Molecular epidemiology of Japanese encephalitis in pigs and risk factors associated with causing Japanese encephalitis in pigs of Lakhimpur, the first case reported in the district of North East India.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is a major public health problem in India. The first outbreaks of JE have been reported from the North-eastern regions of Assam, particularly from the Lakhimpur district of Assam between July-August 1989. In Assam every year many people died due to JE. This study was performed to investigate the molecular epidemiology of JE in pigs in Lakhimpur district of Assam and the risk factors associated with causing Japanese encephalitis in pigs. This study will help to map out the endemic regions and to know where and when to apply the most control strategies towards the prevention and control of the disease. METHODS: A total of 342 serum samples from pigs were collected from 10 organized and 20 unorganized farms from 9 blocks and recorded to age, sex and breed and tested by RT-PCR. Pig farms and the surrounding environment were studied for assessment of farm-level risk factors responsible for JEV infection in pigs. RESULTS: Out of 342 samples tested for detection of the E gene of JEV, 14 samples were found to be positive with a prevalence rate of 4.09%. Age, sex and breed-wise higher cases were found in at the age group above 12 months, sex wise female and breed-wise local pigs. Pig farms less than 500 meters from risk factors like rice field, stagnant water source, wild bird exposure to farm and mosquito exposure at farm/ bite to pigs, found to be more numbers of JE cases. INTERPRETATION & CONCLUSION: Molecular epidemiology of JE in pigs, and humans; positive at Lakhimpur recommend the need for uninterrupted surveillance of this virus in pigs specially those areas where pig population is more and all risk factors are present.

Genome-wide expression analysis of the heat stress response in dermal fibroblasts of Tharparkar (zebu) and Karan-Fries (zebu × taurine) cattle.

The present study sought to evaluate mRNA expression profiles in the cultured dermal fibroblasts of Tharparkar (zebu) and Karan-Fries (zebu, Tharparkar × taurine, Holstein Friesian) cattle in response to heat stress. Bioinformatics' analysis identified temperature-regulated biological processes and pathways. Biological processes overrepresented among the earliest genes induced by temperature stress include regulation of stress responses, protein repair, metabolism, protein transport, cell division, and apoptosis. The present microarray platform contains 51,338 synthesized oligonucleotide probes corresponding to at least 36,713 unigenes. A total of 11,183 and 8126 transcripts were differentially expressed with a fold change of ≥ 2 in Tharparkar and Karan-Fries cattle, respectively. Randomly selected real-time validation showed 83.33% correlation with microarray data. Functional annotation and pathway study of the differentially expressed transcripts or genes (DEGs) reveal that upregulated genes significantly (P < 0.05) affect protein processing and NOD-like receptor pathways (NLRs), while downregulated genes were significantly (P < 0.05) found to be associated with cell cycle, metabolism, and protein transport. Gene expression changes include activation of heat shock factors (HSFs), increased expression of heat shock proteins (HSPs), and apoptosis, while decreasing protein synthesis and another metabolism. These findings provide insights into the underlying mechanism of the physiology of heat stress in Tharparkar and Karan-Fries cattle. Understanding the biology and mechanisms of heat stress is critical to developing approaches to ameliorate current production issues for improving animal performance and agriculture economics in tropical climatic conditions. In conclusion, the present study indicates that heat stress differentially affects the expression of the significant number of genes associated with stress response, metabolism, apoptosis, and protein transport in dermal fibroblasts of Tharparkar and Karan-Fries cattle.

Effect of thermal stress on HSP70 expression in dermal fibroblast of zebu (Tharparkar) and crossbred (Karan-Fries) cattle.

