Novel pathological findings and immunohistochemical detection of FMDV antigens in the brain of calves naturally infected with foot-and-mouth disease.

Foot-and-mouth disease (FMD) is an extremely contagious and economically devastating viral disease of cloven-hoofed domestic and wildlife animals. The disease is endemic in India and other developing countries of the world. The disease is mainly characterized by the presence of vesicular lesions and "tigroid heart" in calves. The current report describes the novel pathologic findings along with the distribution of FMDV antigens in brain of young calves naturally infected with FMDV. The carcasses of 37 calves suspected to have died from FMD were presented for postmortem investigation. Out of 37 dead calves, 10 calves showed the clinical signs of neurological abnormalities like opisthotonos, muscle twitching and tremor in hind limbs, stiffening of the neck followed by death. Microscopically, the meninges were congested, hemorrhagic, and infiltrated with mononuclear cells. The various sub anatomical sites of the brain showed the varying degrees of vascular changes, perivascular cuffing, focal to diffuse gliosis as well as degeneration and neuronal necrosis, indicating the nonsuppurative encephalitis. The immunolabeling of FMDV antigen was demonstrated in the neurons, inflammatory cells, and microglial cells besides its typical locations. The neurons of the brain also showed strong immunopositivity for caspase-3, caspase-9 and p53 and negative for Bcl-2 and apoptosis-inducing factor (AIF) by both immunohistochemistry and western blotting indicating the role of caspase mediated intrinsic, and p53 dependent apoptotic pathway. Further, the TUNEL assay also confirmed the apoptosis in the neurons and glial cells of the brain of naturally infected calves. This study in calves establishes a basis for resemblance to other members of Picornaviruses, such as Enterovirus 71 and Coxsackievirus of humans and showing the neuropathological alterations along with the distribution of FMDV antigens associated with apoptosis in younger calves.

Development of novel iron-regulated Pasteurella multocida B: 2 bacterin and refinement of vaccine quality in terms of minimum variation in particle size and distribution vis-a-vis critical level of iron in media.

To enhance the qualitative bacterial biomass per unit of media and to overcome the limitations of the existing haemorrhagic septicaemia (HS) vaccines, a comprehensive study was undertaken encompassing the role of iron on the bacterial biomass of Pasteurella multocida B: 2 to vaccine development. Trypsin digested hydrochloric acid-treated sheep blood (THSB) as a novel iron rich supplement had been devised for the first time for augmenting the qualitative bacterial biomass per unit of media which was evident with growth kinetic study. The higher recovery of iron from THSB became evident via atomic absorbance spectrophotometry. The critical level of iron in the media as well as mode of iron supplementation showed a major impact on the outer membrane protein profile of P. multocida B:2 and variation in droplet size and particle-size distribution of formulated vaccine. Immune response study against iron-regulated bacterin adjuvanted with aluminum hydroxide gel in mouse model showed that 3% THSB supplementation of casein sucrose yeast (CSY) not only augmented the growth of P. multocida B:2 significantly but conferred highest pre-challenged ELISA IgG titer and protection against pasteurellosis. Thus, THSB supplementation of CSY can resolve existing up-scaling and immunogenic potential problems of HS vaccine production.

Expression kinetics of ISG15, IRF3, IFNγ, IL10, IL2 and IL4 genes vis-a-vis virus shedding, tissue tropism and antibody dynamics in PPRV vaccinated, challenged, infected sheep and goats.

Here, we studied the in vivo expression of Th1 (IL2 and IFN gamma) and Th2 (IL4 and IL10) - cytokines and antiviral molecules - IRF3 and ISG15 in peripheral blood mononuclear cells in relation to antigen and antibody dynamics under Peste des petits ruminants virus (PPRV) vaccination, infection and challenge in both sheep and goats. Vaccinated goats were seropositive by 9 days post vaccination (dpv) while in sheep idiosyncratic response was observed between 9 and 14 dpv for different animals. Expression of PPRV N gene was not detected in PBMCs of vaccinated and vaccinated challenged groups of both species, but was detected in unvaccinated infected PBMCs at 9 and 14 days post infection. The higher viral load at 9 dpi coincided with the peak clinical signs of the disease. The peak in viral replication at 9 dpi correlated with significant expression of antiviral molecules IRF3, ISG15 and IFN gamma in both the species. With the progression of disease, the decrease in N gene expression also correlated with the decrease in expression of IRF3, ISG15 and IFN gamma. In the unvaccinated infected animals ISG15, IRF3, IFN gamma and IL10 expression was higher than vaccinated animals. The IFN gamma expression predominated over IL4 in both vaccinated and infected animals with the infected exhibiting a stronger Th1 response. The persistent upregulation of this antiviral molecular signature - ISG15 and IRF3 even after 2 weeks post vaccination, presumably reflects the ongoing stimulation of innate immune cells.

