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Sample records for thermostable newcastle disease

  1. Thermostability of reconstituted newcastle disease virus strains at 36 ...

    African Journals Online (AJOL)

    Haemagglutination (HA) test was employed to determine the stability of HA titers of reconstituted form of Hitchner – B1 (B1), LaSota (L) and Komarov (K) strains of Newcastle Disease Vaccine (NDV) at 360c. The temperature treatment method was through incubation (in water bath) of the reconstituted vaccines at selected ...

  2. Recovery of avirulent, thermostable Newcastle disease virus strain NDV4-C from cloned cDNA and stable expression of an inserted foreign gene

    NARCIS (Netherlands)

    Zhang, X.; Liu, H.; Liu, P.; Peeters, B.P.H.; Zhao, C.; Kong, X.

    2013-01-01

    A reverse genetics system for thermostable Newcastle disease virus (NDV) is not currently available. In this study, we developed a reverse genetics system for the avirulent and thermostable NDV4-C strain. Successful recovery of NDV4-C was achieved by using either T7 RNA polymerase or cellular RNA

  3. PLAQUE ASSAY OF NEWCASTLE DISEASE VIRUS

    Directory of Open Access Journals (Sweden)

    B. Sardjono

    2012-09-01

    Full Text Available The Newcastle disease virus (NDV was isolated from a 3 months-old indigenous chicken (buras or kampung chicken which showed clinical signs of Newcastle disease (ND. For viral isolation a small part of the spleen and lung were inoculated into 10 days-old embryonated chicken eggs. The physical characteristics of the isolate (A/120 were studied. The hemagglutination of chicken red blood cell showed slow elution, thermostability of hemagglutinin at 56°C was 120 minutes. The vims was able to agglutinate horse erythrocytes but not those of sheep. The biological characteristics on mean death time (MDT of embryonated chicken egg and plaque morphology on chicken embryo fibroblast (CEF primary cell cultures were studied. The MDT was 56 hours, the isolate was velogenic NDV. There were three different plaque morphologies on CEF : 2 mm clear plaques, 1 mm clear plaques, and minute clear plaques which were visible only with microscopic examination.

  4. Response of chickens to oral vaccination with Newcastle disease ...

    African Journals Online (AJOL)

    Thermostable Newcastle disease (ND) vaccine virus strain I2 was investigated for its efficacy as foodborne vaccine, using maize offal as the vehicle. Immune response to vaccination and resistance to challenge were assessed by standard methods. Results showed that following primary vaccination, 40 (64.5%) out of the 62 ...

  5. Control of Newcastle disease virus

    Science.gov (United States)

    Newcastle disease virus (NDV), also know as avian paramyxovirus serotype 1, is an important poultry pathogen worldwide. In naive poultry, the virulent forms of the virus cause high mortality. Because of this the virus is reportable to the World Organization for Animal Health and can be an important ...

  6. Comparative evaluation on some available Newcastle disease ...

    African Journals Online (AJOL)

    Newcastle disease vaccines from three different countries were compared in terms of the level of protection conferred on domestic chickens against Newcastle disease (ND) following routine vaccinations. One hundred and fifty day old chicks were acquired and divided into three groups, with each group of fifty chickens ...

  7. Detection of serum antibody levels against newcastle disease in ...

    African Journals Online (AJOL)

    Poultry diseases are one of the main factors constraining poultry practice in most developing countries. Newcastle disease (ND) is a highly contagious and commonly fatal viral poultry disease caused by Newcastle disease virus (NDV). Detection of antibodies to Newcastle disease virus in 300 blood samples from local ...

  8. Optimization of Newcastle disease virus production in T-flask

    African Journals Online (AJOL)

    GREGORY

    2011-12-16

    Dec 16, 2011 ... In the present study, the production of lentogenic Asplin F strain of Newcastle disease virus by ... future live Newcastle disease vaccine production in larger ..... Production of yellow fever virus in microcarrier-based Vero cell ...

  9. Avian influenza, Newcastle and Gumboro disease antibodies and ...

    African Journals Online (AJOL)

    Studies on avian influenza and Newcastle disease focus on waterfowls, considered natural reservoirs of these viruses. This study surveyed avian influenza (AI), Gumboro and Newcastle disease antibodies and antigens in birds in live wild bird markets (LWBMs), live poultry markets (LPMs) and free flying in Kaduna State ...

  10. 9 CFR 113.205 - Newcastle Disease Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ... Virus. 113.205 Section 113.205 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.205 Newcastle Disease Vaccine, Killed Virus. Newcastle Disease Vaccine...

  11. Role of Maternally Derived Antibody in Newcastle Disease ...

    African Journals Online (AJOL)

    Newcastle disease affects different age group of birds despite vaccination from day old. In order to determine the influence of maternally derived antibody (MDA) on chicks in Newcastle disease (ND) vaccination, a total of 100 broiler chicks were divided into four equal groups, A, B, C and D. Different ND vaccination program ...

  12. RNA Sequence of Spleen of Newcastle Disease Infected Chickens

    Data.gov (United States)

    US Agency for International Development — At 21 days of age, chickens were infected with Newcastle Disease virus (or a mock injection as controls), and spleens were harvested at 2 and 6 days post infection....

  13. Retrospective analysis of Newcastle disease diagnosed at the ...

    African Journals Online (AJOL)

    Newcastle disease (ND) is a highly contagious viral disease of domestic and wild birds with devastating impact on poultry health and production. Many vaccines and vaccination schedules are in use in controlling the disease but prevention and control are still a problem. A ten-year retrospective study (2002-2011) of ...

  14. Evaluation of Some Fowl Pox, Gumboro and Newcastle Disease ...

    African Journals Online (AJOL)

    The quality of live commercial fowl pox (FP), Gumboro disease (GD) and Newcastle disease (ND) vaccines manufactured by four laboratories and on sale in Nigeria were tested. One of the nine vaccines yielded Aspergillus sp., two Salmonella sp. and three Escherichia coli when grown on culture media. All the four ND ...

  15. Epizootiology of Newcastle disease in two live bird markets in ...

    African Journals Online (AJOL)

    Newcastle disease (ND) is a devastating viral disease of poultry worldwide. This study was therefore undertaken to understand the role of live bird markets (LBMs) in the epizootiology of ND in Nigeria. A structured questionnaire was administered to poultry dealers and cloacal swab sampling of live birds in two LBMs in ...

  16. Prevalence of Newcastle disease virus antibodies in sera and eggs ...

    African Journals Online (AJOL)

    ADEYEYE

    2016-03-07

    Mar 7, 2016 ... The seroprevalence and maternal antibody profiles to Newcastle disease virus infection of guinea fowls were studied using ..... gallisepticum. Avian diseases, 28 (4): 877-883. Sa'idu L, Tekdek LB & Abdu PA (2004). Prevalence of ND antibodies in domestic and semi domestic birds in Zaria, Nigeria.

  17. A Serological Survey for Newcastle Disease Virus Antibobies in ...

    African Journals Online (AJOL)

    A Serological Survey for Newcastle Disease Virus Antibobies in Village Poultry in Yobe State, Nigeria. ... The PDF file you selected should load here if your Web browser has a PDF reader plug-in installed (for example, a recent version of Adobe Acrobat Reader). If you would like more information about how to print, save, ...

  18. Molecular screening and isolation of Newcastle disease virus from ...

    African Journals Online (AJOL)

    Molecular screening and isolation of Newcastle disease virus from live poultry markets and chickens from commercial poultry farms in Zaria, Kaduna state, Nigeria. ... The PDF file you selected should load here if your Web browser has a PDF reader plug-in installed (for example, a recent version of Adobe Acrobat Reader).

  19. Serological and Virological Study of Newcastle Disease and Avian ...

    African Journals Online (AJOL)

    Serological survey on the prevalence of Newcastle disease (NCD) virus antibodies using haemagglutination inhibition test (HI) and virological detection by RT-PCR of highly pathogenic avian influenza (HPAI) H5N1, were carried out in 6 regions of Senegal from June to November 2008. Rural chickens were raised in free ...

  20. 9 CFR 113.329 - Newcastle Disease Vaccine.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Newcastle Disease Vaccine. 113.329 Section 113.329 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF.... Challenge virus shall be provided or approved by Animal and Plant Health Inspection Service. (4) If at least...

  1. vaccination with newcastle disease vaccines strain i2 and lasota

    African Journals Online (AJOL)

    UP Employee

    mash feed as vaccine carriers was conducted. Newcastle disease vaccine strain I2 and. NDV La Sota vaccines provided protection to commercial and local chickens vaccinated through i/o, i/m or dw. No significant difference (P≤0.05) was observed in the antibody titre of commercial or local chickens vaccinated with either ...

  2. Quality control assessment of two lentogenic newcastle disease ...

    African Journals Online (AJOL)

    Control of Newcastle Disease is principally by vaccination. Both imported and locally produced vaccines are in use in Nigeria. Comparison was made between two lentogenic vaccines imported from France, India and the Vom-produced Lasota in terms of physical outlook, sterility viral viability, immunogenicity and safety.

  3. A Serological Survey for Newcastle Disease Virus Antibobies in ...

    African Journals Online (AJOL)

    Abstract. A serological survey to detect the presence of antibodies to Newcastle disease virus (NDV) in village poultry was conducted in 17 villages of Yobe State, Nigeria. The aim of the study was to investigate the prevalence of NDV using haemaggluttination inhibition test. Ten households were sampled from each village.

  4. The effects of Newcastle Disease Vaccine (Komarov) on ...

    African Journals Online (AJOL)

    Clinical results show that 30% of the 20 birds vaccinated with Newcastle Disease Vaccine (Komarov) showed characteristic greenish yellowish diarrhoea, cumulative sharp drop in egg production (60%) while respiratory signs like gasping, sneezing and coughing were noticed in the 60% of the birds. Statistical analysis ...

  5. Microculture system for detection of Newcastle disease virus antibodies.

    Science.gov (United States)

    Wooley, R E; Brown, J; Gratzek, J B; Kleven, S H; Scott, T A

    1974-05-01

    A microculture system utilizing cytopathic effect (CPE) and hemadsorption (HAd) end points was effective in determining the level of Newcastle disease virus (NDV) antibodies. The microculture system was of comparable sensitivity to the plaque reduction test for the detection of NDV antibodies. The standards by which the CPE and HAd microculture tests would be considered reproducible were defined. The results indicate that the CPE and HAd microculture tests are reproducible within one twofold dilution.

  6. Newcastle disease: An in-depth review including epidemiology and molecular diagnostics

    Science.gov (United States)

    Infections of birds with strains of avian paramyxovirus serotype 1 (APMV-1), (synonyms: Newcastle disease virus (NDV), pigeon PMV-1 (PPMV-1)) are associated with two clinical outcomes: 1) Newcastle disease (ND) results from infections with virulent APMV-1, and is also called Exotic ND (END) in U. S...

  7. The pathogenesis of Newcastle disease: A comparison of selected Newcastle disease virus wild-type strains and their infectious clones

    International Nuclear Information System (INIS)

    Wakamatsu, Nobuko; King, Daniel J.; Seal, Bruce S.; Samal, Siba K.; Brown, Corrie C.

    2006-01-01

    The effect of mutations of Newcastle disease virus (NDV) fusion (F) gene, hemagglutinin-neuraminidase (HN) gene, and phosphoprotein (P) gene and HN chimeras between the virulent Beaudette C and low virulence LaSota strains on pathogenesis and pathogenicity was examined in fully susceptible chickens. A virulent F cleavage site motif within a LaSota backbone increased pathogenicity and severity of clinical disease. A LaSota HN within a Beaudette C backbone decreased pathogenicity indices and disease severity. A Beaudette C HN within a LaSota backbone did not change either pathogenicity indices or severity of disease in chickens. Loss of glycosylation at site 4 of the HN or modified P gene of Beaudette C decreased pathogenicity indices and caused no overt clinicopathologic disease in chickens. Both pathogenicity indices and clinicopathologic examination demonstrated that the F, HN, and P genes of NDV collectively or individually can contribute to viral virulence

  8. International biological engagement programs facilitate Newcastle disease epidemiological studies

    Directory of Open Access Journals (Sweden)

    Patti J. Miller

    2015-10-01

    Full Text Available Infections of poultry species with virulent strains of Newcastle disease virus (NDV cause Newcastle disease (ND, one of the most economically significant and devastating diseases for poultry producers worldwide. Biological engagement programs (BEP between the Southeast Poultry Research Laboratory (SEPRL of the United States Department of Agriculture and laboratories from Russia, Pakistan, Ukraine, Kazakhstan and Indonesia collectively have produced a better understanding of the genetic diversity and evolution of the viruses responsible for ND, which is crucial for the control of the disease. The data from Kazakhstan, Russia and Ukraine identified possible migratory routes for birds that may carry both virulent NDV (vNDV and NDV of low virulence into Europe. In addition, related NDV strains were isolated from wild birds in Ukraine and Nigeria, and from birds in continental USA, Alaska, Russia, and Japan, identifying wild birds as a possible mechanism of intercontinental spread of NDV of low virulence. More recently, the detection of new sub-genotypes of vNDV suggests that a new, fifth, panzootic of ND has already originated in Southeast Asia, extended to the Middle East, and is now entering into Eastern Europe. Despite expected challenges when multiple independent laboratories interact, many scientists from the collaborating countries have successfully been trained by SEPRL on molecular diagnostics, best laboratory practices, and critical biosecurity protocols, providing our partners the capacity to further train other employees and to identify locally the viruses that cause this OIE listed disease. These and other collaborations with partners in Mexico, Bulgaria, Israel, and Tanzania have allowed SEPRL scientists to engage in field studies, to elucidate more aspects of ND epidemiology in endemic countries, and to understand the challenges that the scientists and field veterinarians in these countries face on a daily basis. Finally, new viral

  9. International Biological Engagement Programs Facilitate Newcastle Disease Epidemiological Studies

    Science.gov (United States)

    Miller, Patti J.; Dimitrov, Kiril M.; Williams-Coplin, Dawn; Peterson, Melanie P.; Pantin-Jackwood, Mary J.; Swayne, David E.; Suarez, David L.; Afonso, Claudio L.

    2015-01-01

    Infections of poultry species with virulent strains of Newcastle disease virus (NDV) cause Newcastle disease (ND), one of the most economically significant and devastating diseases for poultry producers worldwide. Biological engagement programs between the Southeast Poultry Research Laboratory (SEPRL) of the United States Department of Agriculture and laboratories from Russia, Pakistan, Ukraine, Kazakhstan, and Indonesia collectively have produced a better understanding of the genetic diversity and evolution of the viruses responsible for ND, which is crucial for the control of the disease. The data from Kazakhstan, Russia, and Ukraine identified possible migratory routes for birds that may carry both virulent NDV (vNDV) and NDV of low virulence into Europe. In addition, related NDV strains were isolated from wild birds in Ukraine and Nigeria, and from birds in continental USA, Alaska, Russia, and Japan, identifying wild birds as a possible mechanism of intercontinental spread of NDV of low virulence. More recently, the detection of new sub-genotypes of vNDV suggests that a new, fifth, panzootic of ND has already originated in Southeast Asia, extended to the Middle East, and is now entering into Eastern Europe. Despite expected challenges when multiple independent laboratories interact, many scientists from the collaborating countries have successfully been trained by SEPRL on molecular diagnostics, best laboratory practices, and critical biosecurity protocols, providing our partners the capacity to further train other employes and to identify locally the viruses that cause this OIE listed disease. These and other collaborations with partners in Mexico, Bulgaria, Israel, and Tanzania have allowed SEPRL scientists to engage in field studies, to elucidate more aspects of ND epidemiology in endemic countries, and to understand the challenges that the scientists and field veterinarians in these countries face on a daily basis. Finally, new viral characterization tools

  10. International Biological Engagement Programs Facilitate Newcastle Disease Epidemiological Studies.

    Science.gov (United States)

    Miller, Patti J; Dimitrov, Kiril M; Williams-Coplin, Dawn; Peterson, Melanie P; Pantin-Jackwood, Mary J; Swayne, David E; Suarez, David L; Afonso, Claudio L

    2015-01-01

    Infections of poultry species with virulent strains of Newcastle disease virus (NDV) cause Newcastle disease (ND), one of the most economically significant and devastating diseases for poultry producers worldwide. Biological engagement programs between the Southeast Poultry Research Laboratory (SEPRL) of the United States Department of Agriculture and laboratories from Russia, Pakistan, Ukraine, Kazakhstan, and Indonesia collectively have produced a better understanding of the genetic diversity and evolution of the viruses responsible for ND, which is crucial for the control of the disease. The data from Kazakhstan, Russia, and Ukraine identified possible migratory routes for birds that may carry both virulent NDV (vNDV) and NDV of low virulence into Europe. In addition, related NDV strains were isolated from wild birds in Ukraine and Nigeria, and from birds in continental USA, Alaska, Russia, and Japan, identifying wild birds as a possible mechanism of intercontinental spread of NDV of low virulence. More recently, the detection of new sub-genotypes of vNDV suggests that a new, fifth, panzootic of ND has already originated in Southeast Asia, extended to the Middle East, and is now entering into Eastern Europe. Despite expected challenges when multiple independent laboratories interact, many scientists from the collaborating countries have successfully been trained by SEPRL on molecular diagnostics, best laboratory practices, and critical biosecurity protocols, providing our partners the capacity to further train other employes and to identify locally the viruses that cause this OIE listed disease. These and other collaborations with partners in Mexico, Bulgaria, Israel, and Tanzania have allowed SEPRL scientists to engage in field studies, to elucidate more aspects of ND epidemiology in endemic countries, and to understand the challenges that the scientists and field veterinarians in these countries face on a daily basis. Finally, new viral characterization tools

  11. Immune responses of poultry to Newcastle disease virus.

    Science.gov (United States)

    Kapczynski, Darrell R; Afonso, Claudio L; Miller, Patti J

    2013-11-01

    Newcastle disease (ND) remains a constant threat to poultry producers worldwide, in spite of the availability and global employment of ND vaccinations since the 1950s. Strains of Newcastle disease virus (NDV) belong to the order Mononegavirales, family Paramyxoviridae, and genus Avulavirus, are contained in one serotype and are also known as avian paramyxovirus serotype-1 (APMV-1). They are pleomorphic in shape and are single-stranded, non-segmented, negative sense RNA viruses. The virus has been reported to infect most orders of birds and thus has a wide host range. Isolates are characterized by virulence in chickens and the presence of basic amino acids at the fusion protein cleavage site. Low virulent NDV typically produce subclinical disease with some morbidity, whereas virulent isolates can result in rapid, high mortality of birds. Virulent NDV are listed pathogens that require immediate notification to the Office of International Epizootics and outbreaks typically result in trade embargos. Protection against NDV is through the use of vaccines generated with low virulent NDV strains. Immunity is derived from neutralizing antibodies formed against the viral hemagglutinin and fusion glycoproteins, which are responsible for attachment and spread of the virus. However, new techniques and technologies have also allowed for more in depth analysis of the innate and cell-mediated immunity of poultry to NDV. Gene profiling experiments have led to the discovery of novel host genes modulated immediately after infection. Differences in virus virulence alter host gene response patterns have been demonstrated. Furthermore, the timing and contributions of cell-mediated immune responses appear to decrease disease and transmission potential. In view of recent reports of vaccine failure from many countries on the ability of classical NDV vaccines to stop spread of disease, renewed interest in a more complete understanding of the global immune response of poultry to NDV will be

  12. Defective interfering particles in monolayer-propagated Newcastle disease virus

    International Nuclear Information System (INIS)

    Roman, J.M.; Simon, E.H.

    1976-01-01

    Newcastle disease virus (NDV) serially passaged in chick embryo fibroblasts (M-NDV) gives rise to defective interfering (NDV-DI) particles, while NDV passaged in embryonated eggs (E-NDV) does not. Co-infection with these particles and infectious virions results in a 99 percent reduction in yield. Interference is not due to interferon or to prevention of absorption of infectious virions and is specific for NDV. The particles mediating interference sediment at the same velocity as infectious virions. The accumulation of NDV-DI particles in monolayers but not in eggs may be a consequence of the fact that M-NDV virions are larger and probably contain more RNA, or it may reflect differences in NDV replicative processes in eggs and monolayers, or both

  13. Potent Inhibitors against Newcastle Disease Virus Hemagglutinin-Neuraminidase.

    Science.gov (United States)

    Rota, Paola; La Rocca, Paolo; Piccoli, Marco; Montefiori, Marco; Cirillo, Federica; Olsen, Lars; Orioli, Marica; Allevi, Pietro; Anastasia, Luigi

    2018-02-06

    Neuraminidase activity is essential for the infection and propagation of paramyxoviruses, including human parainfluenza viruses (hPIVs) and the Newcastle disease virus (NDV). Thus, many inhibitors have been developed based on the 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid inhibitor (DANA) backbone. Along this line, herein we report a series of neuraminidase inhibitors, having C4 (p-toluenesulfonamido and azido substituents) and C5 (N-perfluorinated chains) modifications to the DANA backbone, resulting in compounds with 5- to 15-fold greater potency than the currently most active compound, the N-trifluoroacetyl derivative of DANA (FANA), toward the NDV hemagglutinin-neuraminidase (NDV-HN). Remarkably, these inhibitors were found to be essentially inactive against the human sialidase NEU3, which is present on the outer layer of the cell membrane and is highly affected by the current NDV inhibitor FANA. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Biological and phylogenetic characterization of a genotype VII Newcastle disease virus from Venezuela: Efficacy of vaccination

    Science.gov (United States)

    Here we describe the characterization a virulent genotype VII Newcastle disease virus (NDV) from Venezuela and evaluate the efficacy of heterologous genotype commercial vaccination under field and controlled rearing conditions. Biological pathotyping and molecular analysis were applied. Results sh...

  15. Genetic Modification of Oncolytic Newcastle Disease Virus for Cancer Therapy.

    Science.gov (United States)

    Cheng, Xing; Wang, Weijia; Xu, Qi; Harper, James; Carroll, Danielle; Galinski, Mark S; Suzich, JoAnn; Jin, Hong

    2016-06-01

    Clinical development of a mesogenic strain of Newcastle disease virus (NDV) as an oncolytic agent for cancer therapy has been hampered by its select agent status due to its pathogenicity in avian species. Using reverse genetics, we have generated a lead candidate oncolytic NDV based on the mesogenic NDV-73T strain that is no longer classified as a select agent for clinical development. This recombinant NDV has a modification at the fusion protein (F) cleavage site to reduce the efficiency of F protein cleavage and an insertion of a 198-nucleotide sequence into the HN-L intergenic region, resulting in reduced viral gene expression and replication in avian cells but not in mammalian cells. In mammalian cells, except for viral polymerase (L) gene expression, viral gene expression is not negatively impacted or increased by the HN-L intergenic insertion. Furthermore, the virus can be engineered to express a foreign gene while still retaining the ability to grow to high titers in cell culture. The recombinant NDV selectively replicates in and kills tumor cells and is able to drive potent tumor growth inhibition following intratumoral or intravenous administration in a mouse tumor model. The candidate is well positioned for clinical development as an oncolytic virus. Avian paramyxovirus type 1, NDV, has been an attractive oncolytic agent for cancer virotherapy. However, this virus can cause epidemic disease in poultry, and concerns about the potential environmental and economic impact of an NDV outbreak have precluded its clinical development. Here we describe generation and characterization of a highly potent oncolytic NDV variant that is unlikely to cause Newcastle disease in its avian host, representing an essential step toward moving NDV forward as an oncolytic agent. Several attenuation mechanisms have been genetically engineered into the recombinant NDV that reduce chicken pathogenicity to a level that is acceptable worldwide without impacting viral production in

  16. Experimental infection with Brazilian Newcastle disease virus strain in pigeons and chickens

    Directory of Open Access Journals (Sweden)

    Adriano de Oliveira Torres Carrasco

    2016-03-01

    Full Text Available Abstract This study was designed with the goal of adding as much information as possible about the role of pigeons (Columba livia and chickens (Gallus gallus in Newcastle disease virus epidemiology. These species were submitted to direct experimental infection with Newcastle disease virus to evaluate interspecies transmission and virus-host relationships. The results obtained in four experimental models were analyzed by hemagglutination inhibition and reverse transcriptase polymerase chain reaction for detection of virus shedding. These techniques revealed that both avian species, when previously immunized with a low pathogenic Newcastle disease virus strain (LaSota, developed high antibody titers that significantly reduced virus shedding after infection with a highly pathogenic Newcastle disease virus strain (São Joao do Meriti and that, in chickens, prevent clinical signs. Infected pigeons shed the pathogenic strain, which was not detected in sentinel chickens or control birds. When the presence of Newcastle disease virus was analyzed in tissue samples by RT-PCR, in both species, the virus was most frequently found in the spleen. The vaccination regimen can prevent clinical disease in chickens and reduce viral shedding by chickens or pigeons. Biosecurity measures associated with vaccination programs are crucial to maintain a virulent Newcastle disease virus-free status in industrial poultry in Brazil.

  17. Isolation and molecular characterization of Newcastle disease viruses from raptors.

    Science.gov (United States)

    Jindal, Naresh; Chander, Yogesh; Primus, Alexander; Redig, Patrick T; Goyal, Sagar M

    2010-12-01

    The present study was undertaken to detect and characterize Newcastle disease virus (NDV) in raptors. Cloacal and oropharyngeal swab samples were collected from 60 casualty raptors during January to March 2009 in Minnesota. Inoculation of all these samples (n=120) in 9-day-old embryonated hens' eggs resulted in isolation of haemagglutinating viruses in three samples from two bald eagles and one great horned owl. These three haemagglutinating viruses were confirmed as NDV by reverse transcription-polymerase chain reaction (RT-PCR) using fusion gene-specific primers, and were negative for avian influenza virus by RT-PCR. Further characterization revealed that all three possessed (112)GKQGRL(117) at the fusion gene cleavage site, indicating that they were lentogenic strains. Phylogenetic analysis revealed that all three isolates clustered with published class II genotype II NDVs. The nucleotide sequence homology of the three NDV isolates among themselves was 98.4 to 99.6% and the sequence homology with lentogenic strains from wild birds used for comparison varied between 94.5 and 100%. Detection of NDV strains from raptors merits further epidemiological studies to determine the prevalence of different NDV strains in raptors and their impact in relation to transmission to domestic poultry.

  18. The role of vaccination in risk mitigation and control of Newcastle disease in poultry.

    Science.gov (United States)

    Mayers, Jo; Mansfield, Karen L; Brown, Ian H

    2017-10-20

    Newcastle disease is regarded as one of the most important avian diseases throughout the world and continues to be a threat and economic burden to the poultry industry. With no effective treatment, poultry producers rely primarily on stringent biosecurity and vaccination regimens to control the spread of this devastating disease. This concise review provides an historical perspective of Newcastle disease vaccination and how fundamental research has paved the way for the development of instrumental techniques which are still in use today. Although vaccination programmes have reduced the impact of clinical disease, they have historically been ineffective in controlling the spread of virulent viruses and therefore do not always offer an adequate solution to the world's food security problems. However, the continued development of novel vaccine technology and improved biosecurity measures through education may offer a solution to help reduce the global threat of Newcastle disease on the poultry industry. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

  19. Genetic and Biological Changes of Newcastle Disease Virus Due to The Development of Chicken Production System

    Directory of Open Access Journals (Sweden)

    Sudarisman

    2009-09-01

    Full Text Available In many countries, Newcastle Disease (ND is one of the most important diseases of poultry. It causes serious economic losses in poultry industry. Newcastle Disease or pseudo-fowl pest is a highly infectious viral disease that causes very high mortality (up to 100% in severe epidemics in poultry and wild birds around the world. Newcastle Disease remains endemic in many regions and continues to severely limit poultry production in some developing countries. The disease is currently being controlled by routine vaccinations in many countries. However, it was reported that outbreaks of ND in vaccinated flocks often occur on the field may not only be due to differences in the antigenicity of the NDV wild field strains and vaccine strains, but could also be as a result of differences in pathogenicity and virulence between different strains used as vaccine seed in NDV vaccine production.

  20. Therapeutic potential of oncolytic Newcastle disease virus: a critical review.

    Science.gov (United States)

    Tayeb, Shay; Zakay-Rones, Zichria; Panet, Amos

    2015-01-01

    Newcastle disease virus (NDV) features a natural preference for replication in many tumor cells compared with normal cells. The observed antitumor effect of NDV appears to be a result of both selective killing of tumor cells and induction of immune responses. Genetic manipulations to change viral tropism and arming the virus with genes encoding for cytokines improved the oncolytic capacity of NDV. Several intracellular proteins in tumor cells, including antiapoptotic proteins (Livin) and oncogenic proteins (H-Ras), are relevant for the oncolytic activity of NDV. Defects in the interferon system, found in some tumor cells, also contribute to the oncolytic selectivity of NDV. Notwithstanding, NDV displays effective oncolytic activity in many tumor types, despite having intact interferon signaling. Taken together, several cellular systems appear to dictate the selective oncolytic activity of NDV. Some barriers, such as neutralizing antibodies elicited during NDV treatment and the extracellular matrix in tumor tissue appear to interfere with spread of NDV and reduce oncolysis. To further understand the oncolytic activity of NDV, we compared two NDV strains, ie, an attenuated virus (NDV-HUJ) and a pathogenic virus (NDV-MTH-68/H). Significant differences in amino acid sequence were noted in several viral proteins, including the fusion precursor (F0) glycoprotein, an important determinant of replication and pathogenicity. However, no difference in the oncolytic activity of the two strains was noted using human tumor tissues maintained as organ cultures or in mouse tumor models. To optimize virotherapy in clinical trials, we describe here a unique organ culture methodology, using a biopsy taken from a patient's tumor before treatment for ex vivo infection with NDV to determine the oncolytic potential on an individual basis. In conclusion, oncolytic NDV is an excellent candidate for cancer therapy, but more knowledge is needed to ensure success in clinical trials.

  1. Effect of low dose gamma-radiation upon Newcastle disease virus antibody level in chicken

    International Nuclear Information System (INIS)

    Vilic, M.; Gottstein, Z.; Ciglar Grozdanic, I.; Matanovic, K.; Miljanic, S.; Mazija, H.; Kraljevic, P.

    2009-01-01

    The specific antibody response against Newcastle disease virus in the blood serum of chickens hatched from eggs exposed to low dose gamma-radiation was studied. Materials and methods: Two groups of eggs of commercial meat chicken lines were irradiated with the dose of 0.30 Gy 60 Co gamma-rays before incubation and on the 19 th day of incubation, respectively. The same number of eggs unexposed to gamma-radiation served as controls. After hatching the group of chicken hatched from eggs irradiated on the 19 th day of incubation was not vaccinated while the group of chicken hatched from eggs irradiated before incubation was vaccinated on the 14 day. Specific serum anti-Newcastle disease virus antibodies were quantified by the hemagglutination inhibition assay with 4 HA units of Newcastle disease virus La Sota strain. Result: Specific antibody titres against Newcastle disease virus in the blood serum of chickens hatched from eggs irradiated before incubation and vaccinated on the 14 th day significantly increased on the 28 th day. Specific antibody titre against Newcastle disease virus in the blood serum of chickens hatched from eggs irradiated on the 19 th day of incubation and non-vaccinated was significantly higher on the 1 st and 14 th day. Conclusion: Acute irradiation of heavy breeding chicken eggs with the dose of 0.30 Gy 60 Co gamma-rays before incubation and on the 19 th day of incubation could have a stimulative effect on humoral immunity in chickens.

  2. Derivation of chicken induced pluripotent stem cells tolerant to Newcastle disease virus-induced lysis through multiple rounds of infection

    Science.gov (United States)

    Background: Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a devastating disease of poultry and wild birds. ND is prevented by rigorous biocontainment and vaccination. One potential approach to prevent spread of the virus is production of birds that show innate resistance to NDV...

  3. CLINICO-PATHOLOGICAL OBSERVATIONS OF PIGEONS (COLUMBA LIVIA SUFFERING FROM NEWCASTLE DISEASE

    Directory of Open Access Journals (Sweden)

    S. Shaheen, A. D. Anjum and F. Rizvi

    2005-01-01

    Full Text Available A survey was conducted to study clinical signs, gross and histopathological lesions in pigeons with naturally occurring Newcastle disease. For this purpose, 30 pigeon lofts were visited. Among these, 14 lofts showed clinical signs of Newcastle disease, including mainly greenish white mucoid diarrhoea and nervous signs with high morbidity and mortality. Postmortem examination of affected birds showed lesions mainly in brain, liver, kidneys and spleen. Amongst various organs, kidneys were more frequently involved. Histopathological changes were also observed in lungs, liver, kidneys, brain and spleen. The results showed that the Newcastle disease virus was widespread in pigeons locally and caused heavy mortality. No preventive measures or vaccination is being adopted by pigeon fanciers to control the disease.

  4. Molecular epidemiology of Newcastle disease in Mexico and the potential spillover of viruses from poultry into wild bird species.

    Science.gov (United States)

    Cardenas Garcia, Stivalis; Navarro Lopez, Roberto; Morales, Romeo; Olvera, Miguel A; Marquez, Miguel A; Merino, Ruben; Miller, Patti J; Afonso, Claudio L

    2013-08-01

    Newcastle disease, one of the most important health problems that affects the poultry industry around the world, is caused by virulent strains of Newcastle disease virus. Newcastle disease virus is considered to be endemic in several countries in the Americas, including Mexico. In order to control Newcastle disease outbreaks and spread, intensive vaccination programs, which include vaccines formulated with strains isolated at least 60 years ago, have been established. These vaccines are dissimilar in genotype to the virulent Newcastle disease viruses that had been circulating in Mexico until 2008. Here, 28 isolates obtained between 2008 and 2011 from different regions of Mexico from free-living wild birds, captive wild birds, and poultry were phylogenetically and biologically characterized in order to study the recent epidemiology of Newcastle disease viruses in Mexico. Here we demonstrate that, until recently, virulent viruses from genotype V continued to circulate and evolve in the country. All of the Newcastle disease viruses of low virulence, mostly isolated from nonvaccinated free-living wild birds and captive wild birds, were highly similar to LaSota (genotype II) and PHY-LMV42 (genotype I) vaccine strains. These findings, together with the discovery of two virulent viruses at the Mexican zoo, suggest that Newcastle disease viruses may be escaping from poultry into the environment.

  5. Infection and transmission of live recombinant Newcastle disease virus vaccines in Rock Pigeons, European House Sparrows, and Japanese Quail

    Science.gov (United States)

    In China and Mexico, engineered recombinant Newcastle disease virus (rNDV) strains are used as live vaccines for the control of Newcastle disease and as vectors to express the avian influenza virus hemagglutinin (HA) gene to control avian influenza in poultry. In this study, non-target species wer...

  6. Phylogenetic analysis of Newcastle disease viruses isolated from commercial poultry in Mozambique, 2011 to 2016

    International Nuclear Information System (INIS)

    Mapaco, L.P.; Monjane, I.V.A.; Nhamusso, A.E.; Viljoen, G.J; Dundon, W.G.; Achá, S.J.

    2016-01-01

    Full text: The complete sequence of the fusion (F) protein gene from eleven Newcastle disease viruses (NDV) isolated from commercial poultry in Mozambique between 2011 and 2016 has been generated. The F gene cleavage site motif for all eleven isolates was 112RRRKRF117 indicating that the viruses are virulent. A phylogenetic analysis using the full F gene sequence revealed that the viruses clustered within genotype VIIh and showed a higher similarity to NDVs from South Africa, China and Southeast Asia than to viruses previously described in Mozambique in 1994 to 1995 and 2005. The characterization of these new NDVs has important implications for Newcastle disease management and control in Mozambique. (author)

  7. Newcastle disease and infectious bursal disease among free range village chickens in Tanzania

    International Nuclear Information System (INIS)

    Yongolo, M.G.S.; Maeda Machangu, A.; Minga, U.M.

    2002-01-01

    Newcastle disease in free-range village chickens was confirmed by retrospective data analysis and epidemiological cross-sectional studies. The combination of serological survey and virus isolation and characterisation established seasonal occurrence of Newcastle disease (ND) in free-range village chickens. The highest sero-prevalence (81.5) and virus isolation frequency (18/27) were found in the period between June and October. The field isolates of Newcastle virus (NDV) were confirmed to be PMV-1 serotype by polyclonal PMV-1 antiserum and monoclonal antibody (mAb) U85. All isolates were not inhibited by mAb 716/161 specific for pigeon panzootic NDV, showing that the current Tanzanian field isolates have antigenic variation and were not involved in the recent pigeon NDV panzootic. Mean death time determination characterised isolates into velogenic, mesogenic and lentogenic pathotypes. Isolation of NDV from apparently healthy ducks revealed the role of ducks in the epidemiology of ND in free-range village chickens in Tanzania. Studies are recommended to determine the similarities of the field isolates from different sources within Tanzania and to panzootic NDV from other countries. Strategic control of ND in free-range village chickens is recommended taking into consideration the presence of different age groups. Infectious bursal disease was histologically diagnosed in free-range village chickens. Therefore, there is a need of carrying out research on the role of other diseases and determine their prevalence and their contribution to the mortalities experienced in the free-range village chickens. (author)

  8. Complete Genome Sequence of Genotype VI Newcastle Disease Viruses Isolated from Pigeons in Pakistan

    OpenAIRE

    Wajid, Abdul; Rehmani, Shafqat Fatima; Sharma, Poonam; Goraichuk, Iryna V.; Dimitrov, Kiril M.; Afonso, Claudio L.

    2016-01-01

    Two complete genome sequences of Newcastle disease virus (NDV) are described here. Virulent isolates pigeon/Pakistan/Lahore/21A/2015 and pigeon/Pakistan/Lahore/25A/2015 were obtained from racing pigeons sampled in the Pakistani province of Punjab during 2015. Phylogenetic analysis of the fusion protein genes and complete genomes classified the isolates as members of NDV class II, genotype VI.

  9. Identification and Complete Genome Sequence Analysis of a Genotype XIV Newcastle Disease Virus from Nigeria

    OpenAIRE

    Shittu, Ismaila; Sharma, Poonam; Volkening, Jeremy D.; Solomon, Ponman; Sulaiman, Lanre K.; Joannis, Tony M.; Williams-Coplin, Dawn; Miller, Patti J.; Dimitrov, Kiril M.; Afonso, Claudio L.

    2016-01-01

    The first complete genome sequence of a strain of Newcastle disease virus (NDV) from genotype XIV is reported here. Strain duck/Nigeria/NG-695/KG.LOM.11-16/2009 was isolated from an apparently healthy domestic duck from a live bird market in Kogi State, Nigeria, in 2009. This strain is classified as a member of subgenotype XIVb of class II.

  10. An outbreak of Newcastle disease in free-living pheasants (Phasianus colchicus)

    DEFF Research Database (Denmark)

    Jørgensen, Poul Henrik; Handberg, Kurt; Ahrens, Peter

    1999-01-01

    The epidemiology of an outbreak of Newcastle disease in a population of approximately 12 000 free-living pheasants (Phasianus colchicus) on the island of Faeno in Denmark in 1996 is described. The mortality epizootic demonstrated over an observation period of 3 weeks. A total of 70 avian paramyxo...... to the pheasants by feral birds....

  11. Stability of Newcastle Disease Virus Strain V4-UPM Coated on ...

    African Journals Online (AJOL)

    Protection of village chickens against Newcastle disease (ND) is considered feasible through food-delivered vaccines. Vaccine virus strain V4-UPM coated on cassava granules with or without additive (2% gelatin) was tested for stability at room temperature (RT) for 8 weeks and 40oC for 12 hours at weekly and two hourly ...

  12. Virosome and ISCOM vaccines against Newcastle disease: preparation, characterization and immunogenicity

    NARCIS (Netherlands)

    Homhuan, A.; Prakongpan, S.; Poomvises, P.; Maas, H.A.; Krommelin, D.; Kersten, G.; Jiskoot, W.

    2004-01-01

    The purpose of this study was to prepare and characterize virosomes and ISCOMs containing envelope proteins of Newcastle disease virus (NDV) and to evaluate their immunogenicity in target animals (chickens). Virosomes were prepared by solubilization of virus with either Triton X-100 or octyl

  13. Antibody Level Upon Newcastle Disease Virus In Chicken After Exposure To GAMMA Radiation

    International Nuclear Information System (INIS)

    Pejakovic-Hlede, J.; Dotur, J.; Pasic, S.; Gottstein, Z.; Majer, M.; Vilic, M.

    2015-01-01

    The aim of this study was to investigate the effect of gamma radiation upon Newcastle disease virus antibody level after acute exposure with dose of 0.05 Gy and 0.8 Gy gamma radiation. The experiment was made on light chicken breeds irradiated with dose of 0.05 Gy and 0.8 Gy gamma radiation with dose rate of 0.0117 Gy/s on the first and on the third day after hatching. Chicken were vaccinated by nebulization on the first day after hatching. Antibody level upon Newcastle disease in blood serum of chicken was quantified by hemagglutination inhibition assay on 1th, 7th, 14th and 28th day after vaccination. Results demonstrate that antibody titre against Newcastle disease in blood serum of chicken irradiated with dose of 0.05 Gy and 0.8 Gy gamma radiation on the first and on the third day after hatching was not statistically significant. Therefore, these results suggest that irradiation of light chicken breeds on the first and third day after vaccination with dose of 0.05 Gy and 0.8 Gy does not change antibody titre upon Newcastle disease. (author).

  14. The Carrier Rate of Newcastle Disease Virus in Pigeons in Owerri ...

    African Journals Online (AJOL)

    Two positive results were recorded for white pigeons, while only one black pigeon showed evidence of NDV. From this study, the carrier rate of NDV in pigeons in Owerri area of Imo State is estimated at 5% Keywords: Carrier Rate, Newcastle Disease Virus, Pigeons. Journal of Medical Laboratory Sciences Vol. 14 (1) 2005: ...

  15. Optimization of Newcastle disease virus production in T-flask | Arifin ...

    African Journals Online (AJOL)

    In the present study, the production of lentogenic Asplin F strain of Newcastle disease virus by using cell culture method was studied. Experiments were carried out in T-flasks to investigate the effects of serum concentration in the culture medium during virus replication phase and multiplicity of infection (MOI) on ND virus ...

  16. Development of Recombinant Newcastle Disease Viruses Expressing the Glycoprotein (G) of Avian Metapneumovirus as Bivalent Vaccines

    Science.gov (United States)

    Using reverse genetics technology, Newcastle disease virus (NDV) LaSota strain-based recombinant viruses were engineered to express the glycoprotein (G) of avian metapneumovirus (aMPV), subtype A, B or C, as bivalent vaccines. These recombinant viruses were slightly attenuated in vivo, yet maintaine...

  17. Pathogenesis of new strains of Newcastle disease virus from Israel and Pakistan

    Science.gov (United States)

    In the past few years, Newcastle disease virus (NDV) strains with epizootic characteristics belonging to subgenotypes VIIi and XIIIb emerged in the Middle East and Asia. In this study, 2 NDV strains—1 representative of subgenotype VIIi isolated in Israel (Kvuzat/13) and 1 representative of subgenoty...

  18. Complete genome sequence of a recent panzootic virulent Newcastle disease virus from Pakistan

    Science.gov (United States)

    Complete genome sequence of a new strain of Newcastle disease virus (NDV) (chicken/Pak/Lahore-611/2013) is reported. The strain was isolated from a vaccinated chicken flock in Pakistan in 2013 and has panzootic features. The genome is 15192 nucleotides in length and is classified as sub-genotype V...

  19. Presence of virulent Newcastle disease virus in vaccinated chickens in farms in Pakistan

    Science.gov (United States)

    The sites where virulent Newcastle disease virus persists in endemic countries are unknown. Evidence presented here shows that the same strain that caused a previous outbreak was present in both apparently healthy and sick vaccinated birds from multiple farms that had high average specific antibody...

