001795   SUBRAHMANYA R M, PRASAD S V, PRASAD R B, MOGRA S, SHETTY V, RAO V (Orthodontics and Dentofacial Orthopaedics Dep, Nitte Deemed to Be Univ, Mangalore, Karnataka, Email: drmsravi@gmail.com) : Mutations in FGFR3 gene associated with maxillary retrognathism. Indian J Dent Res 2019, 30(2), 185-90.

Understanding the role of fibroblast growth factor receptor (FGFR) in the regulation of bone development and disease will ultimately lead to better prevention and treatment of related bone deformities and disorders. To evaluate the role of gene FGFR3 in individuals with retrognathic maxilla by  polymerase chain reaction (PCR) technique at molecular level and evaluate the significance of the same. Hospital based fundamental research involving individuals having maxillary retrognathism. A total of 62 individuals (30M and32F) who were willing to take part in the study were selected from cephalometric measurements of N I A and the length PNS to ANS. The institution based basic genetic research study involved collection of fresh blood samples, DNA extraction, PCR analysis, and amplification using the specifically designed forward and reverse primers for targeting the commonly occurring mutations in FGFR3 gene. Further the products were sequenced to evaluate the presence of any novel mutations. The  targeted single‑nucleotide polymorphisms, at position 1138 in exon 10 of the FGFR3 gene were not identified in the analyzed blood samples. The detailed sequencing of full gene revealed the presence of 2 novel mutations, Exon 3: A213G and Exon 3: A223A/G in one individual. The present study indicated 2 novel mutations in gene FGFR3 in individual with maxillary retrognathism. The genetic–environmental interactions might have played a significant role in the expression of retrognathic maxilla.

3 illus, 4 tables, 16 ref

000627   JOICE A, JOSE D, BASHINI J M, SENTHILKUMAAR P (Biotechnology Dep, St. Joseph Arts and Science Coll, Kovur, Chennai, Email: manjubashinij82@gmail.com) : Effect of apoptosis on human breast cancer cell line (MCF-7 and MDA-MB231) using Curcuma longa. Int J Pharm Biol Sci 2019, 9(1), 73-84.

Cancer cells usually have increased cell proliferation; have ability to survive in unique environment and decreased apoptosis. Decreased apoptosis gives the cancer cells a survival advantage. All cells showed a small base line apoptotic level. Treatment of cancer cells with curcumin significantly increased apoptosis by Caspase 3/7 assay, Annexin IV assay and tunel assay suggesting that both early and late apoptotic events are triggered by curcumin. Increased apoptosis by curcumin may be used to kill the cancer cells and thereby help in treatment of cancer. The overall results obtained in the study point out that the active molecule present in Curcuma longa have considerable consequence on the survival of human breast cancer cell lines. Finally, it is concluded that curcuminoids are a group of phenolic compounds isolated from the rhizome of Curcuma longa has various pharmacological properties. They exhibit growth inhibitory effects on a broad range of tumors and act as potent anticancer, anti-inflammatory and analgesic agent and more research should be carried out and this data should be made accessible for both health care providers and patients for safe anticancer treatments.

35 ref

000620   BASKAR G, LALITHA K, GEORGE G B (Biotechnology Dep, St. Joseph’s Coll of Engineering, Chennai - 600 119, Email: basg2004@gmail.com) : Synthesis, characterization and anticancer activity of selenium nanobiocomposite of L-asparaginase. Bull Mater Sci 2019, 42(1), 4.

Selenium is a rare earth nonmetal and an essential micronutrient for animals, and a trace nutrient for humans. It is known to function as a cofactor for reduction of antioxidant enzymes, such as glutathione peroxidase. The selenium nanobiocomposite synthesized using a co-precipitation method with sizes 20–30 nm were confirmed using scanning electron microscopy and they showed maximum absorption between 300 and 400 nm in the ultra-violet spectrum. Fourier transform infrared and H-nuclear magnetic resonance analysis revealed the involvement of various functional groups in the binding of asparaginase to selenium nanoparticles to form the selenium nanobiocomposite. X-ray diffraction analysis confirmed the hexagonal structure of the selenium nanobiocomposite. Methylthiazolyl diphenyl-tetrazolium bromide assay on HT-29, MG-63 and HEPTO cells loaded with the selenium nanobiocomposite revealed a toxicity of 93.62, 94.24 and 87.68 %, respectively, for a selenium nanobiocomposite concentration of 1000 μg ml⁻¹. The selenium nanobiocomposite of l-asparaginase opens a new arena in cancer treatment.

7 illus, 21 ref

000619   AMARESHWARI P, VENKATESH K, Roja Rani A (Genetics and Biotechnology Dep, Osmania Univ, Hyderabad, Telangana) : Effect of hygromycin on peanut germination and its application in Agrobacterium-mediated genetic transformation. J Pharmacogn Phytochem 2019, 8(1), 263-7.

To overcome the laborious screening tasks in in planta transformation, Agrobacterium infected seed parts mainly embryonated cotyledons and embryos can be passed through the selection of antibiotics for a very short duration for successful identification of transformants. Hygromycin B more toxic than the existing antibiotics and has been used as a selective agent in many crop plants. With the increase in hygromycin concentrations, a decline in the survival frequency of explants was observed. The threshold concentration of hygromycin for the selection of transformants was optimized using embryonated cotyledon and embryo on MS media containing different concentrations of hygromycin. It was observed that 20 mg/l hygromycin was threshold concentration for the selection of embryonated cotyledons and 15 mg/l for embryos. These concentrations suppress the growth of peanut plants. Therefore, hygromycin at 20 mg/l for embryonated cotyledons and embryos at 15 mg/l could screen hygromycin-tolerant transgenic peanuts.

3 illus, 30 ref

000634   SINGH M, BALA M (Floriculture and Landscaping Dep, Punjab Agricultural Univ, Ludhiana - 141 001, Email: madhu-flori@pau.edu) : Induction of radiomutants in Chrysanthemum morifolium Ramat. cv. Gul-e-Sahir for novel traits. Indian J Exp Biol 2019, 57(1), 50-4.

Chrysanthemum is one of the most popular ornamental group of plants worldover. Its wide range of brilliant colour, shapes, size, long lasting flower life, etc makes it quite competitive in commercial floriculture. Here, we tried to estimate the efficiency of different doses of gamma rays to induce novel mutations in chrysanthemum cv. "Gul-e-Sahir" using terminal rooted cuttings as experimental material. The rooted cuttings were exposed to different doses of γ-rays (0, 10, 20 or 30 Gy) using a ⁶⁰Co source and transplanted in earthen pots (8”). Marked variations were recorded in leaf colour, leaf shape, flower size, shape and colour between the mutated and control populations. Four new flower colour variants with altered or novel flower colours were isolated that were unique and different from original flower colour. The original flower colour of Gul-e-Sahir is yellow whereas, flower colour of mutated population was of nearest shades of green yellow group as per RHS colour chart. The ray florets were normal in control whereas, in mutated population ray florets were spoon shaped, narrow, broad, flat and tubular in shape. These mutants were further multiplied on large scale and evaluated to check their stability. This study developed a mutagenesis protocol that could be used to develop novel colour mutants in chrysanthemum.

3 illus, 2 tables, 10 ref

000624   EMAMI S S, NEKOUIAN R, AKBARI A, FARAJI A, ABBASI V, AGAH S (Medical Biotechnology Dep, Iran Univ of Medical Sciences, Tehran, Iran, Email: nekouian.r@iums.ac.ir) : Evaluation of circulating miR‑21 and miR‑222 as diagnostic biomarkers for gastric cancer. J Can Res Ther 2019, 15(1), 115-9.

Gastric cancer is responsible for a large number of death worldwide and its high death rate is associated with a lack of noninvasive tools in GC diagnosis. MicroRNAs (miRNAs), as gene regulators, were shown to dysregulate in different types of cancer. Moreover, it is proven that miRNAs are stable in serum/plasma, so they can be used as a potential noninvasive marker in GC diagnosis. The objective of this study is to investigate the plasma miRNA expression in GC samples compared to controls as a potential biomarker in cancer diagnosis. Expression levels of miR‑21 and miR‑222 were assessed using quantitative real‑time polymerase chain reaction in plasma of 30 GC patients and 30 healthy controls. Diagnostic value of selected miRNAs was evaluated using receiver operating characteristic curve. Target prediction was done using bioinformatics tools to investigate the signaling pathways and function of the selected miRNAs. Our results demonstrated that the expression levels of miR‑21 and miR‑222 were significantly higher in GC plasma than in the controls (P < 0.0001, P = 0.043). The sensitivity and specificity for miR-21 and in plasma were 86.7 % and 72.2 % and for miR-222 were 62.5 % and 56.2 %, respectively. Bioinformatics analysis revealed that most target genes of miR‑21 and miR‑222 are involved in cancer‑related signaling pathway such as tumor initiation and progression. Our results indicated that miR‑21 and miR‑222 in plasma samples can be served as a potential noninvasive tool in GC detection. Furthermore, the miRNA target prediction manifested that miR-21 and miR-222 involve in key processes associated with GC initiation and development.

2 illus, 2 tables, 48 ref

000628   MUHAMMAD A, TOUFEEQ S, YU H-Z, WANG J, ZHANG S-Z, LI B, LI Z, YANG L-A, HU P, MA Y, XU J-P (Anhui Agricultural Univ, Hefei, China, Email: jiapingxu@163.com) : Molecular characterization of two mitogen-activated protein kinases: p38 map kinase and ribosomal s6 kinase from Bombyx mori (Lepidoptera: Bombycidae), and insight into their roles in response to BmNPV infection. J Insect Sci 2019, 19(1), 15.

Proteins p38 map kinase and ribosomal S6 kinase (S6K) as members of mitogen-activated protein kinases (MAPKs) play important roles against pathogens. In this study, Bmp38 and BmS6K were identified as differentially expressed proteins from iTRAQ database. Bmp38 and BmS6K were expressed, and recombinant proteins were purified. The bioinformatics analysis showed that both proteins have serine/threonine-protein kinases, catalytic domain (S_ TKc) with 360 and 753 amino acids, respectively. The real-time quantitative polymerase chain reaction (RT-qPCR) results suggest that Bmp38 and BmS6K had high expression in the midgut and hemolymph. The comparative expression level of Bmp38 and BmS6K in BC9 was upregulated than in P50 in the midgut after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Western bolt results showed a positive correlation between RT-qPCR and iTRAQ data for Bmp38, but BmS6K data showed partial correlation with iTRAQ. Injection of anti-Bmp38 and antiBmS6K serum suggested that Bmp38 may be involved against BmNPV infection, whereas BmS6K may require phosphorylation modification to inhibit BmNPV infection. Taken together, our results suggest that Bmp38 and BmS6k might play an important role in innate immunity of silkworm against BmNPV.

8 illus, 2 tables, 41 ref

000621   CHANG H, GUO J-L, FU X-W, WANG M-L, HOU Y-M, WU K-M (Chinese Academy of Agricultural Sciences, Beijing, China, Email: wukongming@caas.cn) : Molecular characterization and expression profiles of cryptochrome genes in a long-distance migrant, Agrotis segetum (Lepidoptera: Noctuidae). J Insect Sci 2019, 19(1), 8.

