severity of the disease in equines, and potential of transmission to human beings, glanders is recognised as a ‘notifiable’ disease in many countries. An increasing number of glanders outbreaks throughout the Asian continents, including India, have been noticed recently. In view of the recent re-emergence of the disease, the present study was undertaken to estimate the prevalence of glanders among indigenous equines from different parts of India. Serum samples were analysed by complement fixation test (CFT) and ELISA for the detection of B mallei specific antibodies. A total of 7794 equines, which included 4720 horses, 1881 donkeys and 1193 mules were sampled from April 2011 to December 2014 from 10 states of India. Serologically, 36 equines (pony=7, mules=10, horses=19) were found to be positive for glanders by CFT and indirect-ELISA. The highest number of cases were detected in Uttar Pradesh (n=31) followed by Himachal Pradesh (n=4) and Chhattisgarh (n=1). Isolation of B mallei was attempted from nasal and abscess swabs collected from seropositive equines. Four isolates of B mallei were cultured from nasal swabs of two mules and two ponies. Identity of the isolates was confirmed by PCR and sequencing of fliP gene fragment. The study revealed circulation of B mallei in northern India and the need for continued surveillance to support the eradication.

Serosurveillance for Japanese encephalitis virus infection among equines in India.

The seroprevalence of Japanese encephalitis virus (JEV) among equines was evaluated from January 2006 to December 2009 in 13 different states of India by hemagglutination inhibition (HI) test and virus neutralization test (VNT). Antibodies against JEV were detected in 327 out of 3,286 (10%) equines with a maximum prevalence reported in the state of Manipur (91.7%) followed by Gujarat (18.5%), Madhya Pradesh (14.4%), and Uttar Pradesh (11.6%). Evidence of JEV infection was observed in equines in Indore (Madhya Pradesh) where a 4-fold or higher rise in antibody titer was observed in 21 out of 34 horses in November 2007 to October 2006. In March 2008, seven of these horses had a subsequent 4-fold rise in JEV antibody titers while this titer decreased in nine animals. JEV-positive horse sera had a JEV/WNV (West Nile virus) ratio over 2.0 according to the HI and/or VNT. These results indicated that JEV is endemic among equines in India.

Development of EMA-2 recombinant antigen based enzyme-linked immunosorbent assay for seroprevalence studies of Theileria equi infection in Indian equine population

Equine piroplasmosis is a tick-transmitted protozoan disease caused by Theileria equi and/or Babesia caballi. In the present study, we expressed a 53kDa protein from the truncated EMA-2 gene of T. equi (Indian strain) and developed EMA-2ELISA using this expressed protein. This ELISA is able to detect T. equi-specific antibodies in experimentally infected animals as early as 9 days post-infection. The assay developed was validated with the OIE recommended competitive ELISA (cELISA) on 120 serum samples and significant agreement (kappa=0.93) was observed between results of both the ELISAs which indicates suitability of EMA-2ELISA for use in sero-diagnosis. Diagnostic sensitivity and specificity of EMA-2ELISA - as compared with cELISA - were 0.97 and 0.96, respectively. Analysis of 5651 equine serum samples - collected during 2007-2012 from 12 states of India representing eight agro-climatic zones - by EMA-2ELISA revealed 32.65% seroprevalence of T. equi in India. In conclusion, the EMA-2ELISA developed using the T. equi EMA-2 recombinant protein as antigen for detecting T. equi-specific antibodies has good diagnostic potential for sero-epidemiological surveys.

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.

Optimization and Validation of Indirect ELISA Using Truncated TssB Protein for the Serodiagnosis of Glanders amongst Equines

Objective. To express truncated TssB protein of Burkholderia mallei and to evaluate its diagnostic efficacy for serological detection of glanders among equines. Materials and Methods. In an attempt to develop recombinant protein based enzyme-linked immunosorbent assay (ELISA), N-terminal 200 amino acid sequences of B. mallei TssB protein—a type 6 secretory effector protein—were expressed in prokaryotic expression system. Diagnostic potential of recombinant TssB protein was evaluated in indirect ELISA using a panel of glanders positive (n = 49), negative (n = 30), and field serum samples (n = 1811). Cross-reactivity of the assay was assessed with equine disease control serum and human melioidosis positive serum. Results. In comparison to CFT, diagnostic sensitivity and specificity of ELISA were 99.7% and 100%, respectively. Conclusions. The indirect ELISA method using the truncated TssB offered safer and more rapid and efficient means of serodiagnosis of glanders in equines. These data highlight the use of TssB as potential diagnostic antigen for serological diagnosis of glanders.

Re-emergence of glanders in India - Report of Maharashtra state.

Glanders, a notifiable highly contagious disease primarily of equids, is a disease of high zoonotic importance. Caused by gram-negative bacillus, Burkholderia mallei, the disease was restricted to certain pockets of India with sporadic cases. Recently, a major outbreak of glanders occurred in India starting from Maharashtra. Following clinical signs and symptoms and laboratory investigations on serum, nasal swab and pus swab samples, it was confirmed as glanders among equines in Pune and Panchgani areas of Maharashtra. One pus sample and three nasal swabs yielded B. mallei isolates while 23 serum samples were found positive for glanders by complement fixation test (CFT). The disease was successfully controlled in the state by following strategies for prevention of spread of the disease to other areas in accordance with Glanders and Farcy Act, 1899. Follow up of the occurrence in Maharashtra revealed negative status based on testing and physical surveillance on more than 3,500 equines thereafter. Investigations indicated that the nidus of infection may be present elsewhere in North India.