  20. Concurrent outbreak of Newcastle disease and Trichomoniasis in pigeons of Tehran

    Directory of Open Access Journals (Sweden)

    nariman sheykhi

    2014-08-01

    Full Text Available Newcastle disease is the most important viral disease that affected pigeons. The disease is characterized by sudden onset of anorexia and neurological symptoms in pigeon. Trichomonas gallinae causes trichomoniasis of pigeons in the upper gastrointestinal tract and the respiratory system. The symptoms of this disease include yellowish green fetid discharge from the mouth, diarrhea, emaciation, severe weakness and death. In the first 6 months of 1392, from a total of 32      suspicious cases from Tehran and its surrounding, swab samples of the mouth, pharynx and larynx of birds were prepared. The samples were studied for trichomonas infection. At necropsy, foci of white to cream color in the oral mucosa, pharynx, larynx and pharyngeal and tracheal mucous congestion associated with the presence of fetid fluid in the crop were observed. Also, general congestion of the carcass, urate deposition in the ureters, and the emptiness gastrointestinal tract was observed. For detection of Newcastle disease virus (NDV, samples of the trachea and spleen were collected and RT-PCR experiments were performed on the samples. Trichomonas was observed in the samples under the microscope. All of the 19 samples studied were considered positive to the presence of high virulence strain of the virus. Metronidazole and supportive therapies were used for treatment. Adherence to the principles of biosecurity, treatment or removal of trichomoniasis infected birds, and annual Newcastle disease vaccine are essential for the prevention of concurrent outbreak of these two diseases.

  1. Lung Transcriptome of Newcastle Disease Virus Infected Chickens--Different Immune Response in Two Types of Chicken

    Data.gov (United States)

    US Agency for International Development — Males and females from resistant Fayoumi and susceptible Leghorn chicken lines were either challenged with a lentogenic strain of Newcastle Disease virus or given a...

  2. JST Thesaurus Headwords and Synonyms: Newcastle disease virus [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term Newcastle disease virus 名詞 一般 * *... * * ニューカッスル病ウイルス ニューカッスルビョウウイルス ニューカッスルビョーウイルス Thesaurus2015 200906007314327218 C LS07 UNKNOWN_2 Newcastle disease virus

  3. The Effect of Tsukamurella inchonensis Bacterin on the Immune Response Against Influenza and Newcastle Disease Vaccines in Broiler Chickens

    Directory of Open Access Journals (Sweden)

    Forough Talazadeh

    2016-11-01

    Full Text Available Background: In poultry production, improving immunity is very important to prevent infectious diseases. One solution to improve the immunity of animals and to decrease their susceptibility to infectious disease is administration of immunostimulants. Surveys have indicated that some bacteria can work as immunomodulators such as Mycobacterium vaccae and can promote Th1-mediated mechanisms, and switch off pre-existing Th2 preponderance (1. Objectives: The aim of this study was to examine the effect of Tsukamurella inchonensis bacterin on the immune response against Influenza and Newcastle disease vaccine in broiler chickens . Materials and Methods: A total of 170 day-old broiler chicks were purchased and divided randomly into 5 equal groups. Chickens of group A received 106 bacterin subcutaneously on two days before vaccination against Newcastle disease and avian influenza. Chickens of group B received 106 bacterin subcutaneously on six days after the first injection of bacterin. Chickens of group C received 106bacterin subcutaneously on six days after the second injection of bacterin. Chickens of group D, vaccinated against Newcastle disease and avian influenza but did not receive bacterin. Chickens of group E, did not vaccinate against Newcastle disease and avian influenza and did not receive bacterin. All groups except group E, were vaccinated with live Newcastle vaccine and AI-ND killed vaccine (subtype H9N2. Blood samples were collected and antibody titer against Newcastle disease vaccine and avian influenza vaccine was determined by HI test. Results: The results of present study showed that receiving of Tsukamurella inchonensis bacterin for 3 times, significantly increased the specific antibody response to avian influenza subtype H9N2 vaccine. Also about Newcastle vaccine, significantly increased the specific antibody response to Newcastle vaccine at 21 and 28 days after vaccination. Conclusions: Receiving of Tsukamurella inchonensis bacterin

  4. Newcastle disease virus surveillance in Hong Kong on local and imported poultry.

    Science.gov (United States)

    Shortridge, K F; Alexander, D J

    1978-09-01

    Surveillance of apparently healthy ducks, geese and fowl originating in Hong Kong and the People's Republic of China at a poultry dressing plant in Hong Kong yielded 67 isolates of Newcastle disease virus. More than twice as many viruses were isolated from the cloaca than from the trachea. Twelve representative isolates were examined in virulence tests--all six of the fowl isolates and two of five duck isolates behaved as velogenic strains, the other four were lentogenic.

  5. Complete Genome Sequence of Genotype VI Newcastle Disease Viruses Isolated from Pigeons in Pakistan

    Science.gov (United States)

    Wajid, Abdul; Rehmani, Shafqat Fatima; Sharma, Poonam; Goraichuk, Iryna V.; Dimitrov, Kiril M.

    2016-01-01

    Two complete genome sequences of Newcastle disease virus (NDV) are described here. Virulent isolates pigeon/Pakistan/Lahore/21A/2015 and pigeon/Pakistan/Lahore/25A/2015 were obtained from racing pigeons sampled in the Pakistani province of Punjab during 2015. Phylogenetic analysis of the fusion protein genes and complete genomes classified the isolates as members of NDV class II, genotype VI. PMID:27540069

  6. Identification and Complete Genome Sequence Analysis of a Genotype XIV Newcastle Disease Virus from Nigeria

    Science.gov (United States)

    Shittu, Ismaila; Sharma, Poonam; Volkening, Jeremy D.; Solomon, Ponman; Sulaiman, Lanre K.; Joannis, Tony M.; Williams-Coplin, Dawn; Miller, Patti J.; Dimitrov, Kiril M.

    2016-01-01

    The first complete genome sequence of a strain of Newcastle disease virus (NDV) from genotype XIV is reported here. Strain duck/Nigeria/NG-695/KG.LOM.11-16/2009 was isolated from an apparently healthy domestic duck from a live bird market in Kogi State, Nigeria, in 2009. This strain is classified as a member of subgenotype XIVb of class II. PMID:26823576

  7. Pathotyping of a Newcastle disease virus isolated from peacock (Pavo cristatus).

    Science.gov (United States)

    Vijayarani, K; Muthusamy, S; Tirumurugaan, K G; Sakthivelan, S M; Kumanan, K

    2010-03-01

    This report describes Newcastle disease in peacock and the isolation and characterization of the virus. The virus had an intracerbral pathogenicity index of 1.71 and mean death time of 47 h. The isolate had multiple basic amino acids at the fusion protein cleavage site sequence ((110)GGRRQRRFIG(119)) with a phenylalanine at residue 117. Biological and molecular characterization revealed that the virus is velogenic. Phylogenetic analysis placed the isolate in genotype II.

  8. Complete Genome Sequence of a Newcastle Disease Virus Isolated from Wild Peacock (Pavo cristatus) in India.

    Science.gov (United States)

    Khulape, Sagar A; Gaikwad, Satish S; Chellappa, Madhan Mohan; Mishra, Bishnu Prasad; Dey, Sohini

    2014-06-05

    We report here the complete genome sequence of a Newcastle disease virus (NDV) isolated from a wild peacock. Phylogenetic analysis showed that it belongs to genotype II, class II of NDV strains. This study helps to understand the ecology of NDV strains circulating in a wild avian host of this geographical region during the outbreak of 2012 in northwest India. Copyright © 2014 Khulape et al.

  9. Strategies for vaccination of family poultry against Newcastle disease in Africa

    International Nuclear Information System (INIS)

    Alders, R.G.

    2002-01-01

    Criteria for the selection of vaccines against Newcastle disease (ND) appropriate for use in village chickens are discussed. Emphasis is given to the need to ensure that the selected vaccine is used successfully in the field. Those implementing ND control activities are encouraged to collaborate with all stakeholders and to develop comprehensive training and extension programs for field workers and farmers. Issues of cost-recovery and cost-minimisation are also discussed. (author)

  10. Presence of Vaccine-Derived Newcastle Disease Viruses in Wild Birds.

    Directory of Open Access Journals (Sweden)

    Andrea J Ayala

    Full Text Available Our study demonstrates the repeated isolation of vaccine-derived Newcastle disease viruses from different species of wild birds across four continents from 1997 through 2014. The data indicate that at least 17 species from ten avian orders occupying different habitats excrete vaccine-derived Newcastle disease viruses. The most frequently reported isolates were detected among individuals in the order Columbiformes (n = 23, followed in frequency by the order Anseriformes (n = 13. Samples were isolated from both free-ranging (n = 47 and wild birds kept in captivity (n = 7. The number of recovered vaccine-derived viruses corresponded with the most widely utilized vaccines, LaSota (n = 28 and Hitchner B1 (n = 19. Other detected vaccine-derived viruses resembled the PHY-LMV2 and V4 vaccines, with five and two cases, respectively. These results and the ubiquitous and synanthropic nature of wild pigeons highlight their potential role as indicator species for the presence of Newcastle disease virus of low virulence in the environment. The reverse spillover of live agents from domestic animals to wildlife as a result of the expansion of livestock industries employing massive amounts of live virus vaccines represent an underappreciated and poorly studied effect of human activity on wildlife.

  11. Presence of Vaccine-Derived Newcastle Disease Viruses in Wild Birds

    Science.gov (United States)

    Ayala, Andrea J.; Dimitrov, Kiril M.; Becker, Cassidy R.; Goraichuk, Iryna V.; Arns, Clarice W.; Bolotin, Vitaly I.; Ferreira, Helena L.; Gerilovych, Anton P.; Goujgoulova, Gabriela V.; Martini, Matheus C.; Muzyka, Denys V.; Orsi, Maria A.; Scagion, Guilherme P.; Silva, Renata K.; Solodiankin, Olexii S.; Stegniy, Boris T.; Miller, Patti J.; Afonso, Claudio L.

    2016-01-01

    Our study demonstrates the repeated isolation of vaccine-derived Newcastle disease viruses from different species of wild birds across four continents from 1997 through 2014. The data indicate that at least 17 species from ten avian orders occupying different habitats excrete vaccine-derived Newcastle disease viruses. The most frequently reported isolates were detected among individuals in the order Columbiformes (n = 23), followed in frequency by the order Anseriformes (n = 13). Samples were isolated from both free-ranging (n = 47) and wild birds kept in captivity (n = 7). The number of recovered vaccine-derived viruses corresponded with the most widely utilized vaccines, LaSota (n = 28) and Hitchner B1 (n = 19). Other detected vaccine-derived viruses resembled the PHY-LMV2 and V4 vaccines, with five and two cases, respectively. These results and the ubiquitous and synanthropic nature of wild pigeons highlight their potential role as indicator species for the presence of Newcastle disease virus of low virulence in the environment. The reverse spillover of live agents from domestic animals to wildlife as a result of the expansion of livestock industries employing massive amounts of live virus vaccines represent an underappreciated and poorly studied effect of human activity on wildlife. PMID:27626272

  12. Recombinant Newcastle disease virus-vectored vaccines against human and animal infectious diseases.

    Science.gov (United States)

    Duan, Zhiqiang; Xu, Houqiang; Ji, Xinqin; Zhao, Jiafu

    2015-01-01

    Recent advances in recombinant genetic engineering techniques have brought forward a leap in designing new vaccines in modern medicine. One attractive strategy is the application of reverse genetics technology to make recombinant Newcastle disease virus (rNDV) deliver protective antigens of pathogens. In recent years, numerous studies have demonstrated that rNDV-vectored vaccines can induce quicker and better humoral and mucosal immune responses than conventional vaccines and are protective against pathogen challenges. With deeper understanding of NDV molecular biology, it is feasible to develop gene-modified rNDV vaccines accompanied by good safety, high efficacy, low toxicity and better immunogenicity. This review summarizes the development of reverse genetics technology in using NDV as a promising vaccine vector to design new vaccines for human and animal use.

  13. A retrospective study and predictive modelling of Newcastle Disease trends among rural poultry of eastern Zambia.

    Science.gov (United States)

    Mubamba, C; Ramsay, G; Abolnik, C; Dautu, G; Gummow, B

    2016-10-01

    Newcastle Disease (ND) is a highly infectious disease of poultry that seriously impacts on food security and livelihoods of livestock farmers and communities in tropical regions of the world. ND is a constant problem in the eastern province of Zambia which has more than 740 000 rural poultry. Very few studies give a situational analysis of the disease that can be used for disease control planning in the region. With this background in mind, a retrospective epidemiological study was conducted using Newcastle Disease data submitted to the eastern province headquarters for the period from 1989 to 2014. The study found that Newcastle Disease cases in eastern Zambia followed a seasonal and cyclic pattern with peaks in the hot dry season (Overall Seasonal Index 1.1) as well as cycles every three years with an estimated provincial incidence range of 0.16 to 1.7% per year. Annual trends were compared with major intervention policies implemented by the Zambian government, which often received donor support from the international community during the study period. Aid delivered through government programmes appeared to have no major impact on ND trends between 1989 and 2014 and reasons for this are discussed. There were apparent spatial shifts in districts with outbreaks over time which could be as a result of veterinary interventions chasing outbreaks rather than implementing uniform control. Data was also fitted to a predictive time series model for ND which could be used to plan for future ND control. Time series modelling showed an increasing trend in ND annual incidence over 25 years if existing interventions continue. A different approach to controlling the disease is needed if this trend is to be halted. Conversely, the positive trend may be a function of improved reporting by farmers as a result of more awareness of the disease. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Isolation and characterization of Newcastle disease virus from vaccinated commercial layer chicken

    Directory of Open Access Journals (Sweden)

    P. Balachandran

    2014-07-01

    Full Text Available Aim: Newcastle disease (ND is an infectious, highly contagious and destructive viral disease of poultry and controlled by vaccination. In spite of vaccination, incidence of ND was reported in commercial layers with gastrointestinal lesions. This study was undertaken to assess the prevalence and pathotypes of Newcastle disease virus (NDV involved in gastrointestinal tract abnormalities of vaccinated commercial layer chicken of Namakkal region for a period of three years from 2008 and 2011. Materials and Methods: Pooled tissue (trachea, lung, spleen, proventriculus, intestine and caecal tonsils samples collected from dead birds on postmortem examination from 100 layer flocks above 20 weeks of age with gastrointestinal lesions were subjected to isolation of NDV in embryonated specific pathogen free (SPF chicken eggs. Mean death time (MDT and intracerebral pathogenicity index of the isolates were characterized. Flock details were collected from NDV positive flocks to assess the prevalence and impact of NDV on vaccinated commercial layer chicken. Results: Among the 100 flocks examined Newcastle disease virus was detected in 14 flocks as a single infection and 10 flocks as combined infections with worm infestation, necrotic enteritis and coccidiosis. Chicken embryo mean death time (MDT and intracerebral pathogenicity index (ICPI values ranged from 50.4 to 96.0 hrs and from 0.650 to 1.675 respectively. Affected birds showed anorexia, diarrohea and drop in egg production. Macropathologically, matting of vent feathers, petechial haemorrhage on the tip of proventricular papilla, caecal tonsils and degeneration of ovarian follicles were noticed. The incidence of ND was most commonly noticed in 20-50 wk of age and between the months of September to November. Morbidity rate varied from 5% to 10% in the NDV alone affected flocks and 5 to 15% in NDV with other concurrent infections. Egg production drop from the expected level ranged between 3 to 7 % in ND and

  15. Newcastle disease in Nigeria | Abdu | Nigerian Veterinary Journal

    African Journals Online (AJOL)

    The first documented, confirmed outbreak of ND in Nigeria occurred between December 1952 and February 1953 in and around Ibadan (Hill et al.,1953). Subsequently, the disease appeared to be widespread in local and exotic chickens (Fatumbi and Adene, 1979; Ezeokoli et al.,1984; Gomwalk et al., 1985; Nwosu and ...

  16. Herd immunity to Newcastle disease virus in broiler flocks in Israel.

    Science.gov (United States)

    Wiseman, Anat; Berman, Elyakum M

    2017-08-01

    Due to the ongoing need to protect poultry from virulent Newcastle disease virus, all commercial poultry flocks in Israel are vaccinated according to a defined programme using a combination of live and inactivated vaccines. The vaccination protocol for broilers during the years of the study comprised a live vaccine administered by spray on the day of hatching, inactivated vaccine by subcutaneous injection at 10-12 days of age, and another live vaccine given by aerosol at 17-21 days of age. A cross-sectional study was designed in order to examine the influence of herd immunity on the risk of Newcastle disease outbreak in broiler flocks. The study was based on the extensive field data kept in the Poultry Health Laboratories database. The results of serology tests employing haemagglutination inhibition for Newcastle disease virus were analysed and crossed with the list of flocks that had been diagnosed with ND in the years 2007-2014. At the peak of induced immunization (fifth week of growth), 87.5% of the tested flocks had achieved herd immunity (≥85% of birds in the flock with an HI titre ≥4). Based on a logistic regression model, the odds ratio for ND in flocks without herd immunity was 3.7 (95% CI 1.8-7.3, P-value < 0.001). The higher the percentage of birds with low HI titres the higher the risk of ND outbreak. Under field conditions, herd immunity is an important indicator for the risk of ND outbreak.

  17. In Vitro Synergistic Enhancement of Newcastle Disease Virus to 5-Fluorouracil Cytotoxicity against Tumor Cells

    Directory of Open Access Journals (Sweden)

    Ahmed M. Al-Shammari

    2016-01-01

    Full Text Available Background: Chemotherapy is one of the antitumor therapies used worldwide in spite of its serious side effects and unsatisfactory results. Many attempts have been made to increase its activity and reduce its toxicity. 5-Fluorouracil (5-FU is still a widely-used chemotherapeutic agent, especially in combination with other chemotherapies. Combination therapy seems to be the best option for targeting tumor cells by different mechanisms. Virotherapy is a promising agent for fighting cancer because of its safety and selectivity. Newcastle disease virus is safe, and it selectively targets tumor cells. We previously demonstrated that Newcastle disease virus (NDV could be used to augment other chemotherapeutic agents and reduce their toxicity by halving the administered dose and replacing the eliminated chemotherapeutic agents with the Newcastle disease virus; the same antitumor activity was maintained. Methods: In the current work, we tested this hypothesis on different tumor cell lines. We used the non-virulent LaSota strain of NDV in combination with 5-FU, and we measured the cytotoxicity effect. We evaluated this combination using Chou–Talalay analysis. Results: NDV was synergistic with 5-FU at low doses when used as a combination therapy on different cancer cells, and there were very mild effects on non-cancer cells. Conclusion: The combination of a virulent, non-pathogenic NDV–LaSota strain with a standard chemotherapeutic agent, 5-FU, has a synergistic effect on different tumor cells in vitro, suggesting this combination could be an important new adjuvant therapy for treating cancer.

  18. Evaluation of the infection and transmission of wild type and recombinant strains of Newcastle disease virus in Japanese Quail

    Science.gov (United States)

    Newcastle disease virus (NDV) causes a range of clinical disease ranging from asymptomatic infection to severe disease with high mortality. Vaccination for NDV is practiced almost worldwide in commercial chickens. Attenuated live vaccines are most commonly used, with recombinant vaccines becoming ...

  19. Newcastle disease virus in penguins from King George Island on the Antarctic region.

    Science.gov (United States)

    Thomazelli, Luciano M; Araujo, Jansen; Oliveira, Danielle B; Sanfilippo, Luiz; Ferreira, Carolina S; Brentano, Liana; Pelizari, Vivian H; Nakayama, Cristiane; Duarte, Rubens; Hurtado, Renata; Branco, Joaquim O; Walker, David; Durigon, Edison L

    2010-11-20

    Here we report the isolation of Newcastle disease virus (NDV) from cloacal swabs obtained from penguins in the South Atlantic Antarctic region (62°08S, 58°25W). Samples of 100 penguins from King George Island were tested by real-time PCR, of which 2 (2%) were positive for NDV. The positive samples were isolated in embryonated chicken eggs and their matrix and fusion proteins genes were partially sequenced. This was complemented by the serological study performed on the blood of the same specimens, which resulted in a 33.3% rate of positivity. Copyright © 2010 Elsevier B.V. All rights reserved.

  20. Occurrence of velogenic viscerotropic Newcastle disease in pet and exotic birds in 1991.

    Science.gov (United States)

    Panigrahy, B; Senne, D A; Pearson, J E; Mixson, M A; Cassidy, D R

    1993-01-01

    In 1991, velogenic viscerotropic Newcastle disease (VVND) was diagnosed in domestic psittacine birds in six states: Illinois, Indiana, Michigan, Texas, California, and Nevada. In the first four states, the disease assumed outbreak proportions. The affected psittacine birds--yellow-headed Amazon parrots (Amazona ochrocephala oratrix), yellow-naped Amazon parrots (Amazona ochrocephala auropalliata), cockatiels (Nymphicus hollandicus), and conures (unknown species)--exhibited respiratory and/or central nervous system signs. The velogenic viscerotropic Newcastle disease virus (VVNDV) was isolated from cloacal and tracheal swabs and various tissues, such as the lung, trachea, distal intestine, and spleen. The origin of the birds could not be established. The disease in the six states was promptly controlled, with no evidence that domestic poultry had been exposed. Also, VVNDV was isolated from quarantined birds intended for importation into the United States. Included were 53 moustached parakeets (Psittacula alexandri fasciata), a mynah (Gracula religiosa), a drongo (Dicrurus sp.), and three partridges (family Phasianidae). Groups of birds that yielded VVNDV were denied entry into the United States. Birds that are illegally imported and therefore not tested for the presence of foreign animal pathogens are a potential source of VVNDV and a threat to domestic poultry and caged birds.

  1. A combination in-ovo vaccine for avian influenza virus and Newcastle disease virus.

    Science.gov (United States)

    Steel, John; Burmakina, Svetlana V; Thomas, Colleen; Spackman, Erica; García-Sastre, Adolfo; Swayne, David E; Palese, Peter

    2008-01-24

    The protection of poultry from H5N1 highly pathogenic avian influenza A (HPAI) and Newcastle disease virus (NDV) can be achieved through vaccination, as part of a broader disease control strategy. We have previously generated a recombinant influenza virus expressing, (i) an H5 hemagglutinin protein, modified by the removal of the polybasic cleavage peptide and (ii) the ectodomain of the NDV hemagglutinin-neuraminidase (HN) protein in the place of the ectodomain of influenza neuraminidase (Park MS, et al. Proc Natl Acad Sci USA 2006;103(21):8203-8). Here we show this virus is attenuated in primary normal human bronchial epithelial (NHBE) cell culture, and demonstrate protection of C57BL/6 mice from lethal challenge with an H5 HA-containing influenza virus through immunisation with the recombinant virus. In addition, in-ovo vaccination of 18-day-old embryonated chicken eggs provided 90% and 80% protection against highly stringent lethal challenge by NDV and H5N1 virus, respectively. We propose that this virus has potential as a safe in-ovo live, attenuated, bivalent avian influenza and Newcastle disease virus vaccine.

  2. Spillover of Newcastle disease viruses from poultry to wild birds in Guangdong province, southern China.

    Science.gov (United States)

    Xiang, Bin; Han, Lujie; Gao, Pei; You, Renrong; Wang, Fumin; Xiao, Jiajie; Liao, Ming; Kang, Yinfeng; Ren, Tao

    2017-11-01

    Despite intensive vaccination programs in many countries, including China, Newcastle disease has been reported sporadically and is still a significant threat to the poultry industry in China. Newcastle disease virus (NDV) is infectious for at least 250 bird species, but the role of wild birds in virus epidemiology remains largely unknown. Fourteen NDV isolates were obtained from 2040 samples collected from wild birds or the environment in Guangdong province, southern China, from 2013 to 2015. The isolation rate was the highest in the period of wintering and lowest during the periods of spring migration, nesting, and postnesting. A maximum clade credibility phylogenetic analysis revealed that at least four genotypes circulate in southern China: three class II genotypes (II, VI, and IX) and one class I (1b). We also demonstrated that most isolates from wild birds were highly similar to isolates from poultry, and two isolates were linked to viruses from wild birds in northern China. These data suggested that wild birds could disseminate NDV and poultry-derived viruses may spillover to wild birds. Accordingly, vaccine development and poultry management strategies should be considered to prevent future NDV outbreaks, particularly given the strength of the poultry industry in developing countries, such as China. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Genetically engineered oncolytic Newcastle disease virus mediates cytolysis of prostate cancer stem like cells.

    Science.gov (United States)

    Raghunath, Shobana; Pudupakam, Raghavendra Sumanth; Allen, Adria; Biswas, Moanaro; Sriranganathan, Nammalwar

    2017-10-20

    Oncolytic virotherapy is a promising novel approach that overcomes the limitations posed by radiation and chemotherapy. In this study, the oncolytic efficacy of a recombinant Newcastle disease virus (rNDV) BC-KLQL-GFP, against prostate cancer stem-like/tumor initiating cells was evaluated. Xenograft derived prostaspheres (XPS) induced tumor more efficiently than monolayer cell derived prostaspheres (MCPS) in nude mice. Primary and secondary XPS show enhanced self-renewal and clonogenic potential compared to MCPS. XPS also expressed embryonic stem cell markers, such as Nanog, CD44 and Nestin. Further, prostate specific antigen (PSA) activated recombinant Newcastle Disease Virus (rNDV) was selectively cytotoxic to tumor derived DU145 prostaspheres. An effective concentration (EC 50 ) of 0.11-0.14 multiplicity of infection was sufficient to cause prostasphere cell death in serum free culture. DU145 tumor xenograft derived prostaspheres were used as tumor surrogates as they were enriched for a putative tumor initiating cell population. PSA activated rNDV was efficient in inducing cell death of cells and prostaspheres derived from primary xenografts ex-vivo, thus signifying a potential in vivo efficacy. The EC 50 (∼0.1 MOI) for cytolysis of tumor initiating cells was slightly higher than that was required for the parental cell line, but within the therapeutic margin for safety and efficacy. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Wild Birds in Romania Are More Exposed to West Nile Virus Than to Newcastle Disease Virus.

    Science.gov (United States)

    Paştiu, Anamaria Ioana; Pap, Péter László; Vágási, Csongor István; Niculae, Mihaela; Páll, Emőke; Domşa, Cristian; Brudaşcă, Florinel Ghe; Spînu, Marina

    2016-03-01

    The aim of this study was to evaluate the seroprevalence of West Nile virus (WNV) and Newcastle disease virus (NDV) in wild and domestic birds from Romania. During 2011-2014, 159 plasma samples from wild birds assigned to 11 orders, 27 families, and 61 species and from 21 domestic birds (Gallus gallus domesticus, Anas platyrhynchos domesticus) were collected. The sera were assayed by two commercial competitive enzyme-linked immunosorbent assay (cELISA) kits for antibodies against WNV and NDV. We found a high prevalence of WNV antibodies in both domestic (19.1%) and wild (32.1%) birds captured after the human epidemic in 2010. Moreover, the presence of anti-NDV antibodies among wild birds from Romania (5.4%) was confirmed serologically for the first time, as far as we are aware. Our findings provide evidence that wild birds, especially resident ones are involved in local West Nile and Newcastle disease enzootic and epizootic cycles. These may allow virus maintenance and spread and also enhance the chance of new outbreaks.

  5. Inactive vaccine derived from velogenic strain of local Newcastle disease virus .

    Directory of Open Access Journals (Sweden)

    Darminto

    1996-03-01

    Full Text Available The objective of this research is to evaluate an application of an inactive Newcastle disease (ND vaccine derived from velogenic strain of local Newcastle disease virus (NDV. In this research . the Ira strain of velogenic ND virus was grown in specific pathogen free (SPF eggs and then was inactivated by formalin at a final concentration of 1 :1,000 at 4°C. The inactive antigen was then emulsified with an oil adjuvant or aluminium hydroxide gel before being administered for vaccination in layers and compared to a commercial inactive ND vaccine . Results indicated that application of these inactivated ND vaccines for booster vaccination following vaccination with an active lentogenic ND virus in pullets nearly producing eggs, resulted in high antibody titre which persisted for considerable long period of time and capable of protecting layers from sick of ND and from reducing egg production . Hence, it could be concluded that the inactivated vaccine emulsified in either oil-adjuvant (lanolin-paraffin or aluminium hydroxide gel were considered to be highly immunogenic and capable of protecting layers from sick of ND and from reducing egg production

  6. Characterization of velogenic Newcastle disease viruses isolated from dead wild birds in Serbia during 2007.

    Science.gov (United States)

    Vidanović, Dejan; Sekler, Milanko; Asanin, Ruzica; Milić, Nenad; Nisavić, Jakov; Petrović, Tamas; Savić, Vladimir

    2011-04-01

    Avian paramyxoviruses type 1 or Newcastle disease viruses (NDV) are frequently recovered from wild birds and such isolates are most frequently of low virulence. Velogenic NDV are usually recovered from poultry and only occasionally from wild birds. Five NDV isolates were obtained from carcasses of four wild bird species during 2007 in Serbia: Mallard (Anas platyrhynchos), Eurasian Sparrowhawk (Accipiter nisus), feral Rock Pigeon (Columba livia), and Eurasian Collared Dove (Streptopelia decaocto). All the isolates have a typical fusion protein cleavage site motif of velogenic viruses ((112)R-R-Q-K-R-F(117)). The highest homology (99%) for the nucleotide sequences spanning the M and F gene of the studied isolates was with the genotype VII NDV isolate Muscovy duck/China(Fujian)/FP1/02. Phylogenetic analysis based on a partial F gene sequence showed that the isolates from wild birds cluster together with concurrent isolates from poultry in Serbia within the subgenotype VIId, which is the predominant pathogen involved currently in Newcastle disease outbreaks in poultry worldwide. It is unlikely that the wild birds played an important role in primary introduction or consequent spread of the velogenic NDV to domestic poultry in Serbia, and they probably contracted the virus from locally infected poultry.

  7. Phylogenetic analysis of some Newcastle disease virus isolates from the Sudan

    Directory of Open Access Journals (Sweden)

    N.A. Elmardi

    2016-06-01

    Full Text Available A reverse transcription-polymerase chain reaction (RT-PCR was used to amplify 1412 bp of the fusion protein gene (F gene of four Newcastle disease virus (NDV isolates; two velogenic (TY-1/90 and DIK-90 and two lentogenic isolates (Dongla 88/1 and GD.S.1. Following sequencing, nucleotide sequences were annotated and 894 bp were compared phylogenetically with those from strains previously reported in the Sudan and the virus strains published on the GenBank. It could be demonstrated that TY-1/90 and DIK-90 strains belong to the genotype VI of NDV and are in close genetic relationship to sub- genotype VIb. TY-1/90 and DIK-90 strains were observed to be genetically unrelated to the earlier Sudanese isolates of 1970/80s and the late of 2000s suggesting a different origin. The close genetic relationship to the European and African pigeon paramyxovirus type 1 (PPMV-1 suggests a common ancestor. Dongola, GD.S.1 strains were classified into genotype II that comprises non-pathogenic lentogenic NDV strains. The present genetic classification of NDV isolates of the Sudan provides valuable information on genotypes of NDV. Further molecular epidemiological investigations of the recent outbreaks of Newcastle disease in the Sudan are needed in order to improve the efficiency of control strategies and vaccine development.

  8. Chimeric rabies glycoprotein with a transmembrane domain and cytoplasmic tail from Newcastle disease virus fusion protein incorporates into the Newcastle disease virion at reduced levels.

    Science.gov (United States)

    Yu, Gui Mei; Zu, Shu Long; Zhou, Wei Wei; Wang, Xi Jun; Shuai, Lei; Wang, Xue Lian; Ge, Jin Ying; Bu, Zhi Gao

    2017-08-31

    Rabies remains an important worldwide health problem. Newcastle disease virus (NDV) was developed as a vaccine vector in animals by using a reverse genetics approach. Previously, our group generated a recombinant NDV (LaSota strain) expressing the complete rabies virus G protein (RVG), named rL-RVG. In this study, we constructed the variant rL-RVGTM, which expresses a chimeric rabies virus G protein (RVGTM) containing the ectodomain of RVG and the transmembrane domain (TM) and a cytoplasmic tail (CT) from the NDV fusion glycoprotein to study the function of RVG's TM and CT. The RVGTM did not detectably incorporate into NDV virions, though it was abundantly expressed at the surface of infected BHK-21 cells. Both rL-RVG and rL-RVGTM induced similar levels of NDV virus-neutralizing antibody (VNA) after initial and secondary vaccination in mice, whereas rabies VNA induction by rL-RVGTM was markedly lower than that induced by rL-RVG. Though rL-RVG could spread from cell to cell like that in rabies virus, rL-RVGTM lost this ability and spread in a manner similar to the parental NDV. Our data suggest that the TM and CT of RVG are essential for its incorporation into NDV virions and for spreading of the recombinant virus from the initially infected cells to surrounding cells.

  9. Genetically engineered Newcastle disease virus expressing interleukin-2 and TNF-related apoptosis-inducing ligand for cancer therapy

    Science.gov (United States)

    Recombinant Newcastle disease virus (rNDV) has shown oncolytic therapeutic efficacy in preclinical studies and are currently in clinical trials. In this study, we have evaluated the possibility to enhance the cancer therapeutic potential of NDV by means of inserting both interleukin-2 (IL-2) and tu...

  10. Characterization and Sequencing of a Genotype XII Newcastle Disease Virus Isolated from a Peacock (Pavo cristatus) in Peru.

    Science.gov (United States)

    Chumbe, Ana; Izquierdo-Lara, Ray; Tataje-Lavanda, Luis; Figueroa, Aling; Segovia, Karen; Gonzalez, Rosa; Cribillero, Giovana; Montalvan, Angela; Fernández-Díaz, Manolo; Icochea, Eliana

    2015-07-30

    Here, we report the first complete sequence and biological characterization of a Newcastle disease virus (NDV) isolated from a peacock in South America (NDV/peacock/Peru/2011). This isolate, classified as genotype XII in class II, highlights the need for increased surveillance of noncommercial avian species. Copyright © 2015 Chumbe et al.

  11. Activation of human T cells by a tumor vaccine infected with recombinant Newcastle disease virus producing IL-2

    NARCIS (Netherlands)

    Janke, M.; Peeters, B.; Zhao, H.; Leeuw, O.; Moormann, R.J.M.; Arnold, A.; Ziouta, Y.; Fournier, P.; Schirrmacher, V.

    2008-01-01

    A new recombinant (rec) Newcastle disease virus (NDV) with incorporated human interleukin 2 (IL-2) as foreign therapeutic gene [rec(IL-2)] will be described. The foreign gene in rec(IL-2) did not affect the main features of NDV replication nor its tumor selectivity. Biologically active IL-2 was

  12. Molecular and antigenic characteristics of Newcastle disease virus isolates from domestic ducks in China.

    Science.gov (United States)

    Wu, Wei; Liu, Huairan; Zhang, Tingting; Han, Zongxi; Jiang, Yanyu; Xu, Qianqian; Shao, Yuhao; Li, Huixin; Kong, Xiangang; Chen, Hongyan; Liu, Shengwang

    2015-06-01

    Newcastle disease (ND) is one of the most devastating diseases to the poultry industry. The causative agents of ND are virulent strains of Newcastle disease virus (NDV), which are members of the genus Avulavirus within the family Paramyxoviridae. Waterfowl, such as ducks and geese, are generally considered potential reservoirs of NDV and may show few or no clinical signs when infected with viruses that are obviously virulent in chickens. However, ND outbreaks in domestic waterfowl have been frequently reported in many countries in the past decade. In this study, 18 NDV strains isolated from domestic ducks in southern and eastern China, between 2005 and 2013, were genetically and phylogenetically characterized. The complete genomes of these strains were sequenced, and they exhibited genome sizes of 15,186 nucleotides (nt), 15,192 nt, and 15,198 nt, which follow the "rule of six" that is required for the replication of NDV strains. Based on the cleavage site of the F protein and pathogenicity tests in chickens, 17 of our NDV isolates were categorized as lentogenic viruses, and one was characterized as a velogenic virus. Phylogenetic analysis based on the partial sequences of the F gene and the complete genome sequences showed that there are at least four genotypes of NDV circulating in domestic ducks; GD1, AH224, and AH209 belong to genotypes VIId, Ib, and II of class II NDVs, respectively, and the remaining 15 isolates belong to genotype 1b of class I NDVs. Cross-reactive hemagglutination inhibition tests demonstrated that the antigenic relatedness between NDV strains may be associated with their genotypes, rather than their hosts. These results suggest that though those NDV isolates were from duck, they still don't form a phylogenetic group because they came from the same species; however, they may play an important role in promoting the evolution of NDVs. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption

    Directory of Open Access Journals (Sweden)

    Liu Chengqian

    2011-05-01

    Full Text Available Abstract Reverse transcription-polymerase chain reaction (RT-PCR has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV. One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs. The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses.

  14. Rapid Newcastle Disease Virus Detection Based on Loop-Mediated Isothermal Amplification and Optomagnetic Readout

    DEFF Research Database (Denmark)

    Tian, Bo; Ma, Jing; Zardán Gómez de la Torre, Teresa

    2016-01-01

    Rapid and sensitive diagnostic methods based on isothermal amplification are ideal substitutes for PCR in out-of-lab settings. However, there are bottlenecks in terms of establishing low-cost and user-friendly readout methods for isothermal amplification schemes. Combining the high amplification...... efficiency of loop-mediated isothermal amplification (LAMP) with an optomagnetic nanoparticle-based readout system, we demonstrate ultrasensitive and rapid detection of Newcastle disease virus RNA. Biotinylated amplicons of LAMP and reverse transcription LAMP (RT-LAMP) bind to streptavidin-coated magnetic...... nanoparticles (MNPs) resulting in a dramatical increase in the hydrodynamic size of the MNPs. This increase was measured by an optomagnetic readout system and provided quantitative information on the amount of LAMP target sequence. Our assay resulted in a limit of detection of 10 aM of target sequence...

  15. Characterization of pigeon paramyxoviruses (Newcastle disease virus) isolated in South Africa from 2001 to 2006.

    Science.gov (United States)

    Abolnik, C; Gerdes, G H; Kitching, J; Swanepoel, S; Romito, M; Bisschop, S P R

    2008-06-01

    Pigeon paramyxovirus type 1 (PPMV-1), a variant of Newcastle disease virus that primarily affects doves and pigeons has been isolated in South Africa since the mid-1980s. Phylogenetic evidence indicates that pigeon paramyxovirus type 1 viruses were introduced into South Africa on multiple occasions, based on the presence of two separate lineages, 4bi and 4bii, that have been circulating in Europe and the Far East since the early 1990s. During 2006, a PPMV-1 virus was isolated from an African ground hornbill (Bucorvus leadbeateri) which became acutely infected with PPMV-1 and died, probably after scavenging off infected dove carcasses in the region, since a closely-related PPMV-1 strain was also isolated from doves collected nearby. The hornbill isolate had ICPI and MDT values characteristic of PPMV-1 strains. The threat of PPMV-1 to poultry production and biodiversity in southern Africa highlights the importance of monitoring the spread of this strain.

  16. Avian influenza A virus and Newcastle disease virus mono- and co-infections in birds

    Directory of Open Access Journals (Sweden)

    Iv. Zarkov

    2017-06-01

    Full Text Available The main features of avian influenza viruses (AIV and Newcastle disease virus (APMV-1, the possibilities for isolation and identification in laboratory conditions, methods of diagnostics, main hosts, clinical signs and virus shedding are reviewed in chronological order. The other part of the review explains the mechanisms and interactions in cases of co-infection of AIV and APMV-1, either between them or with other pathogens in various indicator systems – cell cultures, chick embryos or birds. The emphasis is placed on quantitative data on the virus present mainly in the first ten days following experimental infection of birds, the periods of virus carrier ship and shedding, clinical signs, pathological changes, diagnostic challenges

  17. Newcastle Disease Virus infection study on duck and chicken in Subang district

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    Aprizal Panus

    2015-06-01

    Full Text Available The objectives of this research were to study Newcastle Disease Virus (NDV infection in Subang area and to examine the diversity of the circulating NDV. Swabs of cloacal and oropharynx, and serum were sampled from total of 393 chickens and 149 ducks in backyard farms and live bird markets located in 10 subdistricts. Screening of NDV in pool of 5-7 samples by real-time Reverse-Transcription Polymerase Chain Reaction (rRT-PCR matrix (M showed 19/67 (28.3% cloacal and 8/67 (11.9% pharyngeal pools of chicken samples; 18/67 (26.9% of the pools excreted virus via cloaca and oropharynx, while the duck pools of 8/30 (26.7% shed virus from cloaca. Virus isolation attempted on individual sample from positive pools yielded 18 isolates which the majority of the isolates showed homogeneous antigenic character, only some of these showed variations up to 2 Log2 with Lasota and 4 Log2 with Komarov antisera. Majority of isolates had a higher affinity to Komarov indicating their propencity to virulent strains. Pathogenicity examination using elution test showed 3 isolates virus were grouped to mesogenic strains and 15 isolates to velogenic strain, in agreement with rRT-PCR fusion results. HI test on 408 sera showed that NDV antibody was detected in 48 (12% birds with titres ranging from 1 to 8 Log2; only about 13% of vaccinated chickens demonstrated protective antibody titre (≥3 Log2. Newcastle disease is still endemic in Subang with relatively low antigenic variation among circulating strains.

  18. Humoral and Cellular Response of Pheasants Vaccinated against Newcastle Disease and Haemorrhagic Enteritis

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    S. Graczyk

    2006-01-01

    Full Text Available The purpose of the experiment was to define whether and to what extent can prophylactic vaccinations against Newcastle disease (ND and haemorrhagic enteritis (HE affect the humoral and cellular response in pheasants. The evaluation of humoral response was performed on a basis of agglutinin titre after administered antigen and the cellular immunity index was the delayed type hypersensitivity (DTH reaction. The pheasants were prophylactically vaccinated against Newcastle Disease (ND on the 1st, 28th and 56th day of life. Moreover, on the 49th day of life, part of the birds was given in the drinking water a vaccine containing the HEV (Haemorrhagic Enteritis Virus. Fourteen days after the HEV vaccination, the birds were intravenously given 0.5 ml of the 10% SRBC (sheep red blood cells suspension. Simultaneously with the SRBC administration the delayed hypersensitivity test was performed by intradermal administration of phytohaemagglutinin (PHA. It was shown that in pheasants vaccinated with NDV and additionally with HEV, the specific agglutinin anti-SRBC titre was significantly (p < 0.05 lower than in birds vaccinated against ND only. It also appeared that, the antibodies resistant to 2-mercaptoethanol were 43% of the total pool of specific anti-SRBC antibodies in the NDV vaccinated birds, whereas in birds vaccinated also with HEV they were 75%. No significant differences were found in the DTH test. Only in the HEV vaccinated pheasants the tendency to increase the wing index value was noted. The results confirm the observations concerning immunosuppressive effects of simultaneous vaccinations. They also indicate that overloading the pheasants with many antigens (ND and HEV vaccination may weaken the humoral response to administered SRBC.

  19. Heterologous prime-boost immunization of Newcastle disease virus vectored vaccines protected broiler chickens against highly pathogenic avian influenza and Newcastle disease viruses.

    Science.gov (United States)

    Kim, Shin-Hee; Samal, Siba K

    2017-07-24

    Avian Influenza virus (AIV) is an important pathogen for both human and animal health. There is a great need to develop a safe and effective vaccine for AI infections in the field. Live-attenuated Newcastle disease virus (NDV) vectored AI vaccines have shown to be effective, but preexisting antibodies to the vaccine vector can affect the protective efficacy of the vaccine in the field. To improve the efficacy of AI vaccine, we generated a novel vectored vaccine by using a chimeric NDV vector that is serologically distant from NDV. In this study, the protective efficacy of our vaccines was evaluated by using H5N1 highly pathogenic avian influenza virus (HPAIV) strain A/Vietnam/1203/2004, a prototype strain for vaccine development. The vaccine viruses were three chimeric NDVs expressing the hemagglutinin (HA) protein in combination with the neuraminidase (NA) protein, matrix 1 protein, or nonstructural 1 protein. Comparison of their protective efficacy between a single and prime-boost immunizations indicated that prime immunization of 1-day-old SPF chicks with our vaccine viruses followed by boosting with the conventional NDV vector strain LaSota expressing the HA protein provided complete protection of chickens against mortality, clinical signs and virus shedding. Further verification of our heterologous prime-boost immunization using commercial broiler chickens suggested that a sequential immunization of chickens with chimeric NDV vector expressing the HA and NA proteins following the boost with NDV vector expressing the HA protein can be a promising strategy for the field vaccination against HPAIVs and against highly virulent NDVs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Experimental infection of duck origin virulent Newcastle disease virus strain in ducks.

