Characterization of Foot-and-Mouth Disease Virus Serotype O-Specific Single Domain Antibody Expressed in the pET Expression System.

Foot and mouth disease (FMD) is an extremely contagious disease of cloven-hoofed domesticated and wild animals, resulting in significant economic losses in many parts of the world. FMD virus (FMDV) serotype O is responsible for approximately 70% of global outbreaks. For detection of FMDV antigen or antibody, ELISAs are used worldwide and have several limitations, such as batch-to-batch variation in generating immunobiologicals, high production cost and ethical concerns over animal sacrifice. The use of single domain antibody (sdAb) or variable N-terminal domain of the heavy chain of heavy chain antibody (VHH) found naturally in camels has proven their effectiveness in diagnostics and therapeutics. In the present study, the anti-FMDV serotype O-specific VHH-C1 gene sequence (Accession no. KJ751546) was retrieved from the NCBI database. The gene was synthesized commercially in the pBluescript KS+ cloning vector and expressed in E. coli BL21 (DE3) cells using the pET303/CT-His expression system with a C-terminal 6X-His tag. The expressed sdAb, verified by SDS‒PAGE and western blotting, was purified by Ni-chelate chromatography and used as a coating antibody in double antibody sandwich (DAS) ELISA for FMDV detection and typing. The sdAb exhibited a high binding affinity for FMDV serotype O, without any cross-reactivity toward serotypes A and Asia-1. It exhibited better thermostability up to 85 °C than conventional rabbit polyclonal anti-FMDV sera. The potential of sdAbs thus produced without sacrificing lab animals could be explored for replacing polyclonal sera in DAS-ELISA as well as for developing biosensors or lateral flow devices for FMDV type O detection.

The Gender Spectrum of In-hospital Survival Post Primary Percutaneous Coronary Intervention for ST Elevation Myocardial Infarction: Exploring Age-driven Trends.

Background: The study was aimed to evaluate gender difference and age & gender specific interaction of in-hospital outcomes of patients with ST elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI). Methods: This was a prospective cohort study of 1748 patients with STEMI undergoing primary PCI. The study was dichotomised according to gender to evaluate the difference in the outcome. The study was further stratified based on an age cut-off of 75 years to examine the age-specific gender relationship in survival outcomes. Independent variables for in-hospital mortality were analysed through logistic regression. Results: There were 314 (17.96%) females with an average age of 60.80 years and 1434 (82.03%) males with an average age of 54.87 years. The prevalence of diabetes (24.8% vs. 13.2%) and hypertension (33.1% vs. 12.9%) was significantly higher in female patients compared to male patients, whereas the significantly higher number of male patients were smokers. On multivariate analysis, odds of female gender OR = 3.54 (1.37-9.17), killip class >2 OR = 3.05 (1.97-4.71) and baseline creatinine OR = 2.27 (1.22-4.23) were found as significant predictors of in-hospital mortality. The crude odds ratio of 2.35 (1.49-3.72) and adjusted OR of 2.05 (1.27-3.30) for female mortality was significant among patients aged <75-years. While patients with ≥75-years of age, the mortality difference was insignificant. Conclusion: Although the incidence of STEMI was higher in male compared to female patients, female patients had two-fold higher in-hospital mortality than male. Female gender was an independent predictor for in-hospital mortality in patients <75-years of age.

Development of amperometric lysine biosensors based on Au nanoparticles/multiwalled carbon nanotubes/polymers modified Au electrodes.

The construction of two amperometric l-lysine biosensors is described in this study. The construction comprises the covalent immobilization of lysine oxidase (LOx) onto nanocomposite composed of gold nanoparticles (AuNPs) and carboxylated multiwalled carbon nanotubes (c-MWCNT), decorated on (i) polyaniline (PANI) and (ii) poly 1,2 diaminobenzene (DAB), electrodeposited on Au electrodes. The biosensors were characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) and electrochemical impedance spectroscopy (EIS) studies. The optimum response (current) was observed within 2 s at pH 7.0 and 25 °C for LOx/AuNPs/c-MWCNT/PANI/Au, and 4 s at pH 7.0 and 30 °C for LOx/AuNPs/c-MWCNT/DAB/Au electrodes. There was a linear relationship between current and lysine concentration ranging from 5.0 to 600 µM for LOx/AuNPs/c-MWCNT/PANI/Au with a detection limit of 5.0 µM, and 20 to 600 µM for the LOx/AuNPs/c-MWCNT/DAB/Au electrode with a detection limit of 20 µM. The PANI modified electrode was in good agreement with the standard HPLC method, with a better correlation (r = 0.992) compared to the DAB modified electrode (r = 0.986). These observations revealed that the PANI modified Au electrode was better than the DAB modified electrode, and hence it was employed for the determination of lysine in milk, pharmaceutical tablets and sera. The PANI modified electrode showed a half life of 120 days, compared to that of 90 days for the DAB modified electrode, after their 100 uses, when stored at 4 °C.

Fractal Dimension and Radiomorphometric analysis of Orthopanoramic radiographs in patients with tobacco and areca nut associated oral mucosal lesions: A pilot in-vivo study in a North Indian cohort.