Medicinal and Therapeutic Potential of Herbs and Plant Metabolites / Extracts Countering Viral Pathogens - Current Knowledge and Future Prospects

BACKGROUND Recently, there has been a remarkable progress in the field of antiviral herbal therapy owing to increasing concerns about the development of drug resistance and limited advances in the field of antiviral drug discovery. In almost all countries, medicinal plants have been used widely throughout history for the treatment of diseases and infections as traditional healing remedies due to their broad therapeutic spectrum and minimal or no side effects. As synthetic antiviral drugs are not available against most of the viral agents, hence all possible efforts have been focused on the search for new drugs and complementary/alternative medicines from different herbal formulations. METHODS We have retrieved the related information from the online published resources (Medline, PubMed, Pub- Med Central, Science Direct and other scientific databases); which were further analyzed and compiled. RESULTS Medicinal plants contain extractable biochemical and bioactive compounds, which can target certain viruses or can cure or prevent several viral diseases and infections. Despite their long history of use, the research and scientific evidences regarding the use of medicinal plants and natural products as prophylactics, therapeutics, and their health multiple beneficial applications have only gained momentum in past few decades. Many scientific studies have been undertaken, which range from the separation of active substances to the comprehension of the therapeutic mechanisms of antiviral herbs, their potent applications in the neutralization of viral pathogens and clinical trials. Consequently, hundreds of herbs and plant metabolites have been screened, identified, and tested for their antiviral activities; fortunately, some have shown significant medicinal activity in the amelioration or prevention of various viral diseases in both preclinical and clinical studies. CONCLUSION This review addresses the scientific significance of various herbal formulations of different medicinal plants and their extracts, which have shown promise or been proven effective for the treatment of diseases caused by various viral pathogens, including emerging and re-emerging viruses that infect humans, animals, poultry and fish.

Nanodiagnostics: a new frontier for veterinary and medical sciences.

Infectious diseases are one of the greatest threats to animal and human population living in the developing world. These diseases have capacity to instigate in a small area and then open out very fast to the rest of the world and causing a heavy pandemic situation, for example; avian influenza pandemic. Such diseases infect large masses of population and may lead to loss of lives and also incur huge economic losses. Therefore, the best way to control these diseases is by diagnosing it at a very primary level and taking necessary precautionary measures so as to avoid the spread. Since last few years, the diagnostic approach has changed from tedious molecular biological techniques, to easy and rapid diagnostic techniques. Nanotechnology has extended the molecular diagnostics limit to nanoscale. These developed techniques do not require sophisticated laboratories and expert personnel, and hence are a cheap diagnostic approach. These assays can also be performed at the field level where the patient is present and get the results there itself. Hence, they are also called as pen side test or lab on chip diagnostic assays. The biological tests using nanotechnology become quicker, more flexible and more sensitive. These techniques have greatly influenced the diagnostic approach in the veterinary as well as medical field. Especially in the developing countries such as India, where the laboratory services are not Upendra Lambe 1 , Minakshi P 1, *, Basanti Brar 1 , Madhusudan Guray 1 , Ikbal 1 , Koushlesh Ranjan 2 , Nitish Bansal 1 , Sandip Kumar Khurana 3 and Manimegalai J 1

CANINE PARVOVIRUS- AN INSIGHT INTO DIAGNOSTIC ASPECT

Canine parvovirus (CPV) leads to an acute disease, characterized by hemorrhagic gastroenteritis, vomiting and myocarditis in dogs. The disease can affect dogs of any age but is fatal in pups. CPV has undergone genetic variations that have led to emergence of various CPV-2 antigenic variants such as 2a, 2b and 2c with replacement of the original CPV-2 circulating in the dog population. CPV genome is made up of 5.2 Kb nucleotides. Viral protein VP2 plays a very important role in determining antigenicity and host range specificity of CPV. The antigenicity as well as host range of CPV is determined by virus specific VP2 protein. That’s why the mutations that affect the VP2 gene are the main source of different antigenic variants. It spreads rapidly in the wild population of canines as well as domestic animals, infected feces serve as a main source of infection because the virus is shed in large quantity in the feces particularly 4 - 7 days post infection. The present review is focused on various

Development of Lateral Flow Assay for Point-of-Care Diagnosis of Trypanosomosis in Equines

Abstract Trypanosomosis, commonly known as “Surra” in Indian subcontinent caused by Trypanosoma evansi, is a worldwide parasitic disease of wild and domestic animals. The disease is transmitted mechanically by biting and interrupted feeding of infected animals by hematophagous flies. The diagnostic methods available for this disease require high‐end instruments and skilled manpower and are time‐consuming. In the present investigation, we attempted an immunoassay based on gold nanoparticles for diagnosis of T. evansi infection in equines. Antibodies to horse IgG were raised in rabbits and conjugated to gold nanoparticles after purification with the protein‐A column. The soluble whole cell lysate antigen was prepared from purified T. evansi parasites and coated as test line on nitrocellulose membrane laminates, along with control line to detect anti‐Trypanosoma antibodies. The experimental and field serum samples were tested and evaluated to determine the sensitivity and specificity of the assay. The antigen‐specific antibodies were detected in experimental serum samples between 10 and 21 days post infection by the lateral flow assay (LFA). The LFA results showed 96.3% sensitivity and 93.9% specificity comparable with enzyme‐linked immunosorbent assay. The study suggests that the LFA can be used as a point‐of‐care diagnostic method in the field, which does not require any specialized instrument for the detection of T. evansi infection in animals. HighlightsA point‐of‐care diagnostic assay is developed for detection of Trypanosoma evansi infection in equines.The lateral flow assay successfully detected T. evansi–specific antibodies present in serum samples within 15 minutes.The lateral flow assay results showed 96.3% sensitivity and 93.9% specificity comparable with enzyme‐linked immunosorbent assay.