    Science.gov (United States)

    Dai, Yabin; Cheng, Xu; Liu, Mei; Shen, Xinyue; Li, Jianmei; Yu, Shengqing; Zou, Jianmin; Ding, Chan

    2014-07-17

    Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is an acute, highly contagious and fatal viral disease affecting most species of birds. Ducks are generally considered to be natural reservoirs or carriers of NDV while being resistant to NDV strains, even those most virulent for chickens; however, natural ND cases in ducks have been gradually increasing in recent years. In the present study, ducks of different breeds and ages were experimentally infected with duck origin virulent NDV strain duck/Jiangsu/JSD0812/2008 (JSD0812) by various routes to investigate the pathogenicity of NDV in ducks. Six breeds (mallard, Gaoyou, Shaoxing, Jinding, Shanma, and Pekin ducks) were infected intramuscularly (IM) with JSD0812 strain at the dose of 5 × 108 ELD50. Susceptibility to NDV infection among breeds varied, per morbidity and mortality. Mallard ducks were the most susceptible, and Pekin ducks the most resistant. Fifteen-, 30-, 45-, 60-, and 110-day-old Gaoyou ducks were infected with JSD0812 strain at the dose of 5 × 108 ELD50 either IM or intranasally (IN) and intraocularly (IO), and their disease development, viral shedding, and virus tissue distribution were determined. The susceptibility of ducks to NDV infection decreased with age. Most deaths occurred in 15- and 30-day-old ducklings infected IM. Ducks infected IN and IO sometimes exhibited clinical signs, but seldom died. Clinical signs were primarily neurologic. Infected ducks could excrete infectious virus from the pharynx and/or cloaca for a short period, which varied with bird age or inoculation route; the longest period was about 7 days. The rate of virus isolation in tissues from infected ducks was generally low, even in those from dead birds, and it appeared to be unrelated to bird age and infection route. The results confirmed that some of the naturally occurring NDV virulent strains can cause the disease in ducks, and that ducks play an important role in the epidemiology of ND. The

  1. Molecular characterization of velogenic viscerotropic Ranikhet (Newcastle) disease virus from different outbreaks in desi chickens.

    Science.gov (United States)

    Dhaygude, V S; Sawale, G K; Chawak, M M; Bulbule, N R; Moregaonkar, S D; Gavhane, D S

    2017-03-01

    Diagnosis of velogenic viscerotropic Ranikhet disease from six different flocks of desi chicken in and around Mumbai by gross and histopathological examination, isolation of virus and molecular methods. A total of 25 carcasses (varying between 2 and 6 carcasses from each flock) of six different flocks of adult desi chicken were subjected to necropsy examination for diagnosis of the disease during the span of a year (2014-2015). After thorough gross examination, the tissue samples were collected and processed for virus isolation and histopathological examination. The 20% tissue homogenate was inoculated into 9-day-old specific pathogen free (SPF) embryonated eggs. Mean death time (MDT) of embryos after inoculation and intracerebral pathogenicity index (ICPI) were used to judge velogenic nature of the virus. Newcastle disease virus (NDV) was isolated from six cases and confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) targeting the partial fusion protein gene of the viral genome. A total of 25 carcasses (varying between 2 and 6 carcasses from each flock) of six different flocks of desi chicken were presented for postmortem examination to Department of Veterinary Pathology, Bombay Veterinary College, Parel, Mumbai during 2014-2015. The gross and histopathological examination revealed lesions suggestive of viscerotropic velogenic form of the Newcastle disease (ND). The 20% tissue homogenate was inoculated into 9-day-old embryonated eggs from SPF chicken. NDV was isolated from six cases and confirmed by RT-PCR targeting the partial fusion protein gene. MDT of all the isolates was <60 h which indicated velogenic nature of the virus. ICPI of the isolates ranged between the 1.63 and 1.78. In four out of six outbreaks concurrent moderate to heavy infection of Ascardii galli in one flock and Railetina spp. in three flocks was also noted. In this study, viscerotropic velogenic form of ND was confirmed in all six outbreaks by gross and microscopic

  2. Molecular characterization of velogenic viscerotropic Ranikhet (Newcastle disease virus from different outbreaks in desi chickens

    Directory of Open Access Journals (Sweden)

    V. S. Dhaygude

    2017-03-01

    Full Text Available Aim: Diagnosis of velogenic viscerotropic Ranikhet disease from six different flocks of desi chicken in and around Mumbai by gross and histopathological examination, isolation of virus and molecular methods. Materials and Methods: A total of 25 carcasses (varying between 2 and 6 carcasses from each flock of six different flocks of adult desi chicken were subjected to necropsy examination for diagnosis of the disease during the span of a year (2014-2015. After thorough gross examination, the tissue samples were collected and processed for virus isolation and histopathological examination. The 20% tissue homogenate was inoculated into 9-day-old specific pathogen free (SPF embryonated eggs. Mean death time (MDT of embryos after inoculation and intracerebral pathogenicity index (ICPI were used to judge velogenic nature of the virus. Newcastle disease virus (NDV was isolated from six cases and confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR targeting the partial fusion protein gene of the viral genome. Results: A total of 25 carcasses (varying between 2 and 6 carcasses from each flock of six different flocks of desi chicken were presented for postmortem examination to Department of Veterinary Pathology, Bombay Veterinary College, Parel, Mumbai during 2014-2015. The gross and histopathological examination revealed lesions suggestive of viscerotropic velogenic form of the Newcastle disease (ND. The 20% tissue homogenate was inoculated into 9-day-old embryonated eggs from SPF chicken. NDV was isolated from six cases and confirmed by RT-PCR targeting the partial fusion protein gene. MDT of all the isolates was <60 h which indicated velogenic nature of the virus. ICPI of the isolates ranged between the 1.63 and 1.78. In four out of six outbreaks concurrent moderate to heavy infection of Ascardii galli in one flock and Railetina spp. in three flocks was also noted. In this study, viscerotropic velogenic form of ND was confirmed in all

  3. [Experience in using cluster analysis and the prognosis of the stages of epizootic risk for Newcastle disease in poultry].

    Science.gov (United States)

    Elitsina, P; Parannzhilova, M; Vukov, M

    1985-01-01

    An attempt was made to use clastic analysis and projection of the steps of the epizootic hazard with regard to Newcastle disease. There were no records of the disease after 1982 but still it is the object of consideration on the part of specialists. A clastic analysis was made on the base of a factorial model of Newcastle disease in this country for the 1970-1979. Used was a programme already worked out and practised at the Pilot Computor Center of the Medical Academy, Sofia. The districts in the country were grouped in a dendogramme, 16 groups being distinguished out of a total of 27 districts. This showed that regardless of the small territory of the country the districts are sufficiently differing between each other (due to the various degrees of integration) so that they could not be grouped together by similar values of intensity of poultry breeding and epizootic conjuncture with regard to Newcastle disease. Districts of epizootic hazard ranging from 1st to 5th degree substantiate the use of a differential approach in building up the tactics of prophylaxis and control of the disease, while measures concerning the poultry dressing combines should be at a high level regardless of the category of the respective district. The grouping of neighboring districts on a territorial principle disclosed the existence of reasons of climatic-and-geographic nature that could predispose equal degrees of intensity of poultry breeding and epizootic hazard.

  4. Antiviral effect of Anthocleista nobilis root extract on the liver homogenate indices of poultry fowls infected with Newcastle Disease Virus (NDV)

    OpenAIRE

    Ayodele P. O,; Okonko I. O,; Chukwuka K. S,; Odu N. N; Michael V. I

    2011-01-01

    This study reports the preliminary investigation of the antiviral effect of Anthocleista nobilis root extract on the liver homogenate indices of poultry fowls treated for Newcastle Disease (ND). Eighteen (18) weeks-old fowls were used for this study. These were divided into 3 groups, A (infected and with treatment), B (infected and without treatment) and C (control). Groups A and B were challenged with Newcastle disease virus (NDV). Group A and C were given ethanolic root extract of A. nobili...

  5. Determination of the seroprevalence of Newcastle disease virus (avian paramyxovirus type 1 in Zambian backyard chicken flocks

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    Chimuka Musako

    2012-02-01

    Full Text Available A cross-sectional study was conducted in five provinces and 11 districts of Zambia to determine the seroprevalence of Newcastle disease in Zambian backyard chicken flocks. Of the chickens sampled, 73.9% tested positive for avian paramyxovirus type 1 antibodies by means of an enzyme-linked immunosorbent assay. Seroprevalence varied amongst the five provinces sampled, ranging from 82.6% in the Eastern Province to 48.3% in Luapula Province. Seroprevalence also varied amongst the 11 districts sampled, ranging from 91.3% in Monze district of Southern Province to 22.8% in Mufulira district of the Copperbelt province. Overall, the seroprevalence of Newcastle disease in Zambian backyard chicken flocks has increased since the previous study conducted in 1994.

  6. Construction of a recombinant viral vector containing part of the nucleocapsid protein gene of newcastle disease virus

    Energy Technology Data Exchange (ETDEWEB)

    Bader, D.E.

    1995-09-01

    This report describes the procedures used to clone a 673 base pair gene fragment of the major nucleocapsid protein gene of Newcastle disease virus into a viral vector molecule for the purpose of maintaining a stable, long-term, renewable source of this target sequence for gene probe studies. The gene fragment was prepared by reverse-transcription polymerase chain reaction of Newcastle disease virus RNA and was cloned into the viral DNA vector Ml3mp18 RF to produce a recombinant DNA molecule. The cloned fragment was shown to be present in the recombinant clones based on (i) clonal selection on indicator plates; (ii) restriction enzyme analysis; (iii) gene probe analysis and (iv) nested PCR amplification.

  7. Experimental intraocular infection of exotic cockerels with field strain of velogenic Newcastle disease virus in Nigeria

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    Samaila Jonathan Badau

    2015-12-01

    Full Text Available Experimental intraocular (conjunctival infection of exotic cockerels with a new field strain of viscerotropic velogenic Newcastle Disease Virus (NDV was conducted to explore the concurrence of some pathological changes with humoral immune responses. After the NDV infection of 4-week-old cockerels, pathologic changes and antibody responses were observed. The clinical signs observed after the artificial inoculation included inappetence, depression, diarrhea, dyspnea, wing and leg paralysis, torticollis and weight loss. Morbidity due to the NDV was 100%, but mortality was 80% by day 18-21 post-infection. Early hyperthermia followed by terminal hypothermia, decreased packed cell volume (PCV, and 231.4 folds peak-antibody response were observed. Necrotic and/or inflammatory lesions were present in the proventriculus, intestine, liver, spleen, kidney and brain. Neurologic and digestive tract perturbations occurred in 10% and 85% of cases, respectively. The disease consistently caused stunted growth, decreased PCV, and necro-inflammatroy lesions concurrent with antibody response, suggesting probable involvement of immune-mediated mechanisms and cell membrane desialylation by viral neuraminidase in the pathogenesis.

  8. Complete Genome Sequence of a Genotype XVII Newcastle Disease Virus, Isolated from an Apparently Healthy Domestic Duck in Nigeria

    OpenAIRE

    Shittu, Ismaila; Sharma, Poonam; Joannis, Tony M.; Volkening, Jeremy D.; Odaibo, Georgina N.; Olaleye, David O.; Williams-Coplin, Dawn; Solomon, Ponman; Abolnik, Celia; Miller, Patti J.; Dimitrov, Kiril M.; Afonso, Claudio L.

    2016-01-01

    The first complete genome sequence of a strain of Newcastle disease virus (NDV) of genotype XVII is described here. A velogenic strain (duck/Nigeria/903/KUDU-113/1992) was isolated from an apparently healthy free-roaming domestic duck sampled in Kuru, Nigeria, in 1992. Phylogenetic analysis of the fusion protein gene and complete genome classified the isolate as a member of NDV class II, genotype XVII.

  9. Complete Genome Sequence of a Genotype XVII Newcastle Disease Virus, Isolated from an Apparently Healthy Domestic Duck in Nigeria

    Science.gov (United States)

    Shittu, Ismaila; Sharma, Poonam; Joannis, Tony M.; Volkening, Jeremy D.; Odaibo, Georgina N.; Olaleye, David O.; Williams-Coplin, Dawn; Solomon, Ponman; Abolnik, Celia; Miller, Patti J.; Dimitrov, Kiril M.

    2016-01-01

    The first complete genome sequence of a strain of Newcastle disease virus (NDV) of genotype XVII is described here. A velogenic strain (duck/Nigeria/903/KUDU-113/1992) was isolated from an apparently healthy free-roaming domestic duck sampled in Kuru, Nigeria, in 1992. Phylogenetic analysis of the fusion protein gene and complete genome classified the isolate as a member of NDV class II, genotype XVII. PMID:26847901

  10. Assessment of the pathogenicity of cell-culture-adapted Newcastle disease virus strain Komarov.

    Science.gov (United States)

    Visnuvinayagam, Sivam; Thangavel, K; Lalitha, N; Malmarugan, S; Sukumar, Kuppannan

    2015-01-01

    Newcastle disease vaccines hitherto in vogue are produced from embryonated chicken eggs. Egg-adapted mesogenic vaccines possess several drawbacks such as paralysis and mortality in 2-week-old chicks and reduced egg production in the egg-laying flock. Owing to these possible drawbacks, we attempted to reduce the vaccine virulence for safe vaccination by adapting the virus in a chicken embryo fibroblast cell culture (CEFCC) system. Eighteen passages were carried out by CEFCC, and the pathogenicity was assessed on the basis of the mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index, at equal passage intervals. Although the reduction in virulence demonstrated with increasing passage levels in CEFCC was encouraging, 20% of the 2-week-old birds showed paralytic symptoms with the virus vaccine from the 18(th)(final) passage. Thus, a tissue-culture-adapted vaccine would demand a few more passages by CEFCC in order to achieve a complete reduction in virulence for use as a safe and effective vaccine, especially among younger chicks. Moreover, it can be safely administered even to unprimed 8-week-old birds.

  11. Pathotypic characterization of Newcastle disease virus isolated from vaccinated chicken in West Java, Indonesia.

    Science.gov (United States)

    Putri, Dwi Desmiyeni; Handharyani, Ekowati; Soejoedono, Retno Damajanti; Setiyono, Agus; Mayasari, Ni Luh Putu Ika; Poetri, Okti Nadia

    2017-04-01

    This research was conducted to differentiate and characterize eight Newcastle disease virus (NDV) isolates collected from vaccinated chicken at commercial flocks in West Java, Indonesia, in 2011, 2014 and 2015 by pathotype specific primers. A total of eight NDV isolates collected from clinical outbreaks among commercial vaccinated flocks in West Java, Indonesia, in 2011, 2014, and 2015 were used in this study. Reverse transcription-polymerase chain reaction was used to detect and differentiate virulence of NDV strains, using three sets of primers targeting their M and F gene. First primers were universal primers to detect NDV targeting matrix (M) gene. Other two sets of primers were specific for the fusion (F) gene cleavage site sequence of virulent and avirulent NDV strains. Our results showed that three isolates belong to NDV virulent strains, and other five isolates belong to NDV avirulent strains. The nucleotide sequence of the F protein cleavage site showed 112 K/R-R-Q/R-K-R/G-F 117 on NDV virulent strains and 112 G-K/R-Q-G-R-L 117 on NDV avirulent strain. Result from the current study suggested that NDV virulent strain were circulating among vaccinated chickens in West Java, Indonesia; this might possess a risk of causing ND outbreaks and causing economic losses within the poultry industry.

  12. The Role of Non-specific and Specific Immune Systems in Poultry against Newcastle Disease

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    Dyah Ayu Hewajuli

    2015-09-01

    Full Text Available Newcastle disease (ND is caused by avian paramyxovirus-1 which belong to Avulavirus genus and Paramyxoviridae family. The birds have abnormalities in humoral (bursa fabricius and cellular (thymus and spleen lymphoid organs. Lesions decrease the immune system. Immune system consists of non-specific and specific immune systems. The main components of non-specific immunity are physical and chemical barrier (feather and skin or mucosa, phagocytic cells (macrophages and natural killer, protein complement and the mediator of inflammation and cytokines. Interferons (IFNs belong to a group of cytokines that play a major role in the nonspecific or innate (natural immunity. The virulent ND virus encodes protein of V gene can be suppressed IFN type I. This leads to non-specific immune system fail to respond to the virulent strains resulting in severe pathogenicity. The defense mechanism of the host is replaced by specific immunity (adaptive immunity when natural immunity fails to overcome the infection. The specific immune system consists of humoral mediated immunity (HMI and cell-mediated immunity (CMI. The cells of immune system that react specifically with the antigen are B lymphocytes producing the antibodies, T lymphocytes that regulate the synthesis of antibodies and T cells as effector or the direct cytotoxic cells. Both non-specific and specific immunities are complementary against the invasion of ND virus in the birds. The objective of this article is to discuss the role of non specific and specific immune system in ND.

  13. Humoral Immune Response Induced by PLGA Micro Particle Coupled Newcastle Disease Virus Vaccine in Chickens

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    Sanganagouda K

    2014-02-01

    Full Text Available This experiment was conducted for evaluating the humoral immune responses induced by Poly Lactide-co-Glycolide Acid (PLGA microspheres coupled inactivated Newcastle Disease Virus (NDV vaccine in comparison to an ‘in-house’ prepared inactivated and a live commercial vaccine. PLG microparticles containing inactivated NDV were prepared by a double emulsion technique based on solvent evaporation method. The size of the NDV coupled PLG microparticles was determined by Electron Microscopy. NDV coupled PLG microparticles were spherical having smooth surface, hollow core inside with no pores on the surface. The experiment was conducted in four groups of chickens (n=15. The encapsulation efficiency of NDV coupled PLG microparticles was determined by protein estimation and HA activity in elute. The mean (± SE size of PLG microspheres was found to be 2.409 ± 0.65 µm. The mean percent of encapsulation efficiency of PLG microspheres coupled to NDV was assessed based on the total protein content and HA activity in elute was found to be 8.03 ± 0.50 and 12.5 ± 0.00, respectively. In conclusion, the results of the experiment showed that PLGA coupled NDV vaccine elicited stronger and prolonged humoral immune response in chickens, in comparison to the other tested vaccines, as assessed by haemagglutination inhibition and enzyme linked immuno sorbent asaay titers.

  14. Characterization of pigeon paramyxoviruses (Newcastle disease virus isolated in South Africa from 2001 to 2006

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    C. Abolnik

    2008-08-01

    Full Text Available Pigeon paramyxovirus type 1 (PPMV-1, a variant of Newcastle disease virus that primarily affects doves and pigeons has been isolated in South Africa since the mid-1980s. Phylogenetic evidence indicates that pigeon paramyxovirus type 1 viruses were introduced in to South Africa on multiple occasions, based on the presence of two separate lineages, 4bi and 4bii, that have been circulating in Europe and the Far East since the early 1990s. During 2006, a PPMV-1 virus was isolated from an African ground hornbil(l Bucorvus leadbeateri which becamea cutely infected with PPMV-1 and died, probably after scavenging off infected dove carcasses in the region, since a closely-related PPMV-1 strain was also isolated from doves collected nearby. The hornbill isolate had lCPl and MDT values characteristic of PPMV-1s trains. The threat of PPMV-1 to poultry production and biodiversity in southern Africa highlights the importance of monitoring the spread of this strain.

  15. On the origin and diversity of Newcastle disease virus in Tanzania

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    Mmeta G. Yongolo

    2011-09-01

    Full Text Available Free-range rural chickens (FRCs dominate the poultry industry in developing countries and chickens are exposed to multi-host infections, including Newcastle disease virus (NDV. The knowledge about the characteristics of NDV from FRCs is limited. This study investigated the persistence, spread and risks of NDV from FRCs. NDV isolates (n = 21 from unvaccinated FRCs in Tanzania were characterised by conventional intracerebral pathogenicity index (ICPI and sequence analysis of a partial region of the deduced fusion protein encompassing the cleavage site. Results showed that five isolates were screened as lentogenic, nine as mesogenic and six as velogenic. Phylogenetic analysis of the 21 isolates compared to reference sequences revealed three, four, nine and five isolates in genotypes 1, 2, 3c and 4a, respectively. Genotype 3c also included published sequences of Tanzanian isolates obtained from exotic birds and chicken isolates from Uganda. The analysis showed that NDV were persistently present among chicken populations and possibly spread through live chicken markets or migration of wild birds. Differences in amino acid sequences detected around the cleavage site separated the isolates in six types. However, cleavage site pattern could not fully differentiate mesogenic isolates from velogenic isolates.

  16. Pathogenesis of New Strains of Newcastle Disease Virus From Israel and Pakistan.

    Science.gov (United States)

    Pandarangga, P; Brown, C C; Miller, P J; Haddas, R; Rehmani, S F; Afonso, C L; Susta, L

    2016-07-01

    In the past few years, Newcastle disease virus (NDV) strains with epizootic characteristics belonging to subgenotypes VIIi and XIIIb emerged in the Middle East and Asia. In this study, 2 NDV strains-1 representative of subgenotype VIIi isolated in Israel (Kvuzat/13) and 1 representative of subgenotype XIIIb isolated in Pakistan (Karachi/07)-were characterized by intracerebral pathogenicity index and detailed clinicopathologic assessment. The intracerebral pathogenicity index values for Kvuzat/13 and Karachi/07 were 1.89 and 1.85, respectively, classifying these strains as virulent by international standards. In 4-week-old White Leghorn chickens, both strains caused 100% mortality within 4 (Kvuzat/13) and 5 (Karachi/07) days postinfection. Histopathology and immunohistochemistry for NDV nucleoprotein showed that both strains had wide systemic distribution, especially targeting lymphoid organs and mucosa-associated lymphoid tissues in the respiratory and intestinal tracts. Results of the animal experiment confirm that both Kvuzat/13 and Karachi/07 are highly virulent and behaved as velogenic viscerotropic NDV strains. © The Author(s) 2016.

  17. Characterization of Newcastle disease virus isolates obtained from Eurasian collared doves (Streptopelia decaocto) in Italy.

    Science.gov (United States)

    Terregino, Calogero; Cattoli, Giovanni; Grossele, Barbara; Bertoli, Elena; Tisato, Ernesto; Capua, Ilaria

    2003-02-01

    Eurasian collared doves (Streptopelia decaocto) are thought to originate from India and they have colonized, throughout the centuries, the Middle East and, more recently, Mediterranean countries such as Italy and Spain. In the present paper we report of the isolation and characterization of Newcastle disease viruses (NDV) obtained from Eurasian collared doves during 2000-2001, and compare them to isolates obtained from feral pigeons (Columba livia) during the same period. All isolates could be classified as avian paramyxovirus type 1 (APMV1) and belonged to the pigeon variant group (PPMV1), as their haemagglutinating activity was inhibited by mAb 161/617 which is specific for PPMV1. The intracerebral pathogenicity indices ranged from 0.68 to 1.38 and all isolates contained multiple basic amino acids at the deduced cleavage site of the fusion protein, which is a typical feature of virulent viruses. Phylogenetic analysis of the isolates indicate that 18/20 of these form a separate cluster from the isolates obtained from pigeons in the same period. These findings suggest that different lineages are circulating in feral pigeon populations, and that a separate lineage affects Eurasian collared doves.

  18. Use of FTA filter paper for the molecular detection of Newcastle disease virus.

    Science.gov (United States)

    Perozo, Francisco; Villegas, Pedro; Estevez, Carlos; Alvarado, Iván; Purvis, Linda B

    2006-04-01

    The feasibility of using Flinders Technology Associates filter papers (FTA cards) to collect allantoic fluid and chicken tissue samples for Newcastle disease virus (NDV) molecular detection was evaluated. Trizol RNA extraction and one-step reverse transcriptase-polymerase chain reaction (RT-PCR) were used. FTA cards allowed NDV identification from allantoic fluid with a titre of 10(5.8) median embryo lethal doses/ml. The inactivated virus remained stable on the cards for 15 days. NDV was detected from FTA imprints of the trachea, lung, caecal tonsil and cloacal faeces of experimentally infected birds. RT-PCR detection from FTA cards was confirmed by homologous frozen-tissue RT-PCR and virus isolation. Direct nucleotide sequence of the amplified F gene allowed prediction of NDV virulence. No virus isolation was possible from the FTA inactivated samples, indicating viral inactivation upon contact. The FTA cards are suitable for collecting and transporting NDV-positive samples, providing a reliable source of RNA for molecular characterization and a hazard-free sample.

  19. Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication.

    Science.gov (United States)

    Meng, Chunchun; Zhou, Zhizhi; Jiang, Ke; Yu, Shengqing; Jia, Lijun; Wu, Yantao; Liu, Yanqing; Meng, Songshu; Ding, Chan

    2012-06-01

    Newcastle disease virus (NDV) can replicate in tumor cells and induce apoptosis in late stages of infection. However, the interaction between NDV and cells in early stages of infection is not well understood. Here, we report that, shortly after infection, NDV triggers the formation of autophagosomes in U251 glioma cells, as demonstrated by an increased number of double-membrane vesicles, GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) a dot formations, and elevated production of LC3II. Moreover, modulation of NDV-induced autophagy by rapamycin, chloroquine or small interfering RNAs targeting the genes critical for autophagosome formation (Atg5 and Beclin-1) affects virus production, indicating that autophagy may be utilized by NDV to facilitate its own production. Furthermore, the class III phosphatidylinositol 3-kinase (PI3K)/Beclin-1 pathway plays a role in NDV-induced autophagy and virus production. Collectively, our data provide a unique example of a paramyxovirus that uses autophagy to enhance its production.

  20. Genomic and biological characterization of Newcastle disease viruses isolated from migratory mallards (Anas platyrhynchos).

    Science.gov (United States)

    Habib, Momena; Yaqub, Tahir; Nazir, Jawad; Shehzad, Wasim; Aziz-Ul-Rahman; Sohail, Tayyebah; Mukhtar, Nadia; Mehboob, Arsalan; Munir, Muhammad; Shabbir, Muhammad Zubair

    2018-04-30

    Given the global evolutionary dynamics of Newcastle disease viruses (NDVs), it is imperative to continue extensive surveillance, routine monitoring and characterization of isolates originating from natural reservoirs (waterfowls). In this report, we isolated and characterized two virulent NDV strains from clinically healthy mallard (Anas platyrhynchos). Both isolates had a genome of 15,192 nucleotides encoding six genes in an order of 3´-NP-P-M-F-HN-L-5´. The biological characteristics (mean death time: 49.5-50 hr, EID 50 10 8.5  ml -1 ) and presence of a typical cleavage site in the fusion (F) protein (112R-R-Q-K-R↓F117) confirmed the velogenic nature of these isolates. Phylogenetic analysis classified both isolates as members of genotype VII within class-II. Furthermore, based upon the hypervariable region of the F gene (375 nt), isolates showed clustering within sub-genotype VIIi. Similarity index and parallel comparison revealed a higher nucleotide divergence from commonly used vaccine strains; LaSota (21%) and Mukteswar (17%). A comparative residues analysis with representative strains of different genotypes, including vaccine strains, revealed a number of substitutions at important structural and functional domains within the F and hemagglutinin-neuraminidase (HN) proteins. Together, the results highlight consistent evolution among circulating NDVs supporting extensive surveillance of the virus in waterfowl to better elucidate epidemiology, evolutionary relationships and their impacts on commercial and backyard poultry.

  1. Preparation of mucosal nanoparticles and polymer-based inactivated vaccine for Newcastle disease and H9N2 AI viruses

    Directory of Open Access Journals (Sweden)

    Heba M. El Naggar

    2017-02-01

    Full Text Available Aim: To develop a mucosal inactivated vaccines for Newcastle disease (ND and H9N2 viruses to protect against these viruses at sites of infections through mucosal immunity. Materials and Methods: In this study, we prepared two new formulations for mucosal bivalent inactivated vaccine formulations for Newcastle and Avian Influenza (H9N2 based on the use of nanoparticles and polymer adjuvants. The prepared vaccines were delivered via intranasal and spray routes of administration in specific pathogen-free chickens. Cell-mediated and humoral immune response was measured as well as challenge trial was carried out. In addition, ISA71 water in oil was also evaluated. Results: Our results showed that the use of spray route as vaccination delivery method of polymer and nanoparticles MontanideTM adjuvants revealed that it enhanced the cell mediated immune response as indicated by phagocytic activity, gamma interferon and interleukin 6 responses and induced protection against challenge with Newcastle and Avian Influenza (H9N2 viruses. Conclusion: The results of this study demonstrate the potentiality of polymer compared to nanoparticles adjuvantes when used via spray route. Mass application of such vaccines will add value to improve the vaccination strategies against ND virus and Avian influenza viruses.

  2. Temporal, geographic, and host distribution of avian paramyxovirus 1 (Newcastle disease virus)

    Science.gov (United States)

    Dimitrov, Kiril M.; Ramey, Andy M.; Qiu, Xueting; Bahl, Justin; Afonso, Claudio L.

    2016-01-01

    Newcastle disease is caused by virulent forms of avian paramyxovirus of serotype 1 (APMV-1) and has global economic importance. The disease reached panzootic proportions within two decades after first being identified in 1926 in the United Kingdom and Indonesia and still remains endemic in many countries across the world. Here we review information on the host, temporal, and geographic distribution of APMV-1 genetic diversity based on the evolutionary systematics of the complete coding region of the fusion gene. Strains of APMV-1 are phylogenetically separated into two classes (class I and class II) and further classified into genotypes based on genetic differences. Class I viruses are genetically less diverse, generally present in wild waterfowl, and are of low virulence. Class II viruses are genetically and phenotypically more diverse, frequently isolated from poultry with occasional spillovers into wild birds, and exhibit a wider range of virulence. Waterfowl, cormorants, and pigeons are natural reservoirs of all APMV-1 pathotypes, except viscerotropic velogenic viruses for which natural reservoirs have not been identified. Genotypes I and II within class II include isolates of high and low virulence, the latter often being used as vaccines. Viruses of genotypes III and IX that emerged decades ago are now isolated rarely, but may be found in domestic and wild birds in China. Containing only virulent viruses and responsible for the majority of recent outbreaks in poultry and wild birds, viruses from genotypes V, VI, and VII, are highly mobile and have been isolated on different continents. Conversely, virulent viruses of genotypes XI (Madagascar), XIII (mainly Southwest Asia), XVI (North America) and XIV, XVII and XVIII (Africa) appear to have a more limited geographic distribution and have been isolated predominantly from poultry.

  3. Different regions of the newcastle disease virus fusion protein modulate pathogenicity.

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    Sandra Heiden

    Full Text Available Newcastle disease virus (NDV, also designated as Avian paramyxovirus type 1 (APMV-1, is the causative agent of a notifiable disease of poultry but it exhibits different pathogenicity dependent on the virus strain. The molecular basis for this variability is not fully understood. The efficiency of activation of the fusion protein (F is determined by presence or absence of a polybasic amino acid sequence at an internal proteolytic cleavage site which is a major determinant of NDV virulence. However, other determinants of pathogenicity must exist since APMV-1 of high (velogenic, intermediate (mesogenic and low (lentogenic virulence specify a polybasic F cleavage site. We aimed at elucidation of additional virulence determinants by constructing a recombinant virus that consists of a lentogenic NDV Clone 30 backbone and the F protein gene from a mesogenic pigeon paramyxovirus-1 (PPMV-1 isolate with an intracerebral pathogenicity index (ICPI of 1.1 specifying the polybasic sequence R-R-K-K-R*F motif at the cleavage site. The resulting virus was characterized by an ICPI of 0.6, indicating a lentogenic pathotype. In contrast, alteration of the cleavage site G-R-Q-G-R*L of the lentogenic Clone 30 to R-R-K-K-R*F resulted in a recombinant virus with an ICPI of 1.36 which was higher than that of parental PPMV-1. Substitution of different regions of the F protein of Clone 30 by those of PPMV-1, while maintaining the polybasic amino acid sequence at the F cleavage site, resulted in recombinant viruses with ICPIs ranging from 0.59 to 1.36 suggesting that virulence is modulated by regions of the F protein other than the polybasic cleavage site.

  4. Transcriptional Innate Immune Response of the Developing Chicken Embryo to Newcastle Disease Virus Infection

    Science.gov (United States)

    Schilling, Megan A.; Katani, Robab; Memari, Sahar; Cavanaugh, Meredith; Buza, Joram; Radzio-Basu, Jessica; Mpenda, Fulgence N.; Deist, Melissa S.; Lamont, Susan J.; Kapur, Vivek

    2018-01-01

    Traditional approaches to assess the immune response of chickens to infection are through animal trials, which are expensive, require enhanced biosecurity, compromise welfare, and are frequently influenced by confounding variables. Since the chicken embryo becomes immunocompetent prior to hatch, we here characterized the transcriptional response of selected innate immune genes to Newcastle disease virus (NDV) infection in chicken embryos at days 10, 14, and 18 of embryonic development. The results suggest that the innate immune response 72 h after challenge of 18-day chicken embryo is both consistent and robust. The expression of CCL5, Mx1, and TLR3 in lung tissues of NDV challenged chicken embryos from the outbred Kuroiler and Tanzanian local ecotype lines showed that their expression was several orders of magnitude higher in the Kuroiler than in the local ecotypes. Next, the expression patterns of three additional innate-immunity related genes, IL-8, IRF-1, and STAT1, were examined in the highly congenic Fayoumi (M5.1 and M15.2) and Leghorn (Ghs6 and Ghs13) sublines that differ only at the microchromosome bearing the major histocompatibility locus. The results show that the Ghs13 Leghorn subline had a consistently higher expression of all genes except IL-8 and expression seemed to be subline-dependent rather than breed-dependent, suggesting that the innate immune response of chicken embryos to NDV infection may be genetically controlled by the MHC-locus. Taken together, the results suggest that the chicken embryo may represent a promising model to studying the patterns and sources of variation of the avian innate immune response to infection with NDV and related pathogens. PMID:29535762

  5. Detection and molecular characterization of Newcastle disease virus in peafowl (Pavo cristatus) in Haryana State, India.

    Science.gov (United States)

    Kumar, Aman; Maan, Sushila; Mahajan, Nand Kishore; Rana, Virender Pratap; Jindal, Naresh; Batra, Kanisht; Ghosh, Arnab; Mishra, Shiv Kumar; Kapoor, Sanjay; Maan, Narender Singh

    2013-12-01

    Present study was undertaken to investigate the cause of deaths of peafowls in Haryana State. In total, 145 birds were sick and 28 birds were reported dead during July to September 2012. Some of the sick birds were showing signs of shaking of heads, torticollis and paresis. Blood and cloacal swab samples from sick birds along with brain and intestinal tissues from dead birds were collected for further investigation. Although post-mortem examination showed no typical lesions of Newcastle disease virus (NDV) yet raised HI tires against NDV in some serum samples and clinical signs indicated the presence of NDV. One of the brain tissues (NDV/IND2012/01) from the field case was processed and adapted to Vero cell line for virus isolation. The fusion (F) gene based nested RT-PCR (RT-nPCR) confirmed the presence of NDV in all field samples and cell culture isolate. Sequencing of the partial F gene amplicons (216 bp) using the PCR primers as sequencing primers confirmed the PCR results. The deduced amino acid sequences of partial F gene were found to have the amino acid motif (111)GRRQKR/F(117) in the fusion protein cleavage site (FPCS). This amino acid motif is indicative of the velogenic nature of these NDVs. Phylogenetic studies have shown that the virus belonged to class II genotype VII very closely related to virus isolates originated from outbreaks in Western Europe, Israel, Indonesia, Taiwan and India. Phylogenetic grouping of the virus and sequence of FPCS is indicative of pathogenic potential of virus strain circulating in peacocks in Haryana.

  6. Transcriptional Innate Immune Response of the Developing Chicken Embryo to Newcastle Disease Virus Infection

    Directory of Open Access Journals (Sweden)

    Megan A. Schilling

    2018-02-01

    Full Text Available Traditional approaches to assess the immune response of chickens to infection are through animal trials, which are expensive, require enhanced biosecurity, compromise welfare, and are frequently influenced by confounding variables. Since the chicken embryo becomes immunocompetent prior to hatch, we here characterized the transcriptional response of selected innate immune genes to Newcastle disease virus (NDV infection in chicken embryos at days 10, 14, and 18 of embryonic development. The results suggest that the innate immune response 72 h after challenge of 18-day chicken embryo is both consistent and robust. The expression of CCL5, Mx1, and TLR3 in lung tissues of NDV challenged chicken embryos from the outbred Kuroiler and Tanzanian local ecotype lines showed that their expression was several orders of magnitude higher in the Kuroiler than in the local ecotypes. Next, the expression patterns of three additional innate-immunity related genes, IL-8, IRF-1, and STAT1, were examined in the highly congenic Fayoumi (M5.1 and M15.2 and Leghorn (Ghs6 and Ghs13 sublines that differ only at the microchromosome bearing the major histocompatibility locus. The results show that the Ghs13 Leghorn subline had a consistently higher expression of all genes except IL-8 and expression seemed to be subline-dependent rather than breed-dependent, suggesting that the innate immune response of chicken embryos to NDV infection may be genetically controlled by the MHC-locus. Taken together, the results suggest that the chicken embryo may represent a promising model to studying the patterns and sources of variation of the avian innate immune response to infection with NDV and related pathogens.

  7. Generation of Newcastle Disease Virus (NDV) Recombinants Expressing the Infectious Laryngotracheitis Virus (ILTV) Glycoprotein gB or gD as Dual Vaccines.

    Science.gov (United States)

    Zhao, Wei; Spatz, Stephen; Zsak, Laszlo; Yu, Qingzhong

    2016-01-01

    Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infection with infectious laryngotracheitis virus (ILTV), a member of the family Herpesviridae. The current commercial ILT vaccines are either unsafe or ineffective. Therefore, there is a pressing need to develop safer and more efficacious vaccines. Newcastle disease (ND), caused by infection with Newcastle disease virus (NDV), a member of the family Paramyxoviridae, is one of the most serious infectious diseases of poultry. The NDV LaSota strain, a naturally occurring low-virulence NDV strain, has been routinely used as a live vaccine throughout the world. This chapter describes the generation of Newcastle disease virus (NDV) LaSota vaccine strain-based recombinant viruses expressing glycoprotein B (gB) or glycoprotein D (gD) of ILTV as dual vaccines against ND and ILT using reverse genetics technology.

  8. Newcastle Disease Viruses Causing Recent Outbreaks Worldwide Show Unexpectedly High Genetic Similarity to Historical Virulent Isolates from the 1940s

    Science.gov (United States)

    Dimitrov, Kiril M.; Lee, Dong-Hun; Williams-Coplin, Dawn; Olivier, Timothy L.; Miller, Patti J.

    2016-01-01

    Virulent strains of Newcastle disease virus (NDV) cause Newcastle disease (ND), a devastating disease of poultry and wild birds. Phylogenetic analyses clearly distinguish historical isolates (obtained prior to 1960) from currently circulating viruses of class II genotypes V, VI, VII, and XII through XVIII. Here, partial and complete genomic sequences of recent virulent isolates of genotypes II and IX from China, Egypt, and India were found to be nearly identical to those of historical viruses isolated in the 1940s. Phylogenetic analysis, nucleotide distances, and rates of change demonstrate that these recent isolates have not evolved significantly from the most closely related ancestors from the 1940s. The low rates of change for these virulent viruses (7.05 × 10−5 and 2.05 × 10−5 per year, respectively) and the minimal genetic distances existing between these and historical viruses (0.3 to 1.2%) of the same genotypes indicate an unnatural origin. As with any other RNA virus, Newcastle disease virus is expected to evolve naturally; thus, these findings suggest that some recent field isolates should be excluded from evolutionary studies. Furthermore, phylogenetic analyses show that these recent virulent isolates are more closely related to virulent strains isolated during the 1940s, which have been and continue to be used in laboratory and experimental challenge studies. Since the preservation of viable viruses in the environment for over 6 decades is highly unlikely, it is possible that the source of some of the recent virulent viruses isolated from poultry and wild birds might be laboratory viruses. PMID:26888902

  9. Characterization of hemagglutination activity of emerging Newcastle disease virus in Bangladesh

    Directory of Open Access Journals (Sweden)

    Helal Uddin

    2017-06-01

    Full Text Available Aim: Newcastle disease (ND is an important viral disease for poultry caused by avian paramyxovirus which can be identified by its nature of agglutination activity with red blood cell (RBC of different species. The study was aimed to characterize the hemagglutinating (HA activity of ND virus (NDV at three different temperatures using RBC of five avian species, six mammalian species, and eight different human blood groups. Materials and Methods: The study was conducted from January to December 2014 at Chittagong Veterinary and Animal Sciences University. Five avian and six different mammalian species were selected for the study. In each species, two blood samples were collected aseptically. Eight different blood groups (A+, A−, B+, B−, AB+, AB−, O+, and O− were studied in human. HA test was performed using two virus strains ND lasota and field isolate of very virulent NDV (VVNDV with mentioned species of RBC at chilling (4°C, incubating (37°C, and room temperature (24°C. Results: Avian RBC requires less time for agglutination than mammalian RBC. Incubation temperature (37°C requires lowest time and chilling temperature requires highest time for agglutination of RBC. Duck RBC requires lowest time (17.81 min while chicken RBC needs highest (57.5 min time for HA at incubation temperature and at chilling temperature, respectively, against ND lasota virus and with field strain. Goat RBC requires significantly higher time for HA (184.68 min at chilling temperature than other mammalian species. Human RBC requires almost similar time but O+ and O− blood group do not show any HA activity. Significant variation (p<0.05 found in quail RBC at incubation temperature. In mammalian species, a significant difference (p<0.05 has been observed in goat and horse RBC at chilling; horse and dog RBC at incubation; goat, horse, buffalo, and dog RBC at room temperature. In human, significant variation (p<0.05 has been found in A+, A− and B− blood group

  10. Effect of Newcastle Disease Control and Improved Management on the Performance of Indigenous Poultry in Western Kenya

    International Nuclear Information System (INIS)

    Ondwasy, H.O.; Okitoi, L.O.; Obali, M.P.; Mukisira, E.A.; Jong, R.

    1999-01-01

    indigenous poultry farming is an important enterprise for small-scale farmers in the mandate areas of the Regional Research Centre (RRC) Kakamega, A topical Participatory rural appraisal (PRA) study involving 407 farmers identified the main constraints to rural poultry production in the area as Newcastle disease (NCD), predation, and inadequate feeding or supplementation. The objectives of the study were to evaluate the effects of vaccination against Newcastle disease (NCD), daytime housing for chicks and feed supplementation on indigenous poultry production. The trial was implemented in four RRC clusters, each representing a specific agro-ecological zone.The clusters included Sabatia (UM 1 ), Butula (LM 1 ) Malava (LM 2 ), and Uranga (LM 3 ). Each cluster comprised of 22 to 35 experimental farmers in four groups assigned to the various treatments thus: Group 1 vaccination, 2. vaccination and supplementation, 3. vaccination, supplementation and daytime housing of chicks and 4. control group or farmer's practice. Since farmers adopted treatments according to their preference, the composition of each group varied as the experiment progressed. Birds were vaccinated after every three months to prevent NCD attacks,' To' avoid predation, the chicks were housed in movable or permanent structures where supplements such as brewers waste, blood and rumen contents from slaughterhouses were provided. From January 1997, data was collected weekly from individual households by frontline extension staff. Monthly monitoring and data verification on flock composition and dynamics, eggs production and utilisation, feed use and growth rates, was done by researchers and agricultural extensionists. The results demonstrated that Newcastle disease could be controlled by routine vaccination; feed supplementation improved the performance of housed birds in three clusters, but not in Sabatia where scarcity of feed was pronounced. It was further concluded that predation continued to constrain

  11. Molecular detection of infectious bronchitis and Newcastle disease viruses in broiler chickens with respiratory signs using Duplex RT-PCR.

    Science.gov (United States)

    Saba Shirvan, Aylar; Mardani, Karim

    2014-01-01

    Infectious bronchitis (IB) and Newcastle disease (ND) are highly contagious and the most economically important diseases of the poultry affecting respiratory tract and causing economic losses in poultry industry throughout the world. In the present study, the simultaneous detection and differentiation of causative agents of these diseases were investigated using duplex-RT-PCR. RNA was extracted from vaccinal and reference strains of infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) and then cDNA was synthesized. Using two universal primer sets for detection of IBV and NDV, the duplex-RT-PCR was developed. In order to assess the efficiency of the developed duplex RT-PCR, a number of 12 broiler farms with the symptoms of respiratory tract infection was sampled (trachea, lung and kidney were sampled from affected birds suspicious for IBV and NDV infections). After RNA extraction from tissues and cDNA synthesis, the presence of IBV and NDV genome were investigated using duplex-PCR. The results showed that three of twelve examined broiler farms were positive for IBV and two farms were positive for NDV and IBV. The results revealed that the duplex-RT-PCR is a quick and sensitive procedure for simultaneously detecting IBV and NDV in birds with respiratory infections.