OBJECTIVE: The aim of this study was to determine the fractal dimension (FD) and radiomorphometric indices (RMIs) in the mandible from orthopantomographic radiographs in patients with oral lesions associated with smokeless/smoking tobacco (SLT/ST) and areca nut habits in a North Indian cohort. STUDY DESIGN: A prospective, cross-sectional, observational pilot study was conducted of 120 subjects, including controls and 3 study groups of 30 patients each with oral submucous fibrosis, tobacco pouch keratosis, and oral leukoplakia (OL). Two observers calculated FD and the RMIs of mandibular cortical thickness (MCT), panoramic mandibular index (PMI), and mandibular cortical index (MCI). RESULTS: Mean FD was significantly reduced compared to controls with all oral lesions (P < .05) and with all habits in 3 of 4 regions of interest (P < .05). MCT was significantly reduced with OL (P < .005) and in ST users (P < .05). PMI did not differ regarding lesion status or habits. Compared to the controls, MCI C2 type was significantly more common in all oral lesions (P ≤ .005) and all types of habit (P < .005). Inter- and intraobserver agreement was strong to excellent. CONCLUSIONS: FD and RMI values were significantly altered compared to controls in oral lesions associated with tobacco and areca nut habits and in the dominant type of habit.

Hepato-Duodenal Fistula Complicating a Pyogenic Liver Abscess: An Unusual Presentation.

An elderly male with type 2 diabetes mellitus was admitted to the emergency ward with fever and pain over the right hypochondrium since one month. An abdominal ultrasound revealed an ill-defined hypoechoic lesion with multiple air foci measuring 8 x 6 x 4 cm within the left lobe of the liver implicating segments III b and IV. A contrast-enhanced computed tomography (CECT) scan of the abdomen showed a similar lesion with leakage of oral contrast into the dependent areas of the collection from a rent in the antero-inferior aspect of the first part of the duodenum. Hepato-duodenal fistula or entero-hepatic fistula secondary to pyogenic liver abscess is an atypical and unusual complication requiring a high degree of suspicion for its diagnosis. Though optimal therapy for its management is still not known, early diagnosis with prompt initiation of therapy is imperative to reduce mortality.

Nucleotide sequence analysis of VP7 gene of Indian isolates of bluetongue virus vis-à-vis other serotypes from different parts of the world.

Bluetongue virus (BTV), a member of genus Orbivirus, a family Reoviridae, is a non-enveloped with double shelled structure and ten segmented double stranded (ds) RNA genome. The RNA segment S7 encodes an inner capsid serogroup specific viral protein VP7. To amplify coding region of VP7 gene of BTV, new primers, forward primer (18-38 bp) and reverse primer (1156-1136 bp), were designed using VP7 gene sequences available in GenBank. This primer pair successfully amplified cell culture adapted Indian isolates of BTV belonging to two different serotypes 1 and 18. The coding sequences of two Indian isolates of BTV (BTV-1H and BTV-18B) were cloned into pPCR Script-Amp SK (+) plasmid vector and transformed into XL10-Gold Kan ultracompetent E. coli cells. The positive clones selected by blue-white screening and colony touch PCR were sequenced. The sequence analysis revealed that there was 93-97% nucleotide sequence identity in VP7 gene of three different Indian serotypes of BTV. The VP7 gene sequences of Indian isolates have comparatively less sequence homology (< 80%) with American (US), and French isolates compared to South African (SA), Australian (AUS) and Chinese (PRC) isolates. In silico restriction enzyme profile analysis of VP7 gene sequences revealed that Indian isolates of BTV-1 can be differentiated from other BTV-1 isolates reported from SA, AUS and PRC using TaqI. Similarly the Indian isolates of BTV belonging to three different serotypes can be differentiated using EcoRI, Hae III and TaqI restriction enzymes.

Immuno-pathological studies on broiler chicken experimentally infected with Escherichia coli and supplemented with neem (Azadirachta indica) leaf extract.

AIM: The present study was conducted to evaluate the effects of neem leaf extract (NLE) supplementation on immunological response and pathology of different lymphoid organs in experimentally Escherichia coli challenged broiler chickens. MATERIALS AND METHODS: For this study, we procured 192-day-old broiler chicks from local hatchery and divided them into Groups A and Group B containing 96 birds each on the first day. Chicks of Group A were supplemented with 10% NLE in water, whereas chicks of Group B were not supplemented with NLE throughout the experiment. At 7(th) day of age, chicks of Group A were divided into A1 and A2 and Group B into B1 and B2 with 54 and 42 chicks, respectively, and chicks of Groups A1 and B1 were injected with E. coli O78 at 10(7) colony-forming units/0.5 ml intraperitoneally. Six chicks from each group were sacrificed at 0, 2, 4, 7, 14, 21, and 28 days post infection; blood was collected and thorough post-mortem examination was conducted. Tissue pieces of spleen and bursa of Fabricius were collected in 10% buffered formalin for histopathological examination. Serum was separated for immunological studies. RESULT: E. coli specific antibody titer was significantly higher in Group A1 in comparison to Group B1. Delayed-type hypersensitivity response against 2,4 dinirochlorobenzene (DNCB) antigen was significantly higher in Group A1 as compared to Group B1. Pathological studies revealed that E. coli infection caused depletion of lymphocytes in bursa of Fabricius and spleen. Severity of lesions in Group A1 was significantly lower in comparison to Group B1. CONCLUSION: 10% NLE supplementation enhanced the humoral as well as cellular immune responses attributed to its immunomodulatory property in experimentally E. coli infected broiler chicken.