Cryptochromes act as photoreceptors or integral components of the circadian clock that involved in the regulation of circadian clock and regulation of migratory activity in many animals, and they may also act as magnetoreceptors that sensed the direction of the Earth’s magnetic field for the purpose of navigation during animals’ migration. Light is a major environmental signal for insect circadian rhythms, and it is also necessary for magnetic orientation. We identified the full-length cDNA encoding As-CRY1 and As-CRY2 in Agrotis segetum Denis and Schiffermaller (turnip moth  (Lepidoptera: Noctuidae)). The DNA photolyase domain and flavin adenine dinucleotide-binding domain were found in both cry genes, and multiple alignments showed that those domains that are important for the circadian clock and magnetosensing were highly conserved among different animals. Quantitative polymerase chain reaction showed that cry genes were expressed in all examined body parts, with higher expression in adults during the developmental stages of the moths. Under a 14:10 (L:D) h cycle, the expression of cry genes showed a daily biological rhythm, and light can affect the expression levels of As-cry genes. The expression levels of cry genes were higher in the migratory population than in the reared population and higher in the emigration population than in the immigration population. These findings suggest that the two cryptochrome genes characterized in the turnip moth might be associated with the circadian clock and magnetosensing. Their functions deserve further study, especially for potential control of the turnip moth.

6 illus, 1 table, 79 ref

000635   SONTAKKE B R, AMBULKAR P S , TALHAR S, SHIVKUMAR P V, BHARAMBE M S , PAL A (Anatomy Dep, Mahatma Gandhi Institute of Medical Sciences, Wardha - 442 102, Email: drbharatsontakke@gmail.com) : Molecular genetic study to detect prevalence of high risk human papilloma virus strains (type 16 and 18) in cervical lesions and asymptomatic healthy subjects of rural Central India. J Cytol 2019, 36(1), 32-7.

Carcinoma cervix of uterus (CaCx) is the most common malignancy affecting women worldwide. It is an established fact that infection of specific types of human papilloma virus (HPV) is essential for the development of cervical cancer. The present study reports the high‑risk viruses(HPV 16 and 18) type distribution in rural central India, which has unique climatic condition. To our knowledge, no molecular study on HPV prevalence has been done in this region of rural population, this intended us do such study. Sexually active women reporting to the Gynecology were divided in three groups, first being asymptomatic women with normal cervix (52 cases), second group with benign cervical lesion (52 cases), and third group of women with frank cervical malignancy (40 cases). Cervical swabs were collected for HPV DNA sampling. The incidence of HPV positivity was recorded in each group. Fifty‑two women with asymptomatic normal cervix showed 44.23 % positivity for HPV 16 and 5.76 % positivity for HPV 18. Fifty‑two women with benign cervical lesion showed 38.46 % positivity for HPV 16 and 3.84 % positivity for HPV 18. Forty women with frank cervical malignancy were with prevalence of 62.5 % for HPV 16 and 22.5 % for HPV 18.   The results of the study are definitely helpful to know the prevalence of HPV in this region of rural population and will enrich the national epidemiological data related to HPV infection in cervical cancer.

3 illus, 3 tables, 31 ref

000636   TAI Y-S, YANG S-C, HSIEH Y-C, HUANG Y-B, WU P-C, TSAI M-J, TSAI Y-H, LIN M-W (Medical Research Dep, E-Da Hospital, Kaohsiung 824, Taiwan, Email: ta990074@gmail.com) : A novel model for studying voltage-gated ion channel gene expression during reversible ischemic stroke. Int J Med Sci 2019, 16(1), 60-7.

The dysfunction of voltage-gated ion channels contributes to the pathology of ischemic stroke. In this study, we developed rat models of transient ischemic attack (TIA) and reversible ischemic neurological deficit (RIND) that was induced via the injection of artificial embolic particles during full consciousness, that allow us to monitor the neurologic deficit and positron emission tomography (PET) scans in real-time. We then evaluated the infarction volume of brain tissue was confirmed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining, and gene expressions were evaluated by quantitative real-time PCR (qPCR). We found that rats with TIA or RIND exhibited neurological deficits as determined by negative TTC and PET findings. However, the expression of voltage-gated sodium channels in the hippocampus was significantly up-regulated in the qPCR array study. Furthermore, an altered expression of sodium channel β-subunits and potassium channels, were observed in RIND compared to TIA groups. In conclusion, to our knowledge, this is the first report of the successful evaluation of voltage-gated ion channel gene expression in TIA and RIND animal models. This model will aid future studies in investigating pathophysiological mechanisms, and in developing new therapeutic compounds for the treatment of TIA and RIND.

3 illus, 34 ref

000630   RAWAT N, GILHARE V R, KUSHWAHA K K, HATTIMARE D D, KHAN F F, SHENDE R K, JOLHE D K (Veterinary Microbiology Dep, Veterinary Science and Animal Husbandry Coll, Chhattisgarh, Email: dr_nidhirawat@yahoo.com) : Isolation and molecular characterization of Mannheimia haemolytica and Pasteurella multocida associated with pneumonia of goats in Chhattisgarh. Vet World 2019, 12(2), 331-6.

The purpose of this study was to isolate and characterize the Mannheimia haemolytica and Pasteurella multocida from blood, nasal discharge, and lung tissue of pneumonic goats. A total of 14 goats were investigated for pneumonic pasteurellosis. Of 14 goats, nasal swabs and blood samples were collected from 10 clinically diseased animals. Moreover, lung tissue and heart blood samples were collected during necropsy of four goats died with pneumonia. All the samples were processed for the isolation of M. haemolytica and P. multocida in the laboratory. Bacterial isolates were identified by cultural and biochemical characters and 16S rRNA sequence analysis. All the isolates were subjected to susceptibility testing using commonly used antimicrobials. M. haemolytica isolates were characterized by PHSSA gene detection. P. multocida isolates were characterized by KMT1 gene detection and capsule typing. On necropsy of dead goats, the pneumonia was characterized as acute fibrinous bronchopneumonia. Bacterial culture revealed the isolation of M. haemolytica (7) and P. multocida (5) of 10 clinical cases. Moreover, M. haemolytica and P. multocida were coisolated from two of the lung tissues. Furthermore, one of the other two lung tissues showed the isolation of M. haemolytica while the other showed recovery of P. multocida. Bacterial isolates were specifically identified by the 16S rRNA sequence analysis. The isolates showed reduced susceptibility to β-lactams, aminoglycosides, and fluoroquinolones. Moreover, the PHSSA and KMT1 genes were specifically detected among M. haemolytica, and P. multocida isolates, respectively. All P. multocida isolates belonged to serogroup A. The present study reported an occurrence of pneumonic pasteurellosis caused by M. haemolytica and P. multocida in a goat flock.

4 illus, 1 table, 34 ref

000637   WIBOWO M H, GINTING T E, ASMARA W (Microbiology Dep, Gadjah Mada Univ, Yogyakarta 55281, Indonesia, Email: mhwibowo@ugm.ac.id) : Molecular characterization of pathogenic 4/91-like and QX-like infectious bronchitis virus infecting commercial poultry farms in Indonesia. Vet World 2019, 12(2), 277-87.

Existing data on the characteristics of infectious bronchitis virus (IBV) gathered throughout Indonesia have been recognized to indicate variants similar to globally distributed vaccine strains. Despite past and current intensive vaccination programs, IBV infections in the country’s poultry industry have not been effectively controlled. Therefore, this study aimed to investigate the genotype of several isolates based on partial S1 gene sequences. In particular, the investigation is directed to focus on layer chickens in actively vaccinated farms indicating IBV symptoms. Samples were isolated from ten different layer chicken flocks experiencing respiratory problem, drops in egg production, and a “penguin-like” stance, which were collected from commercial poultry farms in Central Java and Yogyakarta regions, Indonesia, within the periods of 2012-2018. Fragment of the S1 gene of IBV sampled from actively vaccinated commercial poultry farms was amplified using primer 5’-aca tgg taa ttt ttc aga tgg-3’ (forward) and 5’-cag att gct tac aac cac c-3’ (reverse) with the length of polymerase chain reaction (PCR) product at 383 bp. The sequence of samples was then compared with the sequence of reference S1 gene nucleotides of IBV from NCBI GenBank database. The amino acid analysis and multiple alignment sequence were conducted using Mega X. During necropsy, enlargement of the oviduct and swollen kidney were observed. Reverse transcription-PCR diagnosis of their 383 bp S1 gene showed that all samples were IBV positive. Phylogenetic analysis of the S1 gene discovered seven samples to be clustered as 4/91-like strains. Meanwhile, the remaining three samples were grouped in QX-like strain cluster. This study is a pioneering report providing molecular evidence of pathogenic QX-like and 4/91-like strains circulating in Indonesia. Findings discovered, in this study, strongly suggested the importance of improving protections by available IBV vaccines through updated circulating strain clusters. It is critical to ensure the delivery of an effective control measurement of and vaccination protocols against IBV infections in the country’s commercial poultry industry in particular and worldwide in general.

4 illus, 61 ref

000625   GHAFAR M W, AMER S A M (Forensic Biology Dep, Naif Arab Univ for Security Sciences, Saudi Arabia, Email: samer@nauss.edu.sa) : A preliminary molecular survey of Babesia divergens and first evidence of Theileria annulata in cattle from Saudi Arabia. Vet World 2019, 12(2), 266-70.

Babesia divergens causes human babesiosis in Europe where the parasite utilizes cattle as animal reservoir and Ixodes ricinus as tick vector. Importation of infected animals and passive carriage of infected ticks through migratory birds can lead to tick/pathogen geographic expansion and emergence of diseases in naïve land. Given the information that Saudi Arabia imports cattle from the European countries and that two global bird flyways pass through the country geographic coordinates, we speculate that B. divergens might be introduced into the Kingdom. Therefore, the aim of this preliminary study was to molecularly detect and characterize B. divergens and other piroplasms (including Theileria spp.) in cattle from Taif district, Kingdom of Saudi Arabia. Blood samples from 20 cattle residing Taif district were collected, and polymerase chain reaction tested using wide and species-specific primers. Amplicons from a positive genus-wide reaction were purified, sequenced, and analyzed. Phylogenetic trees were constructed, and similarity to existing GenBank zoonotic piroplasms was also assessed. All samples were negative for B. divergens, and only one sample proved positive for Theileria annulata in a wide reaction. Phylogeny clustered our strain with T. annulata from Spanish dog and another one detected in a cow from France. BLAST analysis showed genetic distance from zoonotic piroplasms with identity ranged from 88 % to 91 %. Although B. divergens was not detected, we are not able to rule out or affirm the existence of the pathogen in the country. On the other hand, identifying T. annulata strain with a southern European origin strongly supports our speculation that bovine zoonotic Babesia might be introduced into KSA. This study is not only the first molecular survey of B. divergens but also the first report of the molecular identity of T. annulata in Saudi Arabia. A national-wide bovine and tick surveillance are needed to further prove our speculation.