  12. In vitro characterization of the antiviral activity of fucoidan from Cladosiphon okamuranus against Newcastle Disease Virus

    Directory of Open Access Journals (Sweden)

    Elizondo-Gonzalez Regina

    2012-12-01

    Full Text Available Abstract Background Newcastle Disease Virus (NDV causes a serious infectious disease in birds that results in severe losses in the worldwide poultry industry. Despite vaccination, NDV outbreaks have increased the necessity of alternative prevention and control measures. Several recent studies focused on antiviral compounds obtained from natural resources. Many extracts from marine organisms have been isolated and tested for pharmacological purposes, and their antiviral activity has been demonstrated in vitro and in vivo. Fucoidan is a sulfated polysaccharide present in the cell wall matrix of brown algae that has been demonstrated to inhibit certain enveloped viruses with low toxicity. This study evaluated the potential antiviral activity and the mechanism of action of fucoidan from Cladosiphon okamuranus against NDV in the Vero cell line. Methods The cytotoxicity of fucoidan was determined by the MTT assay. To study its antiviral activity, fusion and plaque-forming unit (PFU inhibition assays were conducted. The mechanism of action was determined by time of addition, fusion inhibition, and penetration assays. The NDV vaccine strain (La Sota was used in the fusion inhibition assays. PFU and Western blot experiments were performed using a wild-type lentogenic NDV strain. Results Fucoidan exhibited antiviral activity against NDV La Sota, with an obtained IS50 >2000. In time of addition studies, we observed viral inhibition in the early stages of infection (0–60 min post-infection. The inhibition of viral penetration experiments with a wild-type NDV strain supported this result, as these experiments demonstrated a 48% decrease in viral infection as well as reduced HN protein expression. Ribavirin, which was used as an antiviral control, exhibited lower antiviral activity than fucoidan and high toxicity at active doses. In the fusion assays, the number of syncytia was significantly reduced (70% inhibition when fucoidan was added before cleavage of

  13. Soroepidemiologia da doença de Newcastle em plantéis de avestruzes dos Estados da Bahia e de São Paulo Serologic occurrence of Newcastle disease in ostriches raised in Bahia and São Paulo

    Directory of Open Access Journals (Sweden)

    Lia Muniz Barretto Fernandes

    2010-02-01

    Full Text Available Estudos sorológicos em Avestruzes (Struthio camelus são ferramentas úteis para analisar os riscos relacionados à Doença de Newcastle nesses plantéis e à avicultura nacional. No presente estudo, amostras de sangue foram obtidas de avestruzes de ambos os sexos, de diferentes faixas etárias e sem apresentação de sintomatologia clínica, criadas nos Estados da Bahia e de São Paulo com o objetivo de avaliar a presença de anticorpos contra o vírus da Doença de Newcastle por meio de ELISA indireto. Foram testadas 339 amostras provenientes do Estado da Bahia e 105 amostras do Estado de São Paulo. Apesar de os proprietários afirmarem que não foi utilizada vacina em seus animais, foi verificada positividade na Bahia de 17,9% e de 4,7% em São Paulo, em avestruzes, sugerindo contato com vírus vacinal ou de campo.Serological studies in ostriches (Struthio camelus are important tools to assess the risk of Newcastle disease in these herds and to the national poultry industry. In the present study blood samples were obtained from male and female ostriches without symptoms of the disease, raised in Bahia and São Paulo in order to evaluate the presence of antibodies against Newcastle disease virus using an indirect ELISA. There were collected 339 samples in Bahia and 105 samples in São Paulo. Although the owners guarantee that animals were not vaccinated, it was verified the presence 17,9% positives in Bahia and 4,7% in São Paulo, suggesting contact with vaccinal or field strain.

  14. Antiviral activity of viro care gz-08 against newcastle disease virus in poultry and its in-vitro cytotoxicity assay

    International Nuclear Information System (INIS)

    Rasool, M.H.; Afzal, A.M.

    2014-01-01

    Newcastle disease (ND), one of the most important disease of poultry throughout the World is caused by Newcastle Disease Virus (NDV). It is causing huge economic losses in poultry industry of Pakistan. Regardless of vaccination, other prevention and control measures are necessary to prevent ND outbreaks. Natural resources have been exploited to obtain antiviral compounds in several latest studies. In this study, the antiviral activity of Viro Care GZ-081 was checked up in-vitro, in-ovo and in-vivo. The cytotoxicity assay of the product was performed using Vero cell line. All the trials revealed that the stock solution and 1:2 dilution of GZ-08 had some antiviral activity as well as were cytotoxic. As the concentration decreased, cytotoxicity as well as antiviral activities were lost. Based on these findings, it was concluded that GZ-08 sanitizer or spray can be used as antiviral agent to clean or disinfect some non-living surfaces against different viruses in general and NDV in particular. However, in-vivo use of GZ-08 in poultry against NDV is recommended only as pre-treatment with ND vaccines as it significantly reduced morbidity and mortality as compared to the use of vaccines alone. However, further work is recommended in future on GZ-08 for its use as post-treatment of ND as well as on other antiviral compounds of natural origin to develop a novel antiviral drug against NDV in poultry. (author)

  15. Partial antiviral activities detection of chicken Mx jointing with neuraminidase gene (NA against Newcastle disease virus.

    Directory of Open Access Journals (Sweden)

    Yani Zhang

    Full Text Available As an attempt to increase the resistance to Newcastle Disease Virus (NDV and so further reduction of its risk on the poultry industry. This work aimed to build the eukaryotic gene co-expression plasmid of neuraminidase (NA gene and myxo-virus resistance (Mx and detect the gene expression in transfected mouse fibroblasts (NIH-3T3 cells, it is most important to investigate the influence of the recombinant plasmid on the chicken embryonic fibroblasts (CEF cells. cDNA fragment of NA and mutant Mx gene were derived from pcDNA3.0-NA and pcDNA3.0-Mx plasmid via PCR, respectively, then NA and Mx cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-Mx-NA. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequencing, and it was transfected into the mouse fibroblasts (NIH-3T3 cells. The expression of genes in pVITRO2-Mx-NA were measured by RT-PCR and indirect immunofluorescence assay (IFA. The recombinant plasmid was transfected into CEF cells then RT-PCR and the micro-cell inhibition tests were used to test the antiviral activity for NDV. Our results showed that co-expression vector pVITRO2-Mx-NA was constructed successfully; the expression of Mx and NA could be detected in both NIH-3T3 and CEF cells. The recombinant proteins of Mx and NA protect CEF cells from NDV infection until after 72 h of incubation but the individually mutagenic Mx protein or NA protein protects CEF cells from NDV infection till 48 h post-infection, and co-transfection group decreased significantly NDV infection compared with single-gene transfection group (P<0. 05, indicating that Mx-NA jointing contributed to delaying the infection of NDV in single-cell level and the co-transfection of the jointed genes was more powerful than single one due to their synergistic effects.

  16. Production of Newcastle Disease Virus by Vero Cells Grown on Cytodex 1 Microcarriers in a 2-Litre Stirred Tank Bioreactor

    Directory of Open Access Journals (Sweden)

    Mohd Azmir Arifin

    2010-01-01

    Full Text Available The aim of this study is to prepare a model for the production of Newcastle disease virus (NDV lentogenic F strain using cell culture in bioreactor for live attenuated vaccine preparation. In this study, firstly we investigated the growth of Vero cells in several culture media. The maximum cell number was yielded by culture of Vero cells in Dulbecco's Modified Eagle Medium (DMEM which was 1.93×106 cells/ml. Secondly Vero cells were grown in two-litre stirred tank bioreactor by using several commercial microcarriers. We achieved the maximum cell concentration about 7.95×105 cells/ml when using Cytodex 1. Later we produced Newcastle Disease virus in stirred tank bioreactor based on the design developed using Taguchi L4 method. Results reveal that higher multiplicity of infection (MOI and size of cell inoculums can yield higher virus titer. Finally, virus samples were purified using high-speed centrifugation based on 3∗∗(3-1 Fractional Factorial Design. Statistical analysis showed that the maximum virus titer can be achieved at virus sample concentration of 58.45% (v/v, centrifugation speed of 13729 rpm, and centrifugation time of 4 hours. As a conclusion, high yield of virus titer could be achieved through optimization of cell culture in bioreactor and separation by high-speed centrifugation.

  17. Six-year surveillance of Newcastle disease virus in wild birds in north-eastern Spain (Catalonia).

    Science.gov (United States)

    Napp, Sebastian; Alba, Anna; Rocha, Ana Isabel; Sánchez, Azucena; Rivas, Raquel; Majó, Natalia; Perarnau, Mireia; Massot, Cristina; Miguel, Elena San; Soler, Mercé; Busquets, Núria

    2017-02-01

    Given that Newcastle disease (ND) is one of the major threats for the poultry industry, testing of Newcastle disease virus (NDV) has been carried out since 2010 in cases of mortality in wild birds (passive surveillance) in Catalonia. The objective is to provide an early warning system to prevent the infection of poultry. Since 2010, 35 episodes of mortality in wild birds were attributed to NDV infection. Throughout this period there was a progressive expansion of NDV to new areas, with an increase in the episodes of mortality, although it is not clear whether they were the result of the spread of the virus, or of the improvement of the surveillance. Phylogenetic analyses indicate that two distinct sublineages of NDV, 4a and 4b, were circulating in Catalonia. Both sublineages seem to be endemic in the wild bird population, affecting mainly Eurasian-collared doves, with a clear pattern in relation to its spatial distribution (coincident with the distribution of this species), and its temporal distribution (with the majority of cases between September and February). So far, endemicity in wild birds has not resulted in ND outbreaks in poultry. However, there are still many uncertainties about, for example, whether NDV may expand to new areas of Catalonia (with higher poultry density), or about the threat that the apparently more novel sublineage 4a may represent. Hence, efforts should be made so that measures to prevent infection of poultry farms (particularly in high-risk areas and periods) are encouraged, and surveillance is maintained.

  18. Recombinant Newcastle disease viral vector expressing hemagglutinin or fusion of canine distemper virus is safe and immunogenic in minks.

    Science.gov (United States)

    Ge, Jinying; Wang, Xijun; Tian, Meijie; Gao, Yuwei; Wen, Zhiyuan; Yu, Guimei; Zhou, Weiwei; Zu, Shulong; Bu, Zhigao

    2015-05-15

    Canine Distemper Virus (CDV) infects many carnivores and cause several high-mortality disease outbreaks. The current CDV live vaccine cannot be safely used in some exotic species, such as mink and ferret. Here, we generated recombinant lentogenic Newcastle disease virus (NDV) LaSota expressing either envelope glycoproyein, heamagglutinine (H) or fusion protein (F), named as rLa-CDVH and rLa-CDVF, respectively. The feasibility of these recombinant NDVs to serve as live virus-vectored CD vaccine was evaluated in minks. rLa-CDVH induced significant neutralization antibodies (NA) to CDV and provided solid protection against virulent CDV challenge. On the contrast, rLa-CDVF induced much lower NA to CDV and fail to protected mink from virulent CDV challenge. Results suggest that recombinant NDV expressing CDV H is safe and efficient candidate vaccine against CDV in mink, and maybe other host species. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Phylogenetic relationships and pathogenicity variation of two Newcastle disease viruses isolated from domestic ducks in Southern China.

    Science.gov (United States)

    Kang, Yinfeng; Li, Yanling; Yuan, Runyu; Li, Xianwei; Sun, Minhua; Wang, Zhaoxiong; Feng, Minsha; Jiao, Peirong; Ren, Tao

    2014-08-12

    Newcastle disease (ND) is an OIE listed disease caused by virulent avian paramyxovirus type 1 (APMV-1) strains, which is enzootic and causes large economic losses in the poultry sector. Genotype VII and genotype IX NDV viruses were the predominant circulating genotype in China, which may possibly be responsible for disease outbreaks in chicken flocks in recent years. While ducks and geese usually have exhibited inapparent infections. In the present study, we investigate the complete genome sequence, the clinicopathological characterization and transmission of two virulent Newcastle disease viruses, SS-10 and NH-10, isolated from domestic ducks in Southern China in 2010. F, and the complete gene sequences based on phylogenetic analysis demonstrated that SS-10 (genotype VII) and NH-10 (genotype IX) belongs to class II. The deduced amino acid sequence was (112)R-R-Q-K/R-R-F(117) at the fusion protein cleavage site. Animal experiment results showed that the SS-10 virus isolated from ducks was highly pathogenic for chickens and geese, but low pathogenic for ducks. It could be detected from spleen, lung, kidney, trachea, small intestine, bursa of fabricius, thymus, pancreas and cecal tonsils, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. Moreover, it could transmit to chickens, ducks and geese by naive contact. However, the NH-10 virus isolated from ducks could infect some chickens, ducks and geese, but only caused chickens to die. Additionally, it could transmit to the naive contact chickens, ducks, and geese. The two NDV isolates exhibited different biological properties with respect to pathogenicity and transmission in chickens, ducks and geese. Therefore, no species-preference exists for chicken, duck or goose viruses and more attention should be paid to the trans-species transmission of VII NDVs between ducks, geese and chickens for the control and eradication of ND.

  20. Pathotyping and Phylogenetic Characterization of Newcastle Disease Viruses Isolated in Peru: Defining Two Novel Subgenotypes Within Genotype XII.

    Science.gov (United States)

    Chumbe, Ana; Izquierdo-Lara, Ray; Tataje, Luis; Gonzalez, Rosa; Cribillero, Giovana; González, Armando E; Fernández-Díaz, Manolo; Icochea, Eliana

    2017-03-01

    Infections of poultry with virulent strains of avian paramyxovirus 1 (APMV-1), also known as Newcastle disease viruses (NDVs), cause Newcastle disease (ND). This highly contagious disease affects poultry and many other species of birds worldwide. In countries where the disease is prevalent, constant monitoring and characterization of isolates causing outbreaks are necessary. In this study, we report the results of pathogenicity testing and phylogenetic analyses of seven NDVs isolated from several regions of Peru between 2004 and 2015. Six viruses had intracerebral pathogenicity indices (ICPIs) of between 1.75 and 1.88, corresponding to a velogenic pathotype. The remaining virus had an ICPI of 0.00, corresponding to a lentogenic pathotype. These results were consistent with amino acid sequences at the fusion protein (F) cleavage site. All velogenic isolates had the polybasic amino acid sequence 112 RRQKR↓F 117 at the F cleavage site. Phylogenetic analyses of complete F gene sequences showed that all isolates are classified in class II of APMV-1. The velogenic viruses are classified in genotype XII, while the lentogenic virus is classified in genotype II, closely related to the LaSota vaccine strain. Moreover, tree topology, bootstrap values, and genetic distances observed within genotype XII resulted in the identification of novel subgenotypes XIIa (in South America) and XIIb (in China) and possibly two clades within genotype XIIa. All velogenic Peruvian viruses belonged to subgenotype XIIa. Overall, our results confirm the presence of genotype XII in Peru and suggest that it is the prevalent genotype currently circulating in our country. The phylogenetic characterization of these isolates helps to characterize the evolution of NDV and may help with the development of vaccines specific to our regional necessities.

  1. Complete Genome Sequence of a Virulent Newcastle Disease Virus Strain Isolated from a Clinically Healthy Duck (Anas platyrhynchos domesticus) in Pakistan

    Science.gov (United States)

    Wajid, Abdul; Rehmani, Shafqat F.; Wasim, Muhammad; Basharat, Asma; Bibi, Tasra; Arif, Saima; Dimitrov, Kiril M.

    2016-01-01

    Here, we report the complete genome sequence of a virulent Newcastle disease virus (vNDV) strain, duck/Pakistan/Lahore/AW-123/2015, isolated from apparently healthy laying ducks (Anas platyrhynchos domesticus) from the province of Punjab, Pakistan. The virus has a genome length of 15,192 nucleotides and is classified as member of subgenotype VIIi, class II. PMID:27469959

  2. Complete Genome Sequence of a Virulent Newcastle Disease Virus Strain Isolated from a Clinically Healthy Duck (Anas platyrhynchos domesticus) in Pakistan

    OpenAIRE

    Wajid, Abdul; Rehmani, Shafqat F.; Wasim, Muhammad; Basharat, Asma; Bibi, Tasra; Arif, Saima; Dimitrov, Kiril M.; Afonso, Claudio L.

    2016-01-01

    Here, we report the complete genome sequence of a virulent Newcastle disease virus (vNDV) strain, duck/Pakistan/Lahore/AW-123/2015, isolated from apparently healthy laying ducks (Anas platyrhynchos domesticus) from the province of Punjab, Pakistan. The virus has a genome length of 15,192 nucleotides and is classified as member of subgenotype VIIi, class II.

  3. Recombinant Newcastle disease virus (NDV) with inserted gene coding for GM-CSF as a new vector for cancer immunogene therapy

    NARCIS (Netherlands)

    Janke, M.; Peeters, B.P.H.; Leeuw, de O.S.; Moormann, R.J.M.; Arnold, A.; Fournier, P.; Schirrmacher, V.

    2007-01-01

    This is the first report describing recombinant (rec) Newcastle disease virus (NDV) as vector for gene therapy of cancer. The gene encoding granulocyte/macrophage colony-stimulating factor (GM-CSF) was inserted as an additional transcription unit at two different positions into the NDV genome. The

  4. Isolation and characterization of avian paramyxovirus type 1 (Newcastle disease) viruses from a flock of ostriches (Struthio camelus) and emus (Dromaius novaehollandiae) in Europe with inconsistent serology

    DEFF Research Database (Denmark)

    Jørgensen, Poul Henrik; Herczeg, J.; Lomniczi, B.

    1998-01-01

    of Newcastle disease in back yard poultry ire Denmark. Blood samples were taken from all live birds in the flock after 25 and 95 days of quarantine and all were negative for antibodies to APMV-1 in haemagglutination inhibition tests. All samples taken after 95 days of quarantine were also negative...

  5. Have we found an optimal insertion site in a Newcastle disease virus vector to express a foreign gene for vaccine and gene therapy purposes?

    Science.gov (United States)

    Using reverse genetics technology, many strains of Newcastle disease virus (NDV) have been developed as vectors to express foreign genes for vaccine and gene therapy purposes. The foreign gene is usually inserted into a non-coding region of the NDV genome as an independent transcription unit. Eval...

  6. Influence of a yeast fermented product on the serum levels of the mannan-binding lectin and the antibodies against the Newcastle disease virus in Ross broilers

    DEFF Research Database (Denmark)

    Cortés-Coronado, R F; Gómez-Rosales, S; de L Angeles, M

    2017-01-01

    The objective of this research was to evaluate the serum concentrations of mannan-binding lectin (MBL) at different ages in Ross broilers fed increasing amounts of a yeast-fermented product (YFP) and inoculated with a vaccine against Newcastle disease virus (NDV). Eighty mixed Ross B308 broilers...

  7. Maternally derived antibodies in commercial broiler chickens did not significantly interfere with protection of Newcastle disease virus vectored infectious laryngotracheitis vaccines

    Science.gov (United States)

    Newcastle disease virus (NDV) recombinants expressing the infectious laryngotracheitis virus (ILTV) glycoproteins B and D have previously been demonstrated to confer complete clinical protection against virulent ILTV and NDV challenges in naive chickens. However, there was a general concern that the...

  8. Generation of recombinant newcastle disease viruses, expressing the glycoprotein (G) of avian metapneumovirus, subtype A, or B, for use as bivalent vaccines

    Science.gov (United States)

    Using reverse genetics technology, Newcastle disease virus (NDV) LaSota strain-based recombinant viruses were engineered to express the glycoprotein (G) of avian metapneumovirus (aMPV), subtype A, or B, as bivalent vaccines. These recombinant viruses, rLS/aMPV-A G and rLS/aMPV-B G, were slightly att...

  9. Infection of goose with genotype VIId Newcastle disease virus of goose origin elicits strong immune responses at early stage

    Directory of Open Access Journals (Sweden)

    Qianqian Xu

    2016-10-01

    Full Text Available Newcastle disease (ND, caused by virulent strains of Newcastle disease virus (NDV, is a highly contagious disease of birds that is responsible for heavy economic losses for the poultry industry worldwide. However, little is known about host-virus interactions in waterfowl, goose. In this study, we aim to characterize the host immune response in goose, based on the previous reports on the host response to NDV in chickens. Here, we evaluated viral replication and mRNA expression of 27 immune-related genes in 10 tissues of geese challenged with a genotype VIId NDV strain of goose origin (go/CH/LHLJ/1/06. The virus showed early replication, especially in digestive and immune tissues. The expression profiles showed up-regulation of Toll-like receptor (TLR1–3, 5, 7 and 15, avian β-defensin (AvBD 5–7, 10, 12 and 16, cytokines interleukin (IL-8, IL-18, IL-1β and interferon-γ, inducible NO synthase (iNOS, and MHC class I in some tissues of geese in response to NDV. In contrast, NDV infection suppressed expression of AvBD1 in cecal tonsil of geese. Moreover, we observed a highly positive correlation between viral replication and host mRNA expressions of TLR1-5 and 7, AvBD4-6, 10 and 12, all the cytokines measured, MHC class I, FAS ligand, and iNOS, mainly at 72 h post-infection. Taken together, these results demonstrated that NDV infection induces strong innate immune responses and intense inflammatory responses at early stage in goose which may associate with the viral pathogenesis.

  10. Standardization of the indirect enzyme-linked immunosorbent assay for detection of antibodies against Newcastle disease virus in chickens

    International Nuclear Information System (INIS)

    Della Porta, A.J.; Young, J.; Hansson, E.; Spencer, T.

    1994-01-01

    Newcastle disease is the major viral disease of poultry causing significant economic losses in most countries except Australia and New Zealand. Serological monitoring of poultry has traditionally been carried out using the haemagglutinin-inhibition (HI) test. More recently, ELISA has been used for the same purpose. This paper described the use of an indirect ELISA for assay of antibodies in chickens against Newcastle disease viruses and compares some of the parameters for this test. The sucrose density gradient purified, inactivated, antigen enabled performance of the test without the addition of any blocking agents other than the usual Tween 20. A range of plates was compared and the most suitable plate was found to be a polystyrene haemagglutination plate giving an excellent positive to negative ratio of 33.2, compared with some expensive ELISA plates which gave very low +ve/-ve ratios. Various incubation conditions for the steps in the ELISA were compared and incubation with shaking at room temperature (24 to 28 deg. C) gave adequate reactivity whilst simplifying incubation conditions and speeding up the test. The negative cut-off value was determined by testing 1632 HI negative specific pathogen free sera from birds of a wide age range. The reactivity of sera in the ELISA was standardized using a standard curve on every plate and converting the readings to ELISA units (EUs) in the range of 16 to 512 EUs. The EU values of these sera were not normally distributed and so the 95% cut-off was determined by ranking the values in descending order and retaining only the top 5% of the values as false positives. This resulted in a cut-off value of 33.6 EUs, with few of HI positive sera having values lower than this cut-off. The use of a standard curve on each plate is recommended in order to standardize the assay and to determine the ELISA units for the test sera. (author). 14 refs, 2 figs, 1 tab

  11. Studies on the susceptibility of ostriches (Struthio camelus to the Indonesian velogenic strain of Newcastle disease virus

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    Darminto

    1998-12-01

    Full Text Available Susceptibility of ostriches (Struthio camelus to the Indonesian velogenic strain of Newcastle disease virus (NDV was evaluated by artificial infection . Twelve - 5 to 6 week old ostriches were divided into 3 groups each containing 4 birds . The first group was inoculated through respiratory system by dropping directly the virus solution into the nostrils, while the second group was inoculated through digestive system by dropping directly the virus solution into the oesophagus, with the dose of infection 106ELDSo (50%-embryo lethal dose per bird . Meanwhile, the third group was treated as uninfected control . All infected birds developed antibody responses, but only two inoculated birds from the first group and two inoculated birds from the second group developed clinical signs of Newcastle disease (ND, with no specific pathological alterations . Infected birds, either sicks or healthy, excreted the challenge viruses through the respiratory system and still be detected up to the end of this experiment, ie . 15 days post-inoculation . The challenge viruses can be re-isolated from the brain, trachea, lungs, heart, liver, spleen, kidneys, small intestine, cecal-tonsil, and proventriculus of the infected birds . This study concludes that: (1 the ostriches are susceptible to the infection of the Indonesian velogenic strain ofNDV; (2 all infected birds developed immune responses, but only half of them develops el jtigi aj i disease ; (3 the infected birds excreted the challenge viruses for a considerable long time which may play role as the Mginiseti.ce ofinfectron the other healthy ostriches ; and (4 the challenge viruses can be re-isolated from various organs of the birds . .

  12. Adjuvant Activity of Sargassum pallidum Polysaccharides against Combined Newcastle Disease, Infectious Bronchitis and Avian Influenza Inactivated Vaccines

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    Li-Jie Li

    2012-11-01

    Full Text Available This study evaluates the effects of Sargassum pallidum polysaccharides (SPP on the immune responses in a chicken model. The adjuvanticity of Sargassum pallidum polysaccharides in Newcastle disease (ND, infectious bronchitis (IB and avian influenza (AI was investigated by examining the antibody titers and lymphocyte proliferation following immunization in chickens. The chickens were administrated combined ND, IB and AI inactivated vaccines containing SPP at 10, 30 and 50 mg/mL, using an oil adjuvant vaccine as a control. The ND, IB and AI antibody titers and the lymphocyte proliferation were enhanced at 30 mg/mL SPP. In conclusion, an appropriate dose of SPP may be a safe and efficacious immune stimulator candidate that is suitable for vaccines to produce early and persistent prophylaxis.

  13. The response of ducks to V4 Newcastle disease virus and its transmission to contact ducks and domestic chickens

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    Majid Bouzari

    2014-06-01

    Full Text Available Experimental infection of Muscovy ducks with V4 strain of Newcastle disease virus was undertaken to determine the response of the ducks to the virus and the possibility of virus transmission to ducks and chickens in village like conditions. Twelve ducks were randomly and equally divided into three groups of control, inoculated and in-contact. Additionally, the chickens were placed into two groups of four animals each, namely in-contact and control. The inoculated and in-contact ducks and in-contact chickens were kept together. The eye drop route was used for inoculation and hemagglutination inhibition (HI antibodies were measured for assessment of antibody response and cloacal and pharyngeal swabs were used for detection of the virus. The primary antibody response of inoculated ducks was very high and rapid (geometric mean titers [Log base 2] of up to 5.75 ± 0.50. The in-contact ducks showed antibody response with the same pattern but lower titers than the inoculated ducks (geometric mean titers [Log base 2] of up to 3.25 ± 1.70. The in-contact chickens showed a slight increase of HI antibody (geometric mean titers [Log base 2] of up to 2.25 ± 1.25 while the control chickens did not show any increase. The antibody response indicated the transmission of the virus to contact ducks and chickens. A single isolation of virus confirmed the ability of ducks to excrete the virus. It was concluded that the V4 strain of Newcastle disease virus was highly antigenic for ducks, and ducks can transmit it to other ducks and also in-contact chickens.

  14. Dose-dependent Effect of T-2 Toxin on the Immunity against Newcastle Disease Virus in Chickens

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    M. Weber

    2006-01-01

    Full Text Available The effect of 2.35 (a and 4.18 (b mg kg-1 feed T-2 toxin dose for 14 days on the haemagglutination inhibition titres against Newcastle disease virus was investigated in broiler chickens. The animals were divided into four groups and two separate experiments were carried out (a, b: (1 intact control group; (2 birds were fed with T-2 toxin contaminated feed and not vaccinated; (3 repeatedly vaccinated (on day 23 of age control group which received uncontaminated feed; (4 birds were both repeatedly vaccinated and fed the T-2 toxin contaminated diet. Blood samples, from which sera titres were measured, were taken on days 7 and 14 of the experiments. It was found that heamagglutination titres were different in the two experiments even in the control (1 group because of the different efficiency of the first immunization at the hatchery. Titres on day 7 showed increases in all groups except for the group fed lower T-2 contaminated diet (a, group 2 but during the second week they increased only in the groups fed the diet with a lower dose of T-2 toxin. On the contrary, a higher dose of T-2 toxin contamination of the diet resulted in a dramatic decrease during the second week (b, groups 2 and 4. The results suggested that contrary to most of the previously published data, feeding of T-2 toxin contaminated feed with an amount of 2.35 mg.kg-1 did not decrease, but increase the antibody formation against attenuated Newcastle disease virus even without a second vaccination on day 1 of the experiment, whereas a higher amount of T-2 toxin (4.18 mg kg-1 decreased to day 14 after the repeated vaccination.

  15. Immunogenicity of commercial, formaldehyde and binary ethylenimine inactivated Newcastle disease virus vaccines in specific pathogen free chickens

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    Razmaraii, N.

    2012-06-01

    Full Text Available Newcastle disease (ND is one of the most important diseases that affect birds; the epizootic nature of the disease has caused severe economic losses in the poultry industry worldwide. In this experiment ND virus (NDV was inactivated by two different chemicals binary ethylenimine (BEI and formaldehyde. Formaldehyde was used at 0.1%, while BEI was used at concentrations of 1 to 4 mM. NDV inactivation with BEI was done in various incubation temperatures and periods and the best result (30 °C, 4 mM BEI and 21 hrs treatment used as an experimental vaccine. Prepared inactivated NDV vaccines and a commercial vaccine were tested for their efficiency in generating humoral immune response in different groups of specific pathogen free (SPF chicks. Test groups received 0.2 ml formaldehyde inactivated NDV (NDVF, BEI inactivated NDV (NDVEI and Razi institute produced NDV vaccine (NDVR subcutaneously respectively. HI Log 2 total mean titer of NDVEI group (8.42 ± 0.12 were significantly higher than NDVF (7.64 ± 0.16 and NDVR (7.86 ± 0.11 groups (p<0.05. BEI-inactivated vaccine gave higher antibody titers than formaldehyde-inactivated vaccine and preserves both structural integrity and antigenicity of the virus. Thus, it might be possible to use these compounds as an inactivator agent for commercial NDV inactivated vaccines in future.

  16. Genomic selection for the improvement of antibody response to Newcastle disease and avian influenza virus in chickens.

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    Tianfei Liu

    Full Text Available Newcastle disease (ND and avian influenza (AI are the most feared diseases in the poultry industry worldwide. They can cause flock mortality up to 100%, resulting in a catastrophic economic loss. This is the first study to investigate the feasibility of genomic selection for antibody response to Newcastle disease virus (Ab-NDV and antibody response to Avian Influenza virus (Ab-AIV in chickens. The data were collected from a crossbred population. Breeding values for Ab-NDV and Ab-AIV were estimated using a pedigree-based best linear unbiased prediction model (BLUP and a genomic best linear unbiased prediction model (GBLUP. Single-trait and multiple-trait analyses were implemented. According to the analysis using the pedigree-based model, the heritability for Ab-NDV estimated from the single-trait and multiple-trait models was 0.478 and 0.487, respectively. The heritability for Ab-AIV estimated from the two models was 0.301 and 0.291, respectively. The estimated genetic correlation between the two traits was 0.438. A four-fold cross-validation was used to assess the accuracy of the estimated breeding values (EBV in the two validation scenarios. In the family sample scenario each half-sib family is randomly allocated to one of four subsets and in the random sample scenario the individuals are randomly divided into four subsets. In the family sample scenario, compared with the pedigree-based model, the accuracy of the genomic prediction increased from 0.086 to 0.237 for Ab-NDV and from 0.080 to 0.347 for Ab-AIV. In the random sample scenario, the accuracy was improved from 0.389 to 0.427 for Ab-NDV and from 0.281 to 0.367 for Ab-AIV. The multiple-trait GBLUP model led to a slightly higher accuracy of genomic prediction for both traits. These results indicate that genomic selection for antibody response to ND and AI in chickens is promising.

  17. Phylogenetic and Pathotypic Characterization of a Newcastle Disease Virus Strain Isolated from Ducks and Pigeons in Hubei, China

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    Y Wang

    Full Text Available ABSTRACT Newcastle disease is a highly contagious disease responsible for major outbreaks and considerable economic losses in the poultry industry in China. There is still little information available regarding gene characterization of the NDV, especially in ducks and pigeons. Therefore, the aim of this study was to investigate NDV isolated from ducks and pigeons in Hubei, China. In this study, three NDVs from ducks and pigeons were isolated between 2013 and 2015.The fusion protein (F gene of the NDV isolates was sequenced and phylogenetically analyzed. The clinical signs and gross histopathological lesions were examined. Phylogenetic analysis of these strains indicated that all the sequences are classified as genotype II. The isolates shared a 112 G-R-Q-G-R-L 117motif at the F protein cleavage site, indicating that these three isolates strains are lentogenic. Necropsy and histopathology showed the typical pathological changes. It was concluded that commercial ducks and pigeons in Hubei province carry lentogenic NDV strains with regular genetic divergence, indicating that these species may act as the main reservoirs of NDV in poultry. Therefore, strategies and surveillance should be undertaken to reduce the risk of ND outbreaks.

  18. Effects of Dietary Supplemental Vitamins and Periods of Administration on Growth Performance and Antibody Titre of Broiler Chickens Vaccinated against Newcastle Disease

    OpenAIRE

    Odutayo, O. J.; Sogunle, O. M.; Adeyemi, O.A.; Sonibare, A.O.; Oluwayinka, E.B.; Ekunseitan, D.A.; Safiyu1, K. K.

    2016-01-01

    This study investigated the effects of supplemental vitamins and varying administration periods on growth performance and antibody titre of broiler chickens vaccinated against Newcastle Disease (ND). A total of 300 unvaccinated against ND Arbor Acre day-old chicks were used for the study for 8 wk. Birds were brooded together on day 1 of age, and 30 chicks were selected randomly for evaluating the maternally derived antibody titre against ND. At 2 days of age, the remaining 270 chicks were div...

  19. Sequence and phylogenetic analysis of virulent Newcastle disease virus isolates from Pakistan during 2009–2013 reveals circulation of new sub genotype

    International Nuclear Information System (INIS)

    Siddique, Naila; Naeem, Khalid; Abbas, Muhammad Athar; Ali Malik, Akbar; Rashid, Farooq; Rafique, Saba; Ghafar, Abdul; Rehman, Abdul

    2013-01-01

    Despite observing the standard bio-security measures at commercial poultry farms and extensive use of Newcastle disease vaccines, a new genotype VII-f of Newcastle disease virus (NDV) got introduced in Pakistan during 2011. In this regard 300 ND outbreaks recorded so far have resulted into huge losses of approximately USD 200 million during 2011–2013. A total of 33 NDV isolates recovered during 2009–2013 throughout Pakistan were characterized biologically and phylogenetically. The phylogenetic analysis revealed a new velogenic sub genotype VII-f circulating in commercial and domestic poultry along with the earlier reported sub genotype VII-b. Partial sequencing of Fusion gene revealed two types of cleavage site motifs; lentogenic 112 GRQGRL 117 and velogenic 112 RRQKRF 117 along with some point mutations indicative of genetic diversity. We report here a new sub genotype of virulent NDV circulating in commercial and backyard poultry in Pakistan and provide evidence for the possible genetic diversity which may be causing new NDV out breaks. - Highlights: • The first report of isolation of new genotype VII-f of virulent Newcastle disease virus (NDV) in Pakistan. • We report the partial Fusion gene sequences of new genotype VII-f of virulent NDV from Pakistan. • We report the phylogenetic relationship of new NDV strains with reported NDV strains. • Provide outbreak history of new virulent NDV strain in commercial and backyard poultry in Pakistan. • We provide possible evidence for the role of backyard poultry in NDV outbreaks

  20. Two avirulent, lentogenic strains of Newcastle disease virus are cytotoxic for some human pancreatic tumor lines in vitro.

    Science.gov (United States)

    Walter, Robert J; Attar, Bashar M; Rafiq, Asad; Delimata, Megan; Tejaswi, Sooraj

    2012-09-10

    Pancreatic cancer is the fourth leading cause of cancer death in the U.S. Highly infectious Newcastle disease virus (NDV) strains are known to be very cytotoxic for an array of human tumor cell types in vitro and in vivo but the effects of these and avirulent NDV strains on pancreatic neoplasms are little known. Here, the direct cytolytic effects of the avirulent Hitchner-B1 (B1) and Ulster (U) NDV strains on 7 human pancreatic tumor cell lines and 4 normal human cell lines were studied. Cytotoxicity assays used serially diluted NDV to determine minimum cytotoxic plaque forming unit (PFU) doses. For NDV-B1, normal human cells were killed only by relatively high doses (range: 471-3,724 PFU) whereas NDV-U killed these cells at low PFU (range: 0.32-1.60 PFU). Most pancreatic cancer cell types were killed by much lower NDV-B1 doses (range: 0.40-2.60 PFU) while NDV-U killed Capan-1 and SU.86.86 cultures at very low doses (0.00041 PFU and 0.0034 PFU, respectively). On average, 1,555 times more NDV-B1 was needed to kill normal cells than most pancreatic tumor cells and 558 times more NDV-U to kill the two most sensitive pancreatic cancer lines. These innately-targeted lentogenic viruses may have meaningful potential in treating pancreatic cancer.

  1. Perturbations in the antioxidant metabolism during Newcastle disease virus (NDV) infection in chicken. Protective role of vitamin E

    Science.gov (United States)

    Subbaiah, Kadiam C. Venkata; Raniprameela, D.; Visweswari, Gopalareddygari; Rajendra, Wudayagiri; Lokanatha, Valluru

    2011-12-01

    The aim of the present study was to investigate the effect of vitamin E on pro/anti-oxidant status in the liver, brain and heart of Newcastle disease virus (NDV) infected chickens. Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione- S-transferase (GST) and the levels of reduced glutathione and malonaldehyde were estimated in selected tissues of uninfected, NDV-infected and NDV + vit. E-treated chickens. A significant increase in MDA levels in brain and liver ( p neuronal necrosis and degeneration of Purkinje cells were observed in brain and moderate infiltration of inflammatory cells was observed in heart. However such histological alterations were not observed in NDV + vit. E-treated animals. The results of the present study, thus demonstrated that antioxidant defense mechanism is impaired after the induction of NDV, suggesting its critical role in cellular injury in brain and liver. Further, the results also suggest that vitamin E treatment will ameliorate the antioxidant status in the infected animals. The findings could be beneficial to understand the role of oxidative stress in the pathogenesis of NDV and therapeutic interventions of antioxidants.

  2. The application of gene-based technologies in the study of Newcastle disease virus isolates from Uganda

    International Nuclear Information System (INIS)

    Otim, M.O.; Bisgaard, M.; Christensen, H.; Jorgensen, P.; Handberg, K.

    2005-01-01

    Molecular techniques were used to characterize 16 Newcastle disease (ND) Virus (NDV) isolates from ND outbreaks in chickens in eastern Uganda in 2001, and evaluate ND epidemiology, with emphasis on molecular aspects. F and HN genes, which are the major determinants of virulence, were studied. Strain pathogenicity was derived from genetic analysis of the F gene sequence and intracerebral pathogenicity index (ICPI). Comparative genetic and phylogenetic tree analyses were performed on the HN genes of the isolates and some strains selected from GenBank. ClustalX 1.81 and Phylip were used for gene alignment analysis and the final phylogeny was produced by the neighbour-joining method. F gene cleavage site sequence analysis, phylogenetic analysis and biological characterization showed that the strains were very virulent and closely related, being of common ancestry. All the Ugandan NDV isolates formed separate clades from the currently known genotypes, suggesting that they are a novel genotype, unrelated to those that have caused previous pandemics. (author)

  3. The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication.

    Science.gov (United States)

    Duan, Zhiqiang; Chen, Jian; Xu, Haixu; Zhu, Jie; Li, Qunhui; He, Liang; Liu, Huimou; Hu, Shunlin; Liu, Xiufan

    2014-03-01

    The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30-60 of M and amino acids 188-245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Effects of Newcastle Disease Virus Strains AF2240 and V4-UPM on Cytolysis and Apoptosis of Leukemia Cell Lines

    Science.gov (United States)

    Alabsi, Aied M.; Bakar, Siti Aishah Abu; Ali, Rola; Omar, Abdul Rahman; Bejo, Mohd Hair; Ideris, Aini; Ali, Abdul Manaf

    2011-01-01

    Newcastle disease virus (NDV) is used as an antineoplastic agent in clinical tumor therapy. It has prompted much interest as an anticancer agent because it can replicate up to 10,000 times better in human cancer cells than in most normal cells. This study was carried out to determine the oncolytic potential of NDV strain AF2240 and V4-UPM on WEHI-3B leukemia cell line. Results from MTT cytotoxicity assay showed that the CD50 values for both strains were 2 and 8 HAU for AF2240 and V4-UPM, respectively. In addition, bromodeoxyuridine (BrdU) and trypan blue dye exclusion assays showed inhibition in cell proliferation after different periods. Increase in the cellular level of caspase-3 and detection of DNA laddering using agarose gel electrophoresis on treated cells with NDV confirmed that the mode of cell death was apoptosis. In addition, flow-cytometry analysis of cellular DNA content showed that the virus caused an increase in the sub-G1 region (apoptosis peaks). In conclusion, NDV strains AF2240 and V4-UPM caused cytolytic effects against WEHI-3B leukemic cell line. PMID:22272097

  5. Molecular characterization of Newcastle disease viruses in Ostriches (Struthio camelus L.): further evidences of recombination within avian paramyxovirus type 1.

    Science.gov (United States)

    Yin, Yanbo; Cortey, Martí; Zhang, Yi; Cui, Shangjin; Dolz, Roser; Wang, Jianlin; Gong, Zhenhua

    2011-05-05

    Newcastle disease virus (NDV) strains isolated from ostriches have been genotyped for the first time by partial sequencing of the F gene to determine the epidemiologic role that this species can play within ND outbreaks. Fifteen additional NDV strains, mostly isolated from chickens but also from pigeons and penguins, were also included in the study to determine genetic relationships with ostriches NDV isolates. High genetic diversity was demonstrated in ostrich NDV isolates, as the 10 isolates were grouped in four distinct NDV genotypes. In agreement with the results obtained when chicken isolates have been molecularly characterized, the predominant genotype in ostriches was the genotype VII. More interestingly, evidences of recombination between genotype II and VII were observed in one ostrich isolate and in two further chicken isolates. Therefore, it seems that ostriches may play a relevant role in the ecology and epidemiology of ND particularly in those regions where they have an increasing farming importance as minor poultry species. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Patho-epidemiological study on Genotype-XIII Newcastle disease virus infection in commercial vaccinated layer farms

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    J. H. Khorajiya

    2015-03-01

    Full Text Available Aim: The present research work was carried out to study the patho-epidemiological aspects of Genotype-XIII Newcastle disease virus (NDV infection in commercial layer in and around Anand, Gujarat. As the outbreaks have reported in vaccinated flocks, it was felt necessary to study the disease with respect to its changing pathogenicity and relevant aspects. Materials and Methods: The study comprised of patho-epidemiology of Newcastle disease (ND by information collected from different layer farms suffering from the disease in relation to incidence pattern and mortality, duration of mortality, susceptible age, and loss due to production performance. Clinical signs were recorded based on observations. During postmortem, gross lesions were also recorded. For histopathological examination visceral organs according to lesions were collected in 10% formalin and processed slide stained by hematoxylin and eosin for microscopic examination. Cultivation of virus was done in embryonated specific pathogen-free (SPF eggs of 9-11 days and isolation of virus was done for haemagglutination (HA and haemagglutination inhibition (HI test and to identify pathotype of virus by intracerebral pathogenicity index (ICPI test to determine the virulence of virus. The Genotype-XIII NDV was confirmed by F gene sequence and whole genome sequence. Results: During the study mortality due to ND was recorded in 13 layer flocks in spite of routine vaccination, which usually contain Genotype-II strain of virus. The mortality was observed as high as above 50% with an average of 21.21%. The susceptible age for disease was found to be 6-14 weeks. The duration of mortality observed was 23 days. The disease resulted in a significant reduction in body weight, feed intake and drop in egg production. Majority of the outbreaks appeared during extremely hot months of April to June. Greenish diarrhoea was frequently seen in birds that survived early in infection. Mortality continued for 2

  7. Virus interference between H7N2 low pathogenic avian influenza virus and lentogenic Newcastle disease virus in experimental co-infections in chickens and turkeys

    OpenAIRE

    Costa-Hurtado, Mar; Afonso, Claudio L; Miller, Patti J; Spackman, Erica; Kapczynski, Darrell R; Swayne, David E; Shepherd, Eric; Smith, Diane; Zsak, Aniko; Pantin-Jackwood, Mary

    2014-01-01

    International audience; Low pathogenicity avian influenza virus (LPAIV) and lentogenic Newcastle disease virus (l NDV) are commonly reported causes of respiratory disease in poultry worldwide with similar clinical and pathobiological presentation. Co-infections do occur but are not easily detected, and the impact of co-infections on pathobiology is unknown. In this study chickens and turkeys were infected with a l NDV vaccine strain (LaSota) and a H7N2 LPAIV (A/turkey/VA/SEP-67/2002) simultan...