Genome sequencing of an Indian peste des petits ruminants virus isolate, Izatnagar/94, and its implications for virus diversity, divergence and phylogeography.

Peste des petits ruminants is an important transboundary disease infecting small ruminants. Genome or gene sequence analysis enriches our knowledge about the evolution and transboundary nature of the causative agent of this disease, peste des petits ruminants virus (PPRV). Although analysis using whole genome sequences of pathogens leads to more precise phylogenetic relationships, when compared to individual genes or partial sequences, there is still a need to identify specific genes/genomic regions that can provide evolutionary assessments consistent with those predicted with full-length genome sequences. Here the virulent Izatnagar/94 PPRV isolate was assembled and compared to all available complete genome sequences (currently in the NCBI database) to estimate nucleotide diversity and to deduce evolutionary relationships between genes/genomic regions and the full length genomes. Our aim was to identify the preferred candidate gene for use as a phylogenetic marker, as well as to predict divergence time and explore PPRV phylogeography. Among all the PPRV genes, the H gene was identified to be the most diverse with the highest evolutionary relationship with the full genome sequences. Hence it is considered as the most preferred candidate gene for phylogenetic study with 93% identity set as a nucleotide cutoff. A whole genome nucleotide sequence cutoff value of 94% permitted specific differentiation of PPRV lineages. All the isolates examined in the study were found to have a most recent common ancestor in the late 19th or in the early 20th century with high posterior probability values. The Bayesian skyline plot revealed a decrease in genetic diversity among lineage IV isolates since the start of the vaccination program and the network analysis localized the ancestry of PPRV to Africa.

Changes in rumen protozoal community by condensed tannin fractions of different molecular weights from a Leucaena leucocephala hybrid in vitro.

AIMS: To evaluate the effects of condensed tannins (CTs) fractions of differing molecular weights (MWs) from a Leucaena leucocephala hybrid-Rendang on the rumen protozoal community in vitro. METHODS AND RESULTS: The effects of unfractionated CTs (F0) and CT fractions of different MWs (F1 > F2 > F3 > F4 > F5) on protozoal population and community were evaluated in vitro using rumen microbes and ground guinea grass as the substrate. Higher-MW CT fractions F1 and F2 significantly (P < 0·05) decrease the number of ciliate protozoa. The real-time PCR analysis showed that the total protozoa was significantly (P < 0·05) lower in F0 and all CTs with fractions F1 and F2 having the lowest value. High-throughput sequencing of the partial 18S rRNA gene showed that the genus Entodinium significantly (P < 0·05) decreased with increasing MWs of CT, whereas Anoplodinium-Diplodinium were significantly (P < 0·05) increased. Inclusion of the highest MW CT fraction F1 decreased the relative abundance of the minor genera such as Eudiplodinium and Polyplastron compared to the control and CT fractions F2-F5. CONCLUSION: CTs of differing MWs could reduce and alter the rumen protozoa population in vitro. This effect was more pronounced for higher-MW CTs. SIGNIFICANCE AND IMPACT OF THE STUDY: The high MW CTs should be considered as a feed supplement in the ruminant diet to reduce the protozoal population which are known to be associated with methanogens as a means to mitigate methane production in the rumen.

Correlation of inducible nitric oxide synthase (iNOS) inhibition with TNF-α, caspase-1, FasL and TLR-3 in pathogenesis of rabies in mouse model.

