Dot-ELISA based on recombinant Hypodermin C of Przhevalskiana silenus for field diagnosis of goat warble fly infestation.

Goat warble fly infestation (GWFI) is an economically important myiasis caused by larvae of Przhevalskiana silenus (Diptera, Oestridae), prevalent in countries of the Mediterranean Basin and Indian subcontinent. GWFI is characterized by the presence of subcutaneous warbles at the lumbar and sacral region of dorsum in the infested animal. The early larval instars (L1 and L2) remain inaccessible to physical detection due to their small size and subcutaneous presence thus causing hindrance in the diagnosis. The objective of present study was to develop a field applicable early diagnostic intervention for GWFI monitoring and prophylactic management for effective control of the disease. Recombinant Hypodermin C (rHyC) antigen of P. silenus was expressed in Escherichia coli. The purified protein was used for optimizing dot-ELISA in a checkerboard titration using goat warble fly infested serum as known positive. The optimized assay was further tested for lower temperature (18°C) and incubation time (30 min). The optimized assay was assessed for inter-rater reliability and field samples. The optimized conditions require 188 ng of protein/dot, 1:800 dilution of serum sample, 1:4000 dilution of anti-goat IgG conjugate and 5% skim milk powder in phosphate buffer saline as blocking buffer. The assay was found to have a diagnostic sensitivity and specificity of 97.3% and 95.8%, respectively. The inter-rater reliability of dot ELISA with rHyC indirect ELISA was found to be almost perfect with a Cohen's kappa index of 0.973. Further testing at ambient temperature (18°C) and shorter incubation steps (30 min) supported suitability of the assay for field diagnosis of GWFI. The present study provides the first report of a sensitive and specific dot-ELISA for early diagnosis of GWFI which is rapid and cost effective. The test may provide an effective field applicable tool for sustainable control of GWFI.

Genomic characterization of Lumpy Skin Disease virus (LSDV) from India: Circulation of Kenyan-like LSDV strains with unique kelch-like proteins.

Lumpy skin disease (LSD) is an economically important poxviral disease endemic to Asia, Europe, and Africa. Recently, LSD has spread to naïve countries, including India, China, Bangladesh, Pakistan, Myanmar, Vietnam, and Thailand. Here, we describe the complete genomic characterization of LSDV from India, LSDV-WB/IND/19 isolated from an LSD affected calf in 2019 determined by Illumina next-generation sequencing (NGS). The LSDV-WB/IND/19 has a genome size of 150,969 bp encoding 156 putative ORFs. Phylogenetic analysis based on complete genome sequence suggested that LSDV-WB/IND/19 is closely related to Kenyan LSDV strains with 10-12 variants with non-synonymous changes confined to LSD_019, LSD_049, LSD_089, LSD_094, LSD_096, LSD_140, and LSD_144 genes. In contrast to complete kelch-like proteins in Kenyan LSDV strains, LSDV-WB/IND/19 LSD_019 and LSD_144 genes were found to encode truncated versions (019a, 019b, and 144a, 144b). LSD_019a and LSD_019b proteins of LSDV-WB/IND/19 resemble that of wild-type LSDV strains based on SNPs and the C-terminal part of LSD_019b except for deletion at K229, whereas the LSD_144a and LSD_144b proteins resemble that of Kenyan LSDV strains based on SNPs, however, C-terminal part of LSD_144a resembles that of vaccine-associated LSDV strains due to premature truncation. The NGS findings were confirmed by Sanger sequencing of these genes in Vero cell isolate as well as in the original skin scab along with similar findings in another Indian LSDV from scab specimen. LSD_019 and LSD_144 genes are thought to modulate virulence and host range in capripoxviruses. This study demonstrates the circulation of unique LSDV strains in India and highlights the importance of constant monitoring of the molecular evolution of LSDV and associated factors in the region in light of the emergence of recombinant LSDV strains.

An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats.