  8. Chicken galectin-1B inhibits Newcastle disease virus adsorption and replication through binding to hemagglutinin-neuraminidase (HN) glycoprotein.

    Science.gov (United States)

    Sun, Junfeng; Han, Zongxi; Qi, Tianming; Zhao, Ran; Liu, Shengwang

    2017-12-08

    Galectin-1 is an important immunoregulatory factor and can mediate the host-pathogen interaction via binding glycans on the surface of various viruses. We previously reported that avian respiratory viruses, including lentogenic Newcastle disease virus (NDV), can induce up-regulation of chicken galectin (CG)-1B in the primary target organ. In this study, we investigated whether CG-1B participated in the infectious process of NDV in chickens. We demonstrated that velogenic NDV induced up-regulation of CG-1B in target organs. We also found that CG-1B directly bound to NDV virions and inhibited their hemagglutination activity in vitro We confirmed that CG-1B interacted with NDV hemagglutinin-neuraminidase (HN) glycoprotein, in which the specific G4 N -glycans significantly contributed to the interaction between CG-1B and HN glycoprotein. The presence of extracellular CG-1B, rather than the internalization process, inhibited adsorption of NDV. The interaction between intracellular CG-1B and NDV HN glycoproteins inhibited cell-surface expression of HN glycoprotein and reduced the titer of progeny virus in NDV-infected DF-1 cells. Significantly, the replication of parental and HN glycosylation mutant viruses in CG-1B knockdown and overexpression cells demonstrated that the replication of NDV was correlated with the expression of CG-1B in a specific glycan-dependent manner. Collectively, our results indicate that CG-1B has anti-NDV activity by binding to N -glycans on HN glycoprotein. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Phylogenetic and pathotypic characterization of Newcastle disease viruses circulating in South China and transmission in different birds

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    Yinfeng eKang

    2016-02-01

    Full Text Available Although Newcastle disease virus (NDV with high pathogenicity has frequently been isolated in poultry in China since 1948, the mode of its transmission among avian species remains largely unknown. Given that various wild bird species have been implicated as sources of transmission, in this study we genotypically and pathotypically characterized 23 NDV isolates collected from chickens, ducks, and pigeons in live bird markets (LBMs in South China as part of an H7N9 surveillance program during December 2013–February 2014. To simulate the natural transmission of different kinds of animals in LBMs, we selected three representative NDVs—namely, GM, YF18, and GZ289—isolated from different birds to evaluate the pathogenicity and transmission of the indicated viruses in chickens, ducks, and pigeons. Furthermore, to investigate the replication and shedding of NDV in poultry, we inoculated the chickens, ducks, and pigeons with 106 EID50 of each virus via intraocular and intranasal routes. Eight h after infection, the naïve contact groups were housed with those inoculated with each of the viruses as a means to monitor contact transmission. Our results indicated that genetically diverse viruses circulate in LBMs in South China’s Guangdong Province and that NDV from different birds have different tissue tropisms and host ranges when transmitted in different birds. We therefore propose the continuous epidemiological surveillance of LBMs to support the prevention of the spread of these viruses in different birds, especially chickens, and highlight the need for studies of the virus–host relationship.

  10. Lack of detection of host associated differences in Newcastle disease viruses of genotype VIId isolated from chickens and geese

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    Wang Yuyang

    2012-09-01

    Full Text Available Abstract Background The goose is usually considered to be resistant even to strains of Newcastle disease virus (NDV that are markedly virulent for chickens. However, ND outbreaks have been frequently reported in goose flocks in China since the late 1990s with the concurrent emergence of genotype VIId NDV in chickens. Although the NDVs isolated from both chickens and geese in the past 15 years have been predominantly VIId viruses, published data comparing goose- and chicken-originated ND viruses are scarce and controversial. Results In this paper, we compared genotype VIId NDVs originated from geese and chickens genetically and pathologically. Ten entire genomic sequences and 329 complete coding sequences of individual genes from genotype VIId NDVs of both goose- and chicken-origin were analyzed. We then randomly selected two goose-originated and two chicken-originated VIId NDVs and compared their pathobiology in both geese and chickens in vivo and in vitro with genotype IV virus Herts/33 as a reference. The results showed that all the VIId NDVs either from geese or from chickens shared high sequence homology and characteristic amino acid substitutions and clustered together in phylogenetic trees. In addition, geese and chickens infected by goose or chicken VIId viruses manifested very similar pathological features distinct from those of birds infected with Herts/33. Conclusions There is no genetic or phenotypic difference between genotype VIId NDVs originated from geese and chickens. Therefore, no species-preference exists for either goose or chicken viruses and more attention should be paid to the trans-species transmission of VIId NDVs between geese and chickens for the control and eradication of ND.

  11. Immune responses of mature chicken bone-marrow-derived dendritic cells infected with Newcastle disease virus strains with differing pathogenicity.

    Science.gov (United States)

    Xiang, Bin; Zhu, Wenxian; Li, Yaling; Gao, Pei; Liang, Jianpeng; Liu, Di; Ding, Chan; Liao, Ming; Kang, Yinfeng; Ren, Tao

    2018-06-01

    Infection of chickens with virulent Newcastle disease virus (NDV) is associated with severe pathology and increased morbidity and mortality. The innate immune response contributes to the pathogenicity of NDV. As professional antigen-presenting cells, dendritic cells (DCs) play a unique role in innate immunity. However, the contribution of DCs to NDV infection has not been investigated in chickens. In this study, we selected two representative NDV strains, i.e., the velogenic NDV strain Chicken/Guangdong/GM/2014 (GM) and the lentogenic NDV strain La Sota, to investigate whether NDVs could infect LPS-activated chicken bone-derived marrow DCs (mature chicken BM-DCs). We compared the viral titres and innate immune responses in mature chicken BM-DCs following infection with those strains. Both NDV strains could infect mature chicken BM-DC, but the GM strain showed stronger replication capacity than the La Sota strain in mature chicken BM-DCs. Gene expression profiling showed that MDA5, LGP2, TLR3, TLR7, IFN-α, IFN-β, IFN-γ, IL-1β, IL-6, IL-18, IL-8, CCL5, IL-10, IL-12, MHC-I, and MHC-II levels were altered in mature DCs after infection with NDVs at all evaluated times postinfection. Notably, the GM strain triggered stronger innate immune responses than the La Sota strain in chicken BM-DCs. However, both strains were able to suppress the expression of some cytokines, such as IL-6 and IFN-α, in mature chicken DCs at 24 hpi. These data provide a foundation for further investigation of the role of chicken DCs in NDV infection.

  12. UV irradiation analysis of complementation between, and replication of, RNA-negative temperature-sensitivie mutants of Newcastle disease virus

    International Nuclear Information System (INIS)

    Peeples, M.E.; Bratt, M.A.

    1982-01-01

    Random uv irradiation-induced lesions destroy the infectivity of Newcastle disease virus (NDV) by blocking downstream transcription from the single viral promoter. The nucleocapsid-associated polypeptides most likely to be involved in RNA synthesis are located at the extreme ends of the genome: NP and P are promoter proximal genes, and L is the most distal gene. We attempted to order the two temperature-sensitive (ts) RNA-negative (RNA-) mutant groups of NDV by determining the uv target sizes for the complementing abilities of mutants A1 and E1. After uv irradiation, E1 was unable to complement A1, a result compatible with the A mutation lying in the L gene. In contrast, after uv irradiation A1 was able to complement E1 for both virus production and viral protein synthesis, with a target size most consistent with the E mutation lying in the P gene. UV-irradiated virus was unable to replicate as indicated by its absence in the yields of multiply infected cells, either as infectious virus or as particles with complementing activity. After irradiation, ts mutant B1ΔP, with a non-ts mutation affecting the electrophoretic mobility of the P protein, complemented E1 in a manner similar to A1, but it did not amplify the expression of ΔP in infected cells. This too is consistent with irradiated virus being unable to replicate despite the presence of the components needed for replication of E1. At high uv doses, A1 was able to complement E1 in a different, uv-resistant manner, probably by direct donation of input polypeptides. Multiplicity reactivation has previously been observed at high-multiplicity infection by uv-irradiated paramyxoviruses. In this case, virions which are noninfectious because they lack a protein component may be activated by a protein from irradiated virions

  13. Molecular characterization of velogenic viscerotropic Ranikhet (Newcastle) disease virus from different outbreaks in desi chickens

    OpenAIRE

    Dhaygude, V. S.; Sawale, G. K.; Chawak, M. M.; Bulbule, N. R.; Moregaonkar, S. D.; Gavhane, D. S.

    2017-01-01

    Aim: Diagnosis of velogenic viscerotropic Ranikhet disease from six different flocks of desi chicken in and around Mumbai by gross and histopathological examination, isolation of virus and molecular methods. Materials and Methods: A total of 25 carcasses (varying between 2 and 6 carcasses from each flock) of six different flocks of adult desi chicken were subjected to necropsy examination for diagnosis of the disease during the span of a year (2014-2015). After thorough gross examination,...

  14. Sero-surveillance and risk factors for avian influenza and Newcastle disease virus in backyard poultry in Oman.

    Science.gov (United States)

    Shekaili, Thunai Al; Clough, Helen; Ganapathy, Kannan; Baylis, Matthew

    2015-11-01

    Avian Influenza (AI) and Newcastle disease (ND) are the most important reportable poultry diseases worldwide. Low pathogenic AI (H9N2) and ND viruses are known to have been circulating in the Middle East, including in Oman, for many decades. However, detailed information on the occurrence of these pathogens is almost completely lacking in Oman. As backyard poultry are not vaccinated against either virus in Oman, this sector is likely to be the most affected poultry production sector for both diseases. Here, in the first survey of AI and ND viruses in backyard poultry in Oman, we report high flock-level seroprevalences of both viruses. Serum and oropharyngeal swabs were taken from 2350 birds in 243 backyard flocks from all regions and governorates of Oman. Information was recorded on location, type of bird and housing type for each sampled farm. Individual bird serum samples were tested using commercial indirect antibody detection ELISA kits. Pooled oropharyngeal samples from each flock were inoculated onto FTA cards and tested by RT-PCR. Samples came from chickens (90.5%), turkeys (2.1%), ducks (6.2%), guinea fowl (0.8%) and geese (0.4%). The bird-level seroprevalence of antibody to AI and ND viruses was 37.5% and 42.1% respectively, and at the flock level it was 84% and 90% respectively. There were statistically significant differences between some different regions of Oman in the seroprevalence of both viruses. Flock-level NDV seropositivity in chickens was significantly associated with AIV seropositivity, and marginally negatively associated with flock size. AIV seropositivity in chickens was marginally negatively associated with altitude. All oropharyngeal samples were negative for both viruses by RT-PCR, consistent with a short duration of infection. This study demonstrates that eight or nine out of ten backyard poultry flocks in Oman are exposed to AI and ND viruses, and may present a risk for infection for the commercial poultry sector in Oman, or wild birds

  15. Sequence and phylogenetic analysis of virulent Newcastle disease virus isolates from Pakistan during 2009–2013 reveals circulation of new sub genotype

    Energy Technology Data Exchange (ETDEWEB)

    Siddique, Naila, E-mail: naila.nrlpd@gmail.com [National Reference Laboratory for Poultry Diseases, Animal Sciences Institute, National Agricultural Research Center, Islamabad (Pakistan); Naeem, Khalid; Abbas, Muhammad Athar; Ali Malik, Akbar; Rashid, Farooq; Rafique, Saba; Ghafar, Abdul; Rehman, Abdul [National Reference Laboratory for Poultry Diseases, Animal Sciences Institute, National Agricultural Research Center, Islamabad (Pakistan)

    2013-09-15

    Despite observing the standard bio-security measures at commercial poultry farms and extensive use of Newcastle disease vaccines, a new genotype VII-f of Newcastle disease virus (NDV) got introduced in Pakistan during 2011. In this regard 300 ND outbreaks recorded so far have resulted into huge losses of approximately USD 200 million during 2011–2013. A total of 33 NDV isolates recovered during 2009–2013 throughout Pakistan were characterized biologically and phylogenetically. The phylogenetic analysis revealed a new velogenic sub genotype VII-f circulating in commercial and domestic poultry along with the earlier reported sub genotype VII-b. Partial sequencing of Fusion gene revealed two types of cleavage site motifs; lentogenic {sup 112}GRQGRL{sup 117} and velogenic {sup 112}RRQKRF{sup 117} along with some point mutations indicative of genetic diversity. We report here a new sub genotype of virulent NDV circulating in commercial and backyard poultry in Pakistan and provide evidence for the possible genetic diversity which may be causing new NDV out breaks. - Highlights: • The first report of isolation of new genotype VII-f of virulent Newcastle disease virus (NDV) in Pakistan. • We report the partial Fusion gene sequences of new genotype VII-f of virulent NDV from Pakistan. • We report the phylogenetic relationship of new NDV strains with reported NDV strains. • Provide outbreak history of new virulent NDV strain in commercial and backyard poultry in Pakistan. • We provide possible evidence for the role of backyard poultry in NDV outbreaks.

  16. Protection by recombinant Newcastle disease viruses (NDV) expressing the glycoprotein (G) of avian metapneumovirus (aMPV) subtype A or B against challenge with virulent NDV and aMPV

    Science.gov (United States)

    Avian metapneumovirus (aMPV) and Newcastle disease virus (NDV) are threatening avian pathogens that cause sporadic but serious respiratory diseases in poultry worldwide. Although, vaccination, combined with strict biosecurity practices, has been the recommendation for controlling these diseases in t...

  17. Generation and evaluation of a recombinant Newcastle disease virus (NDV) expressing the F and G proteins of avian metapneumovirus subtype C (aMPV-C) as a bivalent vaccine against NDV and aMPV-C challenges in turkeys

    Science.gov (United States)

    Virulent strains of Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) can cause serious respiratory diseases in poultry. Vaccination combined with strict biosecurity practices has been the recommendation for controlling NDV and aMPV diseases in the field. Previously we generated a NDV r...

  18. Newcastle disease virus-based H5 influenza vaccine protects chickens from lethal challenge with a highly pathogenic H5N2 avian influenza virus

    OpenAIRE

    Ma, Jingjiao; Lee, Jinhwa; Liu, Haixia; Mena, Ignacio; Davis, A. Sally; Sunwoo, Sun Young; Lang, Yuekun; Duff, Michael; Morozov, Igor; Li, Yuhao; Yang, Jianmei; García-Sastre, Adolfo; Richt, Juergen A.; Ma, Wenjun

    2017-01-01

    Since December 2014, Eurasian-origin, highly pathogenic avian influenza H5 viruses including H5N1, H5N2, and H5N8 subtypes (called H5Nx viruses), which belong to the H5 clade 2.3.4.4, have been detected in U.S. wild birds. Subsequently, highly pathogenic H5N2 and H5N8 viruses have caused outbreaks in U.S. domestic poultry. Vaccination is one of the most effective ways to control influenza outbreaks and protect animal and public health. Newcastle disease virus (NDV)-based influenza vaccines ha...

  19. Recombinant infectious bronchitis virus (IBV) H120 vaccine strain expressing the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) protects chickens against IBV and NDV challenge.

    Science.gov (United States)

    Yang, Xin; Zhou, Yingshun; Li, Jianan; Fu, Li; Ji, Gaosheng; Zeng, Fanya; Zhou, Long; Gao, Wenqian; Wang, Hongning

    2016-05-01

    Infectious bronchitis (IB) and Newcastle disease (ND) are common viral diseases of chickens, which are caused by infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), respectively. Vaccination with live attenuated strains of IBV-H120 and NDV-LaSota are important for the control of IB and ND. However, conventional live attenuated vaccines are expensive and result in the inability to differentiate between infected and vaccinated chickens. Therefore, there is an urgent need to develop new efficacious vaccines. In this study, using a previously established reverse genetics system, we generated a recombinant IBV virus based on the IBV H120 vaccine strain expressing the haemagglutinin-neuraminidase (HN) protein of NDV. The recombinant virus, R-H120-HN/5a, exhibited growth dynamics, pathogenicity and viral titers that were similar to those of the parental IBV H120, but it had acquired hemagglutination activity from NDV. Vaccination of SPF chickens with the R-H120-HN/5a virus induced a humoral response at a level comparable to that of the LaSota/H120 commercial bivalent vaccine and provided significant protection against challenge with virulent IBV and NDV. In summary, the results of this study indicate that the IBV H120 strain could serve as an effective tool for designing vaccines against IB and other infectious diseases, and the generation of IBV R-H120-HN/5a provides a solid foundation for the development of an effective bivalent vaccine against IBV and NDV.

  20. Generation and evaluation of a recombinant genotype VII Newcastle disease virus expressing VP3 protein of Goose parvovirus as a bivalent vaccine in goslings.

    Science.gov (United States)

    Wang, Jianzhong; Cong, Yanlong; Yin, Renfu; Feng, Na; Yang, Songtao; Xia, Xianzhu; Xiao, Yueqiang; Wang, Wenxiu; Liu, Xiufan; Hu, Shunlin; Ding, Chan; Yu, Shengqing; Wang, Chunfeng; Ding, Zhuang

    2015-05-04

    Newcastle disease virus (NDV) and Goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting geese. In this study, we generated a recombinant rmNA-VP3, expressing GPV VP3 using a modified goose-origin NDV NA-1 by changing the multi-basic cleavage site motif RRQKR↓F of the F protein to the dibasic motif GRQGR↓L as that of the avirulent strain LaSota as a vaccine vector. Expression of the VP3 protein in rmNA-VP3 infected cells was detected by immunofluorescence and Western blot assay. The genetic stability was examined by serially passaging 10 times in 10-day-old embryonated SPF chicken eggs. Goslings were inoculated with rmNA-VP3 showed no apparent signs of disease and developed a strong GPV and NDV neutralizing antibodies response. This is the first study demonstrating that recombinant NDV has the potential to serve as bivalent live vaccine against Goose parvovirus and Newcastle disease virus infection in birds. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. In ovo delivery of Newcastle disease virus conjugated hybrid calcium phosphate nanoparticle and to study the cytokine profile induction

    International Nuclear Information System (INIS)

    Viswanathan, Kaliyaperumal; Rathish, P.; Gopinath, V.P.; Janice, R.; Dhinakar Raj, G.

    2014-01-01

    In this report, the hybrid calcium phosphate (CaP) nanoparticles were synthesized and functionalized with Newcastle disease virus (NDV). These nanoparticles were synthesized by a combination of co-precipitation and polymerization process and functionalized with amino propyl triethoxy silane before coupling to NDV. The 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT) assay of chicken spleen cells incubated with these nanoparticles indicated that, these particles did not exert any significant cytotoxicity. The effects of hybrid CaP nanoparticles on cell cycle were assayed using a flow cytometer. The results demonstrated that the cell viability and proliferation capacity of spleen cells were not affected by hybrid CaP nanoparticles compared with their control cells. The hybrid CaP nanoparticles were characterized by scanning/transmission electron microscopy (SEM/TEM); Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction patterns (XRD), Raman spectroscopy and energy-dispersive X-ray spectroscopy (EDX). These methods revealed that NDV was successfully conjugated on nanoparticles. The ability of the hybrid CaP nanoparticles to induce different cytokine mRNAs in the spleen cells of 18-day old embryonated chicken eggs (ECEs) was studied by quantitative real time polymerase chain reaction (qRT-PCR). NDV conjugated particles induced a high expression of Th1 cytokines such as interferon (IFN)-α, tumor necrosis factor (TNF)-α of and Th2 cytokines, interleukin (IL) 6 and IL-10. Uncoupled NDV induced only Th1 cytokines, IFN-α, INF-γ and TNF-α. The hybrid particles alone did not induce any cytokines. This confirmed that nanoparticle coupling could induce differential cytokine profiles and hence can be used as an alternate strategy to direct favorable immune responses in animals or chickens using appropriate vaccination carrier. - Highlights: • NDV conjugated hybrid CaP NP induced differential cytokine profiles in embryonated chicken eggs.

  2. In ovo delivery of Newcastle disease virus conjugated hybrid calcium phosphate nanoparticle and to study the cytokine profile induction

    Energy Technology Data Exchange (ETDEWEB)

    Viswanathan, Kaliyaperumal [Translational Research Platform for Veterinary Biologicals (TRPVB), Tamil Nadu Veterinary and Animal Sciences University, Chennai 600 051, Tamil Nadu (India); Rathish, P.; Gopinath, V.P.; Janice, R. [Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai 600 007 (India); Dhinakar Raj, G., E-mail: dhinakarrajg@tanuvas.org.in [Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai 600 007 (India); Translational Research Platform for Veterinary Biologicals (TRPVB), Tamil Nadu Veterinary and Animal Sciences University, Chennai 600 051, Tamil Nadu (India)

    2014-12-01

    In this report, the hybrid calcium phosphate (CaP) nanoparticles were synthesized and functionalized with Newcastle disease virus (NDV). These nanoparticles were synthesized by a combination of co-precipitation and polymerization process and functionalized with amino propyl triethoxy silane before coupling to NDV. The 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT) assay of chicken spleen cells incubated with these nanoparticles indicated that, these particles did not exert any significant cytotoxicity. The effects of hybrid CaP nanoparticles on cell cycle were assayed using a flow cytometer. The results demonstrated that the cell viability and proliferation capacity of spleen cells were not affected by hybrid CaP nanoparticles compared with their control cells. The hybrid CaP nanoparticles were characterized by scanning/transmission electron microscopy (SEM/TEM); Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction patterns (XRD), Raman spectroscopy and energy-dispersive X-ray spectroscopy (EDX). These methods revealed that NDV was successfully conjugated on nanoparticles. The ability of the hybrid CaP nanoparticles to induce different cytokine mRNAs in the spleen cells of 18-day old embryonated chicken eggs (ECEs) was studied by quantitative real time polymerase chain reaction (qRT-PCR). NDV conjugated particles induced a high expression of Th1 cytokines such as interferon (IFN)-α, tumor necrosis factor (TNF)-α of and Th2 cytokines, interleukin (IL) 6 and IL-10. Uncoupled NDV induced only Th1 cytokines, IFN-α, INF-γ and TNF-α. The hybrid particles alone did not induce any cytokines. This confirmed that nanoparticle coupling could induce differential cytokine profiles and hence can be used as an alternate strategy to direct favorable immune responses in animals or chickens using appropriate vaccination carrier. - Highlights: • NDV conjugated hybrid CaP NP induced differential cytokine profiles in embryonated chicken eggs.

  3. The positive expression of genotype VII Newcastle disease virus (Malaysian isolate in Japanese quails (Coturnix coturnix japonica

    Directory of Open Access Journals (Sweden)

    Lizma Felisha Mazlan

    2017-05-01

    Full Text Available Aim: Genotype VII Newcastle disease virus (NDV is the most predominant NDV strains that circulating in Malaysia; thus, this study was aimed to determine the susceptibility of Japanese quails toward genotype VII NDV. Clinical signs, gross pathological lesions of organs, positive detection of virus in organs and cloacal swabs, as well as the expression of the antibody titer, were used as parameters to assess the susceptibility of Japanese quails following infection of genotype VII NDV. Materials and Methods: About 20 quails were divided into three groups (n=8 for Groups A and B; n=4 for the control group. The quails in the Groups A and B were infected via intraocular route with 0.03 ml of 103.5 ELD50 and 107.0 ELD50 of NDV strain IBS 002, respectively, while the control group received 1x phosphate-buffered saline. Cloacal swabs and necropsy were taken on day 7 post-infection for all quails were subjected to one-step reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR for detection of virus and examination for gross pathological lesion, respectively. Blood serums of infected quails were taken on day 10, 14, and 21 post-day infections and were subjected for hemagglutination inhibition (HI assay. Results: Depression and ruffled feathers, trachea rales, leg paralysis, and torticollis were shown in some of the quails in both infected groups. Based on statistical analysis, there was no significant difference (p>0.05 in clinical signs between the infected groups. The results for RT-qPCR were found to be negative for all groups, and no gross pathological lesions of organs observed for quails in both infected groups. Trachea, proventriculus, and cecal tonsil were taken for the detection of NDV by RT-qPCR, and some of the organ samples showed positive detection of virus in both infected groups. HI assay showed an increase in mean titers of antibody across time and between infected groups. Conclusion: In summary, Japanese quails

  4. Immunoadjuvant activities of a recombinant chicken IL-12 in chickens vaccinated with Newcastle disease virus recombinant HN protein.

    Science.gov (United States)

    Su, Bor Sheu; Yin, Hsien Sheng; Chiu, Hua Hsien; Hung, Li Hsiang; Huang, Ji Ping; Shien, Jui Hung; Lee, Long Huw

    2011-08-05

    Recombinant fowlpox virus (rFPV/HN) expressing Newcastle disease virus (NDV) HN gene and rFPV/HN/chIL-12 co-expressing chicken IL-12 (chIL-12) and HN (rHN/chIL-12) genes have been characterized. rHN/chIL-12 or rchIL-12, expressed by our previous construct rFPV/chIL-12, co-administered with rHN was assessed for adjuvant activities of chIL-12. Chickens were vaccinated with various amounts of rHN/chIL-12 mixed with mineral oil (MO), intramuscularly. Levels of hemagglutination-inhibition (HI) antibody production depended on the concentration of the injected rHN or rHN/chIL-12. The lower HI antibody titers were obtained in chicken groups rHN/chIL-12/7-rHN/chIL-12/9, receiving 60ng rHN/8ng chIL-12 with MO, 30ng rHN/4ng chIL-12 with MO or 15ng rHN/2ng chIL-12 with MO, respectively, compared to those in chicken groups rHN/7-rHN/9, receiving rHN with MO alone. However, chickens in group rHN/chIL-12/7 or rHN/chIL-12/8 and rHN with MO alone showed the same effective protection. Chicken group rHN/chIL-12/9 was even more protective than that in group rHN/9. When rchIL-12 was co-injected with 15ng rHN plus MO, chickens produced low levels of HI antibody titers; while higher levels of IFN-γ production and an effective protection rate (83%) were obtained. On the other hand, low levels of IFN-γ production and low protection response (50%) were obtained in chickens injected with rHN with MO alone. Taken together, when the concentration of rHN decreased to certain levels, rchIL-12 reduced HI antibody production. The increase in the induction of IFN-γ production might suggest the enhancement of the cell-mediated immunity which conferred the protection from the NDV challenge. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Seroprevalence of the Newcastle disease In fighting cocks (Gallus gallus from the Municipality of Saboyá, Boyacá

    Directory of Open Access Journals (Sweden)

    Edgar Javier Briceño Cruspoca

    2012-06-01

    Full Text Available Along the time, the poultry industry has become one of the pillars of the Colombian economy, as a result of its extensive influence in the national gross domestic product and in the livestock field. In fact, the poultry industry significantly contributes to the food security, due to the affordability of poultry and eggs in relation to other protein sources. Due to the current reality of greater economic integration and globalization, it is required to pursue the removal or reduction of barriers such as the health related ones, that could be an obstacle for free trade. The industrial-type poultry industry (broilers, layers, breeders, grandmothers, turkey is one of the leading sectors of the country’s livestock production. Other types of exploitation such as the breeding of fighting cocks, backyard poultry, farm ostrich and quail, among others, represent another important part of this sector. However, a big concern related to these types of practices is the movement of birds and the potential risk of these as distributors of diseases that could potentially affect the national poultry industry.The Newcastle (ENC is among the most important diseases. This is one of the easily transmitted pathologies that causes great economic impact to the poultry industry due to its costs associated to high morbidity and mortality, low in production, high costs of treatment of secondary infections and significant investments in programs targeting its control and eradication. Therefore the objective of the study was to determine the presence of antibodies of the disease in fighting cocks (Gallus gallus in Saboyá, Boyacá. The technique of hemagglutination inhibition was used for this matter. Prior to the study, a census was conducted to determine the population of (N = 1.500 animals. The health, manipulation and biosecurity standards of the subject population were assessed through the use of an epidemiological survey. The size of the study sample which was n

  6. Whole genome sequencing of genotype VI Newcastle disease viruses from formalin-fixed paraffinembedded tissues from wild pigeons reveals continuous evolution and previously unrecognized genetic diversity in the U.S.

    Science.gov (United States)

    Background: Newcastle disease viruses (NDV) are highly contagious and can cause disease in both wild birds and poultry. A pigeon-adapted variant of genotype VI NDV, termed pigeon paramyxovirus 1, is commonly isolated from Columbiform birds in the United States. Complete genomic characterization of t...

  7. Chimeric avian paramyxovirus-based vector immunization against highly pathogenic avian influenza followed by conventional Newcastle disease vaccination eliminates lack of protection from virulent ND virus

    Directory of Open Access Journals (Sweden)

    C. Steglich

    2014-01-01

    Full Text Available Recently, we described a chimeric, hemagglutinin of highly pathogenic avian influenza virus (HPAIV H5 expressing Newcastle disease virus (NDV-based vector vaccine (chNDVFHNPMV8H5 in which NDV envelope glycoproteins were replaced by those of avian paramyxovirus-8 (APMV-8. This chimeric vaccine induced solid protection against lethal HPAIV H5N1 even in chickens with maternal antibodies against NDV (MDA+. However, due to the absence of the major NDV immunogens it failed to induce protection against Newcastle disease (ND. Here, we report on protection of MDA+ chickens against HPAI H5N1 and ND, by vaccination with chNDVFHNPMV8H5 either on day 1 or day seven after hatch, and subsequent immunization with live attenuated NDV seven days later. Vaccination was well tolerated and three weeks after immunization, challenge infections with highly pathogenic NDV as well as HPAIV H5N1 were carried out. All animals remained healthy without exhibiting any clinical signs, whereas non-vaccinated animals showed morbidity and mortality. Therefore, vaccination with chNDVFHNPMV8H5 can be followed by NDV vaccination to protect chickens from HPAIV as well as NDV, indicating that the antibody response against chNDVFHNPMV8H5 does not interfere with live ND vaccination.

  8. Detection of the Newcastle disease virus and its effect on development of post-vaccination immunity in a commercial flock of laying hens

    Directory of Open Access Journals (Sweden)

    Jana Jeřábková

    2012-01-01

    Full Text Available The aim of this study was to monitor the concentration of antibodies against Newcastle disease after vaccination of laying hens at the beginning and in the end of the laying period. The study was carried out in one commercial flock of laying hens in Opatovice in the Czech Republic in the years 2008-2010. A total of 280 samples of blood sera were taken from laying hens coming from four poultry houses. The sera were tested by the haemagglutination inhibition test according to the OIE Manual. Virological testing was conducted as a consequence of atypical results of serological testing. Newcastle disease virus RNA was proved by the RT-nested PCR method in the pooled tissue samples of 5 hens, in the samples of intestines with ileocaecal tonsila, in trachea and also in one swab sample from the environment of one house. Based on sequencing analysis and subsequent phylogenetic analysis, the virus was identified as a low pathogenic strain of paramyxovirus (PMV-1. This low pathogenic strain did not have any impact on the health of laying hens.

  9. Newcastle Disease Virus (PDQ)

    Science.gov (United States)

    ... NIH). NIH is the federal government’s center of biomedical research. The PDQ summaries are based on an independent ... NCCIH) are sponsoring a number of clinical trials (research studies) at medical centers to test CAM therapies for use in cancer. Conventional approaches to cancer ...

  10. Molecular characterization, isolation, pathology and pathotyping of peafowl (Pavo cristatus) origin Newcastle disease virus isolates recovered from disease outbreaks in three states of India.

    Science.gov (United States)

    Desingu, Perumal Arumugam; Singh, Shambhu Dayal; Dhama, Kuldeep; Vinodhkumar, Obli Rajendran; Barathidasan, Rajamani; Malik, Yashpal Singh; Singh, Rajendra; Singh, Raj Kumar

    2016-12-01

    Disease outbreak investigations were carried out in three states of Northern India namely Haryana (Rewari), Uttar Pradesh (Noida) and Delhi, where a total of 110 Indian peafowls (Pavo cristatus) showed sudden onset of nervous signs and died within a period of two weeks during June, 2012. The F (fusion) gene-based RT-PCR detection of Newcastle disease virus (NDV) in affected tissues confirmed the presence of the virus. Three NDV isolates were selected (one from each area under investigation) and further characterized. They were found to be of virulent pathotype (velogenic NDV) based on both pathogenicity assays (MDT, ICPI and IVPI) and partial F gene sequence analysis. Additionally, the phylogenetic analysis revealed that the isolates belonged to the genotype VIIi and XIII of class II avian Paramyxovirus serotype1 (APMV-1) and related closely to new emerging sub-genotypes. This is the first report regarding the presence of the fifth panzootic vNDV genotype VIIi from India. In this scenario, extensive epidemiological studies are suggested for surveillance of NDV genotypes in wild birds and poultry flocks of the country along with adopting suitable prevention and control measures.

  11. Newcastle disease virus (NDV) recombinants expressing infectious laryngotracheitis virus (ILTV) glycoproteins gB and gD protect chickens against ILTV and NDV challenges.

    Science.gov (United States)

    Zhao, Wei; Spatz, Stephen; Zhang, Zhenyu; Wen, Guoyuan; Garcia, Maricarmen; Zsak, Laszlo; Yu, Qingzhong

    2014-08-01

    Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is controlled mainly through biosecurity and vaccination with live attenuated strains of ILTV and vectored vaccines based on turkey herpesvirus (HVT) and fowlpox virus (FPV). The current live attenuated vaccines (chicken embryo origin [CEO] and tissue culture origin [TCO]), although effective, can regain virulence, whereas HVT- and FPV-vectored ILTV vaccines are less efficacious than live attenuated vaccines. Therefore, there is a pressing need to develop safer and more efficacious ILTV vaccines. In the present study, we generated Newcastle disease virus (NDV) recombinants, based on the LaSota vaccine strain, expressing glycoproteins B (gB) and D (gD) of ILTV using reverse genetics technology. These recombinant viruses, rLS/ILTV-gB and rLS/ILTV-gD, were slightly attenuated in vivo yet retained growth dynamics, stability, and virus titers in vitro that were similar to those of the parental LaSota virus. Expression of ILTV gB and gD proteins in the recombinant virus-infected cells was detected by immunofluorescence assay. Vaccination of specific-pathogen-free chickens with these recombinant viruses conferred significant protection against virulent ILTV and velogenic NDV challenges. Immunization of commercial broilers with rLS/ILTV-gB provided a level of protection against clinical disease similar to that provided by the live attenuated commercial vaccines, with no decrease in body weight gains. The results of the study suggested that the rLS/ILTV-gB and -gD viruses are safe, stable, and effective bivalent vaccines that can be mass administered via aerosol or drinking water to large chicken populations. This paper describes the development and evaluation of novel bivalent vaccines against chicken infectious laryngotracheitis (ILT) and Newcastle disease (ND), two of the most economically important infectious

  12. Description and analysis of the poultry trading network in the Lake Alaotra region, Madagascar: implications for the surveillance and control of Newcastle disease.

    Science.gov (United States)

    Rasamoelina-Andriamanivo, H; Duboz, R; Lancelot, R; Maminiaina, O F; Jourdan, M; Rakotondramaro, T M C; Rakotonjanahary, S N; de Almeida, R Servan; Rakotondravao; Durand, B; Chevalier, V

    2014-07-01

    Madagascar's 36.5-million-head poultry industry holds a foremost place in its economy and the livelihood of its people. Unfortunately, regular Newcastle disease outbreaks associated with high mortality causes high losses for smallholders and threatens their livelihood. Therefore, Madagascar is seeking concrete, achievable and sustainable methods for the surveillance and the control of Newcastle disease. In this paper, we present and analyze the results of a field study conducted in Madagascar between December 2009 and December 2010. The study area was the Lac Alaotra region, a landlocked area in the north-eastern part of the country's center. Poultry trading is suspected of playing a major role in the spread of avian diseases, especially in developing countries characterized by many live-bird markets and middlemen. Therefore, the goals of our study were to: (i) describe and analyze smallholders' poultry trading network in the Lake Alaotra region using social network analysis; (ii) assess the role of the network in the spread of Newcastle disease; and (iii) propose the implementation of a targeted disease surveillance based on the characteristics of the poultry trading network. We focused our field study on the harvesting of two data sets. The first is a complete description of the poultry trading network in the landlocked area of Lac Alaotra, including a description of the poultry movements between groups of villages. The second set of data measures the occurrence of outbreaks in the same area by combining a participatory approach with an event-based surveillance method. These data were used to determine the attributes of the network, and to statistically assess the association between the position of nodes and the occurrence of outbreaks. By using social network analysis techniques combined with a classification method and a logistic model, we finally identified 3 nodes (set of villages), of the 387 in the initial network, to focus on for surveillance and control

  13. Evaluation of the U.S. Department of Agriculture's egg pasteurization processes on the inactivation of high pathogenicity avian influenza virus and velogenic Newcastle disease virus in processed egg products

    Science.gov (United States)

    High pathogenicity avian influenza virus (HPAIV) A/chicken/Pennsylvania/1370/1983 (H5N2), and velogenic Newcastle disease virus (vNDV) AMPV-1/California/212676/2002 were inoculated into various egg products then heat treated at various temperatures for 0 to 30 min to determine thermal inactivation p...

  14. Evaluation of a LaSota strain-based recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of avian metapneumovirus (aMPV) subgroup A or B as a bivalent vaccine in turkeys

    Science.gov (United States)

    To develop a bivalent vaccine candidate, a LaSota strain-based recombinant Newcastle disease virus (NDV) clone expressing the glycoprotein (G) of avian metapneumovirus (aMPV) subgroup A or B was generated using reverse genetics. Vaccination of turkeys with the NDV/aMPV-A G or NDV/aMPV-B G recombinan...

  15. Generation and evaluation of recombinant Newcastle disease viruses (NDV) expressing the F and G proteins of avian metapneumovirus subtype C (aMPV-C) as bivalent vaccine against NDV and aMPV challenges in turkeys

    Science.gov (United States)

    Previously we generated a Newcastle disease virus (NDV) LaSota strain-based recombinant virus expressing the glycoprotein (G) of avian metapneumovirus subgroup C (aMPV-C) as a bivalent vaccine, which provided a partial protection against aMPV-C challenge in turkeys. To improve the vaccine efficacy,...

  16. Generation and evaluation of a LaSota strain-based recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of avian metapneumovirus subgroup C (aMPV-C) as a bivalent vaccine

    Science.gov (United States)

    To develop a bivalent vaccine, a recombinant Newcastle disease virus was generated by using the NDV LaSota strain with insertion of the G gene of aMPV-C. The biological assessments of the recombinant virus, rLS/aMPV-CG, by conducting the mean death time, intracerebral pathogenicity index, and growth...

  17. Avian influenza virus and Newcastle disease virus (NDV) surveillance in commercial breeding farm in China and the characterization of Class I NDV isolates.

    Science.gov (United States)

    Hu, Beixia; Huang, Yanyan; He, Yefeng; Xu, Chuantian; Lu, Xishan; Zhang, Wei; Meng, Bin; Yan, Shigan; Zhang, Xiumei

    2010-07-29

    In order to determine the actual prevalence of avian influenza virus (AIV) and Newcastle disease virus (NDV) in ducks in Shandong province of China, extensive surveillance studies were carried out in the breeding ducks of an intensive farm from July 2007 to September 2008. Each month cloacal and tracheal swabs were taken from 30 randomly selected birds that appeared healthy. All of the swabs were negative for influenza A virus recovery, whereas 87.5% of tracheal swabs and 100% cloacal swabs collected in September 2007, were positive for Newcastle disease virus isolation. Several NDV isolates were recovered from tracheal and cloacal swabs of apparently healthy ducks. All of the isolates were apathogenic as determined by the MDT and ICPI. The HN gene and the variable region of F gene (nt 47-420) of four isolates selected at random were sequenced. A 374 bp region of F gene and the full length of HN gene were used for phylogenetic analysis. Four isolates were identified as the same isolate based on nucleotide sequences identities of 99.2-100%, displaying a closer phylogenetic relationship to lentogenic Class I viruses. There were 1.9-9.9% nucleotide differences between the isolates and other Class I virus in the variable region of F gene (nt 47-420), whereas there were 38.5-41.2% nucleotide difference between the isolates and Class II viruses. The amino acid sequences of the F protein cleavage sites in these isolates were 112-ERQERL-117. The full length of HN gene of these isolates was 1851 bp, coding 585 amino acids. The homology analysis of the nucleotide sequence of HN gene indicated that there were 2.0-4.2% nucleotide differences between the isolates and other Class I viruses, whereas there were 29.5-40.9% differences between the isolates and Class II viruses. The results shows that these isolates are not phylogenetically related to the vaccine strain (LaSota). This study adds to the understanding of the ecology of influenza viruses and Newcastle disease viruses in

  18. A cross sectional study of Infectious Bursal Disease and Newcastle Disease in poultry in Narsingdi district of Bangladesh

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    Shariful Islam

    2016-12-01

    Conclusion: IBD and ND are highly prevalent in the study area. Therefore, it is necessary to conduct effective control measures to reduce the prevalence of these diseases. This study can help in designing appropriate control measures considering risk factors of these diseases. [J Adv Vet Anim Res 2016; 3(4.000: 406-412

  19. The role of Nicotiana gluca Graham (paraguayan herbs) as an adjuvant in immunomodulation of Newcastle disease vaccine for broilers Estudo da ação de Nicotiana glauca Graham (erva paraguaia) como coadjuvante em vacina contra a doença de Newcastle em frangos de corte

    OpenAIRE

    Fabiane Pereira Gentilini; Telmo Vidor; Rogério Freitag; Marcos Antônio Anciuti; Caren Gularte Quincozes; Marlete Brum Cleff; Geferson Fischer; Carlos Eduardo Nogueira

    2008-01-01

    The Nicotiana glauca is a native plant species from Argentina, but found all over South América, being used against headaches, rheumatism, injuries, ulcers, and so on. Researchers have considered it as having immunomodulation effect. This study was conducted to investigate the use of a aqueous extract of Nicotiana glauca Graham as an immunomodulator (adjuvant) of a Newcastle disease vaccine.. A total of 56 broilers were distributed into 4 ...

  20. Enhanced Replication of Virulent Newcastle Disease Virus in Chicken Macrophages Is due to Polarized Activation of Cells by Inhibition of TLR7.

    Science.gov (United States)

    Zhang, Pingze; Ding, Zhuang; Liu, Xinxin; Chen, Yanyu; Li, Junjiao; Tao, Zhi; Fei, Yidong; Xue, Cong; Qian, Jing; Wang, Xueli; Li, Qingmei; Stoeger, Tobias; Chen, Jianjun; Bi, Yuhai; Yin, Renfu

    2018-01-01

    Newcastle disease (ND), caused by infections with virulent strains of Newcastle disease virus (NDV), is one of the most important infectious disease affecting wild, peridomestic, and domestic birds worldwide. Vaccines constructed from live, low-virulence (lentogenic) viruses are the most accepted prevention and control strategies for combating ND in poultry across the globe. Avian macrophages are one of the first cell lines of defense against microbial infection, responding to signals in the microenvironment. Although macrophages are considered to be one of the main target cells for NDV infection in vivo , very little is known about the ability of NDV to infect chicken macrophages, and virulence mechanisms of NDV as well as the polarized activation patterns of macrophages and correlation with viral infection and replication. In the present study, a cell culture model (chicken bone marrow macrophage cell line HD11) and three different virulence and genotypes of NDV (including class II virulent NA-1, class II lentogenic LaSota, and class I lentogenic F55) were used to solve the above underlying questions. Our data indicated that all three NDV strains had similar replication rates during the early stages of infection. Virulent NDV titers were shown to increase compared to the other lentogenic strains, and this growth was associated with a strong upregulation of both pro-inflammatory M1-like markers/cytokines and anti-inflammatory M2-like markers/cytokines in chicken macrophages. Virulent NDV was found to block toll-like receptor (TLR) 7 expression, inducing higher expression of type I interferons in chicken macrophages at the late stage of viral infection. Only virulent NDV replication can be inhibited by pretreatment with TLR7 ligand. Overall, this study demonstrated that virulent NDV activates a M1-/M2-like mixed polarized activation of chicken macrophages by inhibition of TLR7, resulting in enhanced replication compared to lentogenic viruses.