2 illus, 1 table, 18 ref

000632   SETIAWATY R, SOEJOEDONO R D, POETRI O N (National Veterinary Drug Assay Laboratory, Bogor 16340, Indonesia, Email: ajengfkh99@gmail.com) : Genetic characterization of S1 gene of infectious bronchitis virus isolated from commercial poultry flocks in West Java, Indonesia. Vet World 2019, 12(2), 231-5.

Infectious bronchitis (IB) is still a major problem among poultry industry in Indonesia, IB outbreaks continue to happen even in vaccinated flocks. The emergence of new IB virus (IBV) variants might lead to mismatching between vaccine virus strain and circulating virus strain, this may be a reason of vaccination failure. Information about circulating IBV in a region is important to decide which IB vaccine should be used. However, information about recent IBV strains which circulated in Indonesia and their genetic characters were limited; therefore, the aim of our research was to determine the genetic characterization of S1 gene of IBV isolated from commercial poultry flocks in West Java, Indonesia. A total of 47 viral isolate samples collected from chickens with clinical sign and reduced in egg production. Six IB live vaccines were used as control, the reference vaccines represent IBV strains including H120, H52, 4/91, CR88, 233A, and 1-96. Primers XCe²⁺ and XCe^2- were used to amplify S1 gene partially. Twenty-six of 47 samples showed positive result to S1 gene of IBV by reverse transcription-polymerase chain reaction. Three IBV isolates, Indonesia/K233A31/18, Indonesia/K4A9/17, and Indonesia/P3/17, were selected for nucleotide sequencing. Phylogenetic analysis of 352 nucleotides of the partial S1 gene shows that isolates Indonesia/K4A9/17 and Indonesia/K233A31/18 have 100 % homology with IBV vaccine strain 4/91, while isolate Indonesia/P3/17 has 100 % homology with IBV vaccine strain 233A. Our result indicates that at least two IBV strains were circulating among poultry in West Java, Indonesia, which is IBV close to vaccine strain 4/91 and 233A. The present study provides updates on the circulating IBV in commercial poultry flocks in West Java, Indonesia, and might use as guidance on selecting a proper IB vaccine strain to improve IB vaccination efficacy in certain region.

2 illus, 1 table, 22 ref

000631   SALEM M A, EL-DEEB W M, ZAGHAWA A A, HOUSAWI F M, ALLUWAIMI A M (Clinical Studies Dep, King Faisal Univ, Al-Hasa, Kingdom of Saudi Arabia, Email: weldeeb@KFU.edu.sa) : Investigation of Mycobacterium paratuberculosis in Arabian dromedary camels (Camelus dromedarius). Vet World 2019, 12(2), 218-23.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease in ruminants. This study aimed to investigate Mycobacterium paratuberculosis infection in clinically infected camels on the immunological, conventional bacteriological, and molecular biological basis. A total of 30 Arabian camels (Camelus dromedarius) were examined in this study. The camels were suffering from signs ranging from mild to severe infections (that did not respond to antibiotic treatment) to chronic or intermittent diarrhea. Camels were grouped into three groups based on their age, sex, and breed. Detection of anti-MAP antibodies in camels’ serum, Ziehl–Neelsen (ZN) staining technique on rectal scraps, direct recognition of MAP in stool and tissue specimens by IS900 polymerase chain reaction (PCR) assay, and finally isolation and molecular description of MAP from fecal and tissue samples were carried out. Five MAP isolates were recovered from these investigated camel samples giving an isolation rate of 16.6 %, while eight camels were identified by PCR (26.6 %). Five camels yielded MAP in their feces by ZN fecal staining (16.6 %), whereas ELISA detected anti-MAP antibodies in nine camels only (30 %). From the obtained results, we concluded that the gold standard for the diagnosis of MAP is the culture method despite its limitations. Molecular diagnosis (PCR) could be a useful tool in the identification of truly positive and negative camels; however, great care should be given regarding the primers specificity and sensitivity.

2 illus, 2 tables, 37 ref

000618   ADAM K Y, ISMAIL A A, MASRI M A, GAMEEL A A (Ministry of Animal Resources, South Darfur State, Sudan, Email: eleilaf@gmail.com) : First report and molecular characterization of Cryptosporidium spp. in humans and animals in Khartoum state, Sudan. Vet World 2019, 12(1), 183-9.

Cryptosporidium is recognized to infect several mammalian species as well as humans, causing substantial economic losses and serious public health concern. Infected animals can be a source of environmental contamination and human infections. In general, the occurrence of Cryptosporidium species in animals and human in Sudan and zoonotic importance is not well documented. This study aimed to identify Cryptosporidium spp. infecting different animal species and humans and to compare between different isolates obtained. To provide molecular information about Cryptosporidium in animals and humans, both modified Ziehl–Neelsen (MZN) specific stain and molecular assay were used. Concentration techniques followed by three protocols of DNA extraction were carried out. After microscopic screening of 263 fecal samples (goats [n=197], cattle [n=12], sheep [n=12], and human [n=42]), 61 positive and 30 negative, randomly selected samples were used in nested polymerase chain reaction (PCR) targeting part of the 18S RNA. Nested PCR amplification confirmed 91.8 % (56/61) of microscopic-positive samples. 8.2 % (5/61) of negative samples by PCR (positive by microscopy) were considered false negatives. Sequencing followed by alignment of the 14 isolates indicated that all samples were identical (100 %) and belonged to Cryptosporidium parvum. MZN staining procedure is reliable for the routine diagnosis of Cryptosporidium; cetyltrimethylammonium bromide extraction buffer and nested PCR targeting 18S rRNA gene are reliable and useful in epidemiological studies of this parasite.

3 illus, 1 tables, 44 ref

000638   YANESTRIA S M, RAHMANIAR R P, WIBISONO F J, EFFENDI M H (Veterinary Public Health Dep, Airlangga Univ, Surabaya, Indonesia, Email: mheffendi@yahoo.com) : Detection of invA gene of Salmonella from milkfish (Chanos chanos) at Sidoarjo wet fish market, Indonesia, using polymerase chain reaction technique. Vet World 2019, 12(1), 170-5.

The study aimed to detect the invA gene in Salmonella isolated from milkfish in the Sidoarjo wet fish market. A total of 84 samples were prepared in enrichment media and isolated on the surface of Salmonella Shigella Agar. Salmonella growth produces transparent colonies with blackish color in the middle due to H₂S gas formation. Samples were identified as Salmonella based on macroscopic colony morphology. Presumptive Salmonella sp. was put on Bismuth Sulfite Agar media. Salmonella was determined based on the results of the biochemical test that has been carried out using Microbact identification kits from negative gram staining. The results of this study indicate that 32 of 84 samples (38.09 %) were Salmonella bacteria. Furthermore, the invA gene detection was carried out using the polymerase chain reaction technique. Electrophoresis results showed four positive samples contained invA gene with a length of 284 bp. Results in this study indicate that contamination of milkfish with Salmonella needs strict hygienic measures to prevent their transmission to human.

6 illus, 1 table, 27 ref

000633   SHEKHAWAT S S, GAURAV A, JOSEPH B, KUMAR H, KUMAR N (Veterinary Public Health and Epidemiology Dep, Veterinary and Animal Sciences Coll, Udaipur, Rajasthan, Email: apsss14@gmail.com) : Random amplified polymorphic DNA-based molecular heterogeneity analysis of Salmonella enterica isolates from foods of animal origin. Vet World 2019, 12(1), 146-54.

This study aims to study the significance of random amplified polymorphic DNA (RAPD) typing in heterogeneity analysis of Salmonella serovars, isolated from foods of animal origin. Salmonella serovars isolated and identified from different foods of animal origin such as meat, milk, and egg by standard bacteriological methods. DNA isolated from all 10 isolates which are confirmed by biochemical and serotyping methods and then RAPD was performed using the primers OPB 10, primer 1290, NSC I, NSC II, and primer 3. Then, RAPD data were analyzed using the BioNumerics software, Belgium, Germany. RAPD polymerase chain reaction (PCR) using five primers, namely OPB 10, primer 1290, NSC I, NSC II, and primer 3, classified the 10 isolates into 9, 10, 10, 7, and 10 RAPD-PCR types with discriminating powers of 0.1987, 0.423, 0.50889, 0.1842, and 0.2582, respectively. The phylogram constructed with NSC I profile classified isolates based on geographical origin. Primer 1290, NSC II, and primer 3 produced some uniform bands in all isolates indicating their binding ability in conserved genomic region. This study revealed that RAPD profile can be best used for finding out the heterogeneity at molecular level of Salmonella isolates in combination with other molecular and phenotypic typing techniques. Thus, our results support earlier observation of its significance by different workers on different Salmonella serotypes. Repeatability of RAPD-PCR is insufficient to distinguish genetic differences among Salmonella serovars.

10 illus, 2 tables, 35 ref

000626   IBRAHIM W A, MAROUF S A, ERFAN A M, NASEF S A, JAKEE J K E (Animal Health Research Institute, 264-Dokki, Giza 12618, Egypt, Email: waleed.abdelfattah@yahoo.com) : The occurrence of disinfectant and antibiotic-resistant genes in Escherichia coli isolated from chickens in Egypt. Vet World 2019, 12(1), 141-5.

This work aimed to determine the occurrence of antibiotic and disinfectant resistance genes in Escherichia coli isolated from chickens in Egypt. Organs (liver, lung, heart, yolk sac, and bone marrow) of 1500 chicken samples were collected from diseased chickens suffered from colibacillosis with PM findings as CRD, diarrhea and omphalitis from different governorates of Egypt as: Giza, EL-Bahira, Fayoum, El-Dakahlia, El-Ismalia, and El-Sharkia during 2015-2016. These samples were labeled and transported immediately on ice to the Reference laboratory for quality control on poultry production (RLQP). The samples were cultured onto MacConkey agar and Eosin Methylene Blue Agar. Isolation and identification of the E. coli were performed based on morphology, cultural, staining, and biochemical properties. Antimicrobial resistance test was carried out using disk diffusion method. The PCR employing tetA, qacED1 and qacA/B were carried out for detection of these genes in isolated E.coli. The prevalence of E. coli in chicken was 34%. Predominant serotypes of E. coli which serologically identified were O128, O111, O44, O158, and O2. Antibiotic susceptibility test of E. coli revealed that 100% of isolates were resistant to ampicillin, erythromycin, and sulfamethoxazole-trimethoprim, while 73.53 % and 38.23 % of them were sensitive for colistin sulfate and levofloxacin, respectively. Antibiotic resistance genes as tetA gene were tested for isolated E. coli and detected by incidence rate of 91.18 %. qac resistance genes resembling as qacED1 and qacA/B genes were detected in isolated E. coli 70.6 % and 14.7 %, respectively. E. coli isolated from chickens in Egypt was carried qac and antibiotic-resistant genes that affect the poultry industry.

3 tables, 38 ref

026691   SWORNAKUMARI C, MEIGNANALAKSHMI S, LEGADEVI R, PALANISAMMI A (Animal Biotechnology Dep, Madras Veterinary Coll, Chennai- 600 007, Email: smeignanalakshmi@gmail.com) : Preparation of microspheres using poly-3-hydroxybutyrate biopolymer and its characterization. J Environ Biol 2018, 39(3), 331-38.