The present studies were conducted to investigate the difference response of dermal fibroblasts to heat stress in Tharparkar and Karan-Fries cattle. Skin is the most important environmental interface providing a protective envelope to animals. In skin, dermal fibroblasts are the most regular cell constituent of dermis that is crucial for temperature homeostasis. The study aimed to examine the reactive oxygen species (ROS) formation, cytotoxicity (%) and heat shock protein 70 (HSP70) genes expression in dermal fibroblast of Tharparkar and Karan-Fries cattle and to assess whether resistance of dermal fibroblast to heat stress is breed specific. Dermal fibroblasts from ear pinna of Tharparkar and Karan-Fries cattle were exposed at 25 °C, 37 °C, 40 °C and 44 °C for 3 h to measure the ROS, cytotoxicity (%) and HSP 70 (HSPA1A, HSPA2 and HSPA8) genes' expression. The results showed that ROS formation at low temperature (25 °C) decreased in both breeds as compared to control (37 °C) and the differences were significant (P<0.0001). Heat stress at 40 °C did not increase ROS formation significantly in Tharparkar but increased significantly (P<0.001) in Karan-Fries cattle. The overall cytotoxicity (%) was also found to be significantly different (P<0.001) between Tharparkar and Karan-Fries cattle, and on exposure to different temperatures (P<0.001). The cytotoxicity (%) in dermal fibroblast cells of Karan-fries cows was more than Tharparkar. The expression studies indicated that all HSP70 genes (HSPA8, HSPA1A and HSPA2) were up-regulated at different temperatures in both breeds. In Tharparkar, the relative mRNA expression of HSPA8 gene was higher but HSPA1A and HSPA2 genes were low as compared to Karan-Fries cattle. At 40 and 44 °C, the relative expressions of inducible HSP 70 genes (HSPA1A and HSPA2) were higher in Karan-Fries than Tharparkar. In summary, dermal fibroblast resistance to heat shock differed between breeds. Dermal fibroblasts of Tharparkar were observed to be more heat tolerant than crossbred Karan-Fries cattle. The study concludes that zebu cattle (Tharparkar) dermal fibroblasts are more adapted to tropical climatic condition than crossbreed cattle (Karan-Fries). Differences exist in dermal fibroblasts of heat adapted and non-adapted cattle.

Effect of actin polymerization inhibitor during oocyte maturation on parthenogenetic embryo development and ploidy in Capra hircus.

This study was designed to observe the effect of cytochalasin B (CCB) concentrations on ploidy and early development of parthenogenetic embryos in a caprine species. Caprine oocytes were matured in the presence of different concentrations of CCB (5, 10, 15, and 20 µg/ml) and activated by 7% ethanol followed by incubation with 2 mM DMAP. For embryos fertilized in vitro, oocytes were matured in maturation medium without CCB. The cleavage rate and further embryo development were significantly higher (P < 0.05) when oocytes were treated in this way. The percentage of embryos showed higher diploid values in 15 µg/ml CCB (83.66 ± 1.13), followed by 20 (72.22 ± 1.22), 10 (68.57 ± 1.17), and 5 µg/ml (62.00 ± 2.48). These results indicate that CCB with a concentration of 15 µg/ml in maturation medium can be used for the production of diploid parthenogenetic embryos in the caprine species.

Identification of suitable housekeeping genes for normalization of quantitative real-time PCR data during different physiological stages of mammary gland in riverine buffaloes (Bubalus bubalis).

Gene expression analysis unravels the complex changes or relations at transcriptomic level. To nullify all type of errors that can be incorporated during any stage of RNA extraction into cDNA synthesis and for reliable results, the data obtained from qPCR have to be normalized using the appropriate/suitable housekeeping genes (HKGs). Unfortunately, till date, no such HKG has been reported for bubaline mammary gland. The objective of the present study was thus to identify and validate the potential HKGs for the gene expression studies in buffalo mammary gland. Mammary tissues from twelve buffaloes during different physiological stages: pre-pubertal (heifer), lactation and involution were obtained for the present study. A total of 16 potential HKGs (GAPDH, ß-actin, UXT, ß2M, A2M, RPl4, RPS9, RPS15A, RPS18, RPS23, HMBS, HPRT1, GTP, EEF1A1, UB1 and RPL22) from different functional classes were evaluated. The analysis revealed that the expression of EEF1A1, RPl4, ß2M and RPS15A was most consistent across different physiological stages of buffalo mammary gland. On the other hand, ß-actin, A2M, RPL22 and GAPDH were the least stable genes making them unsuitable as HKGs. Based on our analysis, we recommend the use of EEF1A1, RPl4, ß2M and RPS15A genes as suitable HKGs for accurate normalization of gene expression data in bubaline mammary gland.

Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus).

We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), ß-actin (ACTB), ß-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, ß-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation.

Cardiomyocytes rhythmically beating generated from goat embryonic stem cell.

The aim of present investigation was isolation, characterization and differentiation into cardiomyocytes of putative goat embryonic stem cells produced from in vitro fertilized goat embryos. Goat blastocysts were produced in vitro by standard methods of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) techniques. The ICMs isolated from IVF blastocysts were cultured on 10 µl/ml mitomycin-C inactivated fetal fibroblast feeder layer with LIF. The putative ES colonies were characterized for extracellular markers like alkaline phosphatase, TRA-1-60, TRA-1-81, SSEA-1, SSEA-4 by immunocytochemistry and intracellular markers like Oct4, Sox2 and Nanog with reverse-transcription-PCR. The ES cells were successfully subcultured up to 22nd passage with feeder layer and LIF and up to 12th passage without feeder layer with LIF only. They exhibited normal karyotyping (20th passage) and maintained the expression of specific surface markers like alkaline phosphatase, SSEA-4, TRA-1-61, TRA-1-81 and intracellular markers Oct4, Sox2 and Nanog. The embryoid bodies (EBs) were generated from goat ES cells of 20th passage and were analyzed with markers like Gata4, BMP4 and Nestin. Differentiation was induced by medium containing 100 ng/ml Activin-A, 10 ng/ml FGF-2 and 100 ng/ml BMP-4. The embryoid bodies were analyzed with markers like Gata4, BMP4 and Nestin. The rhythmic beating of cardiomyocytes was observed after 30 d and the beating was still continuing even after 160 d of culturing. Similarly, 2nd and 3rd batches of EBs were also beating and the beating continues after 75 d and on. The beating cells were observed positive for cardiac specific markers like α Actinin, C-Troponin and α-Myosin heavy chain. Histological studies also revealed morphology similar to cardiomyocytes. Prominent contractions typical of cardiac tissue have been maintained in the differentiated cells up to 160 d and still continuing beating at the rate of 30 beats/min. It could be concluded that ES cells generated from goat embryos were maintained undifferentiated up to 22nd passage on feeder layer and to 12th passage without feed layer using LIF and that the differentiation protocol induced rhythmic beating cells.

Effect of roscovitine treated donor cells and different activation methods on development of handmade cloned goat (Capra hircus) embryos.

The aim of the present investigation was to find out the effects of roscovitine treatment of donor cells and different activation methods on development of HMC goat embryos. Goat fetal fibroblast cells were cultured and divided into three treatment groups-contact inhibition group, roscovitine treatment group and serum starvation group. There was a significant decrease in blastocyst yield in serum starvation group (6.82%) compared to roscovitine treatment group (19.31%) and contact inhibition group (18.52%), however, no significant difference was found between roscovitine treatment group and contact inhibition group. To see the effect of different methods of activation, the reconstructed embryos were randomly divided into two groups and activated by two methods-one half by 2 µM Ca ionophore and another half by 2.31 kV/cm for 15 µSec electrical pulse. Subsequently, cloned embryos were cultured in TCM-199 based embryo development medium supplemented with 10 mg/mL bovine serum albumin in WOW culture system. There was a significant increase in the rate of cleavage and blastocyst production in electric pulse activation of 78.57% and 21.43% than Ca ionophore activation of 62.63% and 10.61% respectively. In conclusion, treatment of donor cells with roscovitine yields a significantly increased blastocyst than serum starved donor cells but equivalent blastocyst to contact inhibition group and electrical pulse activation (EPA) improves the production of HMC goat embryos.

Purification, sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development.

Oviduct-specific glycoprotein (oviductin) plays an important role during fertilization and early embryonic development. The oviductin cDNA was successfully cloned and sequenced in goat, which possessed an open reading frame of 1620 nucleotides representing 539 amino acids. Predicted amino acid sequence showed very high identity with sheep (97%) followed by cow (94%), porcine (77%), hamster (69%), human (66%), rabbit (65%), mouse (64%) and baboon (62%). The bioinformatics analysis of the sequences revealed the presence of a signal sequence of 21 amino acids, one potential N-linked glycosylation site at position 402, 21 potential O-linked glycosylation sites and 36 potential phosphorylation sites. The native oviductin was purified from the oviductal tissue, which showed three distinct bands on SDS-PAGE and western blot (MW ~60-95 kDa). The predicted molecular weight of goat oviductin was 57.5 kDa, calculated from the amino acid sequences. The observed higher molecular weight has been attributed to the presence of large number of potential O-linked glycosylation sites. The lower concentration (10 µg/mL) of oviductin increased the cleavage rate, morula and blastocyst yield significantly (P < 0.05) as compared to higher concentration (100 µg/mL). Goat oviductin retarded the activity of pronase (0.1%) on zona solubility of oocytes significantly (P < 0.01).

Study of the efficiency of chemically assisted enucleation method for handmade cloning in goat (Capra hircus).

The present investigation was carried out to find an efficient chemically assisted procedure for enucleation of goat oocytes related to handmade cloning (HMC) technique. After 22-h in vitro maturation, oocytes were incubated with 0.5 µg/ml demecolcine for 2 h. Cumulus cells were removed by pipetting and vortexing in 0.5 mg/ml hyaluronidase, and zona pellucida were digested with pronase. Oocytes with extrusion cones were subjected to oriented bisection. One-third of the cytoplasm with the extrusion cone was removed with a micro blade. The remaining cytoplasts were used as recipients in HMC. Goat foetal fibroblasts were used as nuclear donors. The overall efficiency measured as the number of cytoplasts obtained per total number of oocytes used was significantly (p < 0.05) higher in chemically assisted handmade enucleation (CAHE) than oriented handmade enucleation without demecolcine (OHE) (80.02 ± 1.292% vs. 72.9 ± 1.00%, respectively, mean ± SEM). The reconstructed and activated embryos were cultured in embryo development medium (EDM) for 7 days. Fusion, cleavage and blastocyst development rate were 71.63 ± 1.95%, 92.94 ± 0.91% and 23.78 ± 3.33% (mean ± SEM), respectively which did not differ significantly from those achieved with random handmade enucleation and OHE. In conclusion, chemically assisted enucleation is a highly efficient and reliable enucleation method for goat HMC which eliminates the need of expensive equipment (inverted fluorescence microscope) and potentially harmful chromatin staining and ultraviolet (UV) irradiation for cytoplast selection.

Stem cell-like outgrowths from in vitro fertilized goat blastocysts.

With an aim to isolate, culture and characterize goat embryonic stem cell-like cells derived from in vitro fertilized goat blastocysts, slaughterhouse derived goat oocytes were in vitro matured in maturation medium in 5% CO2 air at 38.5 degrees C. Matured oocytes were fertilized in vitro with fresh capacitated spermatozoa. Total 636 (36.5%) cleaved embryos were obtained which were further co-cultured with goat oviductal epithelial cells (GOEC) for 7-10 days. GOEC culture system was better for formation of morula (150; 44.3%) and hatched blastocyst (13; 3.8%) than embryo development medium culture system, [morula (69; 23.1%) and hatched blastocyst (5; 1.6%)]. Out of total blastocysts (48) the primary colonies were formed in 23.3% (7/30) blastocysts, and 66.6% (12/18) of hatched blastocysts. The cells of the inner cell mass (ICM) derived primary colonies were small, aggregated and tightly packed in nature forming embryoid bodies on further subculture. The colonies were stained to see the expression of alkaline phosphatase and positive result was obtained. Goat embryonic stem cell like outgrowths were also characterized for Oct-4 expression and positive result was found. It could be concluded that ICM cells were isolated from in vitro fertilized goat blastocysts and cultured for embryonic stem cell-like cells and expression of alkaline phosphatase and Oct-4 in these cells were positive.

Continuous renal replacement therapy in critically ill patients with renal failure.

Continuous arterio-venous and veno-venous haemodiafiltration (CAVHD, CVVHD), combine convection and diffusing solute clearance. We performed CVVHD on critically ill patients with renal failure, of whom 15 were on inotropic support and 10 on ventilators. Satisfactory diafiltration could be performed in all the patients with adequate solute and fluid removal. The main complication was clotting of the filter. The procedure was simple, safe and could be done by staff with no special training in dialysis technology.

Binding studies of four free base porphyrins and six iron (+3) porphyrins with human serum albumin.

The binding stoichiometry and binding equilibria of human serum albumin with water-soluble four free base porphyrins and six chloroiron (+3) porphyrins have been determined in 0.1M phosphate buffer, pH 7.2, by fluorescence quenching and filtration methods. The binding stoichiometry is observed to be 1:1, porphyrin to protein. The dissociation constants, Kd, between the porphyrins and the protein are found to be 1-4 microM. A binding mechanism between the protein and the porphyrins has been presented.

Binding studies of three water-soluble polycationic porphyrins with human serum albumin.

The binding stoichiometry and equilibria of three polycationic water-soluble porphyrins with human serum albumin (HSA) have been determined by fluorescence quenching and filtration methods in 0.1M phosphate buffer, pH 7.2 at 25 degrees. For one porphyrin the binding equilibrium was also measured by measuring the lifetime of tryptophan and also by measuring the polarization of bound porphyrin. Energy transfer between the porphyrin and the tryptophan residue of HSA has been studied.

Binding of some meso-tetraphenyl porphyrins with apohemoglobin and methemoglobin.

Six tetraphenylporphyrins have been studied for their binding with human apohemoglobin and methemoglobin. Four showed binding with apohemoglobin, in a way hemin binds, to form analogs of recondtituted hemoglobin. Five porphyrins bound with methemoglobin, perhaps, through random ionic interaction.