The role of inflammatory cytokines such as interleukin-1α/ß (IL-1α/ß), IL-6, IL-10, tumour necrosis factor-alpha (TNF-α), interferons, nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in pathogenesis of rabies is being actively pursued. Presently, levels of certain immune molecules in pathogenesis of rabies in mice have been investigated. CVS strain of rabies infection resulted in early increase in iNOS, TNF-α, caspase-1, Fas ligand (FasL) and toll-like receptor-3 (TLR-3) mRNA levels in brain, and nitric oxide levels in serum. The severity of clinical signs and microscopic lesions largely correlated with NO levels. Aminoguanidine (AG; iNOS inhibitor) decreased NO production with delay in development of clinical signs and increase in survival time. Prolonged survival time correlated with reduced viral load evident by real-time PCR, reduced fluorescent signals of rabies antigen in brain and reduced immunohistochemistry signals in neuronal cytoplasm. These parameters suggested that nitric oxide did influence the rabies virus replication. Inhibition of iNOS by AG administration led to decreased expression of TNF-α, caspase-1, FasL and TLR-3 mRNA levels suggesting that increase in NO levels in rabies virus infection possibly contributed to development of disease through inflammation, apoptosis and immune-evasive mechanisms.

Nano-quercetin mitigates triazophos-induced testicular toxicity in rats by suppressing oxidative stress and apoptosis.

The present study was designed to evaluate the testicular toxicity of triazophos in rats and to check the ameliorative effect of nano-quercetin against triazophos-induced toxicity. Nano-quercetin was synthesized from quercetin and characterized. Male Wistar rats were divided into seven groups. The control group received olive oil as a vehicle orally. The high-dose triazophos group and the low-dose triazophos group received 1/10th LD50 of triazophos (7.6 mg/kg) and 1/20th LD50 of triazophos (3.8 mg/kg), respectively. Two groups of animals were dosed with quercetin and nano-quercetin, both at 50 mg/kg body weight orally. The final two groups received high-dose triazophos with co-administration of quercetin and nano-quercetin, respectively. Triazophos disrupted the male endocrine axis by reducing the levels of steroidogenic enzymes 3-ß-HSD and 17-ß-HSD in testicular cells, further reducing FSH and testosterone. Also, triazophos increased the reactive oxygen species, induced lipid peroxidation, decreased the mitochondrial membrane potential, and elevated the number of apoptotic cells in rat testes. Nano-quercetin ameliorated the testicular oxidative stress and apoptotic and endocrine parameters more efficiently than quercetin. Besides, nano-quercetin alleviated the histopathological and biochemical alterations of triazophos. It is concluded that nano-quercetin has higher anti-oxidant efficacy than quercetin in protecting rats against triazophos-induced testicular toxicity.

Development of an efficient antitick natural formulation for the control of acaricide-resistant ticks on livestock.

Animal production has a key role in global economic development and food security. Ticks, specifically Rhipicephalus microplus cause substantial economic and health impacts on more than eighty percent of the world cattle population. Though synthetic acaricides play a major role in tick management, their injudicious usage has caused environmental pollution and also promote the establishment of multi-acaricide resistant tick populations which is a matter of great concern. To provide an effective tool for controlling these resistant ticks, the present work was aimed to develop safe and inexpensive antitick natural formulations. Our bioprospection studies of Ageratum conyzoides plant established it as a species potentially having strong acaricidal activity due to the presence of potent acaricidal phyto-chemicals. To develop a suitable antitick natural formulation, 41 samples/fractions/formulations were prepared from the dry powder of the whole aerial part of the A. conyzoides plant using different techniques and delivery matrices. The strongest antitick effect was recorded for formulation ACF6, which demonstrated 87 ± 6% mean mortality with 57 % inhibition of oviposition in treated female ticks. Ticks treated with the ACF6 formulation showed a significant (p < 0.001) reduction in cuticular protein (1.238 ± 0.01 mg/mL) as compared to control ticks (2.928 ± 0.01 mg/mL) but no significant difference in chitin content of treated ticks and control ticks was observed. The formulation was found safe in a rat model as no significant differences in biochemical and haematological parameters among treated and control rats were noted. Histopathological studies indicated no sign of hepatocellular necrosis and no significant changes in the weights of liver and spleen was recorded. The overall in vivo efficacy of the formulation was 85 % for experimentally infested cattle with direct mortality of more than 80 % within 96 h post-application. The lethal effect of the formulation was in the form of drying and dead ticks 1-2 d after application. The developed formulation has the potential to be adopted as an alternative tick control measure in an ecofriendly manner.