Biodegradable poly-3-hydroxybutyrate is one of the most common biopolymer which is used in different fields such as medicine, agriculture, textile, industrial and food packaging. PHB microspheres are useful for targeting drugs to specific infection sites and for prolonged drug release. The present study focus on microsphere preparation for effective controlled drug release using poly-3-hydroxy butyrate biopolymer. In the present study, poly-3-hydroxy butyrate microspheres were prepared using solvent evaporation technique and characterized by SEM and FTIR. Poly-3-hydroxy butyrate microspheres were encapsulated with BSA and gentamicin. Drug encapsulation efficiency was determined. In vitro drug release profile and in vitro cytotoxicity was also studied. The prepared microspheres were of 2µm in size. Microspheres fabricated with 1 % polyvinylalcohol showed encapsulation efficiency of 94.3 % and 90.27 % with BSA and gentamicin, respectively. The in vitro release studies in simulated body fluid, phosphate buffered saline and contact lens solution showed initial burst release followed by controlled release. In vitro cytotoxicity analysis showed 98 % and 95 % viability of cells in 3T3L1 cell line for microsphere encapsulated with BSA and gentamicin, respectively. Poly-3-hydroxy butyrate microspheres were found to release BSA and gentamicin in a controlled manner and were found to be non-toxic by in vitro cytotoxicity studies.

9 illus, 20 ref

026690   SULTANA N, SAHA P (Microbiology Dep, Burdwan Univ, Burdwan- 713 104, Email: psaha@microbio.buruniv.ac.in) : Studies on potential application of crude keratinase enzyme from Stenotrophomonas sp. for dehairing in leather processing industry. J Environ Biol 2018, 39(3), 324-30.

Chicken feathers represent huge pool of waste biomass which can undergo microbial degradation to generate value added products. Chicken feathers can be used as cheap substrate for production of keratinase enzyme which can be effectively used in dehairing of hides. Such enzyme based process is superior over conventional chemical based dehairing process, known to be responsible for pollution of the aquatic ecosystems. Isolation, characterization and identification of keratin degrading bacteria in feather meal broth medium using 0.5 % chicken feather as substrate. The effect of various parameters such as temperature; pH and substrate concentration on enzyme activity was assayed. The crude keratinase was evaluated comparatively with conventional chemical based process for dehairing activities with specific references to goat skin and cow hide. Application of bacterial crude keratinase enzyme towards eco-friendly dehairing process was highlighted from Norja-1, which was identified as Stenotrophomonas sp. by combination of 16S rRNA gene based molecular phylogenetic approach and phenotypic characterization. Keratinolytic activity was qualitatively demonstrated by scanning electron microscopic studies and quantitatively by using keratin azure as substrate. The crude keratinase enzyme showed optimum activity at 55 $°$C, pH 9 and 5 % substrate concentration. So, the enzyme was considered to be thermo-tolerant and alkaliphilic. Conventional chemical based process generated black spot on hide surface with broken hair while enzyme based process produced hides with better texture and intact hair which may be used further for making other value added products (such as wigs, doll's hair, carpets and mattresses). The dehairing activities of crude keratinase enzyme were demonstrated to be better in comparison to conventional chemical methods and might be useful towards development of pollution free dehairing process for leather processing industries. Presence of enzyme activity at wide range of temperatures (10 to 60$°$C) and pH (7.4 to 10.7) makes it a potential candidate for possible biotechnological application towards development of eco-friendly cost effective dehairing process in leather industries.

5 illus, 1 table, 23 ref

026686   PRIYADARSHINI S, MADHUMADHURYA V R, SHWETHA P, KUMAR D J K, MAHESH M (Biotechnology Dep, Government Science Coll, Bangalore - 560 061, Email: priyadarshiniplm@gmail.com) : Strain improvement for the production of antioxidant activity from Bacillus sp. by induced stress. J Appl Nat Sci 2018, 10(2), 614 - 9.

In the present study, water samples were collected from different beaches in Chennai such as Marina beach, Elliott beach, VGP Golden beach and Kovalam beach to evaluate the best source for antioxidants. The bacteria were isolated on Starch Casein Agar media and screened for the antimicrobial activity. Among 11 isolates, 5 isolates showed antimicrobial potential which were further evaluated for the DPPH (2,2-diphenyl-1- picrylhydrazyl) scavenging activity. Among 5 isolates, one sample showed significant DPPH scavenging activity with half minimal inhibitory concentration of (IC50) 344.754 µg/ mL. The maximum antioxidant production was observed at pH 7 and at temperature of 37 ºC with an IC50 of 188.66µg/mL and 293.76µg/mL respectively. The potent antioxidant producing strain was subjected for mutagenesis. In physical mutagenesis, the organism exposed for UV light for 25 minutes showed maximum antioxidant production with an IC50 of 133.55 µg/mL. This mutant strain was then subjected for chemical mutagenesis with the addition of different concentrations of Ethidium bromide such as 10 µL, 20 µL, 30 µL, 40 µL and 50 µL. The mutant strain obtained with the addition of 20 µL Ethidium bromide (EtBr) showed significant antioxidant activity with an IC50 of 325.4 µg/mL. The sample was purified by solvent extraction method and was evaluated for antioxidant production. The analyte was subjected to HPLC (High Performance Liquid Chromatography) analysis to avince the presence of antioxidants. The ethyl acetate extract showed the total phenolic content of 0.892 mg GAE/g of dry extract. It also showed the total flavonoid content of 0.522 mg RE/g of dry extract.

8 illus, 25 ref

026689   SELEIT I, BAKRY O A, GAYED E A E, GAWAD D A E (Dermatology Dep, Menoufiya Univ Hospital, 32817 Menoufiya Governorate, Egypt, Email: olabakry8@gmail.com) : Polymorphism of FAS and FAS ligand genes in Alopecia areata: A case–control study in Egyptian population. Indian J Dermatol 2018, 63(3), 220-6.

Alopecia areata (AA) is a common dermatologic disease with suspected autoimmune etiology. Tumor necrosis factor superfamily member 6 or CD95 (FAS) and FAS ligand (FASL) are proapoptotic proteins. The relationship between apoptosis and autoimmunity is well recognized. Inflammatory T cells in AA are cytotoxic and possess FAS/FASL antigens. This study aims to investigate the association between FAS-670 A/G and FASL-124 A/G gene polymorphisms and AA to clarify if these polymorphisms influence disease occurrence or increase disease risk. case–control study was conducted on sixty patients with AA, and 40 age- and sex-matched healthy subjects, as a control group. Disease severity was assessed by severity of alopecia tool (SALT) Score. FAS 670A/G and FASL 124A/G gene polymorphisms were investigated by the restriction fragment length polymorphism polymerase chain reaction. For FAS gene, G/G genotype was significantly higher in cases than in control group with odds ratio 5.1. G allele was more prevalent among patient group with odds ratio 1.75. For FASL gene, A/G genotype was significantly higher in cases than in control group with odds ratio 4.53. G allele was more prevalent among patient group with odds ratio 1.88. GG genotype of FAS was significantly associated with longer disease duration (P=0.001), recurrent attacks (P=0.01), higher SALT score (P=0.009), alopecia universalis (P=0.002), and severe disease (P=0.006). FAS and FASL gene polymorphisms are associated with AA. Further large-scale studies on different ethnicities are required for more clarification of their role in disease development. Therapeutic modalities based on their inhibition could be promising in the treatment of a common disease like AA.

2 illus, 2 tables, 36 ref

026688   BUTT J, ISHTIAQ S, IJAZ B, MIR Z A, ARSHAD S, AWAIS S (Pharmacognosy Dep, Punjab Univ Coll of Pharmacy, Lahore, Pakistan, Email: juwairiya.zulfiqar@pharm.uol.edu.pk) : Authentication of polyherbal formulations using PCR technique. Ann Phytomed 2018, 7(1), 131-9.

Unprecedented growth in popularity of complementary medicines raised concerns about their quality and safety. So, there is need to develop methods for their sensitive, specific and accurate analysis. Authenticity and similarity of medicinal nutrients and strict procedures of material handling are significant features to maintain the quality of herbal preparations. Genome based methods to authenticate these plants revolutionized the authentication process. Developing DNA molecular markers by sequencing a standard zone of the DNA is the best technique to identify the adulterants as well as to authenticate the required species of medicinal plants. Application of molecular biological technique serves as one of the very consistent system for authentication of natural herbal materials. The progress of authentic analytical methods is a major challenge to scientists as natural products are optimized as drug like molecules.This research work is based on the application of PCR technique for authentication of Glycyrrhiza glabra L. in its crude form as well as in final dosage form, i.e., Hamdard’s Joshanda, Marhaba’s Joshanda, GNC herbal supplement and Joshaba Sadar (Chest tea) available in market, provided isolated DNA from dried roots of the sample was used as templates in PCR. All the products gave the desired results except one. It proved to be a complementary tool to control quality of herbal materials alone and in different marketable herbal product having Glycyrrhiza spp. as their active ingredient.

5 illus, 5 tables, 77 ref

026687   DEVI N, AZMI W (Biotechnology Dep, Himachal Pradesh Univ, Shimla-171 005, Himachal Pradesh, Email: wamikazmi@yahoo.com) : Structural analysis and characterization of a clinically important low molecular weight natural dextran synthesized by Leuconostoc lactis KU665298 dextransucrase. Ann Phytomed 2018, 7(1), 52-62.

Dextransucrases are glucosyltransferases that catalyze the transfer of $\mathrm{\alpha }$-D-glucopyranose residues from sucrose to low molecular weight acceptors, forming oligos and polysaccharides. The main dextransucrase reaction product is dextran, along with the release of fructose. Dextran is a linear polymer of glucose joined mainly by $\mathrm{\alpha }$ (1 $\to$ 6) links and fewer $\mathrm{\alpha }$ (1 $\to$ 2), $\mathrm{\alpha }$ (1 $\to$3) and/or $\mathrm{\alpha }$(1 $\to$ 4) links. The structural characteristics or the frequency and type of linkage in dextran are dependent on the nature of the enzyme and type of producing micro organism. It has acknowledgeable industrial applications in medical, pharmaceutical, food, textile and chemical industries, depending on its structural characteristics. This paper offers the physical and chemical characterization of a clinically important dextran produced by dextransucrase of a newly isolated strain of L. lactis KU665298 in a laboratory fermenter. Structural analysis of dextran by FTIR, ¹HNMR,¹³C NMR confirmed the presence of a large percentage of $\mathrm{\alpha }$-(1 $\to$ 6) linear linkages with veryfew $\mathrm{\alpha }$-(1 3) branch points. The surface morphology and molecular weight distribution of purified dextran has also been included in this study.

11 illus, 68 ref

026685   NAIDU M A, PRASAD Y R (Biotechnology Dep, Acharya Nagarjuna Univ, Guntur, Andhra Pradesh, Email: manaidupharmacy@gmail.com) : Synthesis of novel diarylsulfonylurea-chalcone hybrid molecules with potential in vitro antimicrobial activity. Asian J Pharm 2018, 12(2), 88-93.