  1. Molecular characterization of partial fusion gene and C-terminus extension length of haemagglutinin-neuraminidase gene of recently isolated Newcastle disease virus isolates in Malaysia

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    Berhanu Ayalew

    2010-08-01

    Full Text Available Abstract Background Newcastle disease (ND, caused by Newcastle disease virus (NDV, is a highly contagious disease of birds and has been one of the major causes of economic losses in the poultry industry. Despite routine vaccination programs, sporadic cases have occasionally occurred in the country and remain a constant threat to commercial poultry. Hence, the present study was aimed to characterize NDV isolates obtained from clinical cases in various locations of Malaysia between 2004 and 2007 based on sequence and phylogenetic analysis of partial F gene and C-terminus extension length of HN gene. Results The coding region of eleven NDV isolates fusion (F gene and carboxyl terminal region of haemagglutinin-neuraminidase (HN gene including extensions were amplified by reverse transcriptase PCR and directly sequenced. All the isolates have shown to have non-synonymous to synonymous base substitution rate ranging between 0.081 - 0.264 demonstrating presence of negative selection. Analysis based on F gene showed the characterized isolates possess three different types of protease cleavage site motifs; namely 112RRQKRF117, 112RRRKRF117 and 112GRQGRL117 and appear to show maximum identities with isolates in the region such as cockatoo/14698/90 (Indonesia, Ch/2000 (China, local isolate AF2240 indicating the high similarity of isolates circulating in the South East Asian countries. Meanwhile, one of the isolates resembles commonly used lentogenic vaccine strains. On further characterization of the HN gene, Malaysian isolates had C-terminus extensions of 0, 6 and 11 amino acids. Analysis of the phylogenetic tree revealed that the existence of three genetic groups; namely, genotype II, VII and VIII. Conclusions The study concluded that the occurrence of three types of NDV genotypes and presence of varied carboxyl terminus extension lengths among Malaysian isolates incriminated for sporadic cases.

  2. Molecular characterization of partial fusion gene and C-terminus extension length of haemagglutinin-neuraminidase gene of recently isolated Newcastle disease virus isolates in Malaysia.

    Science.gov (United States)

    Berhanu, Ayalew; Ideris, Aini; Omar, Abdul R; Bejo, Mohd Hair

    2010-08-08

    Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious disease of birds and has been one of the major causes of economic losses in the poultry industry. Despite routine vaccination programs, sporadic cases have occasionally occurred in the country and remain a constant threat to commercial poultry. Hence, the present study was aimed to characterize NDV isolates obtained from clinical cases in various locations of Malaysia between 2004 and 2007 based on sequence and phylogenetic analysis of partial F gene and C-terminus extension length of HN gene. The coding region of eleven NDV isolates fusion (F) gene and carboxyl terminal region of haemagglutinin-neuraminidase (HN) gene including extensions were amplified by reverse transcriptase PCR and directly sequenced. All the isolates have shown to have non-synonymous to synonymous base substitution rate ranging between 0.081 - 0.264 demonstrating presence of negative selection. Analysis based on F gene showed the characterized isolates possess three different types of protease cleavage site motifs; namely 112RRQKRF117, 112RRRKRF117 and 112GRQGRL117 and appear to show maximum identities with isolates in the region such as cockatoo/14698/90 (Indonesia), Ch/2000 (China), local isolate AF2240 indicating the high similarity of isolates circulating in the South East Asian countries. Meanwhile, one of the isolates resembles commonly used lentogenic vaccine strains. On further characterization of the HN gene, Malaysian isolates had C-terminus extensions of 0, 6 and 11 amino acids. Analysis of the phylogenetic tree revealed that the existence of three genetic groups; namely, genotype II, VII and VIII. The study concluded that the occurrence of three types of NDV genotypes and presence of varied carboxyl terminus extension lengths among Malaysian isolates incriminated for sporadic cases.

  3. IMMUNO-MODULATORY EFFECT OF INACTIVATED EIMERIA TENELLA VACCINE AND LIVE IMPPORTED COCCIDIAL VACCINE ON NEWCASTLE DISEASE VIRUS VACCINA TED BROILER CHICKS

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    Muhammad Akram Muneer, Haji Ahmad Hashmi, Masood Rabbani, Zahid Munir Chaudhry and Ali M. Bahrami

    2001-01-01

    Full Text Available A total of 160 one-day-old broiler chicks were used to evaluate the immunomodulatory effects of an inactivated Eimeria tenella vaccine and a live polyvalent imported antiococcidial vaccine (Coccivac. This study indicated that both of these vaccines did not adversely affect the development of serum antibody against Newcastle disease virus (NDV and the chicks vaccinated with either of the anticoccidial vaccines resisted the virulent NDV challenge. A study of the lymphoid organs such as bursa of fabricuis: thymus and spleen from the experimental chicks indicated that those organs were comparable with those from the chicks not vaccinated with these coccidial vaccines. The overall findings of this study indicate that anticoccidial vaccines do not have any effects on the immune functions of the vaccinates. In fact these vaccines prevented the occurrence of clinical coccidiosis in the vaccinates.

  4. The Advance of Technology of Reverse Transcriptase-Polymerase Chain Reaction in Identifying the Genome of Avian Influenza and Newcastle Diseases

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    Dyah Ayu Hewajuli

    2014-03-01

    Full Text Available Avian Influenza (AI viruses are zoonotic and caused death in humans. Newcastle Diseases (ND virus has an economical impact in poultry. Therefore, the identification and characterization of AI and ND viruses that are appropriate, accurate and quick are important to protect human and poultry health. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR was the latest gold standard to detect the genome of AI and ND viruses. Recently, RT-PCR was developed in routine diagnosis and research. RT-PCR is a method to amplify the sequences of DNA genome, preceded by reverse transcriptase process with the primer-mediated enzymatic. Some factors that influenced detection of AI and ND are design primer and probe, types of samples, enzyme, reagent composition, amplification temperature and cycles, technical and non-technical factors such as contamination and trained staff. Modified conventional and real time RT-PCR are able to improve the specificity and sensitivity of the test.

  5. SAT2 Foot-and-Mouth Disease Virus Structurally Modified for Increased Thermostability.

    Science.gov (United States)

    Scott, Katherine A; Kotecha, Abhay; Seago, Julian; Ren, Jingshan; Fry, Elizabeth E; Stuart, David I; Charleston, Bryan; Maree, Francois F

    2017-05-15

    Foot-and-mouth disease virus (FMDV), particularly strains of the O and SAT serotypes, is notoriously unstable. Consequently, vaccines derived from heat-labile SAT viruses have been linked to the induction of immunity with a poor duration and hence require more frequent vaccinations to ensure protection. In silico calculations predicted residue substitutions that would increase interactions at the interpentamer interface, supporting increased stability. We assessed the stability of the 18 recombinant mutant viruses in regard to their growth kinetics, antigenicity, plaque morphology, genetic stability, and temperature, ionic, and pH stability by using Thermofluor and inactivation assays in order to evaluate potential SAT2 vaccine candidates with improved stability. The most stable mutant for temperature and pH stability was the S2093Y single mutant, while other promising mutants were the E3198A, L2094V, and S2093H single mutants and the F2062Y-H2087M-H3143V triple mutant. Although the S2093Y mutant had the greatest stability, it exhibited smaller plaques, a reduced growth rate, a change in monoclonal antibody footprint, and poor genetic stability properties compared to those of the wild-type virus. However, these factors affecting production can be overcome. The addition of 1 M NaCl was found to further increase the stability of the SAT2 panel of viruses. The S2093Y and S2093H mutants were selected for future use in stabilizing SAT2 vaccines. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in cloven-hoofed livestock and wildlife. The control of the disease by vaccination is essential, especially at livestock-wildlife interfaces. The instability of some serotypes, such as SAT2, affects the quality of vaccines and therefore the duration of immunity. We have shown that we can improve the stability of SAT2 viruses by mutating residues at the capsid interface through predictive modeling. This is an important finding for

  6. SURVEILLANCE FOR NEWCASTLE DISEASE VIRUS, AVIAN INFLUENZA VIRUS AND MYCOPLASMA GALLISEPTICUM IN WILD BIRDS NEAR COMMERCIAL POULTRY FARMS SURROUNDED BY ATLANTIC RAINFOREST REMNANTS, SOUTHEASTERN BRAZIL

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    MB Guimarães

    Full Text Available ABSTRACT The geographic overlap between areas of Atlantic rainforest and human activities allows interactions to occur between humans and wild and domestic animals. Despite the great importance of the domestic animal-wildlife-human interface that occurs at poultry farms in terms of public health, economic production and wildlife conservation, there are few studies in Brazil examining the distribution and health of wild birds that interact with poultry farms. From January to December 2010, mist nets were used to capture 166 free-ranging birds that were within close proximity to three poultry farms in Atlantic rainforest remnants in south-eastern Brazil. The species composition was examined, and molecular methods were used to test for avian influenza virus, Newcastle disease virus, and Mycoplasma gallisepticum. The avian communities near the poultry farms were dominated by three synanthropic species, which corresponded to 70% of all captured individuals: house sparrows Passer domesticus (33%, saffron finches (Sicalis flaveola (22%, and ruddy ground-doves (Columbina talpacoti (15%. These predominant bird species were in poor body condition (27%, were infested with feather mites (43%, or presented both conditions (23%. No evidence of infection by avian influenza virus, Newcastle disease virus or M. gallisepticum was identified in any of the studied birds. Although no evidence of the studied pathogens was, our findings demonstrate that differences in the environmental characteristics and biosecurity practices influence the wild bird community near poultry farms, which in turn may affect the health status of these synanthropic birds and strengthen their role in the transmission of pathogens.

  7. The chicken or the egg? Exploring bi-directional associations between Newcastle disease vaccination and village chicken flock size in rural Tanzania.

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    Julia de Bruyn

    Full Text Available Newcastle disease (ND is a viral disease of poultry with global importance, responsible for the loss of a potential source of household nutrition and economic livelihood in many low-income food-deficit countries. Periodic outbreaks of this endemic disease result in high mortality amongst free-ranging chicken flocks and may serve as a disincentive for rural households to invest time or resources in poultry-keeping. Sustainable ND control can be achieved through vaccination using a thermotolerant vaccine administered via eyedrop by trained "community vaccinators". This article evaluates the uptake and outcomes of fee-for-service ND vaccination programs in eight rural villages in the semi-arid central zone of Tanzania. It represents part of an interdisciplinary program seeking to address chronic undernutrition in children through improvements to existing poultry and crop systems. Newcastle disease vaccination uptake was found to vary substantially across communities and seasons, with a significantly higher level of vaccination amongst households participating in a longitudinal study of children's growth compared with non-participating households (p = 0.009. Two multivariable model analyses were used to explore associations between vaccination and chicken numbers, allowing for clustered data and socioeconomic and cultural variation amongst the population. Results demonstrated that both (a households that undertook ND vaccination had a significantly larger chicken flock size in the period between that vaccination campaign and the next compared with those that did not vaccinate (p = 0.018; and (b households with larger chicken flocks at the time of vaccination were significantly more likely to participate in vaccination programs (p < 0.001. Additionally, households vaccinating in all three vaccination campaigns held over 12 months were identified to have significantly larger chicken flocks at the end of this period (p < 0.001. Opportunities to

  8. A serological survey for avian infectious bronchitis virus and Newcastle disease virus antibodies in backyard (free-range) village chickens in Mexico.

    Science.gov (United States)

    Gutierrez-Ruiz, E J; Ramirez-Cruz, G T; Camara Gamboa, E I; Alexander, D J; Gough, R E

    2000-12-01

    The commercial flocks in Yucatan, Mexico are free of Newcastle disease virus (NDV) in its velogenic viscerotropic form, but little is known about the disease status of backyard poultry. A seroprevalence survey in 30 villages using haemagglutination inhibition (HI) tests for infectious bronchitis virus (IBV) and NDV antibodies was carried out from December 1997 to June 1998. The seroprevalences were 56.5% (95% CI 50-63%) for IBV and 2.2% (95% CI 0.5-3.8%) for NDV. All the villages had chickens that were positive for antibodies to IBV and nine of the villages had chickens that were positive for antibodies to NDV. This suggests that IBV may be responsible for a large proportion of the respiratory disease observed in backyard chickens in Yucatan. The implications of these findings are discussed, including the highly susceptible status of the backyard chickens in Yucatan to NDV and the possibility of this virus being one cause of the syndrome known as mortandad by the local people.

  9. Chitosan-coated poly(lactic-co-glycolic acid nanoparticles as an efficient delivery system for Newcastle disease virus DNA vaccine

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    Zhao K

    2014-09-01

    Full Text Available Kai Zhao,1,* Yang Zhang,1,2,* Xiaoyan Zhang,1,* Ci Shi,1,2 Xin Wang,1 Xiaohua Wang,1 Zheng Jin,3 Shangjin Cui2 1Laboratory of Microbiology, School of Life Science, Heilongjiang University, 2Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, 3Key Laboratory of Chemical Engineering Process and Technology for High-efficiency Conversion, Heilongjiang University, Harbin, People’s Republic of China *These authors contributed equally to this work Abstract: We determined the efficacy and safety of chitosan (CS-coated poly(lactic-co-glycolic acid (PLGA nanoparticles (NPs as a delivery system for a vaccine to protect chickens against Newcastle disease virus (NDV. The newly constructed vaccine contained DNA (the F gene of NDV. The Newcastle disease virus (NDV F gene deoxyribonucleic acid (DNA plasmid (pFDNA-CS/PLGA-NPs were spherical (diameter =699.1±5.21 nm [mean ± ­standard deviation] and smooth, with an encapsulation efficiency of 98.1% and a Zeta potential of +6.35 mV. An in vitro release assay indicated that CS controlled the burst release of plasmid DNA, such that up to 67.4% of the entire quantity of plasmid DNA was steadily released from the pFDNA-CS/PLGA-NPs. An in vitro expression assay indicated that the expression of nanoparticles (NPs was maintained in the NPs. In an immunization test with specific pathogen-free chickens, the pFDNA-CS/PLGA-NPs induced stronger cellular, humoral, and mucosal immune responses than the plasmid DNA vaccine alone. The pFDNA-CS/PLGA-NPs did not harm 293T cells in an in vitro assay and did not harm chickens in an in vivo assay. Overall, the results indicated that CS-coated PLGA NPs can serve as an efficient and safe mucosal immune delivery system for NDV DNA vaccine.Keywords: mucosal immune delivery system, immune effect

  10. Thermostating highly confined fluids.

    Science.gov (United States)

    Bernardi, Stefano; Todd, B D; Searles, Debra J

    2010-06-28

    In this work we show how different use of thermostating devices and modeling of walls influence the mechanical and dynamical properties of confined nanofluids. We consider a two dimensional fluid undergoing Couette flow using nonequilibrium molecular dynamics simulations. Because the system is highly inhomogeneous, the density shows strong fluctuations across the channel. We compare the dynamics produced by applying a thermostating device directly to the fluid with that obtained when the wall is thermostated, considering also the effects of using rigid walls. This comparison involves an analysis of the chaoticity of the fluid and evaluation of mechanical properties across the channel. We look at two thermostating devices with either rigid or vibrating atomic walls and compare them with a system only thermostated by conduction through vibrating atomic walls. Sensitive changes are observed in the xy component of the pressure tensor, streaming velocity, and density across the pore and the Lyapunov localization of the fluid. We also find that the fluid slip can be significantly reduced by rigid walls. Our results suggest caution in interpreting the results of systems in which fluid atoms are thermostated and/or wall atoms are constrained to be rigid, such as, for example, water inside carbon nanotubes.

  11. Experimental co-infections of domestic ducks with a virulent Newcastle disease virus and low or highly pathogenic avian influenza viruses.

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    Pantin-Jackwood, Mary J; Costa-Hurtado, Mar; Miller, Patti J; Afonso, Claudio L; Spackman, Erica; Kapczynski, Darrell R; Shepherd, Eric; Smith, Diane; Swayne, David E

    2015-05-15

    Infections with avian influenza viruses (AIV) of low and high pathogenicity (LP and HP) and Newcastle disease virus (NDV) are commonly reported in domestic ducks in many parts of the world. However, it is not clear if co-infections with these viruses affect the severity of the diseases they produce, the amount of virus shed, and transmission of the viruses. In this study we infected domestic ducks with a virulent NDV virus (vNDV) and either a LPAIV or a HPAIV by giving the viruses individually, simultaneously, or sequentially two days apart. No clinical signs were observed in ducks infected or co-infected with vNDV and LPAIV, but co-infection decreased the number of ducks shedding vNDV and the amount of virus shed (Pducks inoculated with only LPAIV compared to ducks co-infected with vNDV. Ducks that received the HPAIV with the vNDV simultaneously survived fewer days (Pducks that received the vNDV two days before the HPAIV. Co-infection also reduced transmission of vNDV to naïve contact ducks housed with the inoculated ducks. In conclusion, domestic ducks can become co-infected with vNDV and LPAIV with no effect on clinical signs but with reduction of virus shedding and transmission. These findings indicate that infection with one virus can interfere with replication of another, modifying the pathogenesis and transmission of the viruses. Published by Elsevier B.V.

  12. Detection of immunoglobulins containing plasma cells in the thymus, bursa of Fabricius and spleen of vaccinated broiler chickens with Newcastle disease virus vaccine

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    Md. Abdul Masum

    2014-12-01

    Full Text Available Mobilization of immunoglobulins (Igs-containing plasma cells (IgA, IgG and IgM in the spleen, bursa of Fabricius and thymus was investigated in broiler chickens that were vaccinated with Newcastle disease virus (NDV vaccine. In the thymus, the Igs-containing plasma cells were distributed in the cortex and medulla. Their frequency and distribution were higher at D14 and at D28. The number of IgG- and IgM-positive cells was greater than IgA-positive cells in thymus. In the bursa of Fabricius, Igs-containing plasma cells were distributed beneath the capsules; within and around the bursal follicles. Their frequency of occurrence significantly peaked at D14 and at D28 in comparison to day-old chickens, and IgG-positive cells were significantly greater than the IgA- and IgM-positive cells in the bursa of vaccinated chickens. In the spleen, Igs-containing plasma cells were distributed in the white pulp, around the trabeculae, and in the periarterial lymphatic sheath. In this secondary lymphatic tissue, IgG- and IgM-positive cell numbers significantly greater than IgA-positive cells. In conclusion, mobilization of more Igs-positive cells in lymphoid tissues of broiler chickens is due to the effect of NDV vaccine as well as the advancement of age.

  13. [Construction and rescue of infectious cDNA clone of pigeon-origin Newcastle disease virus strain JS/07/04/Pi].

    Science.gov (United States)

    Zhu, Yan-Mei; Hu, Zeng-Lei; Song, Qing-Qing; Duan, Zhi-Qiang; Gu, Min; Hu, Shun-Lin; Wang, Xiao-Quan; Liu, Xiu-Fan

    2012-01-01

    Based on the complete genome sequence of pigeon-origin Newcastle disease virus strain JS/07/04/ Pi(genotype VIb), nine overlapped fragments covering its full-length genome were amplified by RT-PCR. The fragments were connected sequentially and then inserted into the transcription vector TVT7/R resulting in the TVT/071204 which contained the full genome of strain JS/07/04/Pi. The TVT/071204 was co-transfected with three helper plasmids pCI-NP, pCI-P and pCI-L into the BSR cells, and the transfected cells and culture supernatant were inoculated into 9-day-old SPF embryonated eggs 60 h post-transfection. The HA and HI tests were conducted following the death of embryonated eggs. The results showed that the allantoic fluids obtained were HA positive and the HA could be inhibited by anti-NDV serum which indicated that the strain JS/07/04/Pi was rescued successfully. The rescued virus rNDV/071204 showed similar growth kinetics to its parental virus in CEF. The successful recovery of this strain would contribute to the understanding of the host-specificity of pigeon-origin NDV and to the development of the novel vaccines against the NDV infection in pigeons.

  14. Prevalence and risk factors for Campylobacter spp., Salmonella spp., Coxiella burnetii, and Newcastle disease virus in feral pigeons (Columba livia) in public areas of Montreal, Canada

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    Gabriele-Rivet, Vanessa; Fairbrother, Julie-Hélène; Tremblay, Donald; Harel, Josée; Côté, Nathalie; Arsenault, Julie

    2016-01-01

    Feral pigeons (Columbia livia) can harbor a range of zoonotic pathogens. A transversal study was undertaken to estimate the prevalence of feral pigeons infected by various pathogens in public areas in Montreal, Quebec. Cloacal swabs from captured birds were cultured for Salmonella spp. and Campylobacter spp. and tested by real-time polymerase chain reaction (RT-PCR) for the detection of Coxiella burnetii. An oropharyngeal swab was also submitted to real-time reverse-transcription polymerase chain reaction (RRT-PCR) for the detection of Newcastle disease virus. Among the 187 pigeons tested from 10 public areas, 9.1% (95% CI: 3.0 to 15.2) were positive for Campylobacter spp. with all strains identified as Campylobacter jejuni. The Campylobacter status of birds was not associated with individual characteristics of birds, with the exception of body score. None of the pigeons tested positive for the other pathogens. Direct or indirect contacts with feral pigeons may constitute a potential risk for Campylobacter infection in humans. PMID:26733736

  15. Evaluating viral interference between Influenza virus and Newcastle disease virus using real-time reverse transcription–polymerase chain reaction in chicken eggs

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    Ge Shengqiang

    2012-07-01

    Full Text Available Abstract Background Simultaneous and sequential allantoic cavity inoculations of Specific-pathogen-free (SPF chicken eggs with Influenza virus (AIV and Newcastle disease virus (NDV demonstrated that the interaction of AIV and NDV during co-infection was variable. Our research revisited the replication interference potential of AIV and NDV using real-time reverse transcription–polymerase chain reaction (real-time RT-PCR for AIV and NDV to specifically detect the viral genomes in mixed infections. Results Data from this survey showed that when different doses of NDV (Lasota or F48E8 and AIV (F98 or H5N1 were simultaneously inoculated into embryonating chicken eggs (ECE, interference with the growth of NDV occurred, while interference with the growth of AIV did not occur. When equal amount of the two viruses were sequentially employed, the degree of interference was dependent upon the time of superinfection and the virulence of virus. Conclusion AIV have a negative impact on NDV growth if they are inoculated simultaneously or sequentially and that the degree of interference depended upon the quantity and relative virulence of the virus strains used; however, interference with AIV was not observed. Only if NDV were inoculated at an earlier time will NDV able to interfere with the growth of AIV.

  16. High levels of virus replication and an intense inflammatory response contribute to the severe pathology in lymphoid tissues caused by Newcastle disease virus genotype VIId.

    Science.gov (United States)

    Hu, Zenglei; Hu, Jiao; Hu, Shunlin; Song, Qingqing; Ding, Pingyun; Zhu, Jie; Liu, Xiaowen; Wang, Xiaoquan; Liu, Xiufan

    2015-03-01

    Some strains of Newcastle disease virus (NDV) genotype VIId cause more-severe tissue damage in lymphoid organs compared to other virulent strains. In this study, we aim to define the mechanism of this distinct pathological manifestation of genotype VII viruses. Pathology, virus replication, and the innate immune response in lymphoid tissues of chickens infected with two genotype VIId NDV strains (JS5/05 and JS3/05), genotype IX NDV F48E8 and genotype IV NDV Herts/33, were compared. Histopathologic examination showed that JS5/05 and JS3/05 produced more-severe lesions in the spleen and thymus, but these four virulent strains caused comparable mild lesions in the bursa. In addition, JS3/05 and JS5/05 replicated at significantly higher levels in the lymphatic organs than F48E8 and Herts/33. A microarray assay performed on the spleens of chickens infected with JS5/05 or Herts/33 revealed that JS5/05 elicited a more potent inflammatory response by increasing the number and expression levels of activated genes. Moreover, cytokine gene expression profiling showed that JS5/05 and JS3/05 induced a stronger cytokine response in lymphoid tissues compared to F48E8 and Herts/33. Taken together, our results indicate that the severe pathology in immune organs caused by genotype VIId NDV strains is associated with high levels of virus replication and an intense inflammatory response.

  17. Pelacakan Secara Imunohistokimiawi Antigen Virus pada Ayam yang Diinfeksi dengan Virus Penyakit Tetelo (IMMUNOHISTOCHEMICAL DETECTION OF VIRAL ANTIGEN IN TISSUE OF CHICKENS EXPERIMENTALLY INFECTED WITH NEWCASTLE DISEASE VIRUS

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    Anak Agung Ayu Mirah Adi

    2013-07-01

    Full Text Available In order to study the distribution of Newcastle disease virus (NDV following infection, chickenswere experimentally infected with visceretropic velogenic NDV isolate. Monoclonal antibodies (mAbsagainst the NDV LaSota vaccine strain were then produced to detect viral antigen in the infectedorgans. The mAbs were firstly tested for their specificity by enzyme linked immunosorbent assay(ELISA using NDV and normal allantoic fluids as antigens. Eight mAbs specific against NDVwere isolated and two mAbs were used for immunodetection of NDV antigen in chicken’s tissues.By immunohistochemistry labeled streptavidin-biotin (LSAB staining NDV–antigen was detectedin paraffin embedded tissues of NDV-infected chickens. NDV antigen was not detected in noninfected chickens. In the infected chickens, high intensity of NDV antigen was detected in thelymphoid tissues, lung and intestine. The NDV antigen with a lesser intensity was detected in thebrain, trachea, liver and myocardium. This study shows that although viscerotropic velogenicNDV isolate can infect almost all organs, the main target of infection are lung, intestine andlymphoids tissues

  18. Immune responses and interactions following simultaneous application of live Newcastle disease, infectious bronchitis and avian metapneumovirus vaccines in specific-pathogen-free chicks.

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    Awad, Faez; Forrester, Anne; Baylis, Matthew; Lemiere, Stephane; Jones, Richard; Ganapathy, Kannan

    2015-02-01

    Interactions between live Newcastle disease virus (NDV), avian metapneumovirus (aMPV) and infectious bronchitis virus (IBV) vaccines following simultaneous vaccination of day old specific pathogen free (SPF) chicks were evaluated. The chicks were divided into eight groups: seven vaccinated against NDV, aMPV and IBV (single, dual or triple) and one unvaccinated as control. Haemagglutination inhibition (HI) NDV antibody titres were similar across all groups but were above protective titres. aMPV vaccine when given with other live vaccines suppressed levels of aMPV enzyme-linked immunosorbent assay (ELISA) antibodies. Cellular and local immunity induced by administration of NDV, aMPV or IBV vaccines (individually or together) showed significant increase in CD4+, CD8+ and IgA bearing B-cells in the trachea compared to the unvaccinated group. Differences between the vaccinated groups were insignificant. Simultaneous vaccination with live NDV, aMPV and IBV did not affect the protection conferred against aMPV or IBV. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. A rapid method of accurate detection and differentiation of Newcastle disease virus pathotypes by demonstrating multiple bands in degenerate primer based nested RT-PCR.

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    Desingu, P A; Singh, S D; Dhama, K; Kumar, O R Vinodh; Singh, R; Singh, R K

    2015-02-01

    A rapid and accurate method of detection and differentiation of virulent and avirulent Newcastle disease virus (NDV) pathotypes was developed. The NDV detection was carried out for different domestic avian field isolates and pigeon paramyxo virus-1 (25 field isolates and 9 vaccine strains) by using APMV-I "fusion" (F) gene Class II specific external primer A and B (535bp), internal primer C and D (238bp) based reverses transcriptase PCR (RT-PCR). The internal degenerative reverse primer D is specific for F gene cleavage position of virulent strain of NDV. The nested RT-PCR products of avirulent strains showed two bands (535bp and 424bp) while virulent strains showed four bands (535bp, 424bp, 349bp and 238bp) on agar gel electrophoresis. This is the first report regarding development and use of degenerate primer based nested RT-PCR for accurate detection and differentiation of NDV pathotypes by demonstrating multiple PCR band patterns. Being a rapid, simple, and economical test, the developed method could serve as a valuable alternate diagnostic tool for characterizing NDV isolates and carrying out molecular epidemiological surveillance studies for this important pathogen of poultry. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Newcastle disease virus infection in sparrows (Passer domesticus, Linneaus, 1758 captured in poultry farms of the agreste region of the State of Pernambuco

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    JSA Silva

    2006-06-01

    Full Text Available Reservoir competence for the Newcastle Disease virus (NDV was evaluated in sparrows (Passer domesticus, Linnaeus 1758 captured on a commercial poultry farm and a chicken hatchery in the State of Pernambuco, Northeastern Brazil. A total number of 103 birds collected from a poultry farm (24/103 and a chicken hatchery (79/103 were examined. Hemagglutination inhibition tests, isolation, and viral characterization were performed in all samples collected from each bird. Titers ranging from 1:2 to 1:64 were detectable in 10.68% of sparrows, but positive serology and viral isolation were obtained only from sparrows captured at the hatchery. Hemagglutination activity was inhibited by anti-avian paramyxovirus serotype 1 (APMV-1 serum, and this sample showed an intracerebral pathogenicity index (ICOI of 0.21, which is similar to the B1 stock vaccine (0.20 used for vaccination in those farms. Therefore, it was concluded that the sparrows were infected by stock vaccine virus, and that these birds could be a reservoir for NDV. However, additional studies involving sequencing of the virus genome of stock vaccine must be carried out.

  1. Generation and evaluation of a recombinant Newcastle disease virus expressing the glycoprotein (G) of avian metapneumovirus subgroup C as a bivalent vaccine in turkeys.

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    Hu, Haixia; Roth, Jason P; Estevez, Carlos N; Zsak, Laszlo; Liu, Bo; Yu, Qingzhong

    2011-11-03

    Virulent strains of Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) can cause serious respiratory diseases in poultry. Vaccination combined with strict biosecurity practices has been the recommendation for controlling both NDV and aMPV diseases in the field. In the present study, an NDV based, LaSota strain recombinant vaccine virus expressing the glycoprotein (G) of aMPV subgroup C (aMPV-C) was generated as a bivalent vaccine using a reverse genetics approach. The recombinant virus, rLS/aMPV-C G was slightly attenuated in vivo, yet maintained similar growth dynamics, cytopathic effects, and virus titers in vitro when compared to the parental LaSota virus. Expression of the aMPV G protein in rLS/aMPV-C G-infected cells was detected by immunofluorescence assay. Vaccination of turkeys with one dose of rLS/aMPV-C G induced moderate aMPV-C-specific immune responses and comparable NDV-specific serum antibody responses to a LaSota vaccination control. Partial protection against pathogenic aMPV-C challenge and complete protection against velogenic NDV challenge was conferred. These results suggest that the LaSota recombinant virus is a safe and effective vaccine vector and that expression of the aMPV-C G protein alone is not sufficient to provide full protection against an aMPV-C infection. Expression of other immunogenic protein(s) of the aMPV-C virus alone or in conjunction with the G protein may be needed to induce a stronger protective immunity against the aMPV-C disease. Published by Elsevier Ltd.

  2. Cloning of fusion protein gene of Newcastle disease virus into a baculovirus derived bacmid shuttle vector, in order to express it in insect cell line

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    Hashemzadeh MS

    2015-05-01

    Full Text Available Abstract Background: Newcastle disease virus (NDV is one of the major pathogens in poultry and vaccination is intended to control the disease, as an effective solution, yet. Fusion protein (F on surface of NDV, has a fundamental role in virus pathogenicity and can induce protective immunity, alone. With this background, here our aim was to construct a baculovirus derived recombinant bacmid shuttle vector (encoding F-protein in order to express it in insect cell line. Materials and Methods: In this experimental study, at first complete F gene from avirulent strain La Sota of NDV was amplified by RT-PCR to produce F cDNA. The amplicon was cloned into T/A cloning vector and afterwards into pFastBac Dual donor plasmid. After the verification of cloning process by two methods, PCR and enzymatic digestion analysis, the accuracy of F gene sequence was confirmed by sequencing. Finally, F-containing recombinant bacmid was subsequently generated in DH10Bac cell and the construct production was confirmed by a special PCR panel, using F specific primers and M13 universal primers. Results: Analysis of confirmatory tests showed that the recombinant bacmid, expressing of F-protein gene in correct sequence and framework, has been constructed successfully. Conclusion: The product of this F-containing recombinant bacmid, in addition to its independent application in the induction of protective immunity, can be used with the other individual recombinant baculoviruses, expressing HN and NP genes to produce NDV-VLPs in insect cell line.

  3. Virus interference between H7N2 low pathogenic avian influenza virus and lentogenic Newcastle disease virus in experimental co-infections in chickens and turkeys.

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    Costa-Hurtado, Mar; Afonso, Claudio L; Miller, Patti J; Spackman, Erica; Kapczynski, Darrell R; Swayne, David E; Shepherd, Eric; Smith, Diane; Zsak, Aniko; Pantin-Jackwood, Mary

    2014-01-06

    Low pathogenicity avian influenza virus (LPAIV) and lentogenic Newcastle disease virus (lNDV) are commonly reported causes of respiratory disease in poultry worldwide with similar clinical and pathobiological presentation. Co-infections do occur but are not easily detected, and the impact of co-infections on pathobiology is unknown. In this study chickens and turkeys were infected with a lNDV vaccine strain (LaSota) and a H7N2 LPAIV (A/turkey/VA/SEP-67/2002) simultaneously or sequentially three days apart. No clinical signs were observed in chickens co-infected with the lNDV and LPAIV or in chickens infected with the viruses individually. However, the pattern of virus shed was different with co-infected chickens, which excreted lower titers of lNDV and LPAIV at 2 and 3 days post inoculation (dpi) and higher titers at subsequent time points. All turkeys inoculated with the LPAIV, whether or not they were exposed to lNDV, presented mild clinical signs. Co-infection effects were more pronounced in turkeys than in chickens with reduction in the number of birds shedding virus and in virus titers, especially when LPAIV was followed by lNDV. In conclusion, co-infection of chickens or turkeys with lNDV and LPAIV affected the replication dynamics of these viruses but did not affect clinical signs. The effect on virus replication was different depending on the species and on the time of infection. These results suggest that infection with a heterologous virus may result in temporary competition for cell receptors or competent cells for replication, most likely interferon-mediated, which decreases with time.

  4. Live bird markets characterization and trading network analysis in Mali: Implications for the surveillance and control of avian influenza and Newcastle disease.

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    Molia, Sophie; Boly, Ismaël Ardho; Duboz, Raphaël; Coulibaly, Boubacar; Guitian, Javier; Grosbois, Vladimir; Fournié, Guillaume; Pfeiffer, Dirk Udo

    2016-03-01

    Live bird markets (LBMs) play an important role in the transmission of avian influenza (AI) and Newcastle disease (ND) viruses in poultry. Our study had two objectives: (1) characterizing LBMs in Mali with a focus on practices influencing the risk of transmission of AI and ND, and (2) identifying which LBMs should be targeted for surveillance and control based on properties of the live poultry trade network. Two surveys were conducted in 2009-2010: a descriptive study in all 96 LBMs of an area encompassing approximately 98% of the Malian poultry population and a network analysis study in Sikasso county, the main poultry supplying county for the capital city Bamako. Regarding LBMs' characteristics, risk factors for the presence of AI and ND viruses (being open every day, more than 2 days before a bird is sold, absence of zoning to segregate poultry-related work flow areas, waste removal or cleaning and disinfecting less frequently than on a daily basis, trash disposal of dead birds and absence of manure processing) were present in 80-100% of the LBMs. Furthermore, LBMs tended to have wide catchment areas because of consumers' preference for village poultry meat, thereby involving a large number of villages in their supply chain. In the poultry trade network from/to Sikasso county, 182 traders were involved and 685 links were recorded among 159 locations. The network had a heterogeneous degree distribution and four hubs were identified based on measures of in-degrees, out-degrees and betweenness: the markets of Medine and Wayerma and the fairs of Farakala and Niena. These results can be used to design biosecurity-improvement interventions and to optimize the prevention, surveillance and control of transmissible poultry diseases in Malian LBMs. Further studies should investigate potential drivers (seasonality, prices) of the poultry trade network and the acceptability of biosecurity and behavior-change recommendations in the Malian socio-cultural context. Copyright

  5. Effects of Dietary Supplemental Vitamins and Periods of Administration on Growth Performance and Antibody Titre of Broiler Chickens Vaccinated against Newcastle Disease

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    Odutayo, O. J.

    2016-12-01

    Full Text Available This study investigated the effects of supplemental vitamins and varying administration periods on growth performance and antibody titre of broiler chickens vaccinated against Newcastle Disease (ND. A total of 300 unvaccinated against ND Arbor Acre day-old chicks were used for the study for 8 wk. Birds were brooded together on day 1 of age, and 30 chicks were selected randomly for evaluating the maternally derived antibody titre against ND. At 2 days of age, the remaining 270 chicks were divided based on weight equalization into 9 treatment groups and replicated thrice. The 9 treatments consisted of a factorial arrangement of 4 supplemental vitamins (A, C, E and combination of A, C, E and 2 periods of administration (3 days pre- and post-ND vaccinations with a control. The birds were managed intensively throughout the experimental period, ND vaccines were administered on the 5th (i/o and 24th (Lasota day of age, respectively. Supplemental combined vitamins A, C and E at 0.15, 16.67 and 3.03 mg/kg, respectively, resulted in higher (P < 0.05 final body weight of 1785.00 g/bird and better feed conversion ratio (FCR of 2.89. Also, birds fed vitamin A supplemented diet 3 d pre-i/o vaccine had higher (p<0.05 serum antibody titre (75.20 against ND while higher (p<0.05 serum antibody titre (741.33 was also obtained in birds fed diet supplemented with vitamin E 3 d post-Lasota vaccination. Conclusively, broiler chickens diets can be supplemented with combined vitamins A, C, and E for better growth performance measured as final body weight and FCR, in addition, vitamins A (0.45mg/kg and E (9.1mg/kg dietary supplementation at 3 d pre-i/o and 3 d post-Lasota vaccines, respectively, can be adopted for improved antibody production.

  6. Recombinant Newcastle disease virus (NDV/Anh-IL-2 expressing human IL-2 as a potential candidate for suppresses growth of hepatoma therapy

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    Yunzhou Wu

    2016-09-01

    Full Text Available Newcastle disease virus (NDV have shown oncolytic therapeutic efficacy in preclinical study and are currently approved for clinical trials. NDV Anhinga strain which is a mesogenic strain should be classified as lytic strain and has a therapeutic efficacy in hepatocellular cancer. In this study, we evaluated the capacity of NDV Anhinga strain to elicit immune reaction in vivo and the possibility for using as a vaccine vector for expressing tumor therapeutic factors. Interleukin-2 (IL-2 could boost the immune response against the tumor cells. Therefore, we use NDV Anhinga strain as backbone to construct a recombinant virus (NDV/Anh-IL-2 expressing IL-2. The virus growth curve showed that the production of recombinant NDV/Anh-IL-2 was slightly delayed compared to the wild type. The NDV/Anh-IL-2 strain could express soluble IL-2 and effectively inhibit the growth of hepatocellular carcinoma in vivo. 60 days post-treatment, mice which were completely cured by previous treatment were well protected when rechallenged with the same tumor cell. From the H&E-stained sections, intense infiltration of lymphocyte was observed in the NDV Anhinga strain treated group, especially in NDV/Anh-IL-2 group. The NDV Anhinga strain could not only kill the tumor directly, but could also elicit immune reaction and a potent immunological memory when killing tumor in vivo. In conclusion, the Anhinga strain could be an effective vector for tumor therapy; the recombinant NDV/Anh-IL-2 strain expressing soluble IL-2 is a promising candidate for hepatoma therapy.

  7. Newcastle disease virus-attenuated vaccine co-contaminated with fowl adenovirus and chicken infectious anemia virus results in inclusion body hepatitis-hydropericardium syndrome in poultry.

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    Su, Qi; Li, Yang; Meng, Fanfeng; Cui, Zhizhong; Chang, Shuang; Zhao, Peng

    2018-05-01

    Inclusion body hepatitis-hydropericardium syndrome (IBH-HPS) induced by fowl adenovirus type 4 (FAdV-4) has caused huge economic losses to the poultry industry of China, but the source of infection for different flocks, especially flocks with high biological safety conditions, has remained unclear. This study tested the pathogenicity of Newcastle disease virus (NDV)-attenuated vaccine from a large-scale poultry farm in China where IBH-HPS had appeared with high mortality. Analysis revealed that the NDV-attenuated vaccine in use from the abovementioned poultry farm was simultaneously contaminated with FAdV-4 and chicken infectious anemia virus (CIAV). The FAdV and CIAV isolated from the vaccine were purified for the artificial preparation of an NDV-attenuated vaccine singly contaminated with FAdV or CIAV, or simultaneously contaminated with both of them. Seven-day-old specific pathogen-free chicks were inoculated with the artificially prepared contaminated vaccines and tested for corresponding indices. The experiments showed that no hydropericardium syndrome (HPS) and corresponding death occurred after administering the NDV-attenuated vaccine singly contaminated with FAdV or CIAV, but a mortality of 75% with IBH-HPS was commonly found in birds after administering the NDV-attenuated vaccine co-contaminated with FAdV and CIAV. In conclusion, this study found the co-contamination of FAdV-4 and CIAV in the same attenuated vaccine and confirmed that such a contaminated attenuated vaccine was a significant source of infection for outbreaks of IBH-HPS in some flocks. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts.

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    Duan, Zhiqiang; Xu, Haixu; Ji, Xinqin; Zhao, Jiafu; Xu, Houqiang; Hu, Yan; Deng, Shanshan; Hu, Shunlin; Liu, Xiufan

    2018-12-31

    The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336-433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.

  9. Protection conferred by a live avian metapneumovirus vaccine when co-administered with live La Sota Newcastle disease vaccine in chicks

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    Kannan Ganapathy

    2014-06-01

    Full Text Available This paper examines the effects on specific pathogen-free (SPF chicks when avian metapneumovirus (aMPV and Newcastle disease virus (NDV La Sota strain vaccines are co-administered. Day-old SPF chicks were divided into five groups. The first group was inoculated with sterile water (SW and the rest of the groups were inoculated with live NDV vaccine VG/GA by the oculo-oral route. At 21 days-old, the unvaccinated chicks were again inoculated with SW. The four VG/GA-vaccinated groups were further inoculated with (i SW, (ii live aMPV vaccine, (iii live NDV La Sota, or (iv combined live NDV La Sota and live aMPV, respectively. Chicks were monitored for post-vaccination reactions and oropharyngeal swabs were collected for vaccines detection. Blood samples were collected to detect aMPV ELISA and NDV haemagglutination-inhibition antibodies. Twenty-one days following the second vaccination, six chicks from each group were challenged with virulent NDV or aMPV respectively. Chicks were monitored for clinical signs and mortality and oropharyngeal swabs collected for aMPV detection. Results showed that, when challenged with a virulent aMPV, both chicks previously vaccinated with VG/GA and subsequently given aMPV vaccine singly or in combination with La Sota were equally protected against clinical signs. Chicks that were vaccinated against NDV either once with VG/GA or followed by La Sota (singly or in combination with aMPV were fully protected when challenged with velogenic NDV. We concluded that simultaneous administration of live aMPV and NDV La Sota vaccines have no adverse effects on protection conferred by either live vaccine.

  10. Isolation and genetic characterization of avian influenza viruses and a Newcastle disease virus from wild birds in Barbados: 2003-2004.

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    Douglas, Kirk O; Lavoie, Marc C; Kim, L Mia; Afonso, Claudio L; Suarez, David L

    2007-09-01

    Zoonotic transmission of an H5N1 avian influenza A virus to humans in 2003-present has generated increased public health and scientific interest in the prevalence and variability of influenza A viruses in wild birds and their potential threat to human health. Migratory waterfowl and shorebirds are regarded as the primordial reservoir of all influenza A viral subtypes and have been repeatedly implicated in avian influenza outbreaks in domestic poultry and swine. All of the 16 hemagglutinin and nine neuraminidase influenza subtypes have been isolated from wild birds, but waterfowl of the order Anseriformes are the most commonly infected. Using 9-to-11-day-old embryonating chicken egg culture, virus isolation attempts were conducted on 168 cloacal swabs from various resident, imported, and migratory bird species in Barbados during the months of July to October of 2003 and 2004. Hemagglutination assay and reverse transcription-polymerase chain reaction were used to screen all allantoic fluids for the presence of hemagglutinating agents and influenza A virus. Hemagglutination positive-influenza negative samples were also tested for Newcastle disease virus (NDV), which is also found in waterfowl. Two influenza A viruses and one NDV were isolated from Anseriformes (40/168), with isolation rates of 5.0% (2/40) and 2.5% (1/40), respectively, for influenza A and NDV. Sequence analysis of the influenza A virus isolates showed them to be H4N3 viruses that clustered with other North American avian influenza viruses. This is the first report of the presence of influenza A virus and NDV in wild birds in the English-speaking Caribbean.

