004290   PAUL R K, KUMAR M, KATARIA M (Animal Biochemistry Div, ICAR-Indian Veterinary Research Institute, Bareilly - 243 122, Uttar Pradesh, Email: drrajani1980@gmail.com) : Mild and transient heat shock enhances DNA integration following lipofection of recombinant plasmids in 4T1 cells. Indian J Biochem Biophys 2019, 56(4), 316-20.

Cancer cells having stably integrated genes encoding tumor-associated antigens could be utilized as a vaccine, in-vitro stimulators of antigen-primed T-cells, and target for cytotoxicity assay, etc. Lipofection is a simple and safer technique for stable transfection of plasmid DNA. However, the poor rate of genomic integration has limited its application. In the current study, the effect of mild and transient heat shock following lipofection on the improvement of genomic integration was evaluated. The cDNA fragments encoding chicken MMP-11peptide (V³² -K ³⁶⁵) and the immunoglobulin-like domain 2 of chicken VEGFR-2 were cloned separately into pcDNA3.1 vector. Lipofection was carried out using Lipofectamine^® 2000 (Life Technologies, USA) in 4T1 cells followed by a heat shock at 42°C for 10 min. Transfected cells were selected for a period of four weeks against 500 µg/mL G418 in RPMI 1640 media supplemented with 10% fetal bovine serum. Distinct G418-resistant colonies appeared after 14 days of selection. Heat shock significantly (P <0.05) increased the number of viable colonies following antibiotic selection. The immunofluorescent study confirmed the stable integration of the target DNAs into the cells. It is concluded that mild and brief heat shock following lipofection improves the stable integration of recombinant pcDNA3.1 plasmids into 4T1 cells.

3 illus, 22 ref

004297   RENUGADEVI K, VALLI NACHIYAR C, PADMAVATHY H, ANJALI DEVI P (Biotechnology Dep, Sathyabama Institute of Science and Technology, Chennai- 600 119, Tamil Nadu, Email: vnachiyar@gmail.com) : Coupling dye degradation and biodiesel production by Geitlerinema sp TRV27. Indian J Biochem Biophys 2019, 56(4), 309-15.

In this study, the dye degrading the ability of marine cyanobacteria, Geitlerinema sp TRV27 was tested against the textile dye Acid black 52. Optimum conditions like pH, temperature, dye concentration for acid black 52 dye degradation were studied and were found to be pH 7, 25±2 °C. More than 50% of degradation was observed for the tested maximum dye concentration, 100 ppm. The degraded dye intermediate was found to be naphthalene by GC-MS analysis and their toxicity on seed germination was studied. The dye treated biomass was used for the production of biodiesel and the physicochemical properties of biofuel were found to be within the standard limits.

1 illus, 3 tables, 33 ref

004286   NAMASIVAYAM S K R, SHANKAR K G, VIVEK J M, NIZAR M, SUDARSAN A V (Biotechnology Dep, Sathyabama Institute of Science and Technology, Chennai- 600 119, Tamil Nadu, Email: biologiask@gmail.com) : In silico and in vitro analysis of quorum quenching active phytochemicals from the ethanolic extract of medicinal plants against quorum sensing mediated virulence factors of Acinetobacter baumannii. Indian J Biochem Biophys 2019, 56(4), 276-86.

Inhibition of quorum sensing called quorum quenching (QQ) is now extensively utilized in the prevention of bacterial infections. In the present study, in silico and in vitro analysis of quorum quenching (QQ) or anti-Quorum sensing (QS) activity of ethanolic extract of medicinal plants against QS mediated virulence factors of human pathogenic bacteria Acinetobacter baumannii has been investigated. The effect of plant extracts on QS by acyl homoserine lactone (AHL) has been carried out by quantification of secreted AHL by high-pressure liquid chromatography (HPLC). Measurement of QQ activity was determined by maximum inhibition of virulence factors and AHL production which was recorded in E. globules and A. indica extracts. In silico analysis was studied with possible bioactive compounds in the ethanolic extract of respective plant material that were characterized by gas chromatography equipped with mass spectroscopy (GCMS) against the enzyme responsible for the production of signaling molecule which mediates QS AHL synthase. Distinct reduction of all the QS-mediated virulence factors was recorded in the E. globules and A. indica. Among the different bioactive compounds, the ethanolic leaf extract of E. globules of GCMS analyzed compound, Hexadeconoic acid, 1-(hydroxymethyl), 1, 2-ethannediyl ester interacted with 1KZF protein (AHL synthase) and showed binding energy of −11.2 kcal/mol to MET 42 and TYR 54. Phytochemicals mediated inhibition of AHL synthase activity which was responsible for AHL production would suggest the possible utilization of plant extracts as an antibacterial agent to fight against disease-causing pathogenic bacteria.

5 illus, 1 table, 32 ref

004288   PANDEY A, THAKUR M S (Animal Genetics and Breeding Dep, Veterinary Coll, Jabalpur-482 001, Madhya Pradesh, Email: akpandey1109@rediffmail.com) : αS2 casein gene polymorphism and association with milk production traits in Malvi and Nimari cattle of Madhya Pradesh. Indian J Anim Res 2019, 53(8), 1002-5.

The aim of this study was to know the polymorphic variants and their association with milk production traits in Malvi and Nimari cattle of Madhya Pradesh at áS2 casein gene (CSN1S2) gene   locus. The PCR amplified products of 1267 base pair (bp) length were digested by restriction endonuclease enzyme EcoRV, which recognizes GAT^ATC sites. The 1267 bp product was cut into two fragments of sizes 1150 bp and 117 bp.  All the tested samples yielded different results. Absence of restriction site at both the alleles that resulted in the appearance of single compact bands of size 1267 bp was referred to as genotype AA. The samples exhibiting three fragments (1267 bp/ 1150 bp/ 117 bp) were denoted as genotype AB.  The RFLP analysis carried out in above both of the breeds of cattle revealed dissimilar genotypic patterns. AA and AB genotypes were observed in Malvi and Nimari . The genotypic and gene frequencies of AA, AB and BB genotypes of áS2-casein gene (CSN1S2)/EcoRV locus was found to be 0.44, 0.56 and 0.00 in Malvi; 0.68, 0.32 and 0.00 in Nimari and the respective gene frequency for A and B alleles were found to be 0.72 and 0.28 in Malvi; 0.84 and 0.16 in Nimari. The frequency of A allele was found to be highest as compared to B allele in all the four breeds of cattle under the study. Chi-square values for testing correspondence between observed and expected genotypic frequencies at this locus were found to be non-significant in Malvi and  Nimari breeds of cattle. The above result indicated that the populations of animals of above both breeds were in Hardy-Weinberg equilibrium at this locus. The association of polymorphic variants of áS2-casein gene (CSN1S2)/EcoRV in Malvi and  Nimari breeds of cattle with milk yield per lactation (L), daily milk yield (L) showed the non-significant difference was observed between mean MY(L) of AA and AB genotype of Malvi and Nimari and with respect of daily milk yield the  Nimari. However, the  Nimari breed showed significantly higher daily milk yield than the Malvi breed of cattle.

10 ref

004299   SAHOO S S, MISHRA C, MOHANTY S T, KAUSHIK R, ROUT P K, SINGH M K, DASS G, SETHY K, BEHERA K, DIGE M S (ICAR-Central Institute for Research on Goats, Mathura-281 122, Uttar Pradesh, Email: maheshdige@gmail.com) : Characterisation of KiSS1R gene and non genetic factors affecting reproductive traits in Indian goats. Indian J Anim Res 2019, 53(7), 979-83.

This study was undertaken to explore the genetic polymorphism in the KiSS1R (GPR54) gene from 80 Black Bengal, 50 Ganjam and 20 Raighar goat. Each of the sampled goats was recorded for its reproductive traits. The genomic DNA was isolated from the collected blood samples. The target 3’ UTR comprising of 246 bp fragment of KiSS1R gene was successfully amplified using the specific primer. Amplified samples were subjected for HRM analysis followed by sequencing. The nucleotide sequence alignment with the retrieved DNA sequence from NCBI BLAST confirmed absence of polymorphic pattern in KiSS1R gene in 3’ UTR. However, in the studied populations breed had significant effect on littersize, kidding interval and age at sexual maturity. It was found that age at sexual maturity and kidding interval were the highest in Ganjam goat population as compared to Raighar and Black Bengal goat population. Litter size was found highest in Black Bengal goat. 

32 ref

004291   PRADHAN S, THANGAVELU A, SRITHAR A, SENTHILKUMAR T M A, KIRUBAHARAN J J (Tamil Nadu Veterinary and Animal Sciences Univ, Chennai-600 051, Tamil Nadu, Email: thangavelu.a@tanuvas.ac.in) : Phosphoprotein gene of Peste des petits ruminants virus: Unsuitable for lineage determination. Indian J Anim Res 2019, 53(7), 949-53.

The nucleotide sequence of phosphorprotein (P) gene of a recent isolate of Peste des petits ruminants virus (PPRV/INDIA/TN/Apampattu/2014) was determined by RT-PCR amplification and sequencing. The sequence analysis showed that phosphoprotein gene sequence of this isolate has 87-98 percent identity with other Indian PPRV. At amino acid level, the identity was 84-98 percent with recent Indian PPRV isolates and 95 percent with Sungri/96 isolate. Surprisingly, when phylogenetic analysis was conducted with all the available ‘P’ gene sequences in genbank along with our sequence, it depicted more than four lineages in the phylogenetic tree. This is not in compliance with the PPRV ‘F’ and ‘N’ gene based and whole genome sequence based phylogenetic analysis. This concluded that PPRV ‘P’ gene based analysis is not suitable for lineage detection. Meanwhile, the PPRV/INDIA/TN/Apampattu/2014 isolate was found to group with Indian isolates coming under lineage IV. Therefore, ’P’ gene based analysis could be of help to cluster new isolates into close related groups. 

22 ref

004284   MA X, SU L, ZHANG P, ZHANG S, TANG B, ZHANG X, LUAN W, LI Z (Jilin Agricultural Univ, Changchun, China, Email: ziyi@jlu.edu.cn) : Expression and characterization of a lysine rich protein in cow milk. Indian J Anim Res 2019, 53(7), 874-9.

The lysine is considered as the most important essential amino acid, because it is the most limiting in the cereals grains. In this study, a lysine-rich (LR) gene, and the expression vector pcDNA3.1-LR and pBC1-LR were constructed. The LR was expressed in 293T cells driven by the vector pcDNA3.1-LR and checked by RT-PCR and WB. The mammary gland tissue-specific expression vector carrying the LR was injected directly into the lactating mammary glands of cows and the milk samples were checked by a complete amino acid analysis. The results showed that the LR protein was expressed successfully in cells and in cow milk; the expression of LR lasted for 6 d, and the lysine level of the injection group was significantly higher than that of negative controls (p <0.05). This study provide a better understanding of how mammary gland expression systems increase the lysine content of milk that can be applied to transgenic dairy cow.

21 ref

004270   BAI J Y, ZHAO Y G, LI G L, YANG Y B, WANG Y Q, WANG X, YANG S (Henan Univ of Science and Technology, Luoyang- 471 003, China, Email: junyanb@163.com) : Analysis of polymorphism of growth hormone secretagogue receptor in goat. Indian J Anim Res 2019, 53(7), 856-9.

Two pairs of primers (GHSR-₃, GHSR-₄) were used to amplify GHSR gene of Boer goat, Yaoshan goat and black goat. Totally two mutation sites (SNP loci), G200A and T628C were detected in amplification fragments of GHSR-₃ and GHSR-₄ respectively. For locus G200A, allele frequencies of G and A in Yaoshan goat, Boer goat and black goat were 0.637/0.595/0.827 and 0.363/0.450/0.173, respectively, which indicated that G was the dominant allele in three goat populations. G200A was synonymous mutation. For locus T628C, allele frequencies of C and T in Yaoshan goat, Boer goat and black goat were 0.466/0.449/0.458 and 0.534/0.551/0.542 respectively. Clustering analysis based on GHSR gene sequences of different species showed that Boer goat, Yaoshan goat and black goat were clustered with Capra hircus firstly, then clustered with Ovis aries and Pantholops hodgsonii, and finally clustered with cattle and whale. 

12 ref

004293   PRASAD A R, SAGAR N G, CHATTERJEE R N, PASWAN C, YADAV S P, BHATTACHARYA T K (ICAR- Directorate of Poultry Research, Hyderabad-500 030, Telangana, Email: bhattacharyatk@gmail.com) : Expression profile of the myoglobin gene in indigenous native chicken. Indian J Anim Res 2019, 53(7), 852-5.

In the present study, we quantified myoglobin (Mb) gene expression in two different indigenous chicken breeds, namely Ghagus and Aseel to discern tissue specific expression pattern in different age groups. Quantitative real-time PCR assay was developed for measurement of the Mb mRNA expression in different tissues in chickens of different age groups (day old and day 28). Overall, the Mb mRNA level displayed a significant difference among all the tissues investigated in the study. Results exhibited that, bursa and heart tissues relatively had the highest expression of Mb related to the other tissues (P<0.05). Further, in gizzard and bursa, a significant difference was found in the expression between both the breeds studied. It is concluded that the expression of Mb gene varied significantly among different tissues and between age groups in chicken.

15 ref

004310   VAID R K, ANAND T, BATRA P, LEGHA R A, TRIPATHI B N (ICAR-National Research Centre on Equines, Hisar- 125 001, Email: rk_vaid@yahoo.com) : Evaluation of DNA extraction methods of mule dung. Def Life Sci J 2019, 4(3), 170-4.

DNA  isolation  is  a  critical  step  in  microbial  community  analysis  of  animal  dung.  DNA  isolation  from  mule dung is challenging due to microbial diversity, composition and chemical nature of mule dung. Therefore, selection of an appropriate DNA isolation method is important to analyse the complete microbial diversity. In the current study, we evaluated the DNA isolation from mule dung samples (n=11) using QiAmp Mini stool kit as per manufacturer’s procedure with modifications. The results suggest that modifications in proprietary column based method improved the  DNA  quality and  quantity suitable for  mule dung  microbial community analyses.

3 illus, 2 tables, 25 ref

004308   SINHA G, TIWARI S, JADHAV S K (Pt. Ravishankar Shukla Univ, Raipur- 492 010, Email: shubhratiwari238@yahoo.co.in) : Simultaneous sachharification and fermentation of rice residues and its comparative analysis for bioethanol production. Def Life Sci J 2019, 4(3), 158-62.

Energy consumption has inflated steadily over the last century because the world population has fully grown and additional countries became industrialised. Bioethanol is an alcohol produced by fermentation of plant biomass, containing carbohydrate and its production depends upon feedstock availability, variability, and sustainability. The selection  of  feedstock  and  its  pretreatment  is  an  important  part  of  bioethanol  production  process.  In  present  work, the  exploration  of  the  potential  of  agro-waste  rice  residues  such  as,  rice  bran  and  rice  husk  was  done,  because  it contains sufficient amount of carbohydrate which can be ferment into bioethanol. The aim of the research was also to investigate how different pretreatment methods with moderate conditions differ in hydrolysis and fermentation efficiencies. Pretreatment plays an important role in the hydrolysis of cellulose and lignocellulose. It was found that biological pretreatment was a most effective method in terms of production of bioethanol and it enhances the production as well as fermentation efficiency.

4 illus, 1 table, 30 ref

004287   NIRANJANA J, GANDHI K A, SUNMATHI D, NANTHAVANAN P (Dr. N.G.P. Arts and Science Coll, Coimbatore, Tamil Nadu, Email: arungandhicbe@gmail.com) : Extraction, characterization and partial purification of L-asparaginase from the leaves of Arachis hypogaea L. Biosci Biotech Res Asia 2019, 16(3), 681-91.

L-asparaginase has been a promising therapeutic agent in the treatment of acute lymphoblastic leukemia and lymphoma. In recent times, due to the side effects of commercially available bacterial L-asparaginase and its unavoidable importance, plants are being explored as the source of L-asparaginase. The enzyme L-asparaginase was partially purified from Arachis hypogaea L. The crude enzyme extract was subjected to different purification steps including ammonium sulphate precipitation, dialysis followed by separation on Sephadex G-100 gel filtration (size exclusion chromatography) to obtain partially pure form of L - asparaginase. The enzyme was partially purified to 118 folds and contained specific activity of 4686.86 U/mg with 9.85% yield. SDS-PAGE electrophoresis of the partially purified enzyme revealed that it was a single protein with molecular weight of 70 kDa. The study on physiochemical properties showed that L - asparaginase from Arachis hypogaea L. was potassium-dependent in nature, where its optimum pH of enzyme activity was found to be 8.0 and temperature as 40°/50°C with reaction time of 15 - 20 minutes. Also it was observed that the L-asparaginase activity increased with the presence of metal ions such as Na⁺, Mg⁺⁺, making it an enzyme dependent on metal ions for its reaction. In addition to this, it was revealed that the enzyme was partially inhibited in presence of certain chelators. The specificity of L-asparaginase obtained from Arachis hypogaea L. with lack of urease activity and minimal glutaminase activity along with less cytotoxicity on human blood indicated it as an efficient chemotherapeutic agent that could be investigated further in future studies.

8 illus, 9 tables, 16 ref

004276   GAUTAM S, AMBWANI S (Molecular Biology & Genetic Engineering Dep, C.B.S.H., G. B. Pant Univ of Agriculture & Technology, Pantnagar- 263 145, Uttarakhand, Email: sg.mbge@gmail.com) : Tissue engineering: New paradigm of biomedicine. Biosci Biotech Res Asia 2019, 16(3), 521-32.

Tissue engineering is a multidisciplinary field of biomedicine that is being used to develop a new tissue or restore the function of diseased tissue/organ. The main objective of tissue engineering is to overcome the shortage of donor organs. Tissue engineering is mainly based on three components i.e. cells, scaffold and growth factors. Among these three components, scaffold is a primary influencing factor that provides the structural support to the cells and helps to deliver the growth factors which stimulate the proliferation and differentiation of cells to regenerate a new tissue. The properties of a scaffold mainly depend upon types of biomaterial and fabrication techniques that are used to fabricate the scaffold. Biofabrication facilitates the construction of three-dimensional complex of living (cells) and non-living (signaling molecules and extracellular matrices polymers etc.) components. Biofabrication has potential application especially in skin and bone tissue regeneration due to its accuracy, reproducibility and customization of scaffolds as well as cell and signaling molecule delivery. In this review article, different types of biomaterials and fabrication techniques have been discussed to fabricate of a nanofibrous scaffold along with different types of cells and growth factor which are used for tissue engineering applications to regenerate a new tissue. Among different techniques to fabricate a scaffold, electrospinning is simple and cost effective technique that has been mainly focused in the review to produce nanofibous scaffold. On the other hand, a tissue might be repair itself and restore to its normal function inside the body by applying the principle of regenerative medicine.

2 illus, 4 tables, 98 ref

004298   RIVAREZ M P, PARAC E (Biotechnology and Systems Biology Dep, National Institute of Biology, Slovenia, Email: msrivarez@up.edu.ph) : Rapid molecular detection and transmission of bacterial leaf streak pathogen, Xanthomonas Oryzae Pv. Oryzicola, in rice seeds. Biosci Biotech Res Asia 2019, 16(3), 509-20.

Xanthomonas oryzae pv. oryzicola (Xoc) is a seed-borne bacterial pathogen of rice. In this study, a rapid molecular detection method in seeds was developed to prevent unwanted movement of infected materials and greater yield loss. Crude DNA from ground seed extract supernatant was precipitated in ethanol and centrifuged. DNA pellets of homogenous subsamples were pooled and werepurified using SDS or a DNA extraction kit. Three pairs of primers previously designed on Xoc isolate BLS-256 genome sequence robustly amplify Xoc-specific DNA sequences. Primer T40 was found to efficiently detect Xoc but only in single primer reactions. While primers 3864 and 3866 can amplify Xoc DNA separately, however, their multiplex PCR reactions were unsuccessful, probably due to substrate competition in the reaction mixture. Optimized PCR mixture and conditions were able to detect as low as 10 cfu/mL Xoc cells and the direct extraction method and conditions were able to detect as low as 104 cfu/mL inoculated cells using primer T40. Lastly, Xoc transmission through seeds was observed only in plant samples which were heavily infected. Recovery from infection, at around 25-50 % was also observed in some cultivars which also manifested good grain filling at 56 days after transplanting.

6 illus, 3 tables, 20 ref

004267   ABULJADAYEL D, ATEF A, AL-MATARY M, EDRIS S, AL-GHAMDI K M, HAJRAH N H, SABIR J S M, HALL N, BAHIELDIN A (Biological Sciences Dep, King Abdulaziz Univ, Jeddah- 21589, Saudi Arabia, Email: abmahmed@kau.edu.sa) : A new PCR-based species genotyping differentiation approach in entamoeaba. Biosci Biotech Res Asia 2019, 16(3), 491-508.

The most commonly used approach for Entamoeba species differentiation up to date is the tRNA-linked STR regions of the parasite’s genome. In the present study, a new reliable, fast and easy molecular tool for species differentiation was developed. DNA was isolated from fecal samples collected from infected subjects with either Entamoeba histolytica (EH) or Entamoeba disper (ED) in Saudi Arabia. Two types of primer sets were compared in which the first targeted tRNA-linked STR regions, while the second was designed after multiple contig alignment of the two genomes using NUCmer program in aligned areas with high similarity (~90 %) and difference between of ~90 bp. The selection criteria secures that designed primers should pair with both EH and ED contig sequences at homologous regions of 200-500 bp of both species except for the presence of indels that result in the recovery of amplicons of two species with different sizes. Banding patterns in the tRNA-linked STR region resulted in the occurrence of several common amplicons. We speculate that primers mismatch with regions other than the specified STR arrays of Entamoeba histolytica or Entamoeba disper with organisms other than Entamoeba existed in the fecal sample. However, the STR-based approach looked very useful in studying strain differentiation and parasite diversity. The results for the new approach complemented those of the STR-based approach, except that the latter failed to detect coinfected subjects. The new approach proved to be useful at the species level, while the tRNA-linked STR approach can still be a good choice for strain differentiation.

6 illus, 4 tables, 20 ref

004307   SINGH R, UPADHYAY S K, AGGARWAL D, SHARMA I, PRASAD N (Biotechnology Dep, Maharishi Markandeshwar (Deemed to be Univ), Mullana-Ambala- 133 207 , Haryana, Email: sushil.upadhyay@mmumullana.org) : A study on hydroponic farming system of wheat, spinach and sword lily for sustainable development of agriculture. Bio-Sci Res Bull 2019, 35(2), 58-63.

The continuous augmented demand of food production is escalating with increase of world population. The traditional farming system will not be able to cover the world's emergent demand for food with rising pollution level and oscillations in climate. The design and development of new farming and planting system technique is urgent requirement to stay away from food catastrophe in future. The present study aimed to examine an efficient lab to land transfer technique for alternative agri-farming system, the hydroponic system. The in vitro data was evaluated and validated through numerical tools for comparative accounts between hydroponics and tap water system against Triticum sp. (Poales: Poaceae), Spinacea sp. (Caryophyllales: Amaranthaceae) and Gladiolus sp. (Asparagales: Iridaceae). The results showed that wheat, spinach and sword lily showed very good growth in Hoagland solution in comparison to tap water. Thus the field application of the proposed hydroponic system for cereals, vegetables and flowering crops will meet the world wide demand of today and future by sustainable agriculture farming approaches.

3 illus, 24 ref

004277   GHAREKHAN C H, UPASANI V N (M. G. Science Institute, Gujarat- 380 009, Email: vnu_halophiles@yahoo.com) : Diatoms from saline ecosystems and biotechnological applications: An overview. Int J Pharm Biol Sci 2019, 9(3), 869-77.

Diatoms are unicellular, eukaryotic, microscopic algae that are abundant and flourish in diverse ecosystems. They are also interesting due to their cellular structure with deposition of silica that is due to biomineralization. There are many reports on the diversity of these unique microbes in both fresh water as well as marine / saline ecosystems. They belong to the phylum Bacillariophyta family Bacillariophyceae. The DiatomBase is an online free database that provides information on the taxonomy, diversity, recognized species, etc. Currently, there are 2,546 accepted species in this database. These unique phototrophs are also found in saline ecosystems and have been explored for basic as well as applied research. They have applications in oil exploration, climate change, biomineralization, designing, nanoscience, forensic science, etc. The present article is a review to present the current information on diatoms from saline environments and their applications in various areas.

1 illus, 2 tables, 84 ref

004296   REGLA-MÁRQUEZ C F, AVILÉS-VIÑAS S A, CANTO-FLICK A, MUÑOZ-RAMÍREZ L S, PEÑA-YAM L P, VALLE-GOUGH R E, OSORIO-MONTALVO P M, PÉREZ-PASTRANA J, SANTANA-BUZZY N (Yucatan Scientific Research Center, Yucatán, México, Email: buzzy@cicy.mx) : Genes involved in the deformations of the shoot apical meristem in somatic embryos of Capsicum chinense Jacq.. J Genet 2019, 98, 70.

Somatic embryos (SE) of habanero pepper (Capsicum chinense Jacq.) represent persistent deformations in the shoot apical meristem (SAM), which inhibits their capacity to form organs and subsequently plants. In dicotyledonous plants, SAM is formed in the apex, between cotyledons and it plays a central role in postembryonic shoot organ formation. Based on the previous knowledge on the role of some families of gene in the formation, organization and maintenance of the SAM, the expression patterns of WUS, WOX2, NAM, STM, PIN1 and PIN7 genes were analysed, which would allow us to elucidate the possible implication of these genes in SAM deformations in the SE of C. chinense. The results show that the expression patterns of STM and PIN1 in the SE were completely opposite to the respective expression pattern obtained in zygotic embryos (ZE). Moreover, NAM and PIN7 showed an over accumulation of transcripts in SE, compared with ZE. This is the first time in the genus Capsicum that alterations in the expression pattern of key genes of the SE development are reported, as well as its possible implication in the persistent deformations of the SAM.

5 illus, 1 table, 37 ref

004313   ZOU L, QI D, SUN J, ZHENG X, PENG M (Henan Agricultural Univ, Zhengzhou 450046, Henan, People’s Republic of China, Email: hello_zx@hotmail.com) : Expression of the cassava nitrate transporter NRT2.1 enables Arabidopsis low nitrate tolerance. J Genet 2019, 98, 74.

The cassava grows well on low-nutrient soils because of its high-affinity to absorb nitrate. However, the molecular mechanisms by which cassava adapts itself to this environment remain elusive, although we have cloned a putative gene named MeNRT2.1which has a crucial role in high-affinity nitrate transporter from cassava seeding. Here, the expression pattern ofMeNRT2.1 was further assessed using the GUS activity driven by MeNRT2.1 promoter in Arabidopsis transformation plants. The GUS activity was monitored over time following the reduction of nitrate supply. The GUS gene expression not only peaked in roots after 12 h in 0.2 mM nitrate media, but also stained stems and leaves. Arabidopsis plants with overexpression of MeNRT2.1 increased the biomass compared to the wild type on rich nitrogen (N-full) media. However, chlorate sensitivity analysis showed that Arabidopsis plants expressing MeNRT2.1 were more susceptable to chlorate than wild type. Significantly, after growing for 15 days on media containing 0.2 mM nitrate concentration, wild-type plants became yellow or died, while the transgenic MeNRT2.1 Arabidopsis plants maintained normal growth. With significant increases in the amount of ¹⁵NO₃⁻ uptake in roots, the MeNRT2.1 plants also increased the contents of chlorophyll and nitrate reductase. Taken together, these results demonstrate that MeNRT2.1 has an important role in adaptation to low nitrate concentration as a nitrate transporter.

4 illus, 62 ref

004269   ANISIMOVA I N, ALPATIEVA N V, KARABITSINA Y I, GAVRILENKO T A (The N. I. Vavilov All-Russian Institute of Plant Genetic Resources, Saint Petersburg- 190 000) : Nucleotide sequence polymorphism in the RFL-PPR genes of potato. J Genet 2019, 98, 87.

Cytoplasmic male sterility (CMS) is widely used for hybrid seed production in cultivated Solanaceae species. However, there is very limited information about CMS-Rf genetic systems in potato (Solanum tuberosum). Studying the CMS-Rf systems in potato is both of theoretical and practical significance due to the emergence of a new revolutionary strategy of reinventing potato as a diploid inbred line-based crop to develop F1 hybrid seed potato breeding (Lindhout et al. 2011; Jansky et al. 2016). To search for potato Rf gene candidates, the comparative genetic approach was applied. Based on similarity to petunia Rf-PPR592 gene, 38 fragments were identified in five loci of the whole-genome nucleotide sequence of the accession DM 1-3 516 R44 S. tuberosum Phureja group (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The putative encoded mitochondrial proteins have 589–597 amino acid residues, similar to RF-PPR proteins of petunia and chili pepper and contain 14 or 15 PPR motifs. Primers have been developed flanking the most variable 782–865 bp regions of the selected loci, and polymorphism of the cloned fragments has been investigated in a subset of nine potato genotypes. The amplified fragments included seven or eight PPR motifs and lacked introns. The SNP frequencies ranged from 7.0 to 19.8 % depending on the locus, while the ratio of nonsynonymous to synonymous substitutions varied between 0.9 and 2.1. Positions 1, 3 and 6 were the most variable in the studied PPR motifs. Our results demonstrated that the analysed sequences belong to the RFL-PPR gene subfamily and may be considered as Rf gene candidates in potato.

2 illus, 4 tables, 41 ref

004281   JAYASIMHA P P, BAISWAR P, KUMAR R, MAJUMDER D, PATRA S (ICAR- Research Complex for NEH Region, Umiam- 793 103) : Molecular characterization of Sclerotium spp in Meghalaya. J Eco-friendly Agric 2019, 14(2), 61-2.

Sclerotium delphinii and S. rolfsii are the major soil borne plant pathogens infecting many plant species worldwide. The fungus S. delphinii closely resembles S. rolfsii and causes similar symptoms. The pathogen S. rolfsii differs from S. delphinii in few aspects but still differentiation based on morphology alone is difficult. Isolations were done from the infected soybean plants showing collar rot symptoms. Molecular identification of Sclerotium isolates using 28s rDNA region revealed that both Sclerotium rolfsii and S. delphinii are present in this region.

1 illus, 10 ref

004271   BAJPAI A, MUTHUKUMAR M (ICAR-Central Institute for Subtropical Horticulture, Lucknow- 226 101) : Advances in genomics for fruit improvement. J Eco-friendly Agric 2019, 14(2), 38-40.

The omic tools are being used for chancing the quality and nutritional composition of fruit, besides they also play a significant role in resistance breeding, shelf life enhancement and productivity. The use of genomics, proteomics, transcriptomics and metabolomics provide insights to the molecular mechanisms of flowering, fruit development, ripening, insect resistance, herbicides tolerance, etc. Genomics and ascompanying technologies enable systems biology approach toward deciphering complex interactions between genes, proteins and metabolites for resulting phenotype.

1 table, 16 ref

004309   SOLEIMANPOUR E, BABAEI E, MOHAMMAD-ALI HOSSEINPOUR-FEIZI, MONTAZERI V (Animal Biology Dep, Tabriz Univ, Tabriz, Iran, Email: pourfeizi@ eastp.ir) : Circulating miR-21 and miR-155 as potential noninvasive biomarkers in Iranian Azeri patients with breast carcinoma. J Cancer Res Ther 2019, 15(5), 1092-7.

Breast cancer (BC) is the most common cause of cancer‑related mortality among women. Despite recent advances in diagnosis and prognosis of breast carcinomas, noninvasive biomarkers have been poorly identified. We evaluated the biomarker potential of miR‑21 and miR‑155 in tissue and plasma specimens of Iranian Azeri patients. Tumor specimens, paired nontumoral adjacent tissues, and matched plasma samples were collected from a number of thirty Iranian Azeri women with breast carcinoma. Plasma of healthy women was used as the control. The relative expression of miR‑21 and miR‑155 was measured by real‑time polymerase chain reaction. Our data revealed that the expression levels of miR‑21 and miR‑155 in tumor tissues are significantly higher than paired nontumoral adjacent specimens (P < 0.05). Furthermore, receiver operating characteristic (ROC) curve analysis of samples showed the area under the ROC curve of 0.81 for miR‑21 and area of 0.83 for miR‑155. In addition, statistical analysis showed that miR‑21 and miR‑155 RNAs are significantly detected in the plasma of BC patients compared to healthy specimens (P < 0.05). Circulating miRNAs yielded area under the ROC curve of 0.99 for miR‑21 and 0.92 for miR‑155. Our data showed that miR‑21 and miR‑155 oncomiRs can be considered as noninvasive biomarkers for monitoring breast carcinomas. However, further investigations are needed to confirm the use of these noncoding RNAs in pathology.

2 illus, 1 table, 56 ref

004273   CHAKRABORTY S, BASU A (National Institute of Biomedical Genomics, Kalyani- 741 251) : Reconstruction of ancestral footfalls in South Asia using genomic data. J Biosci 2019, 44(3), 74.

Due to its unique geographical position, juxtaposed in the middle of south-central Asia, east Asia and Southeast Asia, the South Asian Region (SAS) has repeatedly come into contact with people from adjacent regions throughout history and prehistory. The antiquity of the populations and the intricate history of admixture have shaped SAS as one of the most genetically diverse regions in the world. In this article we review our current understanding of the peopling and populations structure of SAS. We do not attempt to be exhaustive but summarize the salient conclusions that have been reached using genetic data and evaluate their robustness. We also identify the unanswered questions and suggest possible approaches that may lead to their answers.

27 ref

004305   SINGH A, RAI A K, TRIPATHI G D, NAND V, GUPTA N, SRIVASTAVA K (Sri Ramswaroop Memorial Univ, Barabanki, Uttar Pradesh, Email: anupam4uk@gmail.com) : Development of species-specific polymerase chain reaction (PCR) targeting on mitochondrial D-loop for identification of buffalo and goat raw meat. J Biol Engg Res Rev 2019, 6(2), 1-4.

Today's, consumers are concerned about the meat they eat and also demand accurate labeling. Mitochondrial Analysis of DNA was the most frequently used DNA, because of its highly conserved sequences in various organism species. In this study, a rapid, reproducible and simple method for simultaneous identification of multiple meat species in a single step mitochondrial DNA based test has been developed based on the designing of species-specific primer. Meat samples of goat and buffalo were selected to verify the applicability of the technique. A species specific forward and reverse primer was designed with the help of the primer3 tool for amplification of mitochondrial D-loop region. The species-specific primers were verified in silico by SnapGene software. The two pairs of primers amplified the expected fragment of 338 bp for buffalo and 450 bp for goat. The change in the size of the PCR product was due to the existence of highly polymorphic regions within the buffalo and goat D-loop region. The tested species gives a unique band pattern for each species by using successful amplification of these polymorphic regions in the D-loop region. Overall, the simplicity of amplification of mitochondrial D-loop region could make this technique suitable for meat authentication in routine analysis.  

4 illus, 1 table, 15

004274   CHAUHAN P, SINGLA K, RAJBHAR M, SINGH A, DAS N, KUMAR K (Manav Rachna International Institute of Research and Studies, Faridabad, Haryana, Email: kapila.fet@mriu.edu.in) : A systematic review of conventional and advanced approaches for the control of plant viruses. J Appl Biol Biotechnol 2019, 7(4), 89-98.

Viruses are the obligatory intracellular parasites infecting microbes, plants, animals, and humans. They are dead outside host cell but can take-over the host’s cell machinery as soon as they are into it. Several studies on inhibitor compounds have been done for animal viruses including those that are affecting humans, but there is inadequacy in terms of research and literature for plant viruses that are responsible for losses in crop yield and quality loss all across the globe. This could be focal point to study plant viruses, their transmission and pathogenicity, and to establish widely used, effective, and advanced approaches for their control. The purpose of this review is to discuss various approaches to control plant viruses that have been developed and applied to combat plant viral infections. We have divided these approaches into two categories conventional (meristemtip culture, cryotherapy, thermotherapy, and chemotherapy) and advanced (nucleic acid-based approaches like RNA Silencing, cross-protection, transgenic plants, gene pyramiding, and protein-protein interaction). Moreover, we have discussed and compared the principles, methodologies, advantages, and disadvantages of each technique. The approaches have been explored to promote their application in best suited way on various plants to control viral diseases and to improve food crops quality with increase in production.

3 tables, 124 ref

004312   YADAV A N, YADAV N, SACHAN S G, SAXENA A K (Biotechnology Dep, Eternal Univ, Baru Sahib, Himachal Pradesh, Email: ajar@eternaluniversity.edu.in) : Biodiversity of psychrotrophic microbes and their biotechnological applications. J Appl Biol Biotechnol 2019, 7(4), 99-108.

The extreme cold environments harbor novel psychrotrophic microbes. The psychrotrophic microbes have been reported as plant growth promoters and biocontrol agents for sustainable agriculture, in industry as cold-adapted hydrolytic enzymes and in medicine as secondary metabolites and pharmaceutical important bioactive compounds. Inoculation with psychrotrophic/psychrotolerant strains significantly enhanced root/shoot biomass and nutrients uptake as compared to non-bacterized control. The psychrotrophic microbes play important role in alleviation of cold stress in plant growing at high hill and low temperature and conditions. The psychrotrophic microbes have been reported from worldwide from cold habitats and belong to all three domain archaea, bacteria, and eukarya including different phylum such as Actinobacteria, Ascomycota, Bacteroidetes, Basidiomycota, Chloroflexi, Chlamydiae, Planctomycetes, Cyanobacteria, Euryarchaeota, Firmicutes, Gemmatimonadetes, Verrucomicrobia, Mucoromycota, Proteobacteria, Spirochaetes, Thaumarchaeota and Nitrospirae. The most dominant genera belong to Arthrobacter, Bacillus, Exiguobacterium, Paenibacillus, Providencia, Pseudomonas, and Serratia have been reported from the cold habitats. The Psychrotrophic microbes have biotechnological applications in agriculture, medicine, industry, food, and allied sectors.

1 illus, 3 tables, 129 ref

004304   SHARMA N, ANAS M, RABBANI A, BHARTI P, KAUNDAL S, KUMAR V, MAHATO B (Biochemistry Dep, Shri Guru Ram Rai Univ, Dehradun, Uttarakhand, Email: sharmanarotam5@gmail.com) : HLA-B*27 subtypes and its clinical implications characterization by studying sequence-specific alleles. J Appl Biol Biotechnol 2019, 7(4), 84-8.

Ankylosing spondylitis (AS) is a form of spondylitis in which spine is affected primarily along with other joints. The vertebrae fuse together and form a rigid structure. Individuals with AS are also positive for human leukocyte antigen (HLA) B-27. One hundred and eighty-three symptomatic patients of AS were used in the study. Sequence-specific priming (SSP) PCR technique was used to detect the HLA B-27 specific allele. This study showed (out of 183 suspected cases, 45 cases were detected positive with HLA B-27 allele, while the remaining 138 were negative) that the positivity rate for AS with HLA B-27 allele is less. In 183 cases, 63 were females, whereas 7 cases were positive and 56 negative, whereas 38 cases were positive for male and 82 cases were negative. When sample was analyzed in term of age groups, it was found that out of 45 positive samples, the positivity rate was maximum, i.e., 57.14 % in the patient above 37 years of age. Furthermore, it was observed that the males above 30 years are more prone to develop AS with HLA B27 specific allele. For the diagnosis of AS, conventional PCR technology is a promising diagnostic method and can be considered as an important tool along with other diagnostics parameters.

2 illus, 35 ref

004292   PRANAY K, PADMADEO S R, JHA V, PRASAD B (Biochemistry Dep, Patna Univ, Patna, Bihar, Email: kumar.pranay762@gmail.com) : Screening and identification of amylase producing strains of Bacillus. J Appl Biol Biotechnol 2019, 7(4), 57-62.

An attempt was made to isolate and screen efficient amylolytic strains of Bacillus sp. Initial screening based on the starch hydrolysis ratio resulted in the selection of 72 amylolytic bacterial strains. Among these, 18 strains were selected for further studies. Secondary screening based on amylase production in starch broth medium led to the selection of six amylolytic strains of Bacillus sp. The selected strains were grown in four different fermentation media (FMI-FMIV) in order to screen for three most efficient amylolytic strains for optimization and characterization. FMIV was the best basal medium as it provided required nutrients, which stimulated highest amylase production in bacterial strains within shortest incubation time ( 24 hours). Molecular identification based on 16S rDNA sequence revealed that three most efficient strains [BCM36 (KR1), BCM33 (KR2), and BCM25 (KR3)] belonged to Bacillus sp.

2 illus, 3 tables, 34 ref

004283   KAUSHAL S, SIDANA A (Biosciences Dep, Asian Coll, Patiala, Punjab, Email: shiwanikaushal23@gmail.com) : A novel cost-effective medium for in vitro cultivation of Gentiana kurroo Royle using sugarcane bagasse. J Appl Biol Biotechnol 2019, 7(4), 26-31.

To develop a simple, cost-effective, and efficient medium by using sugarcane bagasse (SB) as a base material to replace the conventional Murashige and Skoog (MS) medium.  Water extracts of SB along with some macronutrients and plant growth regulators were gelled with 0.7 % agar-agar powder. Nodal segments of Gentiana kurroo were used as explants and inoculated in the medium and placed in a growth chamber under standard conditions of light and temperature. Out of the tested combinations of plant growth regulators, 0.5 mg/l each of kinetin (KN) and 6-Benzylaminopurine (BAP) showed the excellent shoot multiplication and proliferation rate on the bagasse medium with the same potential as on the MS medium with an average of 5–6 shoots/explant. In vitro rooting was obtained on half strength MS medium supplemented with IBA (0.5 mg/l) with an average length of 7–8 cm and 20–25 roots/explant. The plants were hardened in a mixture of clay loam and farmyard manure in 1:1(w/w) with 70 %–80 % survival rate without any phenotypic aberrations. The results from the present investigation indicate that SB can be used as a cost-effective substitute of MS medium for in vitro propagation of G. kurroo.

3 illus, 2 tables, 20 ref

004301   SAWANT S S, KELKAR-MANE V (Biotechnology Dep, Mumbai Univ, Santacruz, Mumbai, Email: drvkelkar@mu.ac.in) : Study of the changes in the growth, protein, and bioactive profile of Chlorella emersonii KJ725233 in response to sodium and ammonium nitrate. J Appl Biol Biotechnol 2019, 7(4), 19-25.

Chlorella emersonii KJ725233 is a non-fastidious microalga isolated from the western regions of Maharashtra, India. The alterations in its chlorophyll, protein, and antioxidant content in response to cationic stress were studied. Chlorella emersonii KJ725233 (CEK) was subjected to NaNO₃ and NH₄ (NO₃ )² at concentrations equivalent to 0.9 g/l (1×) and 1.8 g/l (2×) in the cultivation media. Qualitative alterations in the bioactives of the microalga were identified by gas chromatography–high-resolution mass spectrometry (GC-HRMS), while the protein, chlorophyll, and antioxidant manipulations were spectrophotometrically quantified. Doubling of the protein content was observed when CEK was grown in 1× NH₄ ⁺ , whereas in 2× NH₄ ⁺ , chlorosis was significant. 2× NH₄ ⁺ also induced oxidative stress on CEK as evident from the 85.72 % ± 6.72 %, 197.47 % ± 7.01 %, 22.24 % ± 1.78 %, and 187.37 % ± 1.88 % increase in antioxidant potential, ferric reducing capacity, radical scavenging potential, and total phenolic content, respectively, as compsared to CEK grown in 1× Na⁺ . These alterations as indicated from the GC-HRMS data correlate to the bioactive inductions/variations of/in Vitamin E, phytol, and its isomer in CEK grown in 2× NH₄ ⁺ . The study thus indicates that manipulations of nitrate salts in the media significantly induce, as well as alter the concentrations of commercially significant compounds like Vitamin E, phytol, etc.

3 illus, 3 tables, 47 ref

004289   PATIL V M, PATOLE K R, PAPRIKAR M S, RAJPUT J C (Biotechnology Div, Nirmal Seeds Pvt., Ltd, Jalgaon, Maharashtra, Email: drjcrajput@nirmalseedsindia.com) : Screening of different Pseudomonas and Bacillus Spp. for production of L-glutamic acid and impact of their cell-free filtrate on growth and yield of brinjal (Solanum melongena). J Appl Biol Biotechnol 2019, 7(4), 14-8.

Amino acids can play a different role in plants such as nitrogen source, hormonal precursor, and stress reducing agents. L-glutamic acid is involved in several plant metabolic processes. The objectives of present work were to evaluate the L-glutamic acid production by different agriculturally important Pseudomonas and Bacillus species and to determine the effects of L-glutamic acid containing cell-free filtrate on the growth and yield of brinjal. An experiment was conducted in a completely randomized block design. Out of eight different strains of Pseudomonas, the highest L glutamic acid was detected in Pseudomonas fluorescens 128 (1.397 g/l) followed by P. fluorescens NSPL07 (1.073 g/l) and Pseudomonas striata RCOF153 (0.563 g/l). Similarly, out of six different species of Bacillus, moderate L-glutamic acid was detected in Bacillus amyloliquefaciens MTCC10439. (0.232 g/l). The growth stimulatory effects of L glutamic acid containing filtrate (200 ppm) were also studied and it was found that all growth parameters of brinjal (plant height, fruits per plant, fruit yield, etc.) improved significantly. Results indicated that bacterial secretion containing L-glutamic acid along with other amino acids has biostimulatory effects and it should be used to enhance the plant growth and yield.

2 tables, 28 ref

004275   FAKOREDE C N, ITAKORODE B O, ODEYEMI O, BABALOLA G (Biological Sciences Dep, Oduduwa Univ Ipetumod, Osun State, Nigeria, Email: olaniretifakoroede@yahoo.com) : Enhancement of pigment production potential of Serratia marcescens (GBB151) through mutation and random amplified polymorphic deoxyribonucleic acid analysis of its mutants. J Appl Biol Biotechnol 2019, 7(4), 1-6.

Serratia marcescens (GBB151) was isolated and genetically modified for high-yielding pigment production capacity that could be employed for industrial purposes. Ethidium bromide-induced mutagenesis of GBB151 resulted in the generation of eight mutant isolates (GBB151Ea-GBB151Eh). The chemical mutants of S. marcescens obtained produced 5-fold more pigment than the wild-type organism. The wild-type GBB151 produced 413.9 unit/cell, while the mutant strains produced pigments with yields ranging from 841.7 to 2008.5 unit/cell. Random amplified polymorphic deoxyribonucleic acid-polymerase chain reaction analysis showed different amplicons patterns of native as well as mutant derivatives. The factorial analysis diagram and the dendrogram showed a degree of dissimilarity among the wild-type bacterial isolate GBB151 and its mutants. Mutant strains GBB151Ec and GBB151Ef were closest to the wild type as they appeared in the same quadrant. GBB151Ed which had lost its ability to produce pigment was farthest and in the different quadrant to the wild type. These study provided insight into improvement in pigment production by manipulating genetic make-up of S. marcescens, thus meeting industrial demand.

5 illus, 1 table, 23 ref

004295   RAKHSHANDEHROO E, RAZAVI S M, FARZANEH R, ESMAILNEJAD A, ASADPOUR M, SHAMS S (Pathobiology Dep, Shiraz Univ, Shiraz, Iran) : Phylogenetic analysis of goat warble fly (Przhevalskiana silenus) based on mitochondrial COI gene. J Parasit Dis 2019, 43(2), 304-7.

The larvae of the genus Przhevalskiana (Diptera: Oestridae) are the causative agents of subcutaneous myiasis in goats. Several species have been grouped under this genus based on the morphology of different larval stages, albeit with a lot of uncertainties. Thus, application of genetic tools seems to be helpful for taxonomy. During this study, the cytochrome oxidase I (COI) gene was targeted for the characterization of larval stages of goat warble fly. Fragments of 606 bp were amplified for all the specimens. Based on the COI gene analysis, all the recovered specimens were identified as larvae of Przhevalskiana silenus. Molecular data on the genus is relatively rare but present isolates revealed about 87–89% identity with previous isolates of P. silenus. According to the phylogenetic data, the present isolates branched (as a sister group) with a number of Hypoderma spp. including H. bovis, H. diana, H. lineatum and  Hypoderma sinense. The present findings confirmed that the COI gene could be a suitable marker for genetic characterization and identification of larvae up to the species level.

3 illus, 1 table, 14 ref

004278   GUDA S J M, SONTAM B, BAGGA B, RANJITH K, SHARMA S, JOSEPH J (L. V. Prasad Eye Institute, Hyderabad, Telangana - 500 034, Email: joveeta@lvpei.org) : Evaluation of multiplex real‑time polymerase chain reaction for the detection of herpes simplex virus‑1 and 2 and varicella-zoster virus in corneal cells from normal subjects and patients with keratitis in India. Indian J Ophthalmol 2019, 67(7), 1040-6.

To determine the presence of herpes simplex virus and varicella zoster virus (HSV 1 and 2, VZV) in the cornea of normal subjects by multiplex real time quantitative (qPCR) assay and evaluate its utility in the diagnosis of viral keratitis. Corneal epithelial cells from 33 eyes of 22 patients undergoing photorefractive keratectomy surgery (controls) and 50 corneal scrapings from 50 patients with suspected HSV keratitis were analyzed for the presence of HSV1 by conventional PCR and for presence of HSV1 and 2 and/or VZV by multiplex real‑time PCR. Corneal scrapings of patients were also tested for HSV1 antigen by immunofluorescence assay (IFA). The results were compared and clinical records reviewed. HSV1 and VZV DNA were detected in 8/33 controls (mean‑14.3 ± 7.96, range: 3‑29.1 copies/mL) and 2/33 controls (mean‑10.7 ± 10.9, range 3‑18.5 copies/ml) respectively. HSV2 was not detected in any of the controls. Copy numbers above the mean + 1SD of controls were considered significant for viral load in patient samples. Significantly higher number of corneal scrapings (39/50, 78 %) from patients were positive for HSV1 (1.2 × 10⁶ copies/mL ± 3.7 × 10⁶ copies/mL) by real time qPCR compared to IFA (11/48, 23 %, P value 0.0001) and conventional PCR (20/50, 40 %, P value 0.0002). Double infection with HSV‑1 (1.5 × 10⁷ copies/ml) and HSV‑2 (3.57 × 10⁴ copies/ml) in one case and VZV infection (1.03 × 10² copies/ml) in another was also detected by the multiplex real‑time PCR. Multiplex real‑time PCR reliably detects HSV1 and 2 and VZV DNA and is ideal for the diagnosis of HSV and VZV keratitis in an ocular microbiology laboratory.

1 illus, 2 tables, 29 ref

004294   PRASAD R, BANERJEE S, KHARSHIING C E, BHATTACHARJEE A, PRASAD S B (Biotechnology and Bioinformatics Dep, North-Eastern Hill Univ, Shillong - 793 022, Email: sbnehu@gmail.com) : Rutin-mediated apoptosis and glutathione changes in ascites Dalton’s lymphoma cells: In silico analysis of rutin interactions with some antiapoptotic and glutathione-related proteins. Indian J Pharm Sci 2019, 81(4), 720-8.

Rutin-induced changes in apoptosis and reduced glutathione levels was studied in murine ascites Dalton’s lymphoma cells under different treatment conditions followed by the binding affinity of rutin and cisdiamminedichloroplatinum (II) with two antiapoptotic proteins and two glutathione-related enzymes was examined through molecular docking analysis. Rutin, as well as cis-diamminedichloroplatinum (II) induced apoptosis and a decrease in reduced glutathione level in Dalton’s lymphoma cells. The molecular interactions of rutin, cis-diamminedichloroplatinum (II) and a known inhibitor of the protein/enzyme showed that the binding free energies (ΔG) with antiapoptotic proteins, Bcl-xL and c-FLIP, and glutathione-related enzymes such as glutathione S-transferase and glutathione reductase were closer and comparable, which might suggest inhibitory potential of rutin on these proteins/enzymes and could play a role in its anticancer activity.

5 illus, 2 tables, 40 ref

004282   KARDAM P, MEHENDIRATTA M, REHANI S, SHARMA R, SAHAY K (Oral Pathology and Microbiology Dep, Sudha Rustagi Coll of Dental Sciences and Research, Faridabad, Haryana, Email: priyankakardam@ gmail.com) : A crude method of DNA extraction and identification from exfoliated human buccal mucosa cells. Indian J Dent Res 2019, 30(4), 595-9.

DNA analysis has a key role in forensic dentistry. However, techniques of DNA extraction and analysis are far from the reach of majority of medical professionals owing to its expensive set up. The present study was aimed at formulating a crude method of extracting DNA from human buccal mucosa cells using materials commonly available in the laboratory so that the medical professionals could get more exposure to molecular biology techniques. The objectives were to identify the DNA and to assess its purity. Buccal mucosa cells from 10 healthy volunteers were taken for DNA extraction following the protocol of cell lysis, purification, and precipitation. DNA was identified using standardized techniques like Diphenylamine test and its purity was assessed using a spectrophotometer. A gel electrophoresis apparatus was also constructed using readily available materials. DNA was extracted from human buccal mucosa cells using a crude method. The standardized tests confirmed the presence of DNA contaminated with proteins. The locally made Gel electrophoresis model exhibited a faint halo around the wells instead of DNA bands. DNA extraction from human buccal mucosa cells was made possible using locally available materials and a crude method, but it was not of high purity.

3 illus, 2 tables, 13 ref

004300   SALMAN H A, SENTHILKUMAR R, MAHMOOD B S, IMRAN K (Biotechnology Dep, Krupanidhi Degree Coll, Bengaluru, Karnataka, Email: qhalidimran@gmail.com) : Detection and characterization of Streptococcus Downei, a rare bacterial species of Mutans streptococci from caries-active patients. Indian J Dent Res 2019, 30(4), 579-82.

The oral bacteria, mutans streptococci (MS), are an etiological agent of dental caries. Of MS, Streptococcus downei are rarely isolated bacteria. The aim of this study was to isolate and characterize S. downei from caries‑active subjects. In all, 65 dental plaque samples were collected from dental caries‑active subjects. All the isolates were further identified and characterized using 16S rDNA sequencing, biochemical tests, antibiogram, and minimum inhibitory concentration (MIC). Five isolates have been identified as S. downei using 16S rDNA sequencing. Phylogenetic analysis showed that S. downei was closely related to S. sobrinus. The biotype traits of these five isolates were IV (n = 3), V (n = 1), and variants (n = 2). The study proposed one new biotype, classified as biotype VIII for the variant strain. The antibiogram tests revealed that all the strains of S. downei were susceptible to all the antibiotics used in the study with higher sensitivity to penicillin and ampicillin. The MIC of ampicillin and erythromycin against S. downei was 0.047 and 0.39 µg/mL, respectively. The study reports the prevalence of S. downei in caries‑active subjects and recommends further investigations to determine its role in the disease.

1 illus, 2 tables, 22 ref

003024   MANOHARLAL R, SAIPRASAD G V S, KOVARÍK A (ITC Life Science and Technology Centre, Bengaluru- 560 058, Karnataka) : Gene-specific DNA demethylation changes associates with ethylene induced germination of soybean [Glycine max (L.) Merrill]. Plant Physiol Rep 2019, 24(2), 272-7.

The present work aims to evaluate epigenetic changes during ethylene (ET) induced germination in soybean [Glycine max (L.) Merrill]. Our results demonstrated that ethephon (2-chloroethylphosphonic acid, an ET-releasing compound) primed seeds exhibited: (1) DNA hypo-methylation of representative candidate genes (viz. ERF1b and AS-α1) and (2) functional restoration of DNA methylation dependent phenotype and expression of transcriptional silenced reporter transgene. Based on these observations, we proposed the novel and generic role of ET as a DNA de-methylating agent during the germination, which may lead to early developmental phenotypes in plants.

2 illus, 41 ref

003022   KUMAR C N P, RAJ E E, KUMAR P M, KUMAR R R, CHANDRASHEKARA K N (Plant Physiology and Biotechnology Div, Tea Research Foundation, Tamil Nadu- 642 127) : Cold treatment prompts embryogenic callus and cytodifferentiation in the anthers of tea (Camellia sp.) genotypes. Plant Physiol Rep 2019, 24(2), 215-24.

The simple and rapid protocol was developed to induce androgenic embryoids from immature anthers of tea genotypes. Anthers from unopened flower buds of ~ 5 to 6 mm size were subjected to two temperature treatments at 4 °C and 25 °C temperatures for 5 days and cultured onto two mediums, half strength MS liquid and 0.8 % agar medium. The inoculated cultures were incubated under complete dark (24 h) and photoperiod (9:15 h) conditions. Anthers of nine tea clones (UPASI-1, UPASI-2, UPASI-3, UPASI-8, UPASI-10, UPASI-20, UPASI-28, and certain estate selections, ATK-1 and CR-6017) initially cultured onto half strength Murashige and Skoog’s (MS) liquid medium supplemented with 2,4-dichlorophenoxy acetic acid (9.05 µM 2,4-D) combined with kinetin (2.44 µM Kn) and 6-benzylamino purine (2.22 µM BAP). The pre-treated anthers at 4 °C for 5 days of genotype UPASI-3 induced significantly higher percentage of callus induction when cultured onto half strength MS agar medium under both continuous dark and photoperiod (9:15 h) conditions. Percent of callus production was significantly (P < 0.001) influenced by the genotypes (G) and plant growth regulators (P) while it was not affected by culture conditions (Dark; D/Photoperiod: L). Irrespective of genotypes studied, medium supplemented with BAP is superior to Kn in inducing androgenic potential and promotes the emergence of embryoid structures in a short time span of 45 days and the emergence of morphologically prominent globular mature embryoid callus was identified for 55 days and subsequent cytodifferentiation were achieved only in medium fortified with 2,4-D along with BAP. The results found that cold treatment and culture medium accelerated the development of embryogenic calli. Histological evidences of embryoids revealed that in vitro cytodifferentiation rendering to cellular totipotency developed different meristematic cells and vascular elements. Anthers provided with cold treatment induced callus in the liquid growth medium. The liquid growth medium formulation used here is suitable for inducing androgenic potential and promoted callus formation from anthers in less time span for 10–15 days. Callus maturation and immature embryoid callus was induced for 45 days and morphologically prominent round mature embryoids were appeared by 55 days. And the histological studies reveal the trend of in vivo cytodifferentiation rendering to cellular totipotency while growing in vitro. Our experiment concludes the profuse callus induction and embryoid induction by using liquid growth medium formulation, which is difficult task in anther culture of tea because of its highly recalcitrant nature. Liquid medium hastens the androgenesis process in tea anthers and induced embryoids when cultured onto same solid agar medium.

1 illus, 5 tables, 63 ref

003025   MANUKA R, KARLE S B, KUMAR K (Biological Sciences Dep, K. K. Birla Goa Campus, Goa- 403 726) : OsWNK9 mitigates salt and drought stress effects through induced antioxidant systems in Arabidopsis. Plant Physiol Rep 2019, 24(2), 168-81.

With No Lysine kinase (WNK) belongs to the ser/thr protein kinases category in which the conserved catalytic lysine (K) residue is present in subdomain I instead of being in subdomain II. WNKs alleviate various abiotic stresses through a variety of signal transduction pathways governing various stress tolerance mechanisms in plants. These mechanisms include accumulation of osmo-regulator entities such as proline, activation of various antioxidant enzymes such as catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD) as well as certain non-enzyme molecules like secondary metabolites. All of these mechanisms flush out harmful reactive species and maintain cellular integrity in plants. The present study evaluated biochemical properties of wild-type (WT) and OsWNK9 transgenic Arabidopsis lines against the salt and drought stress conditions. Transgenic lines showed high levels of proline accumulation, reduced membrane damage and hydrogen peroxide content compared to WT plants. Moreover, the transgenic lines exhibited the improved activity of antioxidant enzymes such as catalase and ascorbate peroxidase along with the dynamism in peroxidase activity. The total antioxidant capacity in terms of DPPH free radical scavenging percentage was highest in the transgenic lines in comparison to the WT. The Na⁺, K⁺ and Na⁺/K⁺ measurements among the WT and transgenic lines suggested that the transgenic lines efficiently maintained the intracellular ionic balance and thereby reduces the extent of the cell death. Altogether, transgenic Arabidopsis lines evinced more tolerance to salinity and drought stress than WT plants signifying the involvement of OsWNK9 gene in the regulation of abiotic stress tolerance.

47 ref

003011   ANUPMA, KALIA V, KAUR S (ICAR-National Research Centre on Plant Biotechnology, New Delhi- 110 012, Email: dr_sarvjeetkaur@yahoo.com) : Efficacy of transgenic tobacco carrying synthetic plant-preferred codon-optimized novel VIP3AA44 gene towards Helicoverpa armigera and Spodoptera litura. Indian J Entomol 2019, 81(2), 325-31.

Vip3A proteins are synthesized during vegetative growth of Bacillus thuringiensis and are toxic against a wide range of lepidopteran insects. Since the mode of action of vip3A toxins is different from Cry proteins, vip3A proteins are good candidates for gene pyramiding in transgenic crops to combat development of resistance against the currently deployed genes. A synthetic plant-preferred codon-optimized novel vip3Aa44 gene (NCBI accession number HQ650163) was cloned into pBINAR plant transformation vector and tobacco explants were transformed with leaf disc co-cultivation method to evaluate toxicity of this gene against Helicoverpa armigera and Spodoptera litura. The putative transgenics were confirmed by PCR and RT-PCR analysis. The bioassays were performed on detached leaves from putative transgenics using lab-grown population of H. armigera and S. litura. Mortality after 72 hr ranged from 30-56 % for H. armigera and 40-60% for S. litura, indicating potential of vip3Aa44 gene against these lepidopteran pests in transgenic development.

4 illus, 34 ref

003021   KHAN M A (Biochemistry Dep, Lovely Professional Univ, Phagwara-144 411, Punjab) : Phylogenetic identification of chitinase secreting soil bacteria antagonistic to Fungus aspergillus. Nat Environ Pollut Technol 2019, 18(2), 633-8.

Economic losses arising from crop diseases caused by phytopathogenic fungi are principally associated with yield reductions. One alternative to fungicides is biocontrol with beneficial bacteria. Next to Pseudomonas, the endospore-forming genus Bacillus is probably the most widely researched and commercialized biocontrol agent. It is generally recognized that biological control agents are safer and more environmentally sound than is reliance on the use of high volumes of pesticides. The primary objective of current work was to isolate the specific bacterial strain that possesses the potential to act as an antagonist against the fungal pathogen Aspergillus which is thought to secrete aflatoxin, the most potent mycotoxin. Many bacterial cells were isolated randomly from the rhizospheric region of groundnut crop by serial dilution method, which were further screened for their antagonistic effect against Aspergillus. Screening results show that few of the isolates exhibit significant antagonism on dual culture media. One of the bacterial isolates SJ 609 exhibiting the maximum antagonistic effect was further selected after optimization for further studies. The mechanism of action of SJ 609 was studied in vitro for the production of hydrolytic enzymes particularly chitinase which hydrolyses chitin, which is the major component of fungal cell wall. Phylogenetic analysis using 16S rDNA amplification was carried out to establish its identity.

5 illus, 14 ref

003013   BHATTACHARJEE I, MAZUMDAR D, SAHA S P (Microbiology Dep, North Bengal Univ, Siliguri, West Bengal) : Microbial amylases and their potential application in industries: A review. Pharma Innov 2019, 8(6), 162-70.

Amylases are starch hydrolysing enzymes which breakdown starch to dextrin and smaller polymers composed of glucose units. Amylases have application in various industries including food, textile, paper, pharmaceutical industries. Enzymes are good alternative over chemical catalysts based on the growing environmental concern. Enzymes from the microbial sources have dominated applications in industries. The native starch has many undesired properties such as tendency to retrograde, instability at high temperature and low pH and poor water solubility. Enzymatic modification of starch may offer a more favourable and linear structure. This review focuses on the structure and mechanism of action of amylases and their various industrial application and enzymatic modification of starch.

7 illus, 2 tables, 86 ref

003029   MOHITE A V, GURAV R V (Botany Dep, Shivaji Univ, Kolhapur- 416 004, Email: botanyraj@rediffmail.com) : Induced mutation using gamma rays in Okra (Abelmoschus esculentus (L.) Moench). J Appl Hortic 2019, 21(3), 205-8.

Abelmoschus esculentus (L.) Moench, popularly known as okra or bhindi, is an important vegetable crop cultivated mostly in tropical and subtropical countries. The present study aims to induce variability in this economically significant crop using gamma rays. Mature, dry and healthy seeds, irradiated with different doses of gamma rays were allowed to grow in the field along with control seeds. The effect of gamma radiations on different agronomic traits and yield contributing characters of okra, including shoot length, root length, number of secondary branches, fruits per plants, seeds per fruit and 100 seed weight were recorded for M1 and M2 generations. The observed agronomic and yield based characters in the M2 mutants at various doses of gamma rays were found to be significantly correlated. The obtained M2 mutants are useful in development of an improved variety of okra, having agronomically significant traits.

1 illus, 2 tables, 16 ref

003039   SIVAJOTHI S, RAYULU V C, RAO T P, JEYABAL L, KUMAR T V S, SUBRAMANYAM K V (Veterinary Parasitology Dep, Sri Venkateswara Veterinary Univ, Tirupati, Andhra Pradesh) : Detection of cysteine proteinase gene hmcp5 of Haemonchus contortus by reverse transcriptionpolymerase chain reaction. Pharma Innov 2019, 8(6), 24-7.

Barber’s pole worm cysteine proteinases are considered as the important candidates in the immunological control of haemonchosis in sheep and goats. The control of Haemonchus contortus is largely based on pasture management and the antihelmintics utilization. Due to indiscriminating use of drugs, occurrence of resistant to anthelmintics and it is need for the development of alternative control methods i.e. vaccine development. Cysteine proteases are considered as the excellent vaccine candidates due to their role in maintenance of worm nutrition as well as immune evasion. Cysteine proteases have been implicated for development of the vaccination because of their affinity purified detergent soluble extracts of Haemonchus contortus. The aim of the present study was to standardize the reverse transcriptionpolymerase chain reaction of Cysteine Proteinase gene hmcp5 of Haemonchus contortus. The characterization of cysteine protease genes in South Indian isolates may give ideas for control of Haemonchus contortus in future. It is essential for the further research protocols in development of the diagnostic or vaccination modalities.

4 illus, 2 tables, 15 ref

003033   RAM V P, RAO L V, RAO T S, SUBRAMANYAM K V, SRINIVAS K (Veterinary Public Health and Epidemiology Dep, NTR Coll of Veterinary Science, Gannavaram, Andhra Pradesh) : Prevalence and virulence gene profiles of Proteus mirabilis isolated from animal, human and water samples in Krishna District, Andhra Pradesh, India. Pharma Innov 2019, 8(6), 19-23.

In the present study, a total of 507 samples comprising food of animal origin (215), faecal swab samples (188), human urine samples (65), human diarrhoeic stool samples (12) as well as water samples (27) were examined. Overall prevalence of P. mirabilis was found to be 34.51% (175/507) by species-specific PCR. Among foods of animal origin, the highest rate of P. mirabilis isolates were recovered from chicken samples (38.7%), followed by pork (37.5%) and mutton samples (28.9%). Among faecal swabs, the highest rate of P. mirabilis isolates were from poultry (49%), followed by pigs (37.8%). Human urine samples showed a prevalence rate of 10.7%. Water samples showed 7.4% prevalence. All the human diarrhoeic stool samples were negative for P. mirabilis. Some of the virulence factors genes were investigated using polymerase chain reaction technique. The genes ureC (80.5%), ureA (72.5%), flaA (28.5%), hpmA (60.5%) and zapA (50.28%) were detected in P. mirabilis isolates.

4 illus, 2 tables, 20 ref

003040   SRINIVAS B, ASWANI K K, PADMAJA K, PUNYAKUMARI B (Veterinary Biochemistry Dep, Sri Venkateswara Veterinary Univ, Tirupati, Andhra Pradesh) : Genotyping and protein profiling of milk β-casein variants (A1 and A2) in of deoni, sahiwal and malnad gidda breeds of milch cattle. Pharma Innov 2019, 8(5), 181-6.

The exotic and cross bred cattle milk contain A1 β-casein variant, whereas indigenous cattle milk contains A2 β-casein variant. They have structural diversity at 67th amino acid residue proline in A2 which is replaced by histidine in A1 during course of evolution. The histidine at 67th amino acid residue generates a proteolytic cleavage site in A1 leading to the production of betacasomorphin-7 (BCM-7) that is known to be a risk factor for several human diseases. The serious health concern associated with A1 milk consumption endures an urgent need to select cattle breeds with higher genotypic and allelic frequencies of A2 and conserve indigenous cattle breeds through breeding programmes. In this context, the present study was undertaken to screen fourty six animals belonging to two indigenous cattle breeds of India viz., Deoni, Sahiwal, Malnad Gidda and Holstein Friesan cross (twelve from each breed and ten from Malnad Gidda) to characterize β-casein variants (A1 and A2) geotypically by PCR-RFLP with TaqαI restriction enzyme and also to study milk β-casein variants (A1 and A2) by IEF. PCR-RFLP with Taqα1 restriction enzyme yielded two genotypes as A1A2 (251 and 213 bp) and A2A2 (251 bp) in all indigenous cattle breeds studied, whereas only heterozygous (A1A2) genotype was found in the HF cross. The genotypic frequency of A1A2 heterozygous variant of exon 7 of CSN2 gene was the highest for HF cross (1.0) followed by Sahiwal (0.75), Deoni (0.58) and Malnad Gidda (0.40). Whereas, for A2A2 homozygous variant was the highest for Malnad Gidda (0.60) followed by Deoni (0.42) and Sahiwal (0.25). The A1 allelic frequency of exon7 of CSN2 gene was the highest in HF cross (0.50) followed by Sahiwal (0.37) Deoni (0.29) and Malnad Gidda (0.20), whereas the highest A2 allelic frequency was observed in Malnad Gidda (0.80) followed by Deoni (0.71), Sahiwal (0.63) and HF cross (0.50). The IEF gel electrophoresis pattern of milk β-casein protein showed two distinct bands corresponding to A1 and A2 β-casein protein variants at pI values 5.22 and 5.14 respectively and this polymorphism correlated positively with the results of PCR-RFLP with TaqI restriction enzyme. The present study unveils the existing fact that there is incorporation of exotic germplasm in native cattle breeds to varying degrees in different breeds and emphasizes an immediate need for genotypic screening of bulls for βcasein gene variant A2 for documentation and further measures to improve the indigenous breeds with appropriate breeding strategies.The exotic and cross bred cattle milk contain A1 β-casein variant, whereas indigenous cattle milk contains A2 β-casein variant. They have structural diversity at 67th amino acid residue proline in A2 which is replaced by histidine in A1 during course of evolution. The histidine at 67th amino acid residue generates a proteolytic cleavage site in A1 leading to the production of betacasomorphin-7 (BCM-7) that is known to be a risk factor for several human diseases. The serious health concern associated with A1 milk consumption endures an urgent need to select cattle breeds with higher genotypic and allelic frequencies of A2 and conserve indigenous cattle breeds through breeding programmes. In this context, the present study was undertaken to screen fourty six animals belonging to two indigenous cattle breeds of India viz., Deoni, Sahiwal, Malnad Gidda and Holstein Friesan cross (twelve from each breed and ten from Malnad Gidda) to characterize β-casein variants (A1 and A2) geotypically by PCR-RFLP with TaqαI restriction enzyme and also to study milk β-casein variants (A1 and A2) by IEF. PCR-RFLP with Taqα1 restriction enzyme yielded two genotypes as A1A2 (251 and 213 bp) and A2A2 (251 bp) in all indigenous cattle breeds studied, whereas only heterozygous (A1A2) genotype was found in the HF cross. The genotypic frequency of A1A2 heterozygous variant of exon 7 of CSN2 gene was the highest for HF cross (1.0) followed by Sahiwal (0.75), Deoni (0.58) and Malnad Gidda (0.40). Whereas, for A2A2 homozygous variant was the highest for Malnad Gidda (0.60) followed by Deoni (0.42) and Sahiwal (0.25). The A1 allelic frequency of exon7 of CSN2 gene was the highest in HF cross (0.50) followed by Sahiwal (0.37) Deoni (0.29) and Malnad Gidda (0.20), whereas the highest A2 allelic frequency was observed in Malnad Gidda (0.80) followed by Deoni (0.71), Sahiwal (0.63) and HF cross (0.50). The IEF gel electrophoresis pattern of milk β-casein protein showed two distinct bands corresponding to A1 and A2 β-casein protein variants at pI values 5.22 and 5.14 respectively and this polymorphism correlated positively with the results of PCR-RFLP with TaqαI restriction enzyme. The present study unveils the existing fact that there is incorporation of exotic germplasm in native cattle breeds to varying degrees in different breeds and emphasizes an immediate need for genotypic screening of bulls for βcasein gene variant A2 for documentation and further measures to improve the indigenous breeds with appropriate breeding strategies.

8 illus, 7 tables, 16 ref

003038   SINGH P, JANA C , KHULAPE S A , GAUTAM S, RAMAKRISHNAN M A (ICAR-Indian Veterinary Research Institute, Mukteswar– 263138, Nainital, Uttarakhand, Email: drjanac@gmail.com) : Pathological investigation and molecular characterization of bovine papilloma virus-1 detected in cutaneous warts of cattle. Indian J Vet Pathol 2019, 43(2), 89-93.

The present investigation describes the genetic characterization of BPV-1 and its pathological features of cutaneous warts in cattle. A total of 7 clinical cases of cutaneous warts on various positions of cattle were studied at Mukteswar, Uttarakhand. The cases were confirmed by histopathology and molecular method. Grossly, dome shaped lesions were observed on nostril, around the eye, shoulder and neck region whereas single long cylindrical and dome shaped protruding growths were observed in teats. Histopathological study showed presence of hyperkeratinization, basket wave appearance, parakeratosis, koilocytosis, rete pegs formation in epidermal layer and fibroblast proliferation in dermal layer and pathologically diagnosed as fibropapilloma. In teat warts, the characteristic pathological lesions were hyperkeratinization, digit like proliferation, parakeratosis, keratohylinization with koilocytes which were diagnosed as papillomas. Two clinical cases of warts on teat revealed hyperplasia with mild cornification and thickening of epidermal layer. Immunohistochemistry, performed by using monoclonal mouse antibody against E2 protein of BPVs, revealed positive immunolabeling in the nucleus, cytoplasm and cytoplasmic membrane of basal, spinosum and granulosum layer. In PCR, 5 clinical samples were positive for BPV and based on sequence analysis it was identified as BPV-1. The phylogenetic analysis of the L1 gene placed the present BPV under a cluster of delta-papillomavirus. In conclusion, the confirmatory detection of BPV-1 in particular herd and its genetic characterization help in molecular epidemiology study and to set appropriate therapeutic and control measures.

7 illus, 1 table, 14 ref

003023   MAHARANA B R, GANGULY A, ARORA D, BISLA R S (Lala Lajpat Rai Univ of Veterinary and Animal Sciences, Karnal, Haryana-132001) : Development and validation of direct PCR-RFLP assay for rapid detection and differentiation of Anaplasma species in naturally infected goats. J Vet Parasitol 2019, 33(1), 12-21.

This study was carried out with the objective to develop a novel, rapid and cost effective direct PCR-RFLP assay for detection and differentiation of two Anaplasma species (A. ovis and A. marginale). Blood samples were collected randomly from 112 goats. DNA was extracted from blood samples and a direct blood polymerase chain reaction (DT-PCR) for amplifying a fragment of the major surface protein 5 (msp5) gene of A. ovis / A. marginale from whole blood was developed and standardized. Additionally, the 16srRNA and msp4 genes were analysed by nested PCR, semi nested PCR and PCRRFLP methods. Blood smear examination revealed 33 samples (29.46%) to be positive for Anaplasma inclusion bodies. On the contrary, 54 (48.21%) samples were positive by DT-PCR. The results revealed that DT- PCR was 100% sensitive and 73.41% specific when compared with microscopy based detection (k =0.63). All DT-PCR positive samples were confirmed as A. ovis by RFLP analysis. DT-PCR showed 94.44% sensitivity and 100% specificity compared to conventional PCR results with suspected blood samples (k=0.94). The phylogenetic tree and comparative sequence analysis revealed msp5 gene of Anaplasma species of Indian isolate had maximum distance from A. phagocytophilum followed by A. centrale and A. marginale and 100% sequence identity with A. ovis isolates of Chinese origin. The in-house standardized DT-PCR- RFLP method based on msp5 gene is an easy and reliable diagnostic tool to identify and discriminate between these closely related pathogens in ruminants. This type of study is the first of its kind as it depicts the molecular detection, differentiation and phylogenetic characterization of Anaplasma ovis among goat flocks in India.

9 illus, 4 tables, 26 ref