To study the antimicrobial effect of novel diarylsulfonylurea-chalcone hybrids with significant associated morbidity and mortality which is mainly due to the development of microbial resistance to the existing antimicrobial agents. The antimicrobial effect of novel diarylsulfonylurea-chalcone hybrid molecules was evaluated by agar well diffusion method against various strains of bacteria and fungi. Most of the compounds showed promising antibacterial and antifungal activity.The results in the present study suggest that novel diarylsulfonylurea-chalcone hybrids can be used in treating diseases caused by the tested organisms.

2 tables, 11 ref

028132   RADAE T (Biotechnology Dep, Adigrat Univ, Ethiopia) : The utilization of mutagenesis using sodium azide to improve sesame (Sesamum indicum L.). J Med Plants Stud 2018, 6(4), 194-8.

Sesame (Sesamum indicum L.) is considered to be an ancient oil seed crop to generate high quality edible oil having nutritional and health related value but still at an early stage inbreeding. Its chromosome number is 2n=26 and it belongs to the Pedaliaceae family and having significant economic value globally as well as in Ethiopia. In general terms Sesame is unimproved and variety of collections which have been generated of land races, with little or no genetic information that can lead to its application in breeding programs. With the objective of improving the performance of sesame (Sesamum indicum L.) by the utilization of mutagenesis using sodium azide, an experiment was conducted. In this experiment, healthy and dry seeds of sesame (Sesamum indicum L.), were treated with sodium azide in ascending mutagen concentrations of 0.0165, 0.033, 0.0625, 0.125, and 0.25% targeted at determining the effects of the chemical mutagens to promote genetic variability in terms of the agro morphological parameters of sesame following the appropriate procedures. A gradual decrease was observed in mean root length percentage with increasing concentrations. Reduction in seed germination was observed at 0.0625 % concentration in T3 when compared with control and the other treatments but it was increased again in T4 and T5. Root growth was high in the control and in 0.0165 % and 0.033 % concentrations, when compared to the higher concentration treated plants. This revealed that the reduction in seed germination, root length and shoot length percentage was associated with the increase in the dose/concentration of mutagens even if it shows some increasing in seed germination in T4 and T5. Since the produced mutants from first generation are not adequate for studying the genetic stability these traits should be investigated for the desired traits in subsequent generations and in the field conditions, developing sesame varieties resistant for different biotic and abiotic stresses and assisting the present work with the recent biomolecular techniques should be future prospects.

3 illus, 23 ref

028133   REDDY G S, MAHENDRAN B, REDDY R S (Biotechnology Dep, K L Univ, Guntur- 522 502, Email: rsr@kluniversity.in) : Screening and optimization of Achromobacter xylosoxidans GSMSR13B producing bacteria. Asian J Chem 2018, 30(7), 1424-30.

Biosurfactants are amphiphilic mixes created by microorganisms as optional metabolite. The unique properties of biosurfactants make them possible to replace or to be added to synthetic surfactants which are mainly used in food, cosmetics, petroleum refining and pharmaceutical industries and in environmental applications. In this study, 25 hydrocarbon-degrading bacteria were screened for biosurfactant production. All of the bacterial isolates were grown in mineral salt medium with addition of 1 % (v/v) vegetable oil as carbon source. The presence of biosurfactant was determined by the blood hemolysis, drop collapse tests, emulsification assay, emulsification index (E24), foaming activity, lipase activity, haemolytic assay, oil spreading and tilted glass slide, microplate analysis and surface tension measurement. Only one isolate, Achromobacter xylosoxidans GSMSR13B was found to be positive for all the qualitative and qualitative tests and reducing the surface tension of the medium to 47.8 dynes/cm with emulsification index of 28.7 %. This isolate produced biosurfactant optimally at pH 8.0 and incubation temperature of 37 °C. Furthermore, Achromobacter xylosoxidans GSMSR13B when grown in mineral salt medium with addition of 1 % (v/v) glycerol and 1.5 g/L NH4NO3 C/N ratio 16:1 produced biosurfactant with percentage of surface tension reduction at 56 % or 28.6 dynes/cm with % EI24 of 36 %. This percentage of surface tension reduction represents an increasing reduction in surface tension of medium by 33 % over the value before optimization. This study showed that Achromobacter xylosoxidans GSMSR13B has the ability to biodegrade hydrocarbon and concurrently produce biosurfactant.

6 illus, 2 tables, 35 ref

028125   LESTARI D A, PURBOWATI E, SUTOPO S, KURNIANTO E (Animal Science Dep, Diponegoro Univ, Indonesia, Email: kurniantoedy17@gmail.com) : Amino acid sequence based on Cytochrome b gene in Kejobong goat and its genetic relationships among several local goats in Asia. Vet World 2018, 11(8), 1196-1202.

This study aimed to analyze the amino acid sequence of Cytochrome b (Cyt b) gene in Kejobong goat and its genetic relationships with local goats located in Asia.  A total of 28 heads of Kejobong goat were purposively sampled. The deoxyribonucleic acid (DNA) was extracted from blood using gSYNC DNA mini kit (Geneaid Biotech Ltd.). Cyt b gene was amplified using polymerase chain reaction (PCR) method with CytbCapF and CytbCapR primers. The amplified PCR products were sequenced for further analysis. There were a total 377 amino acid sequences translated from 1140 base pair (bp) of Cyt b gene, 99.20 % of it were monomorphic, amino acid alterations were found at site 16th, 121st, and 231st, and Kejobong goat was in the same cluster with Southeast Asian local goats. Most of the amino acid sequence on Cyt b gene in Kejobong goat is monomorphic (99.20 %), only a few nucleotide mutations were found that causing amino acid alteration in three sites (0.80 %). Kejobong goat has a close genetic relationship to several local goats in Southeast Asian

8 illus, 42 ref

028148   VANITHA H D, SETHULEKSHMI C, LATHA C (Veterinary Public Health Dep, Veterinary and Animal Sciences Coll, Thrissur - 680 651, Email: vanivet10@gmail.com) : An epidemiological investigation on occurrence of enterohemorrhagic Escherichia coli in raw milk. Vet World 2018, 11(8), 1164-70.

The aim of the present investigation was to study the epidemiology of enterohemorrhagic Escherichia coli (EHEC) in raw milk and molecular characterization of isolates using multiplex polymerase chain reaction (PCR). A total of 125 raw milk samples were subjected to isolation, identification, and confirmation of virulence-associated genes by multiplex PCR (mPCR). The samples were collected from a milk cooperative society of Thrissur district, Kerala. For further epidemiological investigation, samples such as dung (126), hair coat of cow (60), udder swab (60), udder wash (60), milking utensil wash (36), Milker’s hand wash (36), water (36), soil (36), and feed (36) were collected from the households from which the raw milk tested positive for EHEC. The occurrence of EHEC in individual raw milk samples was found to be 8.8%. The major source of contamination to raw milk was found to be dung (19.84 %) followed by udder swab (16.67 %), hair coat of cow (15 %), Milker’s hand and milking utensils and water (11.11 % each), and udder wash and soil (8.33% each). For identification of virulence genes, all the isolates were subjected to mPCR, of 75 isolates 73.33 % of isolates harbored stx 2 gene while 53.33, 36, and 36 % of isolates were encoded by stx 1, eae A, and hly A genes, respectively. On epidemiological survey, the multiple risk factors accountable for occurrence of EHEC in raw milk were found to be the quality of water used, improper and inadequate udder preparation, unhygienic hands of Milker’s, use of insufficiently cleaned milking utensils, and using common utensil for washings of udder and milking purposes. The result of the present study signifies that raw milk was contaminated with EHEC and possesses a high public health threat. As dairy cattle and its environment serve as a potential niche for EHEC, hygienic milking practices should be adopted to curb the occurrence of EHEC in raw milk.

1 illus, 4 tables, 31 ref

028126   LOKAPIRNASARI W P, SAHIDU A M, SOEPRANIANONDO K, SUPRIYANTO A, YULIANTO A B, ARIF A A (Animal Husbandry Dep, Airlangga Univ, Surabaya, Indonesia, Email: widyaparamitalokapirnasari@gmail.com) : Potency of lactic acid bacteria isolated from balinese bovine (Bos sondaicus) intestinal waste from slaughterhouse to improve nutrient content of wheat pollard as animal feedstuff by fermentation process. Vet World 2018, 11(8), 1127-34.

The purpose of this study was to know the genetic and biochemical identification of isolated lactic acid bacteria (LAB) from Balinese bovine (Bos sondaicus) intestinal waste, acidity, and ox bile salts and to inhibit the growth pathogen of Staphylococcus aureus and Escherichia coli and the potential of those isolated to improve nutrient value of wheat pollard as animal feed ingredient by fermentation process. This research was divided into three stages. The first stage, isolated LAB were obtained from the bovine intestines at a slaughterhouse in Indonesia. Small intestinal samples were collected from 10 healthy Balinese beef cattle (B. sondaicus). The isolated LAB were identified by VITEK 2, polymerase chain reaction, and 16S rDNA. The basic local alignment search tool (BLAST) was performed to determine the phylogenetic tree. The second stage, the LAB were screened for their tolerance at pH 2, 3, and 4; bile salt, and antagonistic to enteric pathogen. In the third stage, to determine the potency of this isolate to increase nutrient content of wheat pollard by facultative anaerobe fermentation for 3 and 5 days. The result of the first stage showed that the isolate could be identified as Lactobacillus casei WPL 315. The result of the second stage showed that the isolate tolerance to low pH (pH 2, pH 3, and pH4) for 90 min and 24 h, and this isolate had viability tolerance in 0.3 % bile salt. The isolate can inhibit S. aureus and E. coli. The result of the third stage by proximate analysis showed that crude protein increased by 23.08 % after fermentation, while crude fiber decreased by 61.24 % on the level 0.5 % L. casei subsp. WPL 315 in the 3-day fermentation. Based on the results, it showed that L. casei WPL 315 derived from indigenous intestinal Balinese beef cattle (B. sondaicus) has tolerant characteristic on acidity and ox bile salts, has antagonistic effect against E. coli and S. aureus, and has the ability to increase crude protein and decrease crude fiber content of wheat pollard. It would be interesting to determine whether the strain has a probiotic candidate.

6 tables, 42 ref

028104   ABDULLAH H H A M, EL-SHANAWANY E E, ABOU-ZEINA H A A, ABDEL-SHAFY S, ABDEL-RAHMAN E H (Parasitology and Animal Diseases Dep, National Research Centre, Giza, Egypt, Email: aasobhy@yahoo.com) : Molecular and immunological characterization of Hyalomma dromedarii and Hyalomma excavatum (Acari: Ixodidae) vectors of Q fever in camels. Vet World 2018, 11(8), 1109-19.