The orf virus (ORFV) is an epitheliotropic virus causing a highly contagious skin disease mainly in sheep and goats. Several diagnostics including molecular tools like Loop mediated isothermal amplification (LAMP) assay are available to detect ORFV in affected species. However, the carry-over contamination associated with LAMP as open tube format prevents the assay applicability as point of care test in field diagnostic settings. In this study, the B2L gene based LAMP assay was optimized in a closed tube format using hydroxynaphthol blue (HNB) and calcein as pre-addition dyes and it has shown a clear positive and negative signal at 60 °C using 4 and 5 mM concentrations of MgSO4 respectively for these dyes. Optimitimzed assay that could reveal the result within one hour is highly specific and senstive with a limit of detection at 12.5 femtogram of viral genomic DNA or ~85 virus genome equivalent. This improved method prevented the cross-contamination of future LAMP reactions in the laboratory without compromising diagnostic sensitivity (100%) and specificity (100%) when compared to open tube system. This closed tube LAMP method has potential to act as a simple visual detection assay for the rapid and specific diagnosis of ORFV in sheep and goats.

Prevalence and molecular characterization of Eimeria species affecting backyard poultry of Jammu region, North India.

The present study was conducted from January 2018 to December 2019 to know the prevalence of coccidiosis in backyard poultry in Jammu, Samba, and Udhampur districts of Union Territory of Jammu and Kashmir, North India. A total of 600 pooled fecal samples collected from backyard poultry were examined for presence of Eimeria oocysts. Morphometry and Polymerase Chain Reaction (PCR)-based amplification of ITS-1 gene was carried to characterize the Eimeria species infecting the backyard poultry of the study area. An overall prevalence of 28.5% Eimeria spp. infection among backyard poultry birds was recorded. Among the seasons, highest prevalence was recorded during rainy season (32%) with significantly (p < 0.05) high oocyst excretion (1.77 ± 0.01) and lowest during summer (19.3%) with low oocyst excretion (0.17 ± 0.006). Young birds up to 3 months of age were found to be more susceptible to infection than older birds, with a significantly (p < 0.05) high prevalence percentage of 38.02. Morphometry with COCCIMORPH software revealed presence of Eimeria tenella, Eimeria necatrix, Eimeria acervulina, and Eimeria maxima species with prevalence rates of 27.6%, 21.3%, 16.5%, and 3.6%, respectively. The amplified fragments of ITS-1 gene presented different sizes of Eimeria spp. viz. E. acervulina (321 bp), E. tenella (278 bp), E. maxima (145 bp), and E. necatrix (383 bp). The study concluded that although backyard poultry did not show clinical form of coccidiosis, it may act as source of potential reservoir.

First report of Przhevalskiana silenus derived recombinant hypodermin C based indirect ELISA for serodiagnosis of goat warble fly myiasis.

Goat warble fly infestation (GWFI) is a subcutaneous myiasis caused by larvae of Przhevalskiana silenus, an insect belonging to the order Diptera. The diagnosis of GWFI is challenging in the early larval instars (L1 and L2) as they are occult under the skin and hair coat causing prolonged economic loss in form of meat and hide damage. This necessitates early diagnosis for disease control at herd level and its prophylactic management to prevent economic losses. Hypodermins, a class of serine proteases from Hypoderminae subfamily have been used as serodiagnostic antigens for the past four decades for diagnosis of warble fly myiasis. In this study,the immunodominant antigen Hypodermin C (HyC) from P. silenus has been recombinantly expressed in E. coli and immunogenic characterisation of expressed protein was done. The protein shows hallmark residues in conserved cysteine and catalytic triad typical of serine proteases along with similar profile of immunoreactivity towards Hypoderminae infestation. The present study reports an optimised indirect-ELISA based on recombinant HyC derived from P. silenus for early diagnosis of GWFI. The optimised indirect ELISA provides a sensitive and specific immunodiagnostic for mass surveillance of the GWFI with diagnostic specificity and sensitivity of 96% and 100%, respectively and not showing any cross reactivity against other important parasitic and bacterial diseases of goats. This study presents the first report of indirect ELISA based on recombinant Hypodermin C antigen derived from P. silenus for the serosurveillance of goat warble fly disease.