Molecular phylogeny of Capripoxviruses based on major immunodominant protein (P32) reveals circulation of host specific sheeppox and goatpox viruses in small ruminants of India.

Sheeppox and goatpox are highly contagious viral diseases of small ruminants causing severe economic losses to the livestock farmers. The disease is enzootic in Asia including India, Middle East and African countries. In the present study, a total of 28 isolates from twenty five sheeppox and goatpox disease outbreaks were phylogenetically analyzed based on P32 gene/protein along with homology modeling and docking using heparan sulfate and UDP-glucose. Three distinct lineage-specific clusters as per their host origin were recorded. Multiple sequence analysis of P32 gene revealed that genetically similar sheeppox virus (SPPV) and goatpox virus (GTPV) strains are circulating in India. Phylogenetically, Lumpy skin disease (LSDV) and SPPV had a closer genetic relationship than GTPV. Comparative sequence alignment indicated conservation of various motifs such as glycosaminoglycan (GAG), chemokine like motif (CX3C) and Asp-Glu-any other residue-Asp (D/ExD), as well as viral specific signature residues in SPPV and GTPV isolates. Structurally, P32 protein of SPPV and GTPV with mixed α helices and ß sheets resembled with crystal structure of homologue vaccinia virus H3L protein. Docking studies in P32 protein of SPPV and GTPV revealed conserved binding pattern with heparan sulfate which is involved in the virus attachment and varied glycosyltransferase fold with UDP-glucose. These findings may help in development of suitable vaccines/diagnostics and therapeutics against capripoxviruses.

Virological, immunological and pathological findings of transplacentally transmitted bluetongue virus serotype 1 in IFNAR1-blocked mice during early and mid gestation.

Transplacental transmission (TPT) of wild-type Indian BTV-1 had never been experimentally proved. This study was first time investigated TPT of Indian BTV-1 (isolated from aborted and stillborn goat fetal spleens). The sequential pathology, virological and immune cell kinetics (CD4+, CD8+ T-lymphocytes and NK cells in spleen and PBMCs), and apoptosis in IFNAR1-blocked pregnant mice during early (infected on 1 GD) and mid (infected on 8 GD) gestation have been studied. There was higher rate of TPT during mid stage (71.43%) than early (57.14%) stage. In early stage reduced implantation sites, early embryonic deaths, abortions, and necro-haemorrhagic lesions had observed. Mid stage, congenital defects and neurological lesions in foetuses like haemorrhages, diffuse cerebral edema, necrotizing encephalitis and decreased bone size (Alizarin red staining) were noticed. BTV-1 antigen was first time demonstrable in cells of mesometrium, decidua of embryos, placenta, uterus, ovary, and brain of foetuses by immunohistochemistry and quantified by real-time qRT-PCR. BTV-inoculated mice were seroconverted by 7 and 5 dpi, and reached peak levels by 15 and 9 dpi in early and mid gestation, respectively. CD4+ and CD8+ cells were significantly decreased (increased ratio) on 7 dpi but subsequently increased on 15 dpi in early gestation. In mid gestation, increased CD8+ cells (decreased ratio) were observed. Apoptotic cells in PBMCs and tissues increased during peak viral load. This first time TPT of wild-type Indian BTV-1 deserves to be reported for implementation of control strategies. This model will be very suitable for further research into mechanisms of TPT, overwintering, and vaccination strategies.

Parasitic pneumonia caused by Paragonimus spp. in a wild Royal Bengal Tiger, Mysuru, South India.