  11. Engineered newcastle disease virus expressing the F and G protein of AMPV-C confers protection against challenges in turkeys.

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    Avian metapneumovirus (aMPV) infects the respiratory and reproductive tracts of domestic poultry, which may develop into secondary infections that can result in substantial economic losses for producers. Live attenuated vaccines appear to be the most effective in countries where the disease is prev...

  12. Protective efficacy of Newcastle disease virus expressing soluble trimeric hemagglutinin against highly pathogenic H5N1 influenza in chickens and mice.

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    Lisette A H M Cornelissen

    Full Text Available BACKGROUND: Highly pathogenic avian influenza virus (HPAIV causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV that express influenza hemagglutinin (HA have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5 or a soluble trimeric form thereof (NDV-sH5(3. A single intramuscular immunization with NDV-sH5(3 or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5(3 was less protective than NDV-H5 (50% vs 80% protection when administered via the respiratory tract. The NDV-sH5(3 was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited. CONCLUSIONS/SIGNIFICANCE: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant

  13. ELISA indireto para detecção de IgG antivírus da doença de Newcastle em soro de codorna Indirect ELISA for the detection of IgG specific to Newcastle disease virus in quail serum

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    D.D. Oliveira

    2007-10-01

    Full Text Available An indirect ELISA for the detection of japanese quail IgG specific to Newcastle disease virus (NDV was developed. The secondary anti-quail IgG was produced in Balb/c mice, by inoculating Freund's complete adjuvant emulsified japanese quail-IgG extract. The purification of IgG was achieved using the caprilic acid method. The ELISA was compared to the haemagglutination-inhibition (HI test for antibodies to NDV. ELISA cut-off point was established through TG-ROC analysis. Total correlation was observed between the ELISA and the HI, being the ELISA efficient in the identification of positive and negative sera, with high sensitivity and specificity (100%. These results validate the use of the indirect ELISA as an alternative for the detection of NDV-specific IgG in japanese quail sera, with the advantage of high sensitivity and automation.

  14. Detection and characterization of Newcastle disease virus in clinical samples using real time RT-PCR and melting curve analysis based on matrix and fusion genes amplification

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    Saad Sharawi

    2013-10-01

    Full Text Available Aim: Newcastle disease is still one of the major threats for poultry industry allover the world. Therefore, attempt was made in this study to use the SYBR Green I real-time PCR with melting curves analysis as for detection and differentiation of NDV strains in suspected infected birds. Materials and Methods: Two sets of primers were used to amplify matrix and fusion genes in samples collected from suspectly infected birds (chickens and pigeons. Melting curve analysis in conjunction with real time PCR was conducted for identifying different pathotypes of the isolated NDVs. Clinical samples were propagated on specific pathogen free ECE and tested for MDT and ICIP. Results: The velogenic NDVs isolated from chickens and pigeons were distinguished with mean T 85.03±0.341 and m 83.78±0.237 respectively for M-gene amplification and for F-gene amplification the mean T were 84.04±0.037 and m 84.53±0.223. On the other hand the lentogenic NDV isolates including the vaccinal strains (HB1 and LaSota have a higher mean T (86.99±0.021 for M-gene amplification and 86.50±0.063 for F-gene amplification. The test showed no reaction with m unrelated RNA samples. In addition, the results were in good agreement with both virus isolation and biological pathotyping (MDT and ICIP. The assay offers an attractive alternative method for the diagnosis of NDV that can be easily applied in laboratory diagnosis as a screening test for the detection and differentiation of NDV infections. Conclusion: As was shown by the successful rapid detection and pathotyping of 15 NDV strains in clinical samples representing velogenic and lentogenic NDV strains, and the agreement with the results of virus isolation , biological pathotyping and pathogenicity indices. The results of this report suggests that the described SybrGreen I real-time RT-PCR assay in conjunction with Melting curve analysis used as a rapid, specific and simple diagnostic tools for detection and pathotyping of

  15. Newcastle disease virus-based H5 influenza vaccine protects chickens from lethal challenge with a highly pathogenic H5N2 avian influenza virus.

    Science.gov (United States)

    Ma, Jingjiao; Lee, Jinhwa; Liu, Haixia; Mena, Ignacio; Davis, A Sally; Sunwoo, Sun Young; Lang, Yuekun; Duff, Michael; Morozov, Igor; Li, Yuhao; Yang, Jianmei; García-Sastre, Adolfo; Richt, Juergen A; Ma, Wenjun

    2017-01-01

    Since December 2014, Eurasian-origin, highly pathogenic avian influenza H5 viruses including H5N1, H5N2, and H5N8 subtypes (called H5N x viruses), which belong to the H5 clade 2.3.4.4, have been detected in U.S. wild birds. Subsequently, highly pathogenic H5N2 and H5N8 viruses have caused outbreaks in U.S. domestic poultry. Vaccination is one of the most effective ways to control influenza outbreaks and protect animal and public health. Newcastle disease virus (NDV)-based influenza vaccines have been demonstrated to be efficacious and safe in poultry. Herein, we developed an NDV-based H5 vaccine (NDV-H5) that expresses a codon-optimized ectodomain of the hemagglutinin from the A/chicken/Iowa/04-20/2015 (H5N2) virus and evaluated its efficacy in chickens. Results showed that both live and inactivated NDV-H5 vaccines induced hemagglutinin inhibition antibody titers against the H5N2 virus in immunized chickens after prime and booster, and both NDV-H5 vaccines completely protected chickens from lethal challenge with the highly pathogenic H5N2 A/turkey/Minnesota/9845-4/2015 virus. No clinical signs and only minimal virus shedding was observed in both vaccinated groups. In contrast, all mock-vaccinated, H5N2-infected chickens shed virus and died within 5 days post challenge. Furthermore, one dose of the live NDV-H5 vaccine also provided protection of 90% chickens immunized by coarse spraying; after exposure to H5N2 challenge, sera from vaccinated surviving chickens neutralized both highly pathogenic H5N1 and H5N8 viruses. Taken together, our results suggest that the NDV-based H5 vaccine is able to protect chickens against intercontinental highly pathogenic H5N x viruses and can be used by mass application to protect the poultry industry.

  16. Expression of chicken interleukin-2 by a highly virulent strain of Newcastle disease virus leads to decreased systemic viral load but does not significantly affect mortality in chickens.

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    Susta, Leonardo; Diel, Diego G; Courtney, Sean; Cardenas-Garcia, Stivalis; Sundick, Roy S; Miller, Patti J; Brown, Corrie C; Afonso, Claudio L

    2015-08-08

    In mammals, interleukin 2 (IL-2) has been shown to decrease replication or attenuate pathogenicity of numerous viral pathogens (herpes simplex virus, vaccinia virus, human respiratory syncytial virus, human immunodeficiency virus) by activating natural killer cells (NK), cytotoxic T lymphocytes and expanding subsets of memory cells. In chickens, IL-2 has been shown to activate T cells, and as such it might have the potential to affect replication and pathogenesis of Newcastle disease virus (NDV). To assess the effect of IL-2 during NDV infection in chickens, we produced a recombinant virulent NDV strain expressing chicken IL-2 (rZJ1-IL2). The effects of IL-2 expression were investigated in vivo using the intracerebral pathogenicity index (ICPI) in day-old chicks and pathogenesis experiments in 4-week-old chickens. In these studies, rZJ1-IL2 was compared to a control virus expressing the green fluorescent protein (rZJ1-GFP). Assessed parameters included survival curves, detailed histological and immunohistochemical grading of lesions in multiple organs, and virus isolation in blood, spleen and mucosal secretions of infected birds. At the site of infection (eyelid), expression of IL-2 was demonstrated in areas of rZJ-IL2 replication, confirming IL-2 production in vivo. Compared to rZJ1-GFP strain, rZJ1-IL2 caused milder lesions and displayed decreased viral load in blood, spleen and mucosal secretions of infected birds. In the rZJ1-IL2-infected group, virus level in the blood peaked at day 4 post-infection (pi) (10(3.46) EID50 /0.1 ml) and drastically decreased at day 5 pi (10(0.9) EID50/0.1 ml), while in the rZJ1-GFP-infected group virus levels in the blood reached 10(5.35) EID50/0.1 ml at day 5. However, rZJ1-IL2-infected groups presented survival curves similar to control birds infected with rZJ1-GFP, with comparable clinical signs and 100 % mortality. Further, expression of IL-2 did not significantly affect the ICPI scores, compared to rZJ1-GFP strain. Increased

  17. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  18. Attitude of poultry farmers towards vaccination against newcastle ...

    African Journals Online (AJOL)

    Attitude of poultry farmers towards vaccination against newcastle disease and avian influenza in Ibadan, Nigeria. ... The PDF file you selected should load here if your Web browser has a PDF reader plug-in installed (for example, a recent version of Adobe Acrobat Reader). If you would like more information about how to ...

  19. Thermostable Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [Royal DSM, San Francisco, CA (United States)

    2010-03-24

    These are a set of slides from the conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  20. Thermostable Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [DSM Innovation, Incorporated, San Francisco, CA (United States)

    2010-04-19

    Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  1. Thermostability of biological systems: fundamentals, challenges, and quantification.

    Science.gov (United States)

    He, Xiaoming

    2011-01-01

    This review examines the fundamentals and challenges in engineering/understanding the thermostability of biological systems over a wide temperature range (from the cryogenic to hyperthermic regimen). Applications of the bio-thermostability engineering to either destroy unwanted or stabilize useful biologicals for the treatment of diseases in modern medicine are first introduced. Studies on the biological responses to cryogenic and hyperthermic temperatures for the various applications are reviewed to understand the mechanism of thermal (both cryo and hyperthermic) injury and its quantification at the molecular, cellular and tissue/organ levels. Methods for quantifying the thermophysical processes of the various applications are then summarized accounting for the effect of blood perfusion, metabolism, water transport across cell plasma membrane, and phase transition (both equilibrium and non-equilibrium such as ice formation and glass transition) of water. The review concludes with a summary of the status quo and future perspectives in engineering the thermostability of biological systems.

  2. Synthesis, characterization, and immune efficacy of layered double hydroxide@SiO2 nanoparticles with shell-core structure as a delivery carrier for Newcastle disease virus DNA vaccine

    Directory of Open Access Journals (Sweden)

    Zhao K

    2015-04-01

    Full Text Available Kai Zhao,1,* Guangyu Rong,1,2,* Chen Guo,1 Xiaomei Luo,1 Hong Kang,1 Yanwei Sun,1,3 Chunxiao Dai,4 Xiaohua Wang,1 Xin Wang,1 Zheng Jin,4 Shangjin Cui,3 Qingshen Sun1 1Key Laboratory of Microbiology, School of Life Science, Heilongjiang University, Harbin, People’s Republic of China; 2Department of Avian Infectious Disease, Shanghai Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Shanghai, People’s Republic of China; 3Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, People’s Republic of China; 4Key Laboratory of Chemical Engineering Process and Technology for High-efficiency Conversion, Heilongjiang University, Harbin, People’s Republic of China *These authors contributed equally to this work Abstract: Layered double hydroxide (LDH@SiO2 nanoparticles were developed as a delivery carrier for the plasmid DNA expressing the Newcastle disease virus F gene. The LDH was hydrotalcite-like materials. The plasmid DNA encapsulated in the LDH@SiO2 nanoparticles (pFDNA-LDH@SiO2-NPs was prepared by the coprecipitation method, and the properties of pFDNA-LDH@SiO2-NPs were characterized by transmission electron microscopy, zeta potential analyzer, Fourier transform infrared spectroscopy, and X-ray diffraction analysis. The results demonstrated that the pFDNA-LDH@SiO2-NPs had a regular morphology and high stability with a mean diameter of 371.93 nm, loading capacity of 39.66%±0.45%, and a zeta potential of +31.63 mV. A release assay in vitro showed that up to 91.36% of the total plasmid DNA could be sustainably released from the pFDNA-LDH@SiO2-NPs within 288 hours. The LDH@SiO2 nanoparticles had very low toxicity. Additionally, their high transfection efficiency in vitro was detected by fluorescent microscopy. Intranasal immunization of specific pathogen-free chickens with pFDNA-LDH@SiO2-NPs

  3. The role of Nicotiana gluca Graham (paraguayan herbs as an adjuvant in immunomodulation of Newcastle disease vaccine for broilers Estudo da ação de Nicotiana glauca Graham (erva paraguaia como coadjuvante em vacina contra a doença de Newcastle em frangos de corte

    Directory of Open Access Journals (Sweden)

    Fabiane Pereira Gentilini

    2008-07-01

    Full Text Available The Nicotiana glauca is a native plant species from Argentina, but found all over South América, being used against headaches, rheumatism, injuries, ulcers, and so on. Researchers have considered it as having immunomodulation effect. This study was conducted to investigate the use of a aqueous extract of Nicotiana glauca Graham as an immunomodulator (adjuvant of a Newcastle disease vaccine.. A total of 56 broilers were distributed into 4 experimental groups. Each one of them received 3 dosages of this vaccine with or without the addition of different concentrations of the extract Using hemmoaglutination inhibition techniques , the results have shown differences (P<0.05 in the third sera collection. An increase in the antibody titer with the inclusion of 5 mg/dosage of the extract (Treatment 3 as compared to 1 mg/dosage (Treatment 2 and 10 mg/dosage of the extract (Treatment 4 was observed, However, birds from Treatment 3 did not differ (P> 0.05 from Treatment 1. These results indicated that further investigations are required, including the use of cytotoxicity tests in vitro, to evaluate the immunomodulation effect of this extract.

     

    KEY WORDS: Immunomodulation effect, Nicotiana glauca Graham, vaccine.

    A Nicotiana glauca Graham é uma espécie nativa da Argentina, bem distribuída na América do Sul, sendo empregada, popularmente, contra dores de cabeça, dores reumáticas, cicatrização de feridas e úlceras, entre outros. Pesquisas têm avaliado a sua ação na potencialização da resposta imune. Assim, com este estudo, buscou-se avaliar a ação de um extrato aquoso de Nicotiana glauca Graham como coadjuvante imunológico em uma vacina contra a doença de Newcastle (DNC. Utilizaram-se 56 frangos de corte, distribuídos em quatro grupos experimentais, que receberam tr

  4. Efficacy of single dose of a bivalent vaccine containing inactivated Newcastle disease virus and reassortant highly pathogenic avian influenza H5N1 virus against lethal HPAI and NDV infection in chickens.

    Directory of Open Access Journals (Sweden)

    Dong-Hun Lee

    Full Text Available Highly pathogenic avian influenza (HPAI and Newcastle disease (ND are 2 devastating diseases of poultry, which cause great economic losses to the poultry industry. In the present study, we developed a bivalent vaccine containing antigens of inactivated ND and reassortant HPAI H5N1 viruses as a candidate poultry vaccine, and we evaluated its immunogenicity and protective efficacy in specific pathogen-free chickens. The 6:2 reassortant H5N1 vaccine strain containing the surface genes of the A/Chicken/Korea/ES/2003(H5N1 virus was successfully generated by reverse genetics. A polybasic cleavage site of the hemagglutinin segment was replaced by a monobasic cleavage site. We characterized the reverse genetics-derived reassortant HPAI H5N1 clade 2.5 vaccine strain by evaluating its growth kinetics in eggs, minimum effective dose in chickens, and cross-clade immunogenicity against HPAI clade 1 and 2. The bivalent vaccine was prepared by emulsifying inactivated ND (La Sota strain and reassortant HPAI viruses with Montanide ISA 70 adjuvant. A single immunization with this vaccine induced high levels of hemagglutination-inhibiting antibody titers and protected chickens against a lethal challenge with the wild-type HPAI and ND viruses. Our results demonstrate that the bivalent, inactivated vaccine developed in this study is a promising approach for the control of both HPAI H5N1 and ND viral infections.

  5. Protective Efficacy of Newcastle Disease Virus Expressing Soluble Trimeric Hemagglutinin against Highly Pathogenic H5N1 Influenza in Chickens and Mice

    NARCIS (Netherlands)

    Cornelissen, A.H.M.; Leeuw, de O.S.; Tacken, M.G.J.; Klos, H.C.; Vries, de R.P.; Boer-Luijtze, de E.A.; Zoelen-Bos, van D.J.; Rigter, A.; Rottier, P.J.M.; Moormann, R.J.M.; Haan, de C.A.M.

    2012-01-01

    Background: Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to

  6. Mobilization of immunoglobulin (Ig)-containing plasma cells in Harderian gland, cecal tonsil and trachea of broilers vaccinated with Newcastle Disease Vaccine.

    Science.gov (United States)

    Nasrin, M; Khan, M Z I; Siddiqi, M N H; Masum, M A

    2013-06-01

    Immunohistochemical studies of Harderian gland, cecal tonsil and trachea of various groups of broiler chickens and the response of Baby Chick Ranikhet Disease Vaccines (BCRDV) on the mobilization of Igs-cells during postnatal development of organs was investigated in the Dept. of Anatomy and Histology, Bangladesh Agricultural University, Mymensingh. In this study twelve chickens were grouped into vaccinated broilers (D14 and D28) which had received vaccines first at D3 of age and a booster dose given at D13; and non-vaccinated broilers (D1) which had not been vaccinated. In this study, it was observed that the frequency and distribution of Igs-positive cells were higher at D14 and at D28 rather than D1. Among Igs-positive cells, the IgG-positive cells were significantly higher than IgM and IgA-positive cells in the Harderian gland of D14 and D28 groups of chickens, however, in day-old chickens, the frequency of IgM-positive cells in this gland were greater. In the cecal tonsil, the frequency and distribution of IgG-positive cells were significantly higher than IgA- and IgM-positive cells both at D14 and D28 ages of chicken. On the other hand, in day-old chickens, the frequency and distribution of IgA-positive cells were insignificantly greater, followed by IgM and IgG-positive cells. In the trachea, few immunoglobulin-containing plasma cells were distributed in the subepithelial layer. IgM-positive cells were higher followed by IgG and IgA-positive cells in the trachea in D14 and D28 groups of chickens. In the same organ, IgG-positive plasma cells were greater than IgA and IgM-positive cells at one-day old. When the data for Harderian gland, cecal tonsil and trachea were compared statistically, it was observed that Igs-positive cells were statistically more common in cecal tonsils in day old chickens, and with the advancement of age, Igs-positive cells were found more in the Harderian gland. In conclusion, with the advancement of age in chickens the Harderian gland

  7. Optimization of medium composition for thermostable protease ...

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... Optimization of the fermentation medium for maximization of thermostable neutral protease production by Bacillus sp. ..... Each contour curve represented an infinite number of combinations of two ..... Production in sea-water of.

  8. Purification and Characterization of Thermostable Cellulase from ...

    African Journals Online (AJOL)

    Available online at http://www.tjpr.org ... Methods: Molecular community structure of the newly selected thermophilic bacterial ... Keywords: Thermostable cellulase, Sugarcane bagasse, Purification, Characterization, Hot spring ... Currently, one.

  9. Functional analysis of thermostable proteins involved in carbohydrate metabolism

    NARCIS (Netherlands)

    Akerboom, A.P.

    2007-01-01

    Thermostable proteins can resist temperature stress whilst keeping their integrity and functionality. In many cases, thermostable proteins originate from hyperthermophilic microorganisms that thrive in extreme environments. These systems are generally located close to geothermal (volcanic) activity,

  10. A comparative molecular dynamics study on thermostability of human and chicken prion proteins

    International Nuclear Information System (INIS)

    Ji, Hong-Fang; Zhang, Hong-Yu

    2007-01-01

    To compare the thermostabilities of human and chicken normal cellular prion proteins (HuPrP C and CkPrP C ), molecular dynamics (MD) simulations were performed for both proteins at an ensemble level (10 parallel simulations at 400 K and 5 parallel simulations at 300 K as a control). It is found that the thermostability of HuPrP C is comparable with that of CkPrP C , which implicates that the non-occurrence of prion diseases in non-mammals cannot be completely attributed to the thermodynamic properties of non-mammalian PrP C

  11. Thermostable Carbonic Anhydrases in Biotechnological Applications

    Directory of Open Access Journals (Sweden)

    Anna Di Fiore

    2015-07-01

    Full Text Available Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application.

  12. Thermostable crude endoglucanase produced by Aspergillus ...

    African Journals Online (AJOL)

    Cellulases are used in many industries worldwide and there is an ever increasing need to isolate, produce or develop thermostable cellulases. Manipulation of fermentation techniques in order to obtain desirable product(s) can be one line of action. In this study Aspergillus fumigatus was grown on chopped wheat straw in a ...

  13. Purification and Characterization of Thermostable Cellulase from ...

    African Journals Online (AJOL)

    The thermostable CMCase was purified with ion-exchange and gel filtration chromatography. Results: ... Conclusion: Due to its high temperature stability, the purified XM70-CMCase may be useful for industrial application such as biofuel, animal feed industry, paper industry and clarification of fruit juices. Keywords: ...

  14. Evaluation of thermostable enzymes for bioethanol processing

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia

    of fermentable sugars (glucose) as cellulose is tightly linked to hemicellulose and lignin. Lignocellulose is disrupted during pretreatment, but to degrade cellulose to single sugars, lignocellulolytic enzymes such as cellulases and hemicellulases are needed. Lignocellulolytic enzymes are costly...... for the ioethanol production, but the expenses can be reduced by using thermostable enzymes, which are known for their increased stability and inhibitor olerance. However, the advantage of using thermostable enzymes has not been studied thoroughly and more knowledge is needed for development of bioethanol processes....... Enzymes are added to the bioethanol process after pretreatment. For an efficient sugar and ethanol yield, the solids content of biomass is normally increased, which results in highly viscous slurries that are difficult to mix. Therefore, the first enzymatic challenge is to ensure rapid reduction...

  15. CSBB: synthetic biology research at Newcastle University.

    Science.gov (United States)

    Goñi-Moreno, Angel; Wipat, Anil; Krasnogor, Natalio

    2017-06-15

    The Centre for Synthetic Biology and the Bioeconomy (CSBB) brings together a far-reaching multidisciplinary community across all Newcastle University's faculties - Medical Sciences, Science, Agriculture and Engineering, and Humanities, Arts and Social Sciences. The CSBB focuses on many different areas of Synthetic Biology, including bioprocessing, computational design and in vivo computation, as well as improving understanding of basic molecular machinery. Such breadth is supported by major national and international research funding, a range of industrial partners in the North East of England and beyond, as well as a large number of doctoral and post-doctoral researchers. The CSBB trains the next generation of scientists through a 1-year MSc in Synthetic Biology. © 2017 The Author(s).

  16. Thermostable cellulase from a thermomonospora gene

    Science.gov (United States)

    Wilson, D.B.; Walker, L.P.; Zhang, S.

    1997-10-14

    The invention relates to a gene isolated from Thermomonospora fusca, wherein the gene encodes a thermostable cellulase. Disclosed is the nucleotide sequence of the T. fusca gene; and nucleic acid molecules comprising the gene, or a fragment of the gene, that can be used to recombinantly express the cellulase or a catalytically active polypeptide thereof, respectively. The isolated and purified recombinant cellulase or catalytically active polypeptide may be used to hydrolyze substrate either by itself; or in combination with other cellulases, with the resultant combination having unexpected hydrolytic activity. 3 figs.

  17. Isolation of herpesvirus and Newcastle disease virus from White Storks (Ciconia ciconia) maintained at four rehabilitation centres in northern Germany during 1983 to 2001 and failure to detect antibodies against avian influenza A viruses of subtypes H5 and H7 in these birds.

    Science.gov (United States)

    Kaleta, Erhard F; Kummerfeld, Norbert

    2012-01-01

    Herpesvirus isolations from peripheral white blood cells of 253 White Storks (Ciconia ciconia) were obtained during a long-term study (1983 to 2001). The storks lived for a few months to 20 years at four rehabilitation centres. Isolates were obtained from 83 of 253 storks. This herpesvirus is indigenous for storks and unrelated to any other avian herpesvirus. Significantly more herpesvirus isolates were obtained during spring than in autumn samplings. The intervals between the first and last virus isolation ranged from 1 to 15 years. Herpesvirus isolates were simultaneously obtained from white blood cells and from pharyngeal swabs of four of 34 storks but not from cloacal swabs. Neutralizing antibodies to stork herpesvirus were detected in 178 of 191 examined blood plasma samples. Neutralizing antibodies against stork herpesvirus did not correlate with herpesvirus viraemia. The results further substantiate the persistence of herpesvirus in White Storks and underline the previously unrecorded long periods of virus and antibody presence. Virulent avian paramyxovirus type 1 (APMV-1; Newcastle disease virus) was isolated from white blood cells during 1992 and 1993 from four healthy migrating storks, and possessed virulence markers on the cleavage site of the H and F genes. These properties resemble the NE type of APMV-1. Haemagglutination inhibition antibodies against APMV-1 were detected in 16 of 191 blood plasma samples. Avian influenza A virus was not isolated and antibodies against subtypes H5 and H7 were not detected.

  18. Thermostability in endoglucanases is fold-specific

    Science.gov (United States)

    2011-01-01

    Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy) database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient. PMID:21291533

  19. Thermostability in endoglucanases is fold-specific

    Directory of Open Access Journals (Sweden)

    Wolt Jeffrey D

    2011-02-01

    Full Text Available Abstract Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient.

  20. Approaches for improving thermostability characteristics in cellulases.

    Science.gov (United States)

    Anbar, Michael; Bayer, Edward A

    2012-01-01

    Many efforts have been invested to reduce the cost of biofuel production to substitute renewable sources of energy for fossil-based fuels. At the forefront of these efforts are the initiatives to convert plant-derived cellulosic material to biofuels. Although significant improvements have been achieved recently in cellulase engineering in both efficiency and cost reduction, complete degradation of lignocellulosic material still requires very long periods of time and high enzyme loads. Thermostable cellulases offer many advantages in the bioconversion process, which include increase in specific activity, higher levels of stability, inhibition of microbial growth, increase in mass transfer rate due to lower fluid viscosity, and greater flexibility in the bioprocess. Besides rational design methods, which require deep understanding of protein structure-function relationship, two of the major methods for improvement in specific cellulase properties are directed evolution and knowledge-based library design based on multiple sequence alignments. In this chapter, we provide protocols for constructing and screening of improved thermostable cellulases. Modifications of these protocols may also be used for screening for other improved properties of cellulases such as pH tolerance, high salt, and more. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. The Newcastle geothermal system, Iron County, Utah

    Energy Technology Data Exchange (ETDEWEB)

    Blackett, R.E.; Shubat, M.A.; Bishop, C.E. (Utah Geological and Mineral Survey, Salt Lake City, UT (USA)); Chapman, D.S.; Forster, C.B.; Schlinger, C.M. (Utah Univ., Salt Lake City, UT (USA). Dept. of Geology and Geophysics)

    1990-03-01

    Geological, geophysical and geochemical studies contributed to conceptual hydrologic model of the blind'' (no surface expression), moderate-temperature (greater than 130{degree}C) Newcastle geothermal system, located in the Basin and Range-Colorado Plateau transition zone of southwestern Utah. Temperature gradient measurements define a thermal anomaly centered near the surface trace of the range-bounding Antelope Range fault with and elongate dissipative plume extending north into the adjacent Escalante Valley. Spontaneous potential and resistivity surveys sharply define the geometry of the dominant upflow zone (not yet explored), indicating that most of the thermal fluid issues form a short segment along the Antelope Range fault and discharges into a gently-dipping aquifer. Production wells show that this aquifer lies at a depth between 85 and 95 meter. Electrical surveys also show that some leakage of thermal fluid occurs over a 1.5 km (minimum) interval along the trace of the Antelope Range fault. Major element, oxygen and hydrogen isotopic analyses of water samples indicate that the thermal fluid is a mixture of meteoric water derived from recharge areas in the Pine Valley Mountains and cold, shallow groundwater. A northwest-southeast trending system of faults, encompassing a zone of increased fracture permeability, collects meteoric water from the recharge area, allows circulation to a depth of 3 to 5 kilometers, and intersects the northeast-striking Antelope Range fault. We postulate that mineral precipitates form a seal along the Antelope Range fault, preventing the discharge of thermal fluids into basin-fill sediments at depth, and allowing heated fluid to approach the surface. Eventually, continued mineral deposition could result in the development of hot springs at the ground surface.

  2. Thermostable enzymes as biocatalysts in the biofuel industry.

    Science.gov (United States)

    Yeoman, Carl J; Han, Yejun; Dodd, Dylan; Schroeder, Charles M; Mackie, Roderick I; Cann, Isaac K O

    2010-01-01

    Lignocellulose is the most abundant carbohydrate source in nature and represents an ideal renewable energy source. Thermostable enzymes that hydrolyze lignocellulose to its component sugars have significant advantages for improving the conversion rate of biomass over their mesophilic counterparts. We review here the recent literature on the development and use of thermostable enzymes for the depolymerization of lignocellulosic feedstocks for biofuel production. Furthermore, we discuss the protein structure, mechanisms of thermostability, and specific strategies that can be used to improve the thermal stability of lignocellulosic biocatalysts. Copyright 2010 Elsevier Inc. All rights reserved.

  3. Engineering of pectinolytic enzymes for enhanced thermostability

    DEFF Research Database (Denmark)

    Larsen, Dorte Møller

    Conversion of waste materials into valuable compounds is promising concerning transformation of byproduct streams such as sugar beet and potato pulp. In order to obtain those compounds with reduced energy consumption, carbohydrate active enzymes can be used as catalysts. Sugar beet and potato pulp...... consist of pectin that can be converted into beneficial polymeric and oligomeric carbohydrates requiring enzymes such as pectin lyases, rhamnogalacturonan I (RGI) lyases, polygalacturonases and galactanases. Enzymatic conversion of such pectinaceous biomasses at high temperatures is advantageous...... as it gives rise to lower substrate viscosity, easier mixing, higher substrate solubility and lowers the risk of contamination. The overall objective of this thesis was to discover enzymes for degradation of RGI structures in pectin and further engineer for enhanced thermostability. The hypotheses were...

  4. Thermostable Alginate degrading enzymes and their methods of use

    NARCIS (Netherlands)

    Hreggvidsson, Gudmundur Oli; Jonsson, Oskar W.J.; Bjornsdottir, Bryndis; Fridjonsson, Hedinn O; Altenbuchner, Josef; Watzlawick, Hildegard; Dobruchowska, Justyna; Kamerling, Johannis

    2015-01-01

    The present invention relates to the identification, production and use of thermostable alginate lyase enzymes that can be used to partially degrade alginate to yield oligosaccharides or to give complete degradation of alginate to yield (unsaturated) mono-uronates.

  5. Porinas as an adyuvant of inactivated Newcastle vaccine in broilers

    Directory of Open Access Journals (Sweden)

    Francisco Bustos M.

    2005-05-01

    Full Text Available Three groups of 25 broilers were vaccinated on two opportunities by aerosol using inactivated NC (Newcastle virus and different helper concentrations of porinas (20 ìg, 50 ìg, 125 ìg. A fourth group was injected with live B1 virus (12 and 28 days of age nasally. The NC inactivated virus (La Sota strain was concentrated 10 times with PEG with a final titer of 1:2.056. Twenty serums for each group were taken in order to evaluate NC antibodies using the HI and double immuno-difusion tests for IgA detection at 1, 12, 28 and 42 days of age. During the study the chickens were on a restricted diet in order to control ascites (2.640 mosl. On day 42, two broilers of the fourth group (live virus presented ascites and 1 broiler of group 1 presented lung edema (20 ìg. The geometric mean for NC antibodies titers at 42 days of age was 2 in the groups 1,2,3 and 5.7 in the group 4 (Log 2. For IgA, 180 mg/dl, 135 mg/dl, 120 mg/dl and 176 mg/dl respectively. Three broilers of each group were challenged with a pathogenic strain of NC, at 42 day of age, without signs of disease after 72 hours when the positive control group was dead. Gross and microscopic lesions were not detected in the bursa of Fabricius or thymo. [thymo sounds like short hand for something that should be properly named.] Very good animal weight, conversion and efficiency results were observed in all the groups. New studies using a fixed dose of porinas, larger numbers of broilers and the establishment of protective levels of IgA against NC challenge are recommended.

  6. Highly Efficient Thermostable DSM Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [DSM Innovation, Inc., San Francisco, CA (United States)

    2011-04-26

    These are the slides from this presentation. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  7. Consistent mutational paths predict eukaryotic thermostability

    Directory of Open Access Journals (Sweden)

    van Noort Vera

    2013-01-01

    Full Text Available Abstract Background Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published. Results Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1, we could also characterise the molecular consequences of some of these mutations. Conclusions The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.

  8. Avian paramyxovirus serotype 1 (Newcastle disease virus), avian influenza virus, and Salmonella spp. in mute swans (Cygnus olor) in the Great Lakes region and Atlantic Coast of the United States.

    Science.gov (United States)

    Pedersen, Kerri; Marks, David R; Arsnoe, Dustin M; Afonso, Claudio L; Bevins, Sarah N; Miller, Patti J; Randall, Adam R; DeLiberto, Thomas J

    2014-03-01

    Since their introduction to the United States in the late 19th century, mute swans (Cygnus olor) have become a nuisance species by causing damage to aquatic habitats, acting aggressively toward humans, competing with native waterfowl, and potentially transmitting or serving as a reservoir of infectious diseases to humans and poultry. In an effort to investigate their potential role as a disease reservoir and to establish avian health baselines for pathogens that threaten agricultural species or human health, we collected samples from 858 mute swans and tested them for avian paramyxovirus serotype 1 (APMV-1), avian influenza virus (AIV), and Salmonella spp. when possible. Our results indicate that exposure to APMV-1 and AIV is common (60%, n = 771, and 45%, n = 344, antibody prevalence, respectively) in mute swans, but detection of active viral shedding is less common (8.7%, n = 414, and 0.8%, n = 390, respectively). Salmonella was isolated from three mute swans (0.6%, n = 459), and although the serovars identified have been implicated in previous human outbreaks, it does not appear that Salmonella is commonly carried by mute swans.

  9. Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia; Jørgensen, Henning

    2013-01-01

    the influence of temperature and ethanol on enzyme activity and stability in the distillation step, where most enzymes are inactivated due to high temperatures. Two enzyme mixtures, a mesophilic and a thermostable mixture, were exposed to typical process conditions [temperatures from 55 to 65 °C and up to 5...... % ethanol (w/v)] followed by specific enzyme activity analyses and SDS-PAGE. The thermostable and mesophilic mixture remained active at up to 65 and 55 °C, respectively. When the enzyme mixtures reached their maximum temperature limit, ethanol had a remarkable influence on enzyme activity, e.g., the more...... ethanol, the faster the inactivation. The reason could be the hydrophobic interaction of ethanol on the tertiary structure of the enzyme protein. The thermostable mixture was more tolerant to temperature and ethanol and could therefore be a potential candidate for recycling after distillation....

  10. Thermostability in rubredoxin and its relationship to mechanical rigidity

    Science.gov (United States)

    Rader, A. J.

    2010-03-01

    The source of increased stability in proteins from organisms that thrive in extreme thermal environments is not well understood. Previous experimental and theoretical studies have suggested many different features possibly responsible for such thermostability. Many of these thermostabilizing mechanisms can be accounted for in terms of structural rigidity. Thus a plausible hypothesis accounting for this remarkable stability in thermophilic enzymes states that these enzymes have enhanced conformational rigidity at temperatures below their native, functioning temperature. Experimental evidence exists to both support and contradict this supposition. We computationally investigate the relationship between thermostability and rigidity using rubredoxin as a case study. The mechanical rigidity is calculated using atomic models of homologous rubredoxin structures from the hyperthermophile Pyrococcus furiosus and mesophile Clostridium pasteurianum using the FIRST software. A global increase in structural rigidity (equivalently a decrease in flexibility) corresponds to an increase in thermostability. Locally, rigidity differences (between mesophilic and thermophilic structures) agree with differences in protection factors.

  11. Thermostability in rubredoxin and its relationship to mechanical rigidity

    International Nuclear Information System (INIS)

    Rader, A J

    2010-01-01

    The source of increased stability in proteins from organisms that thrive in extreme thermal environments is not well understood. Previous experimental and theoretical studies have suggested many different features possibly responsible for such thermostability. Many of these thermostabilizing mechanisms can be accounted for in terms of structural rigidity. Thus a plausible hypothesis accounting for this remarkable stability in thermophilic enzymes states that these enzymes have enhanced conformational rigidity at temperatures below their native, functioning temperature. Experimental evidence exists to both support and contradict this supposition. We computationally investigate the relationship between thermostability and rigidity using rubredoxin as a case study. The mechanical rigidity is calculated using atomic models of homologous rubredoxin structures from the hyperthermophile Pyrococcus furiosus and mesophile Clostridium pasteurianum using the FIRST software. A global increase in structural rigidity (equivalently a decrease in flexibility) corresponds to an increase in thermostability. Locally, rigidity differences (between mesophilic and thermophilic structures) agree with differences in protection factors

  12. Comparison of the thermostability of cellulases from various thermophilic fungi

    Energy Technology Data Exchange (ETDEWEB)

    Wojtczak, G; Breuil, C; Yamada, J; Saddler, J N

    1987-10-01

    The cellulase activities of six thermophilic fungi were compared. Although the thermophilic fungi grew at relatively high temperatures (> 45/sup 0/C) the optimum temperatures for assaying the various cellulase activities were only slightly higher than the optimum temperatures for the mesophilic fungi, Trichoderma harzianum. Over prolonged incubation (> 24 h) the thermophilic strains demonstrated a higher hydrolytic potential as a result of the greater thermostability of the cellulase components. Although the extracellular cellulase activities had similar pH and temperature optima, in some cases the thermostability of the extracellular components were considerably lower.

  13. Prediction of Protein Thermostability by an Efficient Neural Network Approach

    Directory of Open Access Journals (Sweden)

    Jalal Rezaeenour

    2016-10-01

    Full Text Available Introduction: Manipulation of protein stability is important for understanding the principles that govern protein thermostability, both in basic research and industrial applications. Various data mining techniques exist for prediction of thermostable proteins. Furthermore, ANN methods have attracted significant attention for prediction of thermostability, because they constitute an appropriate approach to mapping the non-linear input-output relationships and massive parallel computing. Method: An Extreme Learning Machine (ELM was applied to estimate thermal behavior of 1289 proteins. In the proposed algorithm, the parameters of ELM were optimized using a Genetic Algorithm (GA, which tuned a set of input variables, hidden layer biases, and input weights, to and enhance the prediction performance. The method was executed on a set of amino acids, yielding a total of 613 protein features. A number of feature selection algorithms were used to build subsets of the features. A total of 1289 protein samples and 613 protein features were calculated from UniProt database to understand features contributing to the enzymes’ thermostability and find out the main features that influence this valuable characteristic. Results:At the primary structure level, Gln, Glu and polar were the features that mostly contributed to protein thermostability. At the secondary structure level, Helix_S, Coil, and charged_Coil were the most important features affecting protein thermostability. These results suggest that the thermostability of proteins is mainly associated with primary structural features of the protein. According to the results, the influence of primary structure on the thermostabilty of a protein was more important than that of the secondary structure. It is shown that prediction accuracy of ELM (mean square error can improve dramatically using GA with error rates RMSE=0.004 and MAPE=0.1003. Conclusion: The proposed approach for forecasting problem

  14. In ovo Technology and newcastle disease resistance in Japanese Quail

    International Nuclear Information System (INIS)

    Ezzat, I.E.; Abu-Taleb, A.M.; Ali, I.E.; Shabana, M.K.

    2001-01-01

    A total number of 1600 fertile eggs from japanese quail birds were used in this study. The eggs were divided into eight groups then incubated. NDV and vite were administered to the groups of eggs in Ovo injection at day 14 from incubation. Hatchability, body weight, egg production and mortality were recorded weekly for each group. Five blood samples were collected weekly from each group to measure total serum proteins, albumin, globulin, T3, T4 and HI titre. The results of this work revealed an increase in total serum proteins, globulin, and a decrease in t3, T4 and albumin values of the in ove vaccinating groups. Also HI titre recorded higher values due to ovo vaccination alone or combined with vit. E. It was noticed that the group injected by inactive vaccine plus vit. E registered high increases in hatchability, body weight and egg production beside a decrease in mortality

  15. Avian influenza, Gumboro and Newcastle disease antibodies and ...

    African Journals Online (AJOL)

    If you would like more information about how to print, save, and work with PDFs, Highwire Press provides a helpful Frequently Asked Questions about PDFs. Alternatively, you can download the PDF file directly to your computer, from where it can be opened using a PDF reader. To download the PDF, click the Download link ...

  16. Retrospective analysis of Newcastle disease diagnosed at the ...

    African Journals Online (AJOL)

    ADEYEYE

    2015-10-22

    Solomon et al., 2012;. Snoeck et al., 2013). Vaccination is practiced widely and ... books. Postmortem examination, microbiology and haemagglutination activity of tracheal swabs or serology (haemagglutination inhibition test) was.

  17. Production of thermostable and organic solvent-tolerant alkaline ...

    African Journals Online (AJOL)

    An alkaliphilic bacterium producing organic solvent-tolerant and thermostable alkaline protease was isolated from poultry litter site and identified as Bacillus coagulans PSB-07. Protease production under different submerged fermentation conditions were investigated with the aim of optimizing yield of enzyme. B. coagulans ...

  18. Engineering Thermostable Microbial Xylanases Toward its Industrial Applications.

    Science.gov (United States)

    Kumar, Vishal; Dangi, Arun Kumar; Shukla, Pratyoosh

    2018-03-01

    Xylanases are one of the important hydrolytic enzymes which hydrolyze the β-1, 4 xylosidic linkage of the backbone of the xylan polymeric chain which consists of xylose subunits. Xylanases are mainly found in plant cell walls and are produced by several kinds of microorganisms such as fungi, bacteria, yeast, and some protozoans. The fungi are considered as most potent xylanase producers than that of yeast and bacteria. There is a broad series of industrial applications for the thermostable xylanase as an industrial enzyme. Thermostable xylanases have been used in a number of industries such as paper and pulp industry, biofuel industry, food and feed industry, textile industry, etc. The present review explores xylanase-substrate interactions using gene-editing tools toward the comprehension in improvement in industrial stability of xylanases. The various protein-engineering and metabolic-engineering methods have also been explored to improve operational stability of xylanase. Thermostable xylanases have also been used for improvement in animal feed nutritional value. Furthermore, they have been used directly in bakery and breweries, including a major use in paper and pulp industry as a biobleaching agent. This present review envisages some of such applications of thermostable xylanases for their bioengineering.