Monoclonal antibodies against foot-and-mouth disease virus RNA polymerase for detection of virus infection.

Foot-and-mouth disease (FMD) is a major viral disease in farm animals. In the present study, seven monoclonal antibodies (mAbs) were produced against the FMD virus (FMDV)-encoded RNA-dependent RNA polymerase (3D protein) and characterized. Screening of mAb reactivity against three overlapping fragments of the 3D protein expressed in Escherichia coli revealed that the binding sites of all the mAbs were confined to the N-terminal one-third of the 3D protein. A selected mAb was utilized for detecting FMDV in the infected cell culture and tissues obtained from FMDV-infected animals.

Severe Necrotizing Pneumonia in Children: A Challenge to Intensive Care Specialist.

OBJECTIVE: Necrotizing pneumonia (NP) is recently recognized as a complication of pneumonia. The data on NP are scant from developing world and we aimed to describe the characteristic features of NP in our children. STUDY DESIGN: Single center retrospective cohort analysis. PATIENT SELECTION: Institutional database of children treated for pneumonia between September 2014 and May 2018 was searched to identify children with NP. METHODS: The demographic characteristics, laboratory results, and clinical information were recorded for patients selected as NP and analyzed. RESULTS: In total, 10 patients (3.7%) of NP were identified out of 272 patients with pneumonia. Median age was 3 years (range: 3 months to 12years). All cases had severe respiratory distress and 70% required mechanical ventilation and inotropic support. The causative pathogens were identified in 6/10 children (60%) with Staphylococcus aureus being most common (4/10). Pleural effusion and pneumothorax were seen in six cases. Four cases had bilateral pleural effusion and three had bilateral pneumothorax. Intercostal drainage (ICD) was placed in 70% and bilateral ICD was placed in 40% cases. Bronchopleural fistula (BPF) developed in two cases and one had bilateral BPF. Median [inter quartile range] ICD days and hospital stay were 9 (6-14) and 13.5 (7.5-18.5) days, respectively. Mean (±SD) total antibiotic (in hospital plus outpatient) days were 28.8 ± 9.6 days. Four cases had airway hemorrhage and in three cases this was massive and fatal. CONCLUSION: NP is a relatively rare but severe complication of pneumonia distinct from pediatric acute respiratory distress, pleural effusion and empyema. Airway hemorrhage is the most fatal complication.

Severe Sepsis and Septic Shock Associated with Chikungunya Fever in an Adolescent.

Chikungunya is usually a benign disease, and little is known on the occurrence of severe clinical complications. We describe a 12-year-old boy with rapid onset septic shock and multi-organ failure associated with chikungunya fever. Severe sepsis and septic shock can be associated with chikungunya.

Development and evaluation of solid lipid nanoparticles of raloxifene hydrochloride for enhanced bioavailability.

Raloxifene hydrochloride (RL-HCL) is an orally selective estrogen receptor modulator (SERM) with poor bioavailability of nearly 2% due to its poor aqueous solubility and extensive first pass metabolism. In order to improve the oral bioavailability of raloxifene, raloxifene loaded solid lipid nanoparticles (SLN) have been developed using Compritol 888 ATO as lipid carrier and Pluronic F68 as surfactant. Raloxifene loaded SLN were prepared by solvent emulsification/evaporation method, and different concentrations of surfactant, and homogenization speed were taken as process variables for optimization. SLN were characterized for particle size, zeta potential, entrapment efficiency, surface morphology, and crystallinity of lipid and drug. In vitro drug release studies were performed in phosphate buffer of pH 6.8 using dialysis bag diffusion technique. Particle sizes of all the formulations were in the range of 250 to 1406 nm, and the entrapment efficiency ranges from 55 to 66%. FTIR and DSC studies indicated no interaction between drug and lipid, and the XRD spectrum showed that RL-HCL is in amorphous form in the formulation. In vitro release profiles were biphasic in nature and followed Higuchi model of release kinetics. Pharmacokinetics of raloxifene loaded solid lipid nanoparticles after oral administration to Wistar rats was studied. Bioavailability of RL-HCL loaded SLN was nearly five times than that of pure RL-HCL.