Parasitic pneumonia induced by genus Paragonimus involves many species, which affects both humans and animals and it is a food borne zoonotic disease. In this report, we have described the gross and histopathological findings of Paragonimus fluke infection in lungs of tiger. The postmortem examination of sub adult male wild tiger (Panthera tigris tigris) died in captivity was conducted, earlier which was rescued by Forest Department, Mysuru, Karnataka, India. External examination of carcass revealed pale oral and conjunctival mucous membranes with sunken eye balls. During necropsy, moderate congestion, consolidation and numerous transparent to dark encysted lesions were found in the parenchyma of all lobes of lungs visible grossly on pleural surface. Lungs were hemorrhagic with necrotic foci around the cysts. The incision of encysted lesions revealed the presence of flukes (2-3 in numbers) in each cyst with brownish exudate. The lung tissues with lesions were collected in 10% formalin and haematoxylin and eosin staining was done for histopathological evaluation. The flukes were identified as Paragonimus spp. based on the morphology and micrometry. The histopathological examination revealed presence of longitudinal sections of flukes in bronchial lumen (in pair) with tegument and tegumental spines surrounded by connective tissue capsule as cystic encapsulation and numerous eggs in adjacent lung parenchyma. Necrosis and moderate fibrosis of lung parenchyma with infiltration of polymorphonuclear and mononuclear inflammatory cells were observed around fluke as well as eggs. The squamous cell metaplasia of lining bronchial epithelium and atelectasis of alveoli were also prominently seen.

Blue native electrophoresis study on lipases.

We have developed a modified blue native polyacrylamide gel electrophoresis (PAGE) protocol that can overcome aggregation of lipases seen in native PAGE. We have shown that two lipases, Pseudomonas aeruginosa lipase and Candida rugosa lipase, which aggregate in the native gel, can be resolved using our protocol. Activity staining was done to test for the functionality of the two lipases.

Comparative Neuropathology of Major Indian Bluetongue Virus Serotypes in a Neonatal BALB/c Mouse Model.

Bluetongue virus (BTV) is neurotropic in nature, especially in ruminant fetuses and in-utero infection results in abortion and congenital brain malformations. The aim of the present study was to compare the neuropathogenicity of major Indian BTV serotypes 1, 2, 10, 16 and 23 by gross and histopathological lesions and virus distribution in experimentally infected neonatal BALB/c mice. Each BTV serotype (20 µl of inoculum containing 1 × 105 tissue culture infectious dose [TCID]50/ml of virus) was inoculated intracerebrally into 3-day-old mice, while a control group was inoculated with mock-infected cell culture medium. Infection with BTV serotypes 1, 2 and 23 led to 65-70% mortality at 7-9 days post infection (dpi) and caused severe necrotizing encephalitis with neurodegenerative changes in neurons, swelling and proliferation of vascular endothelial cells in the cerebral cortex, cerebellum, midbrain and brainstem. In contrast, infection with BTV serotypes 10 and 16 led to 25-30% mortality at 9-11 dpi and caused mild neuropathological lesions. BTV antigen was detected by immunohistochemistry, direct fluorescence antibody technique and confocal microscopy in the cytoplasm of neuronal cells of the hippocampus, grey matter of the cerebral cortex and vascular endothelial cells in the midbrain and brainstem of BTV-1, -2, -10, -16 and -23 infected groups from 3 to 20 dpi. BTV nucleic acid was detected in the infected brain tissues from as early as 24 h up to 20 dpi by VP7 gene segment-based one-step reverse transcriptase polymerase chain reaction. This study of the relative neurovirulence of BTV serotypes is likely to help design suitable vaccination and control strategies for the disease.

Prevalence of Campylobacter jejuni and Campylobacter coli in captive wildlife species of India.

Campylobacteriosis is an important zoonotic disease and the prevalence of Campylobacter is largely unknown in the wildlife of India. A total of 370 samples, comprising of 314 fresh faecal samples from apparently healthy captive wild animals and birds, 30 stool swabs from animal care takers and 26 samples of the animals' food and water were collected from G. B. Pant High Altitude Zoo, Nainital, Kanpur Zoo, Wildlife Park, IVRI and the Post Graduate Research Institute in Animal Sciences (PGRIAS), Chennai, Tamilnadu from August 2014 to May 2015. Samples were processed for cultural isolation, direct PCR and multiplex PCR for species confirmation. To decipher the genetic diversity, the 16S rRNA gene was amplified, sequenced and analyzed. Based on isolation, the overall occurrence rate of Campylobacter spp. was 0.8% (3/370), being 2.94% (3/102) for captive wild birds. Three Campylobacter jejuni were isolated from silver pheasants, lady amherest pheasants and saras cranes. Direct PCR assay showed the overall occurrence rate of Campylobacter spp. to be 4.77% (15/315), being 1.58% (2/126) for captive wild ruminants, 5.81% (5/86) for non-ruminants and 7.84% (8/102) for birds. All the isolates were identified as C. jejuni.