  19. Thermophilic growth and enzymatic thermostability are polyphyletic traits within Chaetomiaceae.

    Science.gov (United States)

    van den Brink, Joost; Facun, Kryss; de Vries, Michel; Stielow, J Benjamin

    2015-12-01

    Thermophilic fungi have the potential to produce industrial-relevant thermostable enzymes, in particular for the degradation of plant biomass. Sordariales is one of the few fungal orders containing several thermophilic taxa, of which many have been associated with the production of thermostable enzymes. The evolutionary affiliation of Sordariales fungi, especially between thermophiles and non-thermophilic relatives, is however poorly understood. Phylogenetic analysis within the current study was based on sequence data, derived from a traditional Sanger and highly multiplexed targeted next generation sequencing approach of 45 isolates. The inferred phylogeny and detailed growth analysis rendered the trait 'thermophily' as polyphyletic within Chaetomiaceae (Sordariales, Sordariomycetes), and characteristic to: Myceliophthora spp., Thielavia terrestris, Chaetomium thermophilum, and Mycothermus thermophilus. Compared to mesophiles, the isolates within thermophilic taxa produced enzyme mixtures with the highest thermostability of known cellulase activities. Temperature profiles of the enzyme activities correlated strongly with the optimal growth temperatures of the isolates but not with their phylogenetic relationships. This strong correlation between growth and enzyme characteristics indicated that detailed analysis of growth does give predictive information on enzyme physiology. The variation in growth and enzyme characteristics reveals these fungi as an excellent platform to better understand fungal thermophily and enzyme thermostability. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  20. Thermostable endoglucanases in the liquefaction of hydrothermally pretreated wheat straw

    Directory of Open Access Journals (Sweden)

    Siika-aho Matti

    2011-01-01

    Full Text Available Abstract Background Thermostable enzymes have several benefits in lignocellulose processing. In particular, they potentially allow the use of increased substrate concentrations (because the substrate viscosity decreases as the temperature increases, resulting in improved product yields and reduced capital and processing costs. A short pre-hydrolysis step at an elevated temperature using thermostable enzymes aimed at rapid liquefaction of the feedstock is seen as an attractive way to overcome the technical problems (such as poor mixing and mass transfer properties connected with high initial solid loadings in the lignocellulose to ethanol process. Results The capability of novel thermostable enzymes to reduce the viscosity of high-solid biomass suspensions using a real-time viscometric measurement method was investigated. Heterologously expressed enzymes from various thermophilic organisms were compared for their ability to liquefy the lignocellulosic substrate, hydrothermally pretreated wheat straw. Once the best enzymes were identified, the optimal temperatures for these enzymes to decrease substrate viscosity were compared. The combined hydrolytic properties of the thermostable preparations were tested in hydrolysis experiments. The studied mixtures were primarily designed to have good liquefaction potential, and therefore contained an enhanced proportion of the key liquefying enzyme, EGII/Cel5A. Conclusions Endoglucanases were shown to have a superior ability to rapidly reduce the viscosity of the 15% (w/w; dry matter hydrothermally pretreated wheat straw. Based on temperature profiling studies, Thermoascus aurantiacus EGII/Cel5A was the most promising enzyme for biomass liquefaction. Even though they were not optimized for saccharification, many of the thermostable enzyme mixtures had superior hydrolytic properties compared with the commercial reference enzymes at 55°C.

  1. Health assessment for Newcastle Abandoned Container Site, Newcastle, Delaware, Region 3. CERCLIS No. DED058980442. Preliminary report

    International Nuclear Information System (INIS)

    1988-01-01

    The Newcastle Abandoned Container site is listed on the National Priorities List. On February 28, 1986, radioactive, corrosive, and toxic materials, and a large number of containers filled with unknown material, were inventoried, containerized, and removed. On-site contamination consists of polychlorinated biphenyls, trichloroethylene, creosote, chloroform, and di-n-butylphthalate in soil; and tetrachloroethylene, acetone, xylene, trichlorofluoromethane, trichloroethylene, and tris(betachloroisopropyl)phosphate in groundwater. The site is considered to be of potential public health concern because of the risk to human health caused by the possibility of exposure to hazardous substances via groundwater. The potential for contamination of the lower aquifer should be investigated

  2. Development of a thermostable microneedle patch for influenza vaccination

    Science.gov (United States)

    Mistilis, Matthew; Bommarius, Andreas S; Prausnitz, Mark R.

    2017-01-01

    The goal of this study is to develop thermostable microneedle patch formulations for influenza vaccine that can be partially or completely removed from the cold chain. During vaccine drying associated with microneedle patch manufacturing, ammonium acetate and HEPES buffer salts stabilized influenza vaccine, surfactants had little effect during drying, drying temperature had weak effects on vaccine stability, and drying on polydimethylsiloxane led to increased stability compared to drying on stainless steel. A number of excipients, mostly polysaccharides and some amino acids, further stabilized the influenza vaccine during drying. Over longer time scales of storage, combinations of stabilizers preserved the most vaccine activity. Finally, dissolving microneedle patches formulated with arginine and calcium heptagluconate had no significant activity loss for all three strains of seasonal influenza vaccine during storage at room temperature for six months. We conclude that appropriately formulated microneedle patches can exhibit remarkable thermostability that could enable storage and distribution of influenza vaccine outside the cold chain. PMID:25448542

  3. Thermostable Subunit Vaccines for Pulmonary Delivery: How Close Are We?

    DEFF Research Database (Denmark)

    Foged, Camilla

    2016-01-01

    , such as influenza, tuberculosis, and Ebola, for which no good universal vaccines exist. At least two pharmaceutical improvements are expected to help filling this gap: i) The development of thermostable vaccine dosage forms, and ii) the full exploitation of the adjuvant technology for subunit vaccines to potentiate...... strong immune responses. This review highlights the status and recent advances in formulation and pulmonary delivery of thermostable human subunit vaccines. Such vaccines are very appealing from compliance, distribution and immunological point of view: Being non-invasive, inhalable vaccines are self...... immunity. Here, I review state of the art and perspectives in formulation design and processing methods for powder-based subunit vaccines intended for pulmonary administration, and present dry powder inhaler technologies suitable for translating these vaccines into clinical trials....

  4. Thermostable cellulases, and mutants thereof, capable of hydrolyzing cellulose in ionic liquid

    Science.gov (United States)

    Sapra, Rajat; Datta, Supratim; Chen, Zhiwei; Holmes, Bradley M.; Simmons, Blake A.; Blanch, Harvey W.

    2016-04-26

    The present invention provides for a composition comprising an ionic liquid and a thermostable cellulose, and a method of hydrolyzing a cellulose, comprising: (a) providing a composition comprising a solution comprising an ionic liquid and a cellulose, and (b) introducing a thermostable cellulase to the solution, such that the cellulose is hydrolyzed by the cellulase. The present invention also provides for a Thermatoga maritima thermostable cellulase mutant with increased cellulase activity.

  5. The Impact of Making Vaccines Thermostable in Niger’s Vaccine Supply Chain

    Science.gov (United States)

    Lee, Bruce Y.; Cakouros, Brigid E.; Assi, Tina-Marie; Connor, Diana L.; Welling, Joel; Kone, Souleymane; Djibo, Ali; Wateska, Angela R.; Pierre, Lionel; Brown, Shawn T.

    2012-01-01

    Objective Determine the effects on the vaccine cold chain of making different types of World Health Organization (WHO) Expanded Program on Immunizations (EPI) vaccines thermostable. Methods Utilizing a detailed computational, discrete-event simulation model of the Niger vaccine supply chain, we simulated the impact of making different combinations of the six current EPI vaccines thermostable. Findings Making any EPI vaccine thermostable relieved existing supply chain bottlenecks (especially at the lowest levels), increased vaccine availability of all EPI vaccines, and decreased cold storage and transport capacity utilization. By far, the most substantial impact came from making the pentavalent vaccine thermostable, increasing its own vaccine availability from 87% to 97% and the vaccine availabilities of all other remaining non-thermostable EPI vaccines to over 93%. By contrast, making each of the other vaccines thermostable had considerably less effect on the remaining vaccines, failing to increase the vaccine availabilities of other vaccines to more than 89%. Making tetanus toxoid vaccine along with the pentavalent thermostable further increased the vaccine availability of all EPI vaccines by at least 1–2%. Conclusion Our study shows the potential benefits of making any of Niger’s EPI vaccines thermostable and therefore supports further development of thermostable vaccines. Eliminating the need for refrigerators and freezers should not necessarily be the only benefit and goal of vaccine thermostability. Rather, making even a single vaccine (or some subset of the vaccines) thermostable could free up significant cold storage space for other vaccines, and thereby help alleviate supply chain bottlenecks that occur throughout the world. PMID:22789507

  6. The impact of making vaccines thermostable in Niger's vaccine supply chain.

    Science.gov (United States)

    Lee, Bruce Y; Cakouros, Brigid E; Assi, Tina-Marie; Connor, Diana L; Welling, Joel; Kone, Souleymane; Djibo, Ali; Wateska, Angela R; Pierre, Lionel; Brown, Shawn T

    2012-08-17

    Determine the effects on the vaccine cold chain of making different types of World Health Organization (WHO) Expanded Program on Immunizations (EPI) vaccines thermostable. Utilizing a detailed computational, discrete-event simulation model of the Niger vaccine supply chain, we simulated the impact of making different combinations of the six current EPI vaccines thermostable. Making any EPI vaccine thermostable relieved existing supply chain bottlenecks (especially at the lowest levels), increased vaccine availability of all EPI vaccines, and decreased cold storage and transport capacity utilization. By far, the most substantial impact came from making the pentavalent vaccine thermostable, increasing its own vaccine availability from 87% to 97% and the vaccine availabilities of all other remaining non-thermostable EPI vaccines to over 93%. By contrast, making each of the other vaccines thermostable had considerably less effect on the remaining vaccines, failing to increase the vaccine availabilities of other vaccines to more than 89%. Making tetanus toxoid vaccine along with the pentavalent thermostable further increased the vaccine availability of all EPI vaccines by at least 1-2%. Our study shows the potential benefits of making any of Niger's EPI vaccines thermostable and therefore supports further development of thermostable vaccines. Eliminating the need for refrigerators and freezers should not necessarily be the only benefit and goal of vaccine thermostability. Rather, making even a single vaccine (or some subset of the vaccines) thermostable could free up significant cold storage space for other vaccines, and thereby help alleviate supply chain bottlenecks that occur throughout the world. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. EFFECTS OF CHANGING THE INTERACTION BETWEEN SUBDOMAINS ON THE THERMOSTABILITY OF BACILLUS NEUTRAL PROTEASES

    NARCIS (Netherlands)

    EIJSINK, VGH; VRIEND, G; VANDERVINNE, B; HAZES, B; VANDENBURG, B; VENEMA, G

    1992-01-01

    Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to

  8. Useful halophilic, thermostable and ionic liquids tolerant cellulases

    Science.gov (United States)

    Zhang, Tao; Datta, Supratim; Simmons, Blake A.; Rubin, Edward M.

    2016-06-28

    The present invention provides for an isolated or recombinant polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence of a Halorhabdus utahensis cellulase, such as Hu-CBH1, wherein said amino acid sequence has a halophilic thermostable and/or thermophilic cellobiohydrolase (CBH) activity. In some embodiments, the polypeptide has a CBH activity that is resistant to up to about 20% of ionic liquids. The present invention also provides for compositions comprising and methods using the isolated or recombinant polypeptide.

  9. Understanding thermostability and pH dependent properties of proteins

    DEFF Research Database (Denmark)

    Galberg, Pernille

    The work performed in this thesis is part of a larger project (“Computational design of stable enzymes”) involving several research teams, which aimed to improve PROPKA (http://propka.ki.ku.dk) and to provide the scientific community with a computational protocol and associated PROPKA program......, which could be used for predicting mutations with expectation of increased thermostability at a certain pH value or a shifted pH activity optimum. The ability of a Bacillus circulans xylanase (BCX) mutant (N35D/A115E) to induce a decrease in pH activity optimum was evaluated by a pH dependent xylanase...

  10. Molecular Dynamics Approach in Designing Thermostable Aspergillus niger Xylanase

    Science.gov (United States)

    Malau, N. D.; Sianturi, M.

    2017-03-01

    Molecular dynamics methods we have applied as a tool in designing thermostable Aspergillus niger Xylanase, by examining Root Mean Square Deviation (RMSD) and The Stability of the Secondary Structure of enzymes structure at its optimum temperature and compare with its high temperature behavior. As RMSD represents structural fluctuation at a particular temperature, a better understanding of this factor will suggest approaches to bioengineer these enzymes to enhance their thermostability. In this work molecular dynamic simulations of Aspergillus niger xylanase (ANX) have been carried at 400K (optimum catalytic temperature) for 2.5 ns and 500K (ANX reported inactive temperature) for 2.5 ns. Analysis have shown that the Root Mean Square Deviation (RMSD) significant increase at higher temperatures compared at optimum temperature and some of the secondary structures of ANX that have been damaged at high temperature. Structural analysis revealed that the fluctuations of the α-helix and β-sheet regions are larger at higher temperatures compared to the fluctuations at optimum temperature.

  11. Potential and utilization of thermophiles and thermostable enzymes in biorefining

    Directory of Open Access Journals (Sweden)

    Karlsson Eva

    2007-03-01

    Full Text Available Abstract In today's world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing raw materials. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Strategies that enhance thermostablity of enzymes both in vivo and in vitro are also assessed. Moreover, this review deals with efforts made on developing vectors for expressing recombinant enzymes in thermophilic hosts.

  12. Thermostability promotes the cooperative function of split adenylate kinases.

    Science.gov (United States)

    Nguyen, Peter Q; Liu, Shirley; Thompson, Jeremy C; Silberg, Jonathan J

    2008-05-01

    Proteins can often be cleaved to create inactive polypeptides that associate into functional complexes through non-covalent interactions, but little is known about what influences the cooperative function of the ensuing protein fragments. Here, we examine whether protein thermostability affects protein fragment complementation by characterizing the function of split adenylate kinases from the mesophile Bacillus subtilis (AKBs) and the hyperthermophile Thermotoga neapolitana (AKTn). Complementation studies revealed that the split AKTn supported the growth of Escherichia coli with a temperature-sensitive AK, but not the fragmented AKBs. However, weak complementation occurred when the AKBs fragments were fused to polypeptides that strongly associate, and this was enhanced by a Q16L mutation that thermostabilizes the full-length protein. To examine how the split AK homologs differ in structure and function, their catalytic activity, zinc content, and circular dichroism spectra were characterized. The reconstituted AKTn had higher levels of zinc, greater secondary structure, and >10(3)-fold more activity than the AKBs pair, albeit 17-fold less active than full-length AKTn. These findings provide evidence that the design of protein fragments that cooperatively function can be improved by choosing proteins with the greatest thermostability for bisection, and they suggest that this arises because hyperthermophilic protein fragments exhibit greater residual structure compared to their mesophilic counterparts.

  13. A novel non-thermostable deuterolysin from Aspergillus oryzae.

    Science.gov (United States)

    Maeda, Hiroshi; Katase, Toru; Sakai, Daisuke; Takeuchi, Michio; Kusumoto, Ken-Ichi; Amano, Hitoshi; Ishida, Hiroki; Abe, Keietsu; Yamagata, Youhei

    2016-09-01

    Three putative deuterolysin (EC 3.4.24.29) genes (deuA, deuB, and deuC) were found in the Aspergillus oryzae genome database ( http://www.bio.nite.go.jp/dogan/project/view/AO ). One of these genes, deuA, was corresponding to NpII gene, previously reported. DeuA and DeuB were overexpressed by recombinant A. oryzae and were purified. The degradation profiles against protein substrates of both enzymes were similar, but DeuB showed wider substrate specificity against peptidyl MCA-substrates compared with DeuA. Enzymatic profiles of DeuB except for thermostability also resembled those of DeuA. DeuB was inactivated by heat treatment above 80° C, different from thermostable DeuA. Transcription analysis in wild type A. oryzae showed only deuB was expressed in liquid culture, and the addition of the proteinous substrate upregulated the transcription. Furthermore, the NaNO3 addition seems to eliminate the effect of proteinous substrate for the transcription of deuB.

  14. National Uranium Resource Evaluation: Newcastle Quadrangle, Wyoming and South Dakota

    International Nuclear Information System (INIS)

    Santos, E.S.; Robinson, K.; Geer, K.A.; Blattspieler, J.G.

    1982-09-01

    Uranium resources of the Newcastle 1 0 x2 0 Quadrangle, Wyoming and South Dakota were evaluated to a depth of 1500 m (5000 ft) using available surface and subsurface geologic information. Many of the uranium occurrences reported in the literature and in reports of the US Atomic Energy Commission were located, sampled and described. Areas of anomalous radioactivity, interpreted from an aerial radiometric survey, were outlined. Areas favorable for uranium deposits in the subsurface were evaluated using gamma-ray logs. Based on surface and subsurface data, two areas have been delineated which are underlain by rocks deemed favorable as hosts for uranium deposits. One of these is underlain by rocks that contain fluvial arkosic facies in the Wasatch and Fort Union Formations of Tertiary age; the other is underlain by rocks containing fluvial quartzose sandstone facies of the Inyan Kara Group of Early Cretaceous age. Unfavorable environments characterize all rock units of Tertiary age above the Wasatch Formation, all rock units of Cretaceous age above the Inyan Kara Group, and most rock units of Mesozoic and Paleozoic age below the Inyan Kara Group. Unfavorable environments characterize all rock units of Cretaceous age above the Inyan Kara Group, and all rock units of Mesozoic and Paleozoic age below the Inyan Kara Group

  15. National Uranium Resource Evaluation: Newcastle Quadrangle, Wyoming and South Dakota

    Energy Technology Data Exchange (ETDEWEB)

    Santos, E S; Robinson, K; Geer, K A; Blattspieler, J G

    1982-09-01

    Uranium resources of the Newcastle 1/sup 0/x2/sup 0/ Quadrangle, Wyoming and South Dakota were evaluated to a depth of 1500 m (5000 ft) using available surface and subsurface geologic information. Many of the uranium occurrences reported in the literature and in reports of the US Atomic Energy Commission were located, sampled and described. Areas of anomalous radioactivity, interpreted from an aerial radiometric survey, were outlined. Areas favorable for uranium deposits in the subsurface were evaluated using gamma-ray logs. Based on surface and subsurface data, two areas have been delineated which are underlain by rocks deemed favorable as hosts for uranium deposits. One of these is underlain by rocks that contain fluvial arkosic facies in the Wasatch and Fort Union Formations of Tertiary age; the other is underlain by rocks containing fluvial quartzose sandstone facies of the Inyan Kara Group of Early Cretaceous age. Unfavorable environments characterize all rock units of Tertiary age above the Wasatch Formation, all rock units of Cretaceous age above the Inyan Kara Group, and most rock units of Mesozoic and Paleozoic age below the Inyan Kara Group. Unfavorable environments characterize all rock units of Cretaceous age above the Inyan Kara Group, and all rock units of Mesozoic and Paleozoic age below the Inyan Kara Group.

  16. Cooperation between catalytic and DNA binding domains enhances thermostability and supports DNA synthesis at higher temperatures by thermostable DNA polymerases.

    Science.gov (United States)

    Pavlov, Andrey R; Pavlova, Nadejda V; Kozyavkin, Sergei A; Slesarev, Alexei I

    2012-03-13

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

  17. Phosphate and arsenate removal efficiency by thermostable ferritin enzyme from Pyrococcus furiosus using radioisotopes

    KAUST Repository

    Sevcenco, Ana-Maria

    2015-03-13

    Oxo-anion binding properties of the thermostable enzyme ferritin from Pyrococcus furiosus were characterized with radiography. Radioisotopes 32P and 76As present as oxoanions were used to measure the extent and the rate of their absorption by the ferritin. Thermostable ferritin proved to be an excellent system for rapid phosphate and arsenate removal from aqueous solutions down to residual concentrations at the picomolar level. These very low concentrations make thermostable ferritin a potential tool to considerably mitigate industrial biofouling by phosphate limitation or to remove arsenate from drinking water.

  18. Thermostating extended Lagrangian Born-Oppenheimer molecular dynamics.

    Science.gov (United States)

    Martínez, Enrique; Cawkwell, Marc J; Voter, Arthur F; Niklasson, Anders M N

    2015-04-21

    Extended Lagrangian Born-Oppenheimer molecular dynamics is developed and analyzed for applications in canonical (NVT) simulations. Three different approaches are considered: the Nosé and Andersen thermostats and Langevin dynamics. We have tested the temperature distribution under different conditions of self-consistent field (SCF) convergence and time step and compared the results to analytical predictions. We find that the simulations based on the extended Lagrangian Born-Oppenheimer framework provide accurate canonical distributions even under approximate SCF convergence, often requiring only a single diagonalization per time step, whereas regular Born-Oppenheimer formulations exhibit unphysical fluctuations unless a sufficiently high degree of convergence is reached at each time step. The thermostated extended Lagrangian framework thus offers an accurate approach to sample processes in the canonical ensemble at a fraction of the computational cost of regular Born-Oppenheimer molecular dynamics simulations.

  19. Phosphate and arsenate removal efficiency by thermostable ferritin enzyme from Pyrococcus furiosus using radioisotopes

    KAUST Repository

    Sevcenco, Ana-Maria; Paravidino, Monica; Vrouwenvelder, Johannes S.; Wolterbeek, Hubert Th.; van Loosdrecht, Mark C.M.; Hagen, Wilfred R.

    2015-01-01

    Oxo-anion binding properties of the thermostable enzyme ferritin from Pyrococcus furiosus were characterized with radiography. Radioisotopes 32P and 76As present as oxoanions were used to measure the extent and the rate of their absorption

  20. Exogenous cellulases of thermophilic micromycetes. Pt. 2. Thermostability of enzyme preparations

    Energy Technology Data Exchange (ETDEWEB)

    Kvesitadze, G; Gogilashvili, L; Svanidze, R; Buachidze, T; Chirgadze, L; Nizharadze, D

    1986-01-01

    The ability of a large number of higher fungi to form extracellular cellulases is investigated. Some representatives of these fungi grow at 40-50/sup 0/C, and form extracellular cellulases exceeding cellulases of mesophilic fungi in thermostability. It is shown that cellulases of higher thermophilic fungi differ by their thermostability. The temperature optimum of cellulase action of higher fungi occurs within 60-62/sup 0/C.

  1. Purification and Characterization of a Thermostable Lipase from Geobacillus thermodenitrificans IBRL-nra

    Directory of Open Access Journals (Sweden)

    Anuradha Balan

    2012-01-01

    Full Text Available Thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was purified and characterized. The production of thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was carried out in a shake-flask system at 65°C in cultivation medium containing; glucose 1.0% (w/v; yeast extract 1.25% (w/v; NaCl 0.45% (w/v olive oil 0.1% (v/v with agitation of 200 rpm for 24 hours. The extracted extracellular crude thermostable lipase was purified to homogeneity by using ultrafiltration, Heparin-affinity chromatography, and Sephadex G-100 gel-filtration chromatography by 34 times with a final yield of 9%. The molecular weight of the purified enzyme was estimated to be 30 kDa after SDS-PAGE analysis. The optimal temperature for thermostable lipase was 65°C and it retained its initial activity for 3 hours. Thermostable lipase activity was highest at pH 7.0 and stable for 16 hours at this pH at 65°C. Thermostable lipase showed elevated activity when pretreated with BaCl2, CaCl2, and KCl with 112%, 108%, and 106%, respectively. Lipase hydrolyzed tripalmitin (C16 and olive oil with optimal activity (100% compared to other substrates.

  2. Improving Self Service the Six Sigma Way at Newcastle University Library

    Science.gov (United States)

    Kumi, Susan; Morrow, John

    2006-01-01

    Purpose: To report on the collaborative project between Newcastle University Library and 3M which aimed to increase self-issue levels using six sigma methodology. Design/methodology/approach: The six-month long project is outlined and gives an insight into the process improvement methodology called six sigma. An explanation of why we ran the…

  3. Cluster Analysis of the Newcastle Electronic Corpus of Tyneside English: A Comparison of Methods

    NARCIS (Netherlands)

    Moisl, Hermann; Jones, Valerie M.

    2005-01-01

    This article examines the feasibility of an empirical approach to sociolinguistic analysis of the Newcastle Electronic Corpus of Tyneside English using exploratory multivariate methods. It addresses a known problem with one class of such methods, hierarchical cluster analysis—that different

  4. Cluster Analysis of the Newcastle Electronic Corpus of Tyneside English: In A Comparison of Methods

    NARCIS (Netherlands)

    Moisl, Hermann; Jones, Valerie M.

    2005-01-01

    This article examines the feasibility of an empirical approach to sociolinguistic analysis of the Newcastle Electronic Corpus of Tyneside English using exploratory multivariate methods. It addresses a known problem with one class of such methods, hierarchical cluster analysis—that different

  5. Facteurs favorisant l'apparition de la maladie de Newcastle au Tchad

    African Journals Online (AJOL)

    SARAH

    31 oct. 2013 ... 5591. Facteurs favorisant l'apparition de la maladie de. Newcastle au Tchad. BAN-BO Bebanto Antipas1; KEBKIBA Bidjeh2; NADJILEMDigamtar1. 1. Faculté des Sciences ... L'agent causal est un paramyxovirus aviaire 1. (APMV 1) du ... de la République Tchad, Bongor, Fianga (région du Mayo. Kebbi Est) ...

  6. PBC Triggers in Water Reservoirs, Coal Mining Areas and Waste Disposal Sites: From Newcastle to New York

    Directory of Open Access Journals (Sweden)

    Daniel Smyk

    2010-01-01

    Full Text Available Various environmental factors have been proposed as triggers of primary biliary cirrhosis (PBC, a progressive autoimmune cholestatic liver disease which is characterised by the destruction of the small intrahepatic bile ducts. Support for their pathogenic role in PBC is provided by epidemiological studies reporting familial clustering and clusters of the disease within a given geographical area. The seminal study by Triger reporting that the great majority of PBC cases in the English city of Sheffield drank water from a specific water reservoir, has been followed by studies reporting disease 'hot spots' within a restricted geographic region of the former coal mining area of Newcastle. The New York study reporting an increased risk and significant clustering of PBC cases near toxic federal waste disposal sites has added strength to the notion that environmental factors, possibly in the form of infectious agents or toxic/chemical environmental factors in areas of contaminated land, water or polluted air may play a key role in the development of the disease. This review discusses the findings of reports investigating environmental factors which may contribute to the cause of primary biliary cirrhosis.

  7. VapD in Xylella fastidiosa Is a Thermostable Protein with Ribonuclease Activity.

    Science.gov (United States)

    Mendes, Juliano S; Santiago, André da S; Toledo, Marcelo A S; Rosselli-Murai, Luciana K; Favaro, Marianna T P; Santos, Clelton A; Horta, Maria Augusta C; Crucello, Aline; Beloti, Lilian L; Romero, Fabian; Tasic, Ljubica; de Souza, Alessandra A; de Souza, Anete P

    2015-01-01

    Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.

  8. A thermostable serralysin inhibitor from marine bacterium Flavobacterium sp. YS-80-122

    Science.gov (United States)

    Liang, Pengjuan; Li, Shangyong; Wang, Kun; Wang, Fang; Xing, Mengxin; Hao, Jianhua; Sun, Mi

    2018-03-01

    Serralysin inhibitors have been proposed as potent drugs against many diseases and may help to prevent further development of antibiotic-resistant pathogenic bacteria. In this study, a novel serralysin inhibitor gene, lupI, was cloned from the marine bacterium Flavobacterium sp. YS-80-122 and expressed in Escherichia coli. The deduced serralysin inhibitor, LupI, shows <40% amino acid identity to other reported serralysin inhibitors. Multiple sequence alignment and phylogenetic analysis of LupI with other serralysin inhibitors indicated that LupI was a novel type of serralysin inhibitor. The inhibitory constant for LupI towards its target metalloprotease was 0.64 μmol/L. LupI was thermostable at high temperature, in which 35.6%-90.7% of its inhibitory activity was recovered after treatment at 100°C for 1-60 min followed by incubation at 0°C. This novel inhibitor may represent a candidate drug for the treatment of serralysin-related infections.

  9. VapD in Xylella fastidiosa Is a Thermostable Protein with Ribonuclease Activity.

    Directory of Open Access Journals (Sweden)

    Juliano S Mendes

    Full Text Available Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC, a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c and nonpathogenic (XfJ1a12 strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.

  10. Thermostable cross-protective subunit vaccine against Brucella species.

    Science.gov (United States)

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  11. Structure Based Thermostability Prediction Models for Protein Single Point Mutations with Machine Learning Tools.

    Directory of Open Access Journals (Sweden)

    Lei Jia

    Full Text Available Thermostability issue of protein point mutations is a common occurrence in protein engineering. An application which predicts the thermostability of mutants can be helpful for guiding decision making process in protein design via mutagenesis. An in silico point mutation scanning method is frequently used to find "hot spots" in proteins for focused mutagenesis. ProTherm (http://gibk26.bio.kyutech.ac.jp/jouhou/Protherm/protherm.html is a public database that consists of thousands of protein mutants' experimentally measured thermostability. Two data sets based on two differently measured thermostability properties of protein single point mutations, namely the unfolding free energy change (ddG and melting temperature change (dTm were obtained from this database. Folding free energy change calculation from Rosetta, structural information of the point mutations as well as amino acid physical properties were obtained for building thermostability prediction models with informatics modeling tools. Five supervised machine learning methods (support vector machine, random forests, artificial neural network, naïve Bayes classifier, K nearest neighbor and partial least squares regression are used for building the prediction models. Binary and ternary classifications as well as regression models were built and evaluated. Data set redundancy and balancing, the reverse mutations technique, feature selection, and comparison to other published methods were discussed. Rosetta calculated folding free energy change ranked as the most influential features in all prediction models. Other descriptors also made significant contributions to increasing the accuracy of the prediction models.

  12. Official and Vernacular Public History: Historical Anniversaries and Commemorations in Newcastle, NSW

    Directory of Open Access Journals (Sweden)

    Erik Eklund

    2007-09-01

    Full Text Available The city of Newcastle commemorated two bicentenaries within the space of seven years. In 2004, the city marked 200 years since the permanent establishment of the settlement on 30 March 1804. But 2004 was not the city’s first bicentennial. In 1997, Newcastle celebrated the 1797 journey of Lieutenant John Shortland, who named and sketched the Hunter River and brought back samples of coal to Sydney. These anniversaries, and earlier ones such as Newcastle’s centennial in 1897 and its sesqui-centennial in 1947, were crucial moments of history making in the public sphere. History was evoked to celebrate progress, encourage civic loyalty and, more recently, to emphasise the city’s transition into a post-industrial era. This article will explore the way in which commemorative dates in Newcastle’s history were interpreted, utilised and presented to the general public. It will examine how history, heritage, politics and policy come together to use the past in a public way. Utilising US historian John Bodnar’s terms, the shift in the themes and tenor of public history in Newcastle over this period has been from an ‘official’ to a more ‘vernacular’ style. Official public history emphasised unitary notions of progress while vernacular styles presented more diverse and occasionally more critical versions of public history. By the time of the 2004 commemorative events there was more scope for active popular participation. Newcastle public history was being nourished by community groups often with conflicting notions of public history, generating a multivalent, multilayered sense of the past, though older themes persisted with remarkable durability. In a city where ‘history’ has such an ambivalent position, large-scale historical commemorations make for intriguing analysis. After a review of the principal themes in the Newcastle commemorations of 1897, 1947, and 1997, I consider the 2004 ‘Newcastle 200’ programme. In particular, I will

  13. Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756

    Science.gov (United States)

    The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-direc...

  14. Coal trains and air quality in newcastle (trenes de carbón y su influencia en la calidad del aire en Newcastle)

    OpenAIRE

    Piedra Montoya, José Antonio

    2014-01-01

    El presente trabajo estudia la influencia de carbón en la calidad del aire una vez que ha sido extraído y es transportado en vagones desde el sitio de extracción hasta el puerto de exportación, en la localidad de Newcastle-Australia. A través de mediciones con equipos apropiados para calidad de aire, se observó el inclemento en la concentración de Material Particulado (MP 10 y MP 2,5) en el aire. Con la ayuda de software y las mediciones obtenidas se observó que a medida que los trenes que tr...

  15. Structurally stabilized organosilane-templated thermostable mesoporous titania.

    Science.gov (United States)

    Amoli, Vipin; Tiwari, Rashmi; Dutta, Arghya; Bhaumik, Asim; Sinha, Anil Kumar

    2014-01-13

    Structurally thermostable mesoporous anatase TiO2 (m-TiO2) nanoparticles, uniquely decorated with atomically dispersed SiO2, is reported for the first time. The inorganic Si portion of the novel organosilane template, used as a mesopores-directing agent, is found to be incorporated in the pore walls of the titania aggregates, mainly as isolated sites. This is evident by transmission electron microscopy and high-angle annular dark field scanning transmission electron microscopy, combined with electron dispersive X-ray spectroscopy. This type of unique structure provides exceptional stability to this new material against thermal collapse of the mesoporous structure, which is reflected in its high surface area (the highest known for anatase titania), even after high-temperature (550 °C) calcination. Control of crystallite size, pore diameter, and surface area is achieved by varying the molar ratios of the titanium precursor and the template during synthesis. These mesoporous materials retain their porosity and high surface area after template removal and further NaOH/HCl treatment to remove silica. We investigate their performance for dye-sensitized solar cells (DSSCs) with bilayer TiO2 electrodes, which are prepared by applying a coating of m-TiO2 onto a commercial titania (P25) film. The high surface area of the upper mesoporous layer in the P25-m-TiO2 DSSC significantly increases the dye loading ability of the photoanode. The photocurrent and fill factor for the DSSC with the bilayer TiO2 electrode are greatly improved. The large increase in photocurrent current (ca. 56%) in the P25-m-TiO2 DSSC is believed to play a significant role in achieving a remarkable increase in the photovoltaic efficiency (60%) of the device, compared to DSSCs with a monolayer of P25 as the electrode. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Fish Remains from Excavations near the Riverfront at Newcastle upon Tyne, England

    Directory of Open Access Journals (Sweden)

    Rebecca A. Nicholson

    1997-12-01

    Full Text Available The City of Newcastle, situated some 10 miles inland on the River Tyne in north-east England, is not now an important fishing port. Most of the fresh fish marketed in the city has been landed at the nearby coastal ports of North and South Shields. Excavations at two sites behind the present Quayside in Newcastle, however, have yielded quantities of fish bones, representing a wide variety of species. This is in contrast to excavations in other parts of the city, where few fish remains have been recovered, and suggests that the quayside in Newcastle was an important centre for the fishing industry during the medieval period. It seems likely that most of the fish remains represent waste from landing and processing fish on or near the quayside. Yet, when taphonomic factors are taken into account, the limitations of using even large bone assemblages to interpret processing activities is demonstrated. As always, the need for a programme of on-site sieving to obtain representative samples of fish bone is evident.

  17. In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop1[OPEN

    Science.gov (United States)

    Shivhare, Devendra

    2017-01-01

    To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice (Oryza sativa) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca. Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function. PMID:28546437

  18. Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer

    DEFF Research Database (Denmark)

    Huus, Kasper; Havelund, Svend; Olsen, Helle B

    2006-01-01

    The influence of ligand binding and conformation state on the thermostability of hexameric zinc-insulin was studied by differential scanning calorimetry (DSC). The insulin hexamer exists in equilibrium between the forms T6, T3R3, and R6. Phenolic ligands induce and stabilize the T3R3- and R6-stat...

  19. Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis

    Science.gov (United States)

    Cellobiose 2-epimerase from the thermophile Caldicellulosiruptor saccharolyticus (CsCE) catalyzes the isomerization of lactose into lactulose, a non-digestible disaccharide widely used in food and pharmaceutical industries. Semi-rational approaches were applied to enhance the thermostability of CsCE...

  20. CHARACTERIZATION OF A NEW BACILLUS-STEAROTHERMOPHILUS ISOLATE - A HIGHLY THERMOSTABLE ALPHA-AMYLASE-PRODUCING STRAIN

    NARCIS (Netherlands)

    WIND, RD; BUITELAAR, RM; EGGINK, G; HUIZING, HJ; DIJKHUIZEN, L

    A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known alpha-amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable alpha-amylase. The half-time of inactivation of this

  1. Enhanced Thermostability of Arabidopsis Rubisco activase improves photosynthesis and growth rates under moderate heat stress.

    Science.gov (United States)

    Kurek, Itzhak; Chang, Thom Kai; Bertain, Sean M; Madrigal, Alfredo; Liu, Lu; Lassner, Michael W; Zhu, Genhai

    2007-10-01

    Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Deltarca) line. In a long-term growth test at either constant 26 degrees C or daily 4-h 30 degrees C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions.

  2. Not so monofunctional-a case of thermostable Thermobifida fusca catalase with peroxidase activity

    NARCIS (Netherlands)

    Lončar, Nikola; Fraaije, Marco W

    Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and

  3. Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B

    NARCIS (Netherlands)

    Winter, Remko T.; Heuts, Dominic P. H. M.; Rijpkema, Egon M. A.; van Bloois, Edwin; Wijma, Hein J.; Fraaije, Marco W.

    We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene

  4. Evaluating the value proposition for improving vaccine thermostability to increase vaccine impact in low and middle-income countries.

    Science.gov (United States)

    Karp, Christopher L; Lans, Deborah; Esparza, José; Edson, Eleanore B; Owen, Katey E; Wilson, Christopher B; Heaton, Penny M; Levine, Orin S; Rao, Raja

    2015-07-09

    The need to keep vaccines cold in the face of high ambient temperatures and unreliable access to electricity is a challenge that limits vaccine coverage in low and middle-income countries (LMICs). Greater vaccine thermostability is generally touted as the obvious solution. Despite conventional wisdom, comprehensive analysis of the value proposition for increasing vaccine thermostability has been lacking. Further, while significant investments have been made in increasing vaccine thermostability in recent years, no vaccine products have been commercialized as a result. We analyzed the value proposition for increasing vaccine thermostability, grounding the analysis in specific vaccine use cases (e.g., use in routine immunization [RI] programs, or in campaigns) and in the broader context of cold chain technology and country level supply chain system design. The results were often surprising. For example, cold chain costs actually represent a relatively small fraction of total vaccine delivery system costs. Further, there are critical, vaccine use case-specific temporal thresholds that need to be overcome for significant benefits to be reaped from increasing vaccine thermostability. We present a number of recommendations deriving from this analysis that suggest a rational path toward unlocking the value (maximizing coverage, minimizing total system costs) of increased vaccine thermostability, including: (1) the full range of thermostability of existing vaccines should be defined and included in their labels; (2) for new vaccines, thermostability goals should be addressed up-front at the level of the target product profile; (3) improving cold chain infrastructure and supply chain system design is likely to have the largest impact on total system costs and coverage in the short term-and will influence the degree of thermostability required in the future; (4) in the long term, there remains value in monitoring the emergence of disruptive technologies that could remove the

  5. Highly thermostable xylanase production from a thermophilic Geobacillus sp. strain WSUCF1 utilizing lignocellulosic biomass

    Directory of Open Access Journals (Sweden)

    Aditya eBhalla

    2015-06-01

    Full Text Available AbstractEfficient enzymatic hydrolysis of lignocellulose to fermentable sugars requires a complete repertoire of biomass deconstruction enzymes. Hemicellulases play an important role in hydrolyzing hemicellulose component of lignocellulose to xylo-oligosaccharides and xylose. Thermostable xylanases have been a focus of attention as industrially important enzymes due to their long shelf life at high temperatures. Geobacillus sp. strain WSUCF1 produced thermostable xylanase activity (crude xylanase cocktail when grown on xylan or various inexpensive untreated and pretreated lignocellulosic biomasses such as prairie cord grass and corn stover. The optimum pH and temperature for the crude xylanase cocktail were 6.5 and 70ºC, respectively. The WSUCF1 crude xylanase was found to be highly thermostable with half-lives of 18 and 12 days at 60 and 70ºC, respectively. At 70ºC, rates of xylan hydrolysis were also found to be better with the WSUCF1 secretome than those with commercial enzymes, i.e., for WSUCF1 crude xylanase, CellicHTec2, and AccelleraseXY, the percent xylan conversions were 68.9, 49.4, and 28.92, respectively. To the best of our knowledge, WSUCF1 crude xylanase cocktail is among the most thermostable xylanases produced by thermophilic Geobacillus spp. and other thermophilic microbes (optimum growth temperature ≤70ºC. High thermostability, activity over wide range of temperatures, and better xylan hydrolysis than commercial enzymes make WSUCF1 crude xylanase suitable for thermophilic lignocellulose bioconversion processes.

  6. Respons Antibodi Sekunder Terhadap Penyakit Tetelo pada Ayam Petelur Pascavaksinasi Ulangan dengan Vaksin Tetelo Aktif (NEWCASTLE DISEASESECONDARY ANTIBODY RESPONSE AFTER REVACCINATION IN LAYER WITH THE ACTIVE ND VACCINE

    Directory of Open Access Journals (Sweden)

    Andika Budi Kurnianto

    2016-11-01

    Full Text Available Revaccination is required in order to preventNewcastle Disease (ND reccurence inlayers chickens. Oneof vaccine for ND revaccination is freeze-died ND active vaccine containing e” 106,5EID50. Revaccinationisdone to trigger a faster secondary antibody responses in layers and can achieve protective antibody titersagainst ND that can be monitored by a hemagglutinationinhibition (HI. The aim of this study was todetermine the ND secondary antibody responses in layers after revaccination with ND active vaccine.Antibody titer of 20 layers chickens of 20 week old were determined before revaccinations (week 0 andafter revaccinations (week 1 until week 9. The first vaccination was conducted using ND-IB (NewcastleDisease-Infectious Bronchitis at the age of 2 days through eye drops and subcutaneous injection at the ageof 5 days using a dose of 1 ampoule.Vaccination is repeated at the age of 20 weeks at a dose of 1 ½ ampoule through drinking water. Blood samples were collected on the wing vein (venous cutane ulnar and left for 5-10 minutes at room temperature.Sera were then collected and stored at -20oC until use. HI antibody titerwas determined by micro titeration system. The HI mean titers were analyzed by Duncan test. The studyresults showed that antibody titer before revaccination was3,47 HI log 2 and the HI titers after revaccinationwere 4,02; 5,22; 6,52; 7,85; 8,4; 8,6; 7,7; 5,92; dan 3,87 HI log 2 respectivelly at weeks 1, 2, 3, 4, 5, 6, 7, 8, and9.The NDV revaccination with ND active vaccine significantly (P <0.01 increased in antibody titer inlayers starting from week 1 to week 6, but decreased following week 7 to week-9. It can be concluded thatrevaccinantion with ND active vaccine increases the antibody titers in layer chickens.

  7. Molecular characterization of a novel thermostable laccase PPLCC2 from the brown rot fungus Postia placenta MAD-698-R

    Directory of Open Access Journals (Sweden)

    Hongde An

    2015-11-01

    Conclusions: This is the first identified thermo activated and thermostable laccase in brown rot fungi. This investigation will contribute to understanding the roles played by laccases in brown rot fungi.

  8. Screening of strains with the high activity and thermostability nattokinase by {sup 60}Co γ-ray irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Shuying, Li; Ying, Nie; Huan, Du; Xuanming, Tang [Institute of Agro-products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing (China); Zhonglin, Zhao [College of Sciences, Henan Agricultural University, Zhengzhou (China); Xin, Ma [Agricultural Information Institute, Chinese Academic of Agricultural Sciences, Beijing (China); Yan, Li [School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou (China)

    2013-06-15

    In this study, Bacillus natto was irradiated by {sup 60}Co γ-ray, and activity was determined by Casein plate method in order to get high activity and thermostability strains. 60 strains with high activity were obtained through irradiation by 800 Gy {sup 60}Co γ-ray. In this dose, the positive mutation rate was 45%. Then 60 strains was treated by different tempreture and 11 strains showed thermostability at 65℃. (authors)

  9. Screening of strains with the high activity and thermostability nattokinase by "6"0Co γ-ray irradiation

    International Nuclear Information System (INIS)

    Li Shuying; Nie Ying; Du Huan; Tang Xuanming; Zhao Zhonglin; Ma Xin; Li Yan

    2013-01-01

    In this study, Bacillus natto was irradiated by "6"0Co γ-ray, and activity was determined by Casein plate method in order to get high activity and thermostability strains. 60 strains with high activity were obtained through irradiation by 800 Gy "6"0Co γ-ray. In this dose, the positive mutation rate was 45%. Then 60 strains was treated by different tempreture and 11 strains showed thermostability at 65℃. (authors)

  10. Target priority transboundary animal diseases and zoonoses in the ...

    African Journals Online (AJOL)

    Overall, Rift Valley fever had the highest rank, followed by Contagious Bovine Pleuropneumonia, Newcastle Disease, Highly Pathogenic Avian Influenza, Lumpy Skin Disease, Peste des Petits des Ruminants, Rabies, Brucellosis, Bovine Tuberculosis, Foot-and Mouth Disease and Sheep and Goat Pox. In conclusion, the ...

  11. FireProt: web server for automated design of thermostable proteins

    Science.gov (United States)

    Musil, Milos; Stourac, Jan; Brezovsky, Jan; Prokop, Zbynek; Zendulka, Jaroslav; Martinek, Tomas

    2017-01-01

    Abstract There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. Here, we present FireProt, a web server for the automated design of multiple-point thermostable mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen tools and three protein engineering strategies for making reliable protein designs. The server is complemented with interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable mutants. FireProt is freely available at http://loschmidt.chemi.muni.cz/fireprot. PMID:28449074

  12. Thermostability of bovine submaxillary mucin (BSM) in bulk solution and at a sliding interface

    DEFF Research Database (Denmark)

    Madsen, Jan Busk; Pakkanen, Kirsi I.; Lee, Seunghwan

    2014-01-01

    Thermostability of bovine submaxillary mucin (BSM) was studied in terms of its structure, hydrodynamic