Restriction analysis of conserved and variable regions of VP2 gene of Indian isolates of bluetongue virus serotype 1.

Bluetongue virus (BTV) is a member of Orbivirus genus in family Reoviridae. The virus genome is composed of 10 double-stranded RNA segments. The RNA segment L2 encodes an outer capsid viral protein VP2, which is the main determinant of neutralization and serotype-specific immune response. BTV serotype 1 (BTV-1) specific novel primer pair was designed using VP2 gene sequences available in GenBank to amplify 1240-1844 bp region because two hypervariable and three conserved regions have been reported within these 604 nucleotides. This primer pair successfully amplified cell culture adapted six Indian isolates of BTV-1 from different geographical regions of the country. The 604 bp PCR product of VP2 gene of all six BTV-1 yielded two fragments of 273 and 331 bp when digested with Taq1 restriction enzyme. This indicated that there is only one TaqI site at 1513 bp (within 1240-1844 bp region) of VP2 gene of BTV-1 Indian isolates. The in silico restriction analysis revealed that in BTV-1 South African isolate (BTV-1SA) there is no TaqI site while in BTV-1 Australian isolates (BTV-1AUS), there are two TaqI sites (at 1513 and 1567 bp) within 1240-1844 bp region of VP2 gene. The earlier reported VP2 gene based primer pair for BTV-1 was used in the present study to amplify 2242-2933 bp region of six BTV-1 Indian isolates as three conserved regions have been reported within these 691 nucleotides. The digestion of 691 bp PCR products with XmnI yielded three fragments of 364, 173 and 154 bp with all the six Indian isolates of BTV-1 suggesting that there are two XmnI sites within 2242-2933 bp region of VP2 gene. A single XmnI site was observed in silico in BTV-1AUS and BTV-1SA isolates at different positions within this region. The in vitro and in silico restriction profile analyses of partial VP2 gene sequences using TaqI and XmnI restriction enzymes indicated a close relationship of Indian isolates of BTV-1 with BTV-1AUS isolates but not with BTV-1SA isolate.

Fowl adenovirus serotype 4 associated with outbreaks of infectious hydropericardium in Haryana, India.

During 1998, hydropericardium syndrome was observed among 3-to-6-wk-old broilers in 45 different flocks of Haryana, India, with mortality ranging between 10% and 30%. Fowl adenovirus (FAV) was isolated from one of the affected flocks by chicken embryo liver cell culture. Serum neutralization test and polymerase chain reaction assay coupled with restriction enzyme analysis confirmed that the isolated virus belonged to FAV serotype 4. The disease was reproduced in 28-day-old broilers by subcutaneous and oral inoculation of isolated FAV4 alone. Typical hydropericardium and basophilic intranuclear inclusions in hepatocytes were observed in experimental birds by day 4 postinoculation.

VP2 gene based phylogenetic relationship of Indian isolates of Bluetongue virus serotype 1 and other serotypes from different parts of the world.

Bluetongue virus (BTV), a member of genus Orbivirus, family Reoviridae, is non-enveloped with double shelled structure and 10 segmented double stranded RNA genome. The RNA segment L2 encodes an outer capsid serotype specific viral protein VP2. BTV serotype 1 (BTV-1) specific novel primer pair, forward primer (1240-1271 bp) and reverse primer (1844-1813 bp), was designed using VP2 gene sequences available in GenBank to amplify 1240-1844 bp region because two hypervariable and three conserved regions have been reported within these 604 nucleotides. This primer pair successfully amplified cell culture adapted six Indian isolates of BTV-1. The 604 bp PCR product of VP2 gene of BTV-1 Avikanagar (A), Chennai (C) and Sirsa 3 (S3) Indian isolates were cloned in pPCR-Script Amp SK (+) vector and transformed into XL10-Gold Kan ultracompetent Epicurian coli cells. The positive clones selected by blue-white screening and colony touch PCR were sequenced. BTV-1A, C and S3 isolates revealed 99% nucleotide sequence identity within 1304-1844 bp region of VP2 gene. The partial VP2 gene sequences (1240-1844 bp region) revealed that BTV-1 Indian isolates were 89% identical with Australian (AUS) BTV-1 isolates while the identity with South African (SA) BTV-1 isolate was 75%. Phylogenetically, three BTV-1 Indian isolates formed one group which is closely related to BTV-1AUS isolates followed by BTV-1SA, BTV-2, 9, 23, 13, 17, 10 and 11 isolates from different parts of world. Based on partial VP2 gene sequences, it is concluded that Indian isolates of BTV-1 are closely related to BTV-1AUS isolates than BTV-1SA and other serotypes.