Genome wide host gene expression analysis in mice experimentally infected with Pasteurella multocida.

Pasteurella multocida causes acute septicemic and respiratory diseases, including haemorrhagic septicaemia, in cattle and buffalo with case fatality of 100%. In the present study, mice were infected with P. multocida (1.6 × 103 cfu, intraperitoneal) to evaluate host gene expression profile at early and late stages of infection using high throughput microarray transcriptome analyses. Several differentially expressed genes (DEGs) at both the time points were identified in P.multocida infected spleen, liver and lungs. Functional annotation of these DEGs showed enrichment of key pathways such as TLR, NF-κB, MAPK, TNF, JAK-STAT and NOD like receptor signaling pathways. Several DEGs overlapped across different KEGG pathways indicating a crosstalk between them. The predicted protein-protein interaction among these DEGs suggested, that the recognition of P. multocida LPS or outer membrane components by TLR4 and CD14, results in intracellular signaling via MyD88, IRAKs and/or TRAF6 leading to activation of NFκB and MAPK pathways and associated cytokines.

Polyamine structural effects on the induction and stabilization of liquid crystalline DNA: potential applications to DNA packaging, gene therapy and polyamine therapeutics.

DNA undergoes condensation, conformational transitions, aggregation and resolubilization in the presence of polyamines, positively charged organic molecules present in all cells. Under carefully controlled environmental conditions, DNA can also transform to a liquid crystalline state in vitro. We undertook the present work to examine the ability of spermidine, N4-methylspermidine, spermine, N1-acetylspermine and a group of tetramine, pentamine and hexamine analogs of spermine to induce and stabilize liquid crystalline DNA. Liquid crystalline textures were identified under a polarizing microscope. In the absence of polyamines, calf thymus DNA assumed a diffused, planar cholesteric phase with entrapped bubbles when incubated on a glass slide at 37 degrees C. In the presence of spermidine and spermine, the characteristic fingerprint textures of the cholesteric phase, adopting a hexagonal order, were obtained. The helical pitch was 2.5 micro m. The final structures were dendrimeric and crystalline when DNA was treated with spermine homologs and bis(ethyl) derivatives. A cholesteric structure was observed when DNA was treated with a hexamine at 37 degrees C. This structure changed to a hexagonal dendrimer with fluidity on prolonged incubation. These data show a structural specificity effect of polyamines on liquid crystalline phase transitions of DNA and suggest a possible physiological function of natural polyamines.

Role of nitric oxide in the regulation of immune responses during rabies virus infection in mice.

Rabies virus (RABV) stimulates nitric oxide (NO) production, which either triggers T cell differentiation or suppresses T cell function depending on its concentration. Herein, we assessed the potential role of NO in regulation of immune responses during RABV infection in mice model. The experimental animals were divided into four groups and 100LD50 of challenge virus standard (CVS) strain of RABV was inoculated intracerebrally on day 0 and subsequently aminoguanidine (AG; inducible nitric oxide synthase inhibitor) was injected intraperitoneally twice a day, up to 6 days. The samples were collected at 2, 4, 6, 8, 9, 10 and 12 days post infection (DPI). The immune cells including CD4+, CD8+ T lymphocytes and natural killer (NK) cells were estimated from peripheral blood mononuclear cells (PBMCs) and splenocytes. Serum total NO concentration, histopathology, immunohistochemistry, direct fluorescent antibody technique and TUNEL assay was performed. Infection with CVS resulted in significant early increase in CD4+, CD8+ and NK cells in blood and spleen until 2 DPI. From 4 DPI onwards significant reduction was noticed in these parameters which coincided with increased NO on 4 DPI, rising to maximum on 8 DPI, until their death on 10 DPI. Conversely, the CVS-AG treated group showed lower levels of NO and increased number of CD4+, CD8+ and NK cells. Increased number of cells in blood and spleen coincided with increased survival time, delayed development of clinical signs, reduced viral load and less apoptotic cells. NO played important role in regulation of immune responses during RABV infection. The findings of present study confirmed the role of NO and/or iNOS using iNOS inhibitor (aminoguanidine) in immune response during RABV infection, which would further help in understanding the virus immunopathogenesis with adoption of newer antiviral strategies to counter the progression of disease.