Q fever Coxiella burnetii is a worldwide zoonotic disease, and C. burnetii was detected in mammals and ticks. Ticks play an important role in the spread of C. burnetii in the environment. Therefore, the aims of this study were to detect Q fever C. burnetii in camels and ixodid ticks by molecular tools and identification of Hyalomma dromedarii and Hyalomma excavatum using molecular and immunological assays. A total of 113 blood samples from camels and 190 adult ticks were investigated for the infection with C. burnetii by polymerase chain reaction (PCR) and sequencing the targeting IS30A spacer. The two tick species H. dromedarii and H. excavatum were characterized molecularly by PCR and sequencing of 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit-1 (CO1) genes and immunologically by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. A total of 52 camels (46 %) were positive for Q fever infection. Only 10 adult ticks of H. dromedarii were infected with C. burnetii. The IS30A sequence was around 200 bp in length for C. burnetii in H. dromedarii ticks with a similarity of 99 % when compared with reference data in GenBank records. The length of 16S rDNA and CO1 was 440 and 850 bp, respectively, for both H. dromedarii and H. excavatum. The phylogenetic status of H. dromedarii was distant from that of H. excavatum. SDS-PAGE revealed seven different bands in the adult antigens of either H. dromedarii or H. excavatum with molecular weights ranged from 132.9 to 17.7 KDa. In western blot analyses, the sera obtained from either infested camel by H. dromedarii or infested cattle by H. excavatum recognized four immunogenic bands (100.7, 49.7, 43.9, and 39.6 kDa) in H. dromedarii antigen. However, the infested camel sera identified two immunogenic bands (117 and 61.4 kDa) in H. excavatum antigen. Furthermore, the sera collected from cattle infested by H. excavatum recognized three immunogenic bands (61.4, 47.3, and 35 kDa) in H. excavatum antigen. Molecular analyses indicated that both camels and ticks could be sources for infection of animals and humans with Q fever. Furthermore, the molecular analyses are more accurate tools for discriminating H. dromedarii and H. excavatum than immunological tools.

7 illus, 2 tables, 78 ref

028136   SAXENA A, BISWAS S K, CHAND K, NASKAR J, CHAUHAN A, MOHD G, TEWARI N, KURAT-UL-AIN, RAMAKRISHNAN M A, PANDEY A B (Indian Veterinary Research Institute, Bareilly- 243 122, Email: abpandey58@rediffmail.com) : Genetic and phylogenetic analysis of the outer capsid protein genes of Indian isolates of bluetongue virus serotype-16. Vet World 2018, 11(8), 1025-9.

The aim of the study was to characterize bluetongue virus serotype 16 (BTV-16), recently isolated from different states of India. The evolutionary relationship of newly isolated BTV-16 and previously reported Indian and global BTV-16 isolates were compared using molecular analysis. In the present study, five (n=5) BTV-16 isolates were used to amplify gene segment-2 and segment-6 encoding the outer capsid proteins VP2 and VP5, respectively. The amplified products were purified and sequenced by the Sanger sequencing method. The phylogenetic relationship and nucleotide identity of all five BTV-16 isolates were compared with previously reported Indian and global BTV-16 isolates. Nucleotide sequence data were aligned using the CLUSTAL W algorithm implemented in the MegAlign of DNASTAR program package (MegAlign 5.00, DNASTAR Inc., Madison, USA). Phylogenetic analyses were carried out using MEGA version 6.0 software with the best nucleotide substitution model. Phylogenetic analysis based on the VP2 and VP5 encoding genes, segregates Indian BTV-16 isolates in a distinct cluster with proximity to the Eastern topotype. Indian isolates make a monophyletic cluster with Eastern topotypes with Western topotype BTV-16 (BTV-16/NIG/AJ586694) occupying a separate cluster. Indian isolates were found to share 91.5 %- 97.5 % and 96.5 %-98.9 % identity at the nucleotide and deduced amino acid (aa) level, respectively, to the global BTV-16 isolates. There is a high degree of variation with the Nigerian isolate with 27.0-27.7 % and 26.0-26.9 % at the nucleotide and aa sequence level, respectively. These data suggest that Indian BTV-16 isolates might have evolved separately within the Eastern BTV topotype. Phylogenetic analyses and nucleotide identity of BTV-16 isolates at the VP2 and VP5 gene encoded level indicate that isolates used in the present study might have evolved from a common Eastern topotype ancestor. The data presented in this study will be helpful for future selection of reference strains in a serological and molecular epidemiology study.

2 illus, 2 tables, 22 ref

028103   ABBAS A M, EL-MOATY D A M A, ZAKI E S A, EL-SERGANY E F, EL-SEBAY N A , FADL H A, SAMY A A (Genetic Engineering Research Dep, Veterinary Serum and Vaccine Research Institute, Cairo, Egypt, Email: dody.ahmed@gmail.com) : Use of molecular biology tools for rapid identification and characterization of Pasteurella spp.. Vet World 2018, 11(7), 1006-14.

This study aimed to create rapid characterization and genotyping of Pasteurella multocida (PM) protocol using modern molecular biology techniques. Thirty bacterial isolates were characterized by capsular and somatic identification using conventional procedure followed by multiplex polymerase chain reaction (PCR), restriction endonucleases analysis (REA), and finally confirmed by sequence analysis. Two local vaccine strains and two field isolates were identified as PM Type A and B.  A total of 30 isolates were found positive for PM either morphologically and biochemically; however, multiplex PCR technique identified only 22 isolates as Pasteurella species using universal primers while 8 isolates were found negative for PM. 12 of 22 isolates (54 %) were characterized at the same reaction into PM Type A, five isolates (23 %) were Type B and the rest five isolates (23 %) of tested isolates were negative for Types A, B, and D. Hemorrhagic septicemia Type B: 2 or B: 5 could be identified somatically within PM capsular serogroup B using PCR technique. Somatic characterization of PM was done using REA that could identify all PM Type A into A:1 and all PM Type B into B: 2. These protocols were verified for its accuracy and reliability by sequence analysis of two vaccine strains of PM Type A and B that were characterized previously by biochemical and serological methods as well as two selected isolates from the 22 positive isolates representing PM Type A and B. PCR and REA could confirm the identity of PM and provide a rapid and reliable characterization in comparison with biochemical analysis and conventional serotyping that may take up to 2 weeks. Hence, they can reduce the time needed for polyvalent vaccine production and when the reference antisera are unavailable. Moreover, the identity of Omp-H for vaccine and field strains may provide better data to control Pasteurellosis in Egypt.

6 illus, 2 tables, 35 ref

028137   SELIM K M, SELIM A, ARAFA A, HUSSEIN H A, ELSANOUSI A A (Animal Health Research Institute, Egypt, Email: dr.kareemseleem_87@yahoo.com) : Molecular characterization of full fusion protein (F) of Newcastle disease virus genotype VIId isolated from Egypt during 2012-2016. Vet World 2018, 11(7), 930-8.

The aim of this work was to study the full F gene sequence of Newcastle disease virus (NDV) in regard to pathotyping and genotyping and to study the evolution of this NDV in Egypt. The present study was conducted using samples from seven suspected NDV flocks of vaccinated chickens during 2012-2016 from six governorates in Egypt. The NDV was successfully isolated from pathological specimens through inoculation in specific pathogen-free embryonated chicken eggs. Pathogenicity of the NDV isolates has been estimated through intracerebral pathogenicity index and ranged from 1.66 to 1.73 which indicates the velogenic type of NDV isolates. Pathotyping and genotyping of these isolates were done through sequencing of full-length F gene. Results indicated that the seven NDV isolates showed characteristic cleavage site motif (112RRQKRF117) for the velogenic strains of NDV. Phylogenetic analysis of the F gene clustered these isolates within Group I of genotype VIId within Israeli strains NDV/IS/2015, NDV-Ch/SD883, and most of the Middle East strains. Six of seven sequenced isolates have six potential N-linked glycosylation sites. The neutralization epitope on the five antigenic sites of fusion is conserved in all Egyptian strains of this study except NDV-KFR-B7-2012 which has a substitution at D 170 N in epitope A4. In all our strains, 10 cysteine residues are recorded, except one loss of cysteine at residue 370 in both NDV-EG-35-2014 and NDV-GHB-328F-2016. All viruses in this study have 52 amino acid substitutions within fusion gene in compared with Lasota strain that reveals importance for its antigenic and structural function. The present work highlights the important need to sequence F gene of NDV genotype VIId to investigate the evolution of this NDV in Egypt.

4 illus, 1 table, 41 ref

028107   ATA E B, MAHMOUD M A E F, MADBOLI A A (Parasitology and Animal Diseases Dep, National Research Center, Egypt, Email: emadvet2003@gmail.com) : Molecular detection and immunopathological examination of Delta papillomavirus 4 in skin and udder of Egyptian cattle. Vet World 2018, 11(7), 915-20.

Bovine papillomaviruses (BPVs) are the main cause of bovine papillomatosis resulting in cutaneous and/or mucosal benign tumors that could be transformed to malignant ones with marked economic importance, especially in the dairy farms. Molecular, pathological, and immunohistochemical (IHC) diagnosis of bovine papillomatosis cases was conducted to identify and characterize the circulating BPV genotype in some Egyptian governorates. Wart-like lesions in skin, udder, and teats were collected from 123 infected cases in Giza, Beni Suef, and El Menoufia Governorates, Egypt, during 2016-2017. Pathological and IHC characterization, molecular identification, genotyping, and phylogenetic analysis based on the conserved late (L1) gene of the all samples were carried out. 89 of the 123 collected samples (72.3 %) were positively detected by polymerase chain reaction (PCR). The sequence analysis of the obtained PCR amplicons was identical revealing identification and genotyping of only one type (Deltapapillomavirus 4 isolate EGY 2017) with accession number (MG547343) which found to be closely related to the recently detected Deltapapillomavirus 4 isolate 04_asi_UK (accession no. MF384288.1) and isolate Deltapapillomavirus 4 isolate 25_equ_CH (accession no. MF384286.1) with 99 % nucleotide sequence identity. Histopathological examination revealed severe hyperkeratosis in stratum corneum and acanthosis in most of the cases. These tissue changes were confirmed by the presence of golden brown stained proliferating cell nuclear antigen which was localized intranuclear and perinuclear in other cells using IHC Technique. It is the first time to detect and genotype the BPVs in these areas with no record of previous genotyping in the whole country. The obtained results will highlight the importance of this disease.

5 illus, 38 ref

028119   ISHMAYANA S, KENNEDY U, LEARMONTH R P (Southern Queensland Univ, Australia, Email: robert.learmonth@alumni.unimelb.edu.au) : Comparative assessment of yeast fermentation performance, ethanol tolerance and membrane fluidity. Res J Chem Environ 2018, 22(2), 226-35.

Yeast nitrogen base (YNB) is a chemically defined fermentation medium that does not interfere with fluorescence spectroscopy measurements in the UV to blue range. It is a useful medium for in situ monitoring during fermentation of cell physiology by fluorescence methods. However, compared to rich media, it is considered to have poor nutritional availability. Overall nutrition affects yeast ability to convert sugar to ethanol. This study investigated growth and fermentation performance of three Saccharomyces cerevisiae strains in YNB. Three different glucose concentrations ranging from 5 to 15 % (w/v) were applied to investigate the highest concentration of glucose able to be efficiently converted to ethanol by each yeast strain. Growth and fermentation performance of the yeast strains were different. The fermentation performance could be ranked (highest to lowest) as strains A15, A12 and K7, while the growth performance could be ranked K7, A12, and A15. In general, fermentation with 15 % initial sugar in the minimal medium led to lower sugar conversion to ethanol. The medium containing 10 % glucose was considered the best to optimally differentiate fermentation performance of yeast strains.