Conservative management of isolated splenic abscess in children.

INTRODUCTION: Isolated splenic abscesses (SAs) are rare in children. We report a single-center experience with emphasis on their diagnosis, etiology, treatment, and outcome. METHODS: This is a retrospective review. RESULTS: Eighteen children (age, 3-16 years; male-female ratio, 5:1) were managed over a period of 8 years in a tertiary-care institution. Presenting symptoms included fever, abdominal pain, and anorexia. Splenomegaly was present in 12 (67%), leukocytosis in 9 (50%), and thrombocytosis in 12 (67%) patients. Associated diseases were thalassemia (1), tuberculosis (1), and typhoid fever (9). Solitary and multiple SAs were seen in equal numbers. Blood culture grew Salmonella paratyphi A in 1 case. Splenic aspirate culture was positive in 3 (Escherichia coli [1], S paratyphi A [1], Acinetobacter [1]). Widal serology was positive in 9 (50%) patients. Management consisted of intravenous broad-spectrum antibiotic therapy in all patients, together with percutaneous aspiration in 10 (56%) cases where the abscess size was greater than 3 cm. All patients responded, and complete resolution was observed. CONCLUSION: Isolated SA in children responds favorably to conservative treatment with intravenous broad-spectrum antibiotics and percutaneous drainage without the need for splenectomy.

Isolated post-traumatic right-sided diaphragmatic hernia.

A 3-yr-old boy presented with respiratory distress of 2 days duration. There was a history of blunt trauma to the lower chest having occurred 5 days earlier. Although missed initially, serial chest X-rays and a computed tomographic (CT) scan revealed an isolated traumatic right-sided diaphragmatic hernia without any injury to the viscera or the ribcage. Laparotomy with reduction of the herniated right lobe of the liver and the transverse colon was performed. Recovery was uneventful. The presentation, diagnosis and management of this relatively uncommon injury is discussed. The need for a high index of suspicion and critical evaluation of appropriate investigations to prevent diagnostic delay and optimize management in patients with traumatic diaphragmatic injury is emphasized.

Trans-anal protrusion of ventriculo-peritoneal shunt catheter with silent bowel perforation: report of ten cases in children.

Ventriculo-peritoneal (VP) shunting used in the treatment for hydrocephalus is associated with several complications. Mechanical failure of shunt is the commonest complication of all. Visceral/bowel perforation is an unusual but serious complication of VP shunting. This article reports our experience in the management of ten children who had VP Shunt catheter protrusion from anus. This is a retrospective study of ten patients who had VP shunt catheter protrusion from anus, admitted in the department of paediatric surgery between Jan 1996 and Dec 2005. The records of above ten cases were reviewed for their clinical presentation and management, etc. We had performed 398 VP shunt operations in the last 10 years. Two hundred and seventy one (68.09%) VP Shunts were done for congenital hydrocephalus of which 164 were done in infancy/neonatal period and 107 VP shunts were done in the age group of >1-12 years. One hundred and twenty-seven (31.90%) VP shunt operations were done for patients who had hydrocephalus as a complication following tubercular meningitis (TBM). Out of 398 VP shunts, ten patients (2.51%) had protrusion of the distal end of peritoneal catheter from anus without causing/leading to peritonitis. We observed a 08.29% mortality of all VP shunt operations. Protrusion of VP shunt catheter per rectum can occur without producing peritonitis. Formal exploration and localization of entry of VP shunt catheter in bowel is not mandatory. Mini laparotomy and revision of peritoneal part of shunt can be done if there is no shunt infection.