Isolation of bovine coronavirus (bcoV) in vero cell line and its confirmation by direct FAT and RT-PCR.

Bovine Coronavirus (BCoV) is widespread both in dairy and beef cattle throughout the world. The virus is one of the largest RNA virus and has specific tropism for intestinal and pulmonary epithelial cells. It is responsible for huge economic losses by causing winter dysentery in adult dairy cattle and respiratory and intestinal tract infections leading to pneumo-enteritis in young calves. Isolation of BCoV has been reported to be difficult. Studies regarding epidemiology, virus isolation and molecular detection from India are very few. In the present study Vero cell line was used for isolation of the BCoV from Enzyme Linked Immunosorbent Assay (ELISA) positive samples. Direct florescent antibody technique (dFAT) and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to confirm the isolated virus strains at antigenic and genomic levels, respectively. Out of the 15 positive fecal samples, virus from only seven was able to infect vero cell line. Subsequently BCoV got adapted to the vero cell line upto three passages, which was confirmed both at genomic and antigenic levels by dFAT and RT-PCR testing. It can be concluded that vero cell line can be used for isolation of BCoV, however due to the enormous stain diversity of the virus it is possible that many stains can't grow and get adapt in this cell line. Further studies are required for isolation of different viral strains, finding the susceptible cell lines and also to confirm the variations among the BCoV isolates at antigenic/genomic levels.

WITHDRAWN: A strain of Lysinibacillus sphaericus secretes an organic solvent-stable esterase.

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Ionic and structural specificity effects of natural and synthetic polyamines on the aggregation and resolubilization of single-, double-, and triple-stranded DNA.

DNA condensation, precipitation, and aggregation are related phenomena involving DNA-DNA interactions in the presence of multivalent cations, and studied for their potential implications in DNA packaging in the cell. Recent studies have shown that the condensation/aggregation is a prerequisite for the cellular uptake of DNA for gene therapy applications. To elucidate the ionic and structural factors involved in DNA aggregation, we studied the precipitation and resolubilization of high molecular weight and sonicated calf thymus DNA, two therapeutic oligonucleotides, and poly(dA).2Poly(dT) triplex DNA in the presence of the tetravalent polyamine spermine using a centrifugation assay, Tm measurements, and CD spectroscopy. The ability of spermine to provoke DNA precipitation was in the following order: triplex DNA > duplex DNA > single-stranded DNA. In contrast, their resolubilization at high polyamine concentrations followed a reverse order. The effective concentration of spermine to precipitate DNA increased with Na+ in the medium. Tm data indicated the DNA stabilizing effect of spermine even in the resolubilized state. CD spectroscopy revealed a series of sequential conformational alterations of duplex and triplex DNA, with the duplex form regaining the B-DNA conformation at high concentrations (approximately 200 mM) of spermine. The triplex DNA, however, remained in a Psi-DNA conformation in the resolubilized state. Chemical structural specificity effects were exerted by spermidine and spermine analogues in precipitating and resolubilizing sonicated calf thymus DNA, with N4-methyl substitution of spermidine and a heptamethylene separation of the imino groups of spermine having the maximal difference in the precipitating ability of the analogues compared to spermidine and spermine, respectively. Therapeutically important bis(ethyl) substitution reduced the precipitating ability of the analogues compared to spermine. The effect of the cationicity of polyamines was evident with the pentamines being much more efficacious than the tetramines and triamines. These results provide new insights into the mechanism of DNA precipitation by polyamines, and suggest the importance of polyamine structure in developing gene delivery vehicles for therapeutic applications.