7 illus, 2 tables, 31 ref

028114   FEBRIANI F, HELWATI H, VELAYATI M A , IQBALSYAH T M (Syiah Kuala Univ, Indonesia, Email: febriani@unsyiah.ac.id) : Identification of a DNA polymerase I gene fragment from a local isolate (PLS 80) from an underwater hot spring. Res J Chem Environ 2018, 22(2), 789-92.

DNA polymerase is a thermostable enzyme widely used in Polymerase Chain Reaction (PCR) processes. The goal of this research was to amplify of 0.9 kb DNA polymerase I gene fragments of thermo-halophilic bacteria isolated from an underwater hot spring area, namely Pria Laot Sabang (PLS) 80. Chromosomal DNA was amplified using 1 set of primers, FP2 and RP1. The sequences of DNA polymerase I gene fragments from the PLS 80 isolate had a length of 921 bp and were identified using a direct sequencing with the NCBI BLAST tool. The construction of a phylogenetic tree of the 0.9 kb DNA polymerase I fragment, coupled with homological analysis, showed that the PLS 80 isolate had a homology closest (99 % similar) to that of Geobacillus thermoleovorans strain KCTC 3570 from the NCBI GenBank database. The results suggest that amplification of the 0.9 kb DNA polymerase gene fragment from PLS 80 isolate was 5-3’ polymerase domain.

2 illus, 2 tables, 5 ref

028135   SAADAH D R, RISMA W H, AGUS S, ISHMAYANA S (Padjadjaran Univ, Indonesia, Email: ishmayana@unpad.ac.id) : A comparison of the fermentation performance and stress tolerance of baker's yeast cells grown in media with or without magnesium addition. Res J Chem Environ 2018, 22(2), 124-8.

Bioethanol is an alternative fuel that was developed to overcome the depletion of fossil fuels. It is produced by converting sugars to ethanol through fermentation using microorganism activity, especially the yeast Saccharomyces cerevisiae. However, during ethanol fermentation, the yeast cells are exposed to various environmental stress factors which can reduce their performance. Some metal ions are known to have an impact on yeast cell’s tolerance against environmental stress factors. The present study investigates the role of magnesium ions in the fermentation performance and stress tolerance of a baker’s yeast strain. Yeast cells were grown in media with or without magnesium ions. Growth and fermentation kinetic parameters were then monitored every 6 hours during the first 24 hours followed by 12 hours intervals for the rest fermentation. After 24 hours of fermentation, stress tolerance assays were performed against ethanol, hyperosmotic, oxidative and acetic acid stress. The results of the present study indicate that magnesium ions can enhance the fermentation performance of yeast cells as indicated by better specific growth rates, substrate utilization, ethanol productivity and ethanol yield. Magnesium also showed a protective effect against all stress factors tested in this study.

3 illus, 1 table, 20 ref

029178   KOFFI S S, YAO N, SAMPSON G O, OUTTARA H G, BRUNEAU D, KONAN K, DIBY K A (Félix Houphouët-Boigny Univ of Cocody 22 B P 582, Abidjan, Côte d’Ivoire, Email: xastinaka@gmail.com) : Analysis of the performance of a newly designed fermenter built in local materials for improvement of cocoa fermentation, in Ivory Coast. J Appl Biosci 2018, 129, 13708 - 117.

Cocoa beans fermentation is a spontaneous chemical process, traditionally done in box or in a heap. Equipment is also important in achieving the final chocolate aroma. This study analyzes the performances of a new-designed cocoa bean fermenter.  In this study, a new type of fermenter, a rotating cylindrical fermenter (RCF) has been designed in order to improve the fermentation of cocoa that remains difficult to control, because of the spontaneous nature of the microbiota. The performances of this fermenter was analyzed and compared to those of the traditional wooden box (TWB) fermenter that is commonly used on farm. During the 6 days of fermentation in both fermenters, the growth of microorganisms such as yeasts, lactic bacteria, acetic bacteria, bacillus and moulds as well as chemical and physical changes of the fermenting cocoa were monitored. The results showed that in the fermenter (RCF) a rapid temperature increase was observed in the course of the fermentation process with a temperature reaching 51°C within 73 h comparatively to the traditional fermenter (48°C within 118 h). This leads to a higher proportion of brown beans, indicator of a good fermentation from RCF fermenter as assessed by the cut test. This proportion was 94.44 % for RCF and 85.88 % for TWB. Moreover the optimization of heat generated in the RCF fermenter, allowed a normal browning (final gray level was 77 in both fermenters) despite modification of microbiota growth order (early growth of acetic bacteria and stunted growth of yeast in RCF, but not for TWB). The high proportions of brown beans in RCF suggest that this equipment is liable to contribute to the improvement of standard quality of cocoa beans.

5 illus, 4 tables, 23 ref

029176   KHAJEHNASIRI N, KHAZALI H, SHEIKHZADEH F (Animal Sciences and Biotechnology Dep, Shahid Beheshti Univ, Tehran, Iran, Email: homkhazali1@gmail.com) : Various responses of male pituitary–gonadal axis to different intensities of long-term exercise: Role of expression of KNDYrelated genes. J Biosci 2018, 43(4), 569–74.

The essential role of regular physical activity has been emphasized for maintaining a healthy life. However, unfortunately, during the last few decades, the lifestyle of people has led to a decrease in physical activity. Research studies have shown that exercise of different intensities is applied on reproductive performance indices, luteinizing hormone (LH) and testosterone (T), with different effects. Nevertheless, the molecular and cellular mechanisms underlying its function are not completely understood. Therefore, this study aimed to evaluate the role of kisspeptin, neurokinin-B and pro-dynorphin (KNDY) gene-expression changes located in the upstream of GnRH neurons in transferring the effects of different long-term exercise intensities on male reproductive axis. Twenty-one adult Wistar rats were randomly divided into control, 6-month regular moderate exercise (RME-6) and 6-month regular intensive exercise (RIE-6). In moderate and intensive exercise groups, rats were treated 5 days a week for 60 min, at 22 and 35 m/min, respectively. Finally, the hypothalamic arcuate nucleus was isolated and the relative gene expression of kisspeptin (Kiss1), neurokinin-B (Nkb), pro-dynorphin (Pdyn) and gonadotropin-releasing hormone (Gnrh) genes were measured by realtime polymerase chain reaction method. The results showed that RIE-6 treatment decreased Gnrh and increased Pdyn mRNA levels in the arcuate nucleus. Furthermore, although RME-6 treatment decreased Nkb and increased Pdyn mRNA levels, the Gnrh mRNA was not affected. Regarding the Gnrh mRNA levels and serum concentrations of reproductive indices (LH and T), moderate exercise did not impose harmful effects on the hypothalamic–pituitary–gonadal axis than intensive exercise. The different impacts of diverse long-term exercise intensities on the male pituitary–gonadal axis maybe relay by the various changes in hypothalamic Nkb and Pdyn gene expressions.

5 illus, 1 table, 26 ref

029182   LAPIRA J E E, BALBIN M M, BELOTINDOS L P, VILORIA V V, ABES N S, MINGALA C N (Central Luzon State Univ, 3120 Science City of Mun˜oz, Nueva Ecija, Philippines, Email: cnmingala@hotmail.com) : Molecular detection of ephemeral fever virus among large ruminants in the Philippines. Virusdisease 2018, 29(3), 400–4.

In the Philippines, bovine ephemeral fever (BEF) is currently undetected and considered as an exotic disease of both cattle and water buffaloes. The Philippines until now has no official data regarding the occurrence of BEF. There were no existing control programs or vaccine used for the prevention of the disease. However, there are claims of BEF existence in different water buffalo and cattle farms based on the clinical signs but never confirmed using laboratory test yet. Detection of BEF virus in cattle and water buffalo blood samples was conducted using reverse-transcription PCR targeting the glycoprotein (G) gene, a conserved region in the BEF virus genome. The samples were collected from 22 cattle and 50 water buffaloes with clinical signs suggesting of BEF infection. All water buffalo blood samples were negative while four cattle blood samples turned positive for BEF virus. The G gene partial sequence analysis from two BEF virus positive samples showed close relationship to Australian isolates.

2 illus, 14 ref

029202   ULLAH S, KHAN M A, RAHMAN S U, KHAN I, AKBAR F, BABBAR A (Biotechnology Dep, Shaheed Benazir Bhutto Univ, Dir Upper, Pakistan, Email: Anshu.Babbar@outlook.com) : Molecular characterization and clinical epidemiology of HCV in District Dir (Lower), Pakistan. Virusdisease 2018, 29(3), 369–74.

With about 200 million infections globally, Hepatitis C virus (HCV) infection is a major global health threat. The relative prevalence of hepatitis and HCV genotypes/subtypes varies among different geographic regions. Therefore, the present study was conducted to determine the prevalence of hepatitis C and HCV genotypes/subtypes in District Dir (Lower), Pakistan. Blood samples from HCV positive patients were genotyped through multiplex PCR using specific primers for HCV core region. A structured questionnaire was used to obtain information from the patients and data was statistically analyzed for different epidemiological parameters. The molecular evaluation results suggested the prevalence of genotype 3 in this study. The frequency of hepatitis C was found higher in males (P < 0.01). Present study suggests injections received at local clinics as a highly significant mode of HCV transmission to these patients (P < 0.002). These findings might be helpful for clinicians and related health care personnel to assess status of this important disease and highlight the need for more detailed evaluation of this devastating disease in order to frame better treatment strategies.

1 illus, 1 table, 33 ref

029195   REDDY G B M, KRISHNAPPA S, VIANYAGAMURTHY B, SINGH R, SINGH K P, SAMINATHAN M, SAJJANAR B, RAHMAN H (ICAR-NIVEDI, Karnataka - 560 064, Email: gbmpatho@gmail.com) : Molecular epidemiology of rabies virus circulating in domestic animals in India. Virusdisease 2018, 29(3), 362–8.

Rabies is a neglected viral zoonotic disease affecting humans, domestic and wild animals and is endemic in most parts of the India. Dog mediated rabies is more predominant than other forms of rabies and molecular epidemiology is poorly understood in both reservoir and susceptible hosts. In the present study, a total of 140 rabies suspected brain samples from different species of animals from different geographical regions of India were used. The samples were parallelly tested by direct fluorescent antibody test, reverse transcriptase PCR and real-time PCR. Thirty positive samples were subjected for partial nucleoprotein gene sequencing and phylogenetic analysis. On sequence and phylogenetic analysis, it was observed that all Indian rabies viruses belonged to classical rabies virus of genotype 1 of Lyssavirus and formed two distinct groups. The majority of isolates were in group-1 and are closely related to arctic/arctic like lineage, whereas group– II isolated are closely related to cosmopolitan lineage. These results indicated there is simultaneous existence of two distinct lineages of rabies viruses in Indian subcontinent. Further whole genome studies are needed for better understanding of molecular epidemiology of rabies virus circulating in animals for control and prevention of rabies in India.

