Comparative genomic diversity analysis of copy number variations (CNV) in indicine and taurine cattle thriving in Europe and Indian subcontinent.

Copy number variations (CNVs) include deletions, duplications, and insertions that are larger than 50 bp in size causing structural variation responsible for diversity, adaptation, and breed development. Indian cattle breeds are highly diverse from the taurine breeds. The pattern of CNVRs in 191 animals belonging to 39 cattle breeds (four Indicine and 35 Taurine) was studied based on Illumina 777K BovineHD chip data. The Indicine breeds revealed 2590 CNVs and 335 copy number variation regions (CNVRs) in autosomes. Out of the identified CNVs, 50 were found to be novel. Structure analysis revealed admixed nature of Siri. Neighbor joining tree from CNVR data showed that hot (Kankrej and Hallikar) and cold (Ladakhi and Siri) adapted cattle breeds clustered separately. CNVR of Indian and European breeds revealed that Balkan and Italian breeds of Podolian group are admixed with Indian cattle breeds corroborating indicine introgression (6.1-13.5%). CNVRs spanning the regions of olfactory receptors and immune system genes were identified. AMOVA revealed 9% variation among populations which is 2% greater than SNP based studies showing higher inclusion of variation by CNVR. Detailed analysis of CNVs/CNVRs in Indian cattle adapted to hot and cold climate, and their diversity among worldwide cattle is presented in this study.

Genome analyses revealed genetic admixture and selection signatures in Bos indicus.

The genomic diversity and relationship among seven diverse cattle breeds viz. Sahiwal, Tharparkar, Gir, Vechur, Ongole, Kangayam and Hariana were investigated in 132 random samples based on high density SNP array comprising > 777 K SNPs. A total of 1993 SNPs (0.25% of the total) having greater power (FST ≥ 0.20) to differentiate these cattle populations were identified, and utilized to partition genome of each animal into a predefined number of clusters. The structure of these cattle indicated shared ancestry of dairy breeds viz. Gir, Tharparkar and Sahiwal. Most of the animals (> 76%) of different populations under study except Vechur clustered into their own group of animals called breed. Vechur population retained highest rate of admixture, consistent with its crossing with other breeds. Ongole, Kangayam and Hariana shared comparatively less of their genome (≤ 15%) with other breeds. The study indicated that all seven breeds evolved from their independent ancestry but there was intermixing of these breeds in the recent past. The selection signatures identified between draft (Kangayam) and dairy breeds included several genes like FAM19A2, RAB31P, BEST3, DGKA, AHCY, PIGU and PFKP which are involved in immune response, metabolic pathway, transportation of glucose and sugars, signaling pathways, cellular processes, cell division and glycolysis regulation, respectively. Moreover, these genomic regions also harbour QTLs affecting milk performance traits. The signatures were also identified even between the dairy breeds. In comparison to large-sized cattle, there were significant differences in the number of QTLs affecting production (body weight, growth rate etc.) and morphological traits (height) in short-statured Vechur breed. The presence of HMGA2 gene in the selection signature on chromosome 5 may explain the variations in stature between these cattle.

Vaccine approaches for the 'therapeutic management' of Mycobacterium avium subspecies paratuberculosis infection in domestic livestock.

High endemicity of Johne's disease (JD) in herds adversely affects heavy milk yielding breeds by reducing the per animal productivity and 'productive life-span'. This review evaluates different vaccines used for its control and summarizes the benefits of 'global vaccine' in the four major domestic livestock species, namely goat, sheep, buffalo and cattle. Vaccines developed by using 'native strains' revealed both 'therapeutic' and preventive effects in domestic livestock. The 'therapeutic' role of vaccine in animals suffering from clinical JD turned out to be valuable in some cases by reversing the disease process and animals returning back to health and production. Good herd management, improved hygiene, 'test and cull' methodology, proper disposal of animal excreta and monitoring of MAP bio-load were also regarded as crucial in the 'therapeutic' management of JD. Vaccine approaches have been widely adopted in JD control programs and may be considered as a valuable adjunct in order to utilize huge populations of otherwise un-productive livestock. It has been shown that vaccination was the preeminent strategy to control JD, because it yielded approximately 3-4 times better benefit-to-cost ratios than other strategies. Internationally, 146 vaccine trials/studies have been conducted in different countries for the control of JD and have shown remarkable reduction in its national prevalence. It is concluded that for JD, there cannot be global vaccines or diagnostic kits as solutions have to come from locally prevalent strains of MAP. Despite some limitations, vaccines might still be an effective strategy to reduce or eradicate JD.

Evaluation of Bovine High-Density SNP Genotyping Array in Indigenous Dairy Cattle Breeds.

In total 52 samples of Sahiwal ( 19 ), Tharparkar ( 17 ), and Gir ( 16 ) were genotyped by using BovineHD SNP chip to analyze minor allele frequency (MAF), genetic diversity, and linkage disequilibrium among these cattle. The common SNPs of BovineHD and 54K SNP Chips were also extracted and evaluated for their performance. Only 40%-50% SNPs of these arrays was found informative for genetic analysis in these cattle breeds. The overall mean of MAF for SNPs of BovineHD SNPChip was 0.248 ± 0.006, 0.241 ± 0.007, and 0.242 ± 0.009 in Sahiwal, Tharparkar and Gir, respectively, while that for 54K SNPs was on lower side. The average Reynold's genetic distance between breeds ranged from 0.042 to 0.055 based on BovineHD Beadchip, and from 0.052 to 0.084 based on 54K SNP Chip. The estimates of genetic diversity based on HD and 54K chips were almost same and, hence, low density chip seems to be good enough to decipher genetic diversity of these cattle breeds. The linkage disequilibrium started decaying (r2 < 0.2) at 140 kb inter-marker distance and, hence, a 20K low density customized SNP array from HD chip could be designed for genomic selection in these cattle else the 54K Bead Chip as such will be useful.

Immunomodulatory activity of aqueous extract of Nyctanthes arbor-tristis flowers with particular reference to splenocytes proliferation and cytokines induction.

OBJECTIVES: To investigate the immunomodulatory activity of aqueous extract of Nyctanthes arbor-tristis flowers (NAFE) with particular reference to splenocytes proliferation and induction of cytokines. MATERIALS AND METHODS: Antibody titer was determined by tube agglutination and indirect ELISA assay in four groups of mice-control, antigen alone, and NAFE-treated (400 and 800 mg/kg for 21 days) after immunization with Salmonella antigen while cellular immunity was studied in three groups of rats (control and NAFE-treated - 400 and 800 mg/kg) following DNCB application. Splenocytes from untreated and NAFE-treated rats were stimulated using concanavalin-A (Con-A) and optical density (OD) and stimulation index were determined. Splenocytes from control rats were also treated in vitro with NAFE (50-1600 µg/ml) and Con-A to determine the effect on splenocytes proliferation. Interleukin-2 (IL-2) and IL-6 levels in splenocytes supernatant from control and NAFE-treated rats and following in vitro treatment of splenocytes with NAFE (50-1600 µg/ml) were determined using ELISA kits. RESULTS: Marked to a significant increase in antibody titer by both the methods in NAFE-treated mice and a significant increase in skin thickness in rats after challenge with DNCB, respectively suggested humoral and cell-mediated immunostimulant potential of NAFE. Significant increase in OD and stimulation index following e x vivo and in vitro exposure of splenocytes and sensitization with Con-A and significant elevation in IL-2 and IL-6 levels in splenocytes supernantant was also observed after their ex vivo and in vitro exposure to NAFE. CONCLUSION: Humoral and cell-mediated immunostimulant activity of NAFE seems to be mediated through splenocytes proliferation and increased production of cytokines, especially IL-2 and IL-6.

Some newer marker phytoconstituents in methanolic extract of Moringa oleifera leaves and evaluation of its immunomodulatory and splenocytes proliferation potential in rats.

OBJECTIVES: The present study was undertaken to unravel the newer marker phytoconstituents in methanolic extract of Moringa oleifera leaves (MOLE) and evaluation of its immunomodulatory and splenocytes proliferation potential in rats. MATERIALS AND METHODS: Hot methanolic extract of MOLE was subjected to gas chromatography-mass spectrometry (GC-MS) analysis. Immunomodulatory potential was studied in four groups of rats following administration of MOLE at 62.5 and 125 mg/kg for 21 days, followed by immunization with Salmonella typhimurium "O" antigen and antibody titer determined using indirect enzyme-linked immunosorbent assay kit. Total lymphocytes and T- and B-lymphocytes count were determined in control and after MOLE administration (62.5 and 125 mg/kg) to rats for 42 days. Splenocytes (2 × 10(6) spleen cells/ml) from MOLE treated rats were harvested and stimulated using concanavalin A and optical density (OD) and stimulation index were determined. Splenocytes from healthy control rats were also collected and treated in vitro with different concentrations of MOLE (5, 10, 25, 50, and 100 µg/ml) and concanavalin A to determine effect of MOLE on OD and stimulation index. RESULTS: GC-MS analysis revealed presence of 9,12,15-octadecatrienoic acid ethyl ester, 6-octadecenoic acid, cis-vaccenic acid and 2-octyl-cyclopropaneoctanal in MOLE. MOLE at 125 mg/kg increased the antibody titer by 50%. Although there was slight decline in lymphocytes count (total, B- and T-lymphocytes) in MOLE treated rats, percentage of T-lymphocytes was increased nonsignificantly. Ex vivo and in vitro studies revealed marked increase in OD and stimulation index indicating MOLE-induced splenocytes proliferation. CONCLUSION: GC-MS study revealed four new compounds in MOLE apart from promising its immunomodulatory potential based on humoral immune response, percentage increase in T-lymphocytes count, and induction of splenocytes proliferation.

'Bio-load' and bio-type profiles of Mycobacterium avium subspecies paratuberculosis infection in the domestic livestock population endemic for Johne's disease: a survey of 28 years (1985-2013) in India.

Bio-load and bio-profile of Mycobacterium avium subspecies paratuberculosis was studied in the domestic livestock population of the country. Of the 23,429 farm and farmer's animals screened, average bio-load was 23.3% (Period of study; 28 years for goats; 13 years for sheep, cattle and buffaloes). Species-wise, bio-load was 20.1, 32.7, 39.3 and 28.3% in goats, sheep, cattle and buffaloes, respectively. Bio-load was significantly lower in time period A (P < 0.001) and B (P < 0.03), compared with period C. Geographical zone-wise, bio-load of MAP was significantly higher (P < 0.05) in Central zone compared with South, West, East and North zones. Bio-load in 11 states ranged from 16.2 to 87.8%. Of 8450, 5643, 8185 and 1151 samples screened by microscopy, culture, indigenous ELISA and IS900 blood PCR, 20.0, 10.6, 35.1 and 26.6% samples were positive, respectively. Bio-load was 32.8 and 31.6% in farm and farmer's goats and sheep, respectively, and 62.1% in farmer's cattle. MAP bio-load was also monitored in four farm units (three goats and one sheep) for breed improvement and three farm goats units for experimental purposes at Central Institute for Research on Goats in Mathura district. Of the 8025 goats and 1525 sheep that died from 1988 to 2013, 10.9 and 3.0% deaths were due to JD, respectively. On the basis of JD and suspected JD, 10.0 and 28.4% goats and 2.2 and 40.9% sheep, respectively were culled from the farm units in 25 years. Microscopic examination of 214 tissues (mesenteric lymph nodes and intestines) of 107 animals, it was observed that bio-load of MAP was high (25.0-60.0%) in farm animals. 'Indian Bison Type' was the dominant biotype, irrespective of domestic livestock species and the geographical zone.

Detection of rotavirus from hospitalized diarrheic children in uttar pradesh, India.

In the present study 220 stool samples collected from diarrheic children admitted to different hospitals and nursing homes of Uttar Pradesh and Uttarakhand were screened for rotavirus. Of 220 diarrheic samples screened 46 samples were found to be positive for rotavirus by RNA PAGE. All the isolates exhibited 4-2-3-2 migration pattern suggesting group A rotavirus. Both long and short electropherotypes were prevalent in these regions. Six different electropherotypes were detected in this study period. Male diarrheic children were found to be more susceptible to rotavirus infection (22.96 %) than that of the female ones (17.64 %). Viral RNA isolated from stool samples again subjected to VP4 gene amplification by RT-PCR using con2 and con3 primer which resulted 876 bp product suggesting group A rotavirus. Besides virus isolation was successfully done using MA104 cell line.

Development of a capsid based competitive inhibition enzyme-linked immunosorbent assay for detection of bovine immunodeficiency virus antibodies in cattle and buffalo serum.

The aim of this study was to develop a more specific and sensitive competitive inhibition ELISA (CI-ELISA) than the currently used indirect ELISA for detection of antibodies to bovine immunodeficiency virus (BIV) in cattle and buffaloes. Murine monoclonal antibodies (MAbs) were generated against a recombinant capsid (CA) protein of bovine immunodeficiency virus. Of the 13 anti-CA MAbs developed, MAb-9G10 was selected for CI-ELISA based on the maximum inhibition (98%) obtained with reference BIV antibody positive serum. Based on the distribution of percent inhibition of known negative sera (n=50), a cut-off value was set at 40% inhibition. The MAb-based CI-ELISA showed much higher agreement (concordance: 95.4%) than the indirect ELISA (concordance: 77.8%) with Western blot. Out of 672 sera of cattle and buffaloes tested by CI-ELISA from four states of India, 22% (113/516) of cattle and 19% (30/156) of buffalo were sero-positive for BIV with an overall seroprevalence of 21% (143/672) in India.

Characterization of mycobacteria isolated from bovines by PRA-targetting hsp 65 gene region.

Bovine tuberculosis caused by the bacterium Mycobacterium bovis is a major infectious disease of animals and has zoonotic importance for humans. Even though the incidence is believed to be very low in India, human tuberculosis caused by M. bovis has been increasingly recognized in many other countries of the world. As differentiation of mycobacterial species take long time, a method for the rapid identification of mycobacteria isolated from bovine samples to the species level was used, which is based on polymerase chain reaction (PCR) of the gene encoding for the 65-kD protein followed by restriction analysis. The method involves restriction enzyme analysis of PCR products obtained with primers common to all mycobacteria and generate M. tuberculosis complex specific pattern. PRA was performed on 33 bovine isolates of which 90.9% (30/33) isolates were identified clearly as M. tuberculosis complex, M. fortuitum, M. phlei and M. smegmatis using restriction enzyme Hae III.

In vitro microtuberization in potato (Solanum tuberosum L.) cultivars.

Mechanism of microtuberization in three elite cultivars kufri badhsha (KB), kufri chandramukhi (KCM) and kufri jawahar (KJ) of potato was studied. Sprouts of all the three cultivars were used to obtain in vitro shoot cultures. MS medium supplemented with chlorocholine chloride was found to be most suitable for all the cultivars. Maximum tuberization was obtained under incubation conditions of continuous darkness at 20 degrees +/- 1 degrees C. The highest number of micro-tubers per plant basis was produced under continuous darkness and KCM recorded the highest yield of micro-tubers and was found significantly superior to KJ and KB.

Phase-amplitude solution of the Schrödinger equation with application to free-free absorption.

The phase-amplitude method for solving the Schrödinger equation is implemented for free-free absorption in a hot, dense plasma. The method is benchmarked against two independent direct Schrödinger calculations.

(-)-Epigallocatechin-3-gallate in Camellia sinensis leaves from Himalayan region of Sikkim: inhibitory effects against biochemical events and tumor initiation in Sencar mouse skin.

Recently, we and others showed that the components of green tea may be useful cancer chemopreventive agents. It has been suggested that (-)-epigallocatechin-3-gallate (EGCG), the major constituent in green tea, may possess antitumor-promoting and/or anticarcinogenic effects in rodent tumor bioassay systems. During the chemical analysis of various green tea products, we found a traditionally preserved preparation of green tea used by tribes in the Himalayan region of Sikkim, India that was rich in EGCG. EGCG was isolated from this tea product, and its inhibitory effects were evaluated against the binding of topically applied 3H-labeled polycyclic aromatic hydrocarbons (PAHs) to epidermal DNA and 12-O-tetradecanoylphorbol-13-acetate (TPA) caused induction of epidermal ornithine decarboxylase (ODC) activity in Sencar mice, the short-term markers of tumor initiation and tumor promotion, respectively. Preapplication of EGCG resulted in significant inhibition (p less than 0.05) in the binding of [3H]PAH to epidermal DNA. Similarly, the topical application of EGCG resulted in significant inhibition (p less than 0.005) in TPA-caused induction of epidermal ODC activity. In further studies, we assessed the anti-skin tumor-initiating effect of EGCG in Sencar mice in an initiation-promotion protocol. The application of EGCG before challenge with 7,12-dimethylbenz[a]anthracene as tumor initiator resulted in significant reduction both in percentage of mice with tumors and number of tumors per mouse compared with a non-EGCG-pretreated group of animals. The results of the present study suggest that the green tea preparation from Sikkim may be a good source for the isolation of EGCG and that this compound may have significant potential as a cancer chemopreventive agent.