Perumal Arumugam Desingu

A rapid method of accurate detection and differentiation of Newcastle disease virus pathotypes by demonstrating multiple bands in degenerate primer based nested RT-PCR.

A rapid and accurate method of detection and differentiation of virulent and avirulent Newcastle disease virus (NDV) pathotypes was developed. The NDV detection was carried out for different domestic avian field isolates and pigeon paramyxo virus-1 (25 field isolates and 9 vaccine strains) by using APMV-I fusion (F) gene Class II specific external primer A and B (535bp), internal primer C and D (238bp) based reverses transcriptase PCR (RT-PCR). The internal degenerative reverse primer D is specific for F gene cleavage position of virulent strain of NDV. The nested RT-PCR products of avirulent strains showed two bands (535bp and 424bp) while virulent strains showed four bands (535bp, 424bp, 349bp and 238bp) on agar gel electrophoresis. This is the first report regarding development and use of degenerate primer based nested RT-PCR for accurate detection and differentiation of NDV pathotypes by demonstrating multiple PCR band patterns. Being a rapid, simple, and economical test, the developed method could serve as a valuable alternate diagnostic tool for characterizing NDV isolates and carrying out molecular epidemiological surveillance studies for this important pathogen of poultry.

Ebola virus – epidemiology, diagnosis, and control: threat to humans, lessons learnt, and preparedness plans – an update on its 40 year's journey

ABSTRACT Ebola virus (EBOV) is an extremely contagious pathogen and causes lethal hemorrhagic fever disease in man and animals. The recently occurred Ebola virus disease (EVD) outbreaks in the West African countries have categorized it as an international health concern. For the virus maintenance and transmission, the non-human primates and reservoir hosts like fruit bats have played a vital role. For curbing the disease timely, we need effective therapeutics/prophylactics, however, in the absence of any approved vaccine, timely diagnosis and monitoring of EBOV remains of utmost importance. The technologically advanced vaccines like a viral-vectored vaccine, DNA vaccine and virus-like particles are underway for testing against EBOV. In the absence of any effective control measure, the adaptation of high standards of biosecurity measures, strict sanitary and hygienic practices, strengthening of surveillance and monitoring systems, imposing appropriate quarantine checks and vigilance on trade, transport, and movement of visitors from EVD endemic countries remains the answer of choice for tackling the EBOV spread. Herein, we converse with the current scenario of EBOV giving due emphasis on animal and veterinary perspectives along with advances in diagnosis and control strategies to be adopted, lessons learned from the recent outbreaks and the global preparedness plans. To retrieve the evolutionary information, we have analyzed a total of 56 genome sequences of various EBOV species submitted between 1976 and 2016 in public databases.

Spatial molecular epidemiology of carbapenem‐resistant and New Delhi metallo beta‐lactamase (blaNDM)‐producing Escherichia coli in the piglets of organized farms in India

A cross‐sectional study was conducted in 10 government‐organized pig farms between 2014 and 2016 representing seven states of India to understand the epidemiology of carbapenem resistance in the Escherichia coli.

Molecular characterization, isolation, pathology and pathotyping of peafowl (Pavo cristatus) origin Newcastle disease virus isolates recovered from disease outbreaks in three states of India

ABSTRACT Disease outbreak investigations were carried out in three states of Northern India namely Haryana (Rewari), Uttar Pradesh (Noida) and Delhi, where a total of 110 Indian peafowls (Pavo cristatus) showed sudden onset of nervous signs and died within a period of two weeks during June, 2012. The F (fusion) gene-based RT-PCR detection of Newcastle disease virus (NDV) in affected tissues confirmed the presence of the virus. Three NDV isolates were selected (one from each area under investigation) and further characterized. They were found to be of virulent pathotype (velogenic NDV) based on both pathogenicity assays (MDT, ICPI and IVPI) and partial F gene sequence analysis. Additionally, the phylogenetic analysis revealed that the isolates belonged to the genotype VIIi and XIII of class II avian Paramyxovirus serotype1 (APMV-1) and related closely to new emerging sub-genotypes. This is the first report regarding the presence of the fifth panzootic vNDV genotype VIIi from India. In this scenario, extensive epidemiological studies are suggested for surveillance of NDV genotypes in wild birds and poultry flocks of the country along with adopting suitable prevention and control measures.

Development of slide ELISA (SELISA) for detection of four poultry viral pathogens by direct heat fixation of viruses on glass slides.

The development of an easy and simpler method of slide enzyme-linked immunosorbent assay (SELISA) for the diagnosis of four economically important poultry viruses viz., Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV) and egg drop syndrome 76 virus (EDS 76) and the use of SELISA for semi quantitation of NDV are described. The positive signals for viral aggregates were detected under light microscope. This is the first report regarding the development of SELISA based on heat fixation for the diagnosis of viral pathogens.

Impact of Cryopreservation on Caprine Fetal Adnexa Derived Stem Cells and Its Evaluation for Growth Kinetics, Phenotypic Characterization, and Wound Healing Potential in Xenogenic Rat Model

This study was conducted to know the impact of cryopreservation on caprine fetal adnexa derived mesenchymal stem cells (MSCs) on the basic stem cell characteristics. Gravid caprine uteri (2–3 months) were collected from local abattoir to derive (amniotic fluid [cAF], amniotic sac [cAS], Whartons jelly [cWJ], and cord blood [cCB]) MSCs and expanded in vitro. Cells were cryopreserved at 3rd passage (P3) using 10% DMSO. Post‐thaw viability and cellular properties were assessed. Cells were expanded to determine growth kinetics, tri‐lineage differentiation, localization, and molecular expression of MSCs and pluripotency markers; thereafter, these cells were transplanted in the full‐thickness (2u2009×u20092cm2) rat skin wound to determine their wound healing potential. The post‐thaw (pt) growth kinetics study suggested that cWJ MSCs expanded more rapidly with faster population doubling time (PDT) than that of other fetal adnexa MSCs. The relative mRNA expression of surface antigens (CD73, CD90, and CD 105) and pluripotency markers (Oct4, KLF, and cMyc) was higher in cWJ MSCs in comparison to cAS, cAF, and cCB MSCs post‐thaw. The percent wound contraction on 7th day was more than 50% for all the MSC‐treated groups (pre and post‐thaw), against 39.55% in the control group. On day 28th, 99% and more wound contraction was observed in cAF, cAF‐pt, cAS‐pt, cWJ, cWJ‐pt, and cCB, MSCs with better scores for epithelization, neovascularization, and collagen characteristics at a non‐significant level. It is concluded that these MSCs could be successfully cryopreserved without altering their stemness and wound healing properties. J. Cell. Physiol. 232: 2186–2200, 2017.

Phylogenetic analysis of Newcastle disease virus isolates occurring in India during 1989-2013.

The study details characterization of Newcastle disease virus (NDV) isolates recovered from commercial poultry flocks (chicken) and wild birds (crane) of India during the time period from 1989 to 2013. Phylogenetic analysis revealed that most of the NDV isolates belongs to class II, genotype XIIIa and a chicken isolate (108/BAREILLY/AD-IVRI/91) was of genotype VI, where it showed diversity of 3xa0% from the other viruses belonging to same genotype. Another chicken isolate (75/RAMPUR/AD-IVRI/89) grouped in genotype III and showed 4xa0% diversity with viruses of genotype III. The crane origin NDV identified as of genotype II corresponding to the vaccine virus. This appears to be the first report about existence of genotype XIIIa and its ancestral viruses are circulating in India for the last two decades in different species of birds. Furthermore, genetically distinct viruses belonging to genotypes II, III and VI are also circulating in India.

Clinicopathological characterization of experimental infection in chickens with sub-genotype VIIi Newcastle disease virus isolated from peafowl

Newcastle disease (ND) is an economically important viral disease distressing poultry industry across the globe. Herein, we report the clinicopathology of sub-genotype VIIi Newcastle disease virus (NDV) isolated from peafowl in chickens. The virus isolate produced systemic infection with prominent tropism in visceral organs in chicken, confirmed on the basis of gross and microscopic lesions, and immunohistochemistry findings. The experimentally infected chickens exhibited 100% mortality with severe hemorrhagic lesions in the proventriculus and intestine, especially marked lymphocytolysis in spleen and bursa. The virus could be re-isolated from the cloacal swabs of infected chickens during 4th to 6th dpi (on 6th dpi all birds died), and all were tested positive in conventional RT-PCR. This is the first report on clinicopathology of NDV isolated from peafowl and/or sub-genotype VIIi NDV in experimentally infected chickens. Explorative epidemiological and molecular studies are suggested to screen wild peafowls and poultry flocks of the country for establishing the occurrence of this sub-genotype and opting for appropriate prevention and control strategies.

A sensitive haemadsorption technique based RT-PCR for concentration and detection of Newcastle disease virus from clinical samples and allantoic fluid

The present study describes the exploitation of haemadsorption (HAd) property of the Newcastle disease virus (NDV) for the development of a novel sensitive HAd technique based RT-PCR for detection of NDV from clinical samples of virus infected experimental birds. The NDV propagated allantoic fluid from the infected embryonated chicken eggs or supernatant of the processed clinical samples (tissue triturate, cloaca and tracheal swabs) from the experimentally infected birds were added with chicken red blood cells (RBC) to adsorb the virus on RBC’s surface. The virus adsorbed RBCs were subjected to trizol method of RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) for detection of NDV. The HAd based RNA extraction showed better yield of 700–900xa0ng RNA and when subjected to RT-PCR detection revealed a 100 times higher sensitivity than the conventional RNA extraction and RT-PCR detection system. This could be an alternate technique which can be exploited in low NDV load situations in clinical samples.

Reply to "May Newly Defined Genotypes XVII and XVIII of Newcastle Disease Virus in Poultry from West and Central Africa Be Considered a Single Genotype (XVII)?".

We read with great interest the article by Snoeck et al. (1), where the authors reported great genetic diversity among Newcastle disease virus (NDV) strains circulating in the poultry population of West and Central Africa along with two newly defined genotypes, genotypes XVII and XVIII, based on fusion (F) gene sequence analysis. More than 90% identity was noticed between the sequences belonging to genotypes XVII and XVIII in NCBI BLAST analysis. We analyzed the report and determined the mean distance within a genotype, mean distance between the genotypes, and net mean distances between genotypes XVII (n 56) and XVIII (n 15) using complete F gene sequences of NDVs, which were used in the report by Snoeck et al. (1). The mean evolutionary distance was calculated in MEGA6 software as follows. The number of nucleotide base substitutions per site from an estimation of the net average from comparisons between groups of sequences was analyzed using the maximum composite likelihood model (2). The rate of variation among sites was modeled with a gamma distribution (shape parameter 1). The analysis involved 71 gene sequences available in NCBI GenBank. The codon positions included were 1st plus 2nd plus 3rd plus noncoding. The nucleotide positions containing gaps and missing data were eliminated. The standard error estimate was obtained by a bootstrap procedure (500 replicates) (2). The analysis results revealed that the mean distance within genotype XVII is 0.031 (3.1%) with a standard error of 0.002 and within genotype XVII is 0.044 (4.4%) with a standard error of 0.004. The mean distance between genotypes XVII and XVIII was determined to be 0.108 (10.8%) with a standard error of 0.008, whereas the net mean distance between genotypes XVII and XVIII was determined to be 0.071 (7.1%) with a standard error of 0.007. The net mean distances between the genotypes showed agreement with the sequence identity determined in the NCBI BLAST analysis. A minimum of a 10% mean evolutionary distance between NDV genotypes has been recommended to define a new genotype (3). We conclude that newly defined genotypes XVII and XVIII of NDV (1) may be considered a single genotype, genotype XVII.