3 illus, 2 tables, 20 ref

029169   HENRIQUES A M, DUARTE M, BARROS S C, FAGULHA T, RAMOS F, LUI´S T, FEVEREIRO M (Virology Dep, Instituto Nacional de Investigacao Agraria e Veterinaria, Oeiras, 2780-157 Lisbon, Portugal, Email: margarida.henriques@iniav.pt) : Development and validation of a real-time PCR for the detection and quantification of porcine circovirus type 2. Virusdisease 2018, 29(3), 355–61.

Porcine circovirus type 2 (PCV2) is a spherical and non-enveloped virus belonging to the genus Circovirus of the Circoviridae family with a single stranded circular DNA genome. This virus, already detected worldwide, has been associated to several diseases and was implicated as the etiological agent of a disease named postweaning multisystemic wasting syndrome. Several methods have been described for the detection of PCV2, being real-time PCR the most simple and reliable. As far as we know, all the real-time PCR systems described until now are based on ORF2 gene, that exhibit the highest variability. This paper reports the development and validation of a real-time PCR targeted to ORF1 and based on a TaqMan probe for the detection of porcine circovirus type 2 DNA in swine samples. Due to the lack of PCV1 samples, the ability of the test to discriminate between PCV1 and PCV2 positive samples was evaluated in silico. Estimations of 100 % specificity and 100 % sensitivity were obtained based on the qPCR results with panel of 81 swine samples (known PCV2-positive (n = 50); known PCV2-negative (n = 17); samples positive to other common swine viral pathogens (n = 13) and one sample from a BFDV-positive parrot (n = 1)). Intra- and inter-assay coefficients of variation obtained with three positive samples of different viral charges in five replicates or in five independent assays were below the acceptance threshold. The limit of detection determined with a recombinant plasmid containing the amplicon, led to conclude that this assay can detect at least three plasmid copies.

3 illus, 2 tables, 22 ref

029165   GARCIA G G, AQUINO M A D, BALBIN M M, BELOTINDOS L P, SUPNET J G, MINGALA C N (Central Luzon State Univ, 3120 Science City of Mun˜oz, Nueva Ecija, Philippines, Email: cnmingala@hotmail.com) : Characterisation of porcine epidemic diarrhea virus isolates during the 2014-2015 outbreak in the Philippines. Virusdisease 2018, 29(3), 342–8.

The viral agent of the porcine epidemic diarrhea (PED) was investigated during the reported 2014–2015 outbreaks in commercial farms in Central Luzon, Philippines. The study covered detection of PED virus (PEDV) in fecal and intestinal samples through reverse transcription PCR and sequence analysis of the nucleocapsid (N) gene. Results showed that 10 out of 34 fecal and intestinal samples examined were positive for PEDV. The partial nucleotide sequence of the N gene of the field samples showed 98–99 % homologous to PEDV sequences registered in the GenBank. It was also noted that N gene sequences between field samples were 98 % homologous. Interestingly, the partial sequences of the N genes of the field samples were genetically similar to the PEDV isolates from USA, China, Mexico, Canada and Japan. The phylogenetic tree analysis revealed that the Philippine samples clustered in group 2–1 of the PEDV, wherein the isolates of this group were responsible for the outbreaks in Asia and the USA. Analysis of the partial nucleotide and amino acid sequences revealed polymorphisms, deletions and insertions in the N-gene of the PEDV. Amino acid sequence alignment also showed deletions and insertion in the PEDV detected in the Philippines.

2 illus, 47 ref

029151   ABDEL-MAWGOD S, ADEL A, ARAFA A-S, HUSSEIN H A (Animal Health Research Institute, P.O. Box 264, Dokki, Giza 12618, Egypt, Email: drsara_vet2006@yahoo.com) : Full genome sequences of chicken anemia virus demonstrate mutations associated with pathogenicity in two different field isolates in Egypt. Virusdisease 2018, 29(3), 333–41.

Chicken anemia virus (CAV) is an important pathogen associated with immunosuppression in chicken. In this study, out of samples collected from 115 commercial poultry farms, 12 samples were CAV positive by PCR. Partial sequence and phylogenetic analysis of VP1 gene revealed that the detected viruses were clustered to genotype I (n = 3) and genotype II (n = 9). Motifs of both low (E144) and high pathogenic strains (T89, I125, Q141) were found in the three viruses of genotype I. Whereas genotype II viruses demonstrated the characteristic motifs of highly pathogenic strains (I75, T89, I125, Q141, and Q144). Three isolates representative of both genotypes (CAV/CA1, CAV/GZ1 and CAV/SK4) were selected for full genome sequencing and results revealed that the VP2 gene had two substitutions at V153 and E 175, while VP3 gene had only one substitution at C118. To evaluate virus pathogenicity, two isolates from each genotype (CAV/SK4 of genotype I and CAV/CA1 of genotype II) were intramuscularly inoculated in two groups of one-day-old specific pathogen free chicks. Eighteen days post inoculation, PCR detected CAV in 75 and 90 % of chicks in group I and II; respectively. Mortalities in inoculated chicks were 5 and 20 % and packed cell volume values were 0.21 and 0.19; respectively. CAV/CA1 and CAV/SK4 isolates showed pathogenic evidences at the level of genetic (Q141 and 394Q) with variable degree of virulence. In conclusion, the study reports the circulation of at least two genotypes of CAV among chicken population with mutation associated with pathogenicity.

2 illus, 2 tables, 35 ref

029152   AKHONDNEZHAD M, HAGHSHENAS M R, GHASEMI M, MOUSAVI T (Mazandaran Univ of Medical Sciences, Sari, Iran, Email: haghshenas2001@yahoo.com) : The prevalence and genotyping of human papillomavirus in patients with oral tumors in health centers and clinics of Mazandaran in Iran. Virusdisease 2018, 29(3), 297–302.

Oral cancer is one of the most prevalent cancers in the world which contains many kinds of malignant neoplasms in the oral cavity. Due to the carcinogenicity of human papillomavirus (HPV) and its prevalence in cancer, including the oral cancer, this study was aimed at investigate the prevalence of HPV and its genotypes in patients suffering from oral tumors using PCR method. In this study, 83 samples of oral lesions were collected in the form of paraffin-embedded tissue. After extracting the DNA using DNA extraction kits, high-risk HPV positive samples were examined using special kits for genotyping, and lowrisk types were sequenced after nested PCR. The results showed that 13.2 % of samples was HPV positive. The result of PCR using genotyping kit indicated that high-risk types of 18, 31, 16, and 33 appeared in samples with prevalence rate of 27.2, 18.1, 9.09 and 9.09 %, respectively. In this manner, the result of sequence indicated that the prevalence of HPV-6 genotype was 36.3 % in the samples. The results of this study indicated that both low-risk and high-risk types of HPV are associated with the risk of oral tumors, so that Types 6 and 18 were reported as the most prevalent types in the samples.

3 illus, 4 tables, 34 ref

029159   DEESEENTHUM S, LUANG -IN V, JOHN S M, CHOTTANOM P, CHUNCHOM S (Mahasarakham Univ, Maha Sarakham, THAILAND, Email: sirirat.d@msu.ac.th) : Effects of kefir fermentation on antioxidation activities (in vitro) and antioxidative stress (in vivo) of three Thai rice milk varieties prepared by ultrasonication technique. Pharmacogn J 2018, 10(5), 1061-6.

The effects of kefir fermentation were investigated on antioxidation activities (in vitro) and antioxidative stress (in vivo) for different Thai rice; Hawm Nil rice, Red Hawm rice and Khao Dawk Mali 105 rice. Antioxidant activity (in vitro) was investigated using ferric reducing antioxidant power and 2, 2´-diphenyl-1-picrylhydrazyl assays. In addition, antioxidative stress (in vivo) was performed using colitis rat models to study nitric oxide (NO), lipid peroxidation (LPO) and superoxide dismutase (SOD) compared with rats treated with prednisolone and cow’s milk kefir. Antioxidant activity of rice kefir powder from both assays had higher antioxidant activity than cow’s milk kefir powder. NO levels of colitis rats received Hawm Nil rice kefir powder (HNKP) was reduced when compared to phosphate buffered saline (PBS) group. Moreover, colitis rats received HNKP did not differ in NO levels from colitis rats that received prednisolone and non-colitis rats. The result of LPO product malondialdehyde (MDA) indicated that colitis rats treated with HNKP had reduced TBARS compared to PBS group, and did not differ in TBARS levels from rats that received prednisolone and non-colitis rats. Surprisingly, increase in SOD activity was observed in colitis rats that received HNKP compared to PBS, with similar results of increased SOD in rats that received prednisolone and cow’s milk kefir powder. Hawm Nil rice kefir may offer a protective effect for antioxidative stress resulting from chemical induction; it has potential as a supplementary food with high antioxidant activity and is regarded as safe for consumer health.

3 illus, 1 table, 27 ref

029184   MANSAUDA K L R, ANWAR E, NURHAYATI T (Indonesia Univ, West Java, Indonesia-16424, Email: effionora.anwar@farmasi.ui.ac.id) : Antioxidant and anti-collagenase activity of Sargassum plagyophyllum extract as an anti-wrinkle cosmetic ingredient. Pharmacogn J 2018, 10(5), 932-6.

Sea algae are widely used as food and cosmetics in the world. There are several algae including brown algae which are us for human used to maintain health and skin care. Brown algae have various potential biological activities because contain substantial phytochemical constituent. Numerous report has identified phytochemical compound of Sargassum sp. extract but the activity as anti-collagenase almost none. The objective was to study the antioxidant, and anti-collagenase activity of Sargassum plagyophyllum extract as active pharmaceutical ingredient for anti-wrinkle cosmetics. Sargassum plagyophyllum was obtained from Pasauran Beach, Banten, West Java, Indonesia. The extract Sargassum plagyophyllum extracted with three concentration ethanol-water: (E1) ethanol 25 %, (E2) ethanol 50 % and (E3) ethanol 75 %, by using maceration extraction method for 24 h, thrice. The extract was evaluated include total phenolic content, antioxidant activity, and the best extract was tested for the anti-collagenase activity. Total phenol in the extract were 0.588 ± 0.01 (E1), 0.272 ± 0.01 (E2), and 0.220 ± 0.03 (E3) mg PGE/ 100 g extract, respectively. Antioxidant activity of the extract (50 mg/mL) was 41,61 ± 0,02 % (E1), 39,16 ± 0,01 % (E2), 37,58 ± 0,03 % (E3) and ascorbic acid 78.03 ± 0,65 % (22.44 µg/mL) as a standard. The best extract (E1) had inhibited the activity of collagenase by 54.46 ± 0.37%. It can be concluded that the brown seaweed (Sargassum plagyophyllum) extract can be used as an active pharmaceutical ingredient for anti-wrinkles cosmetic

4 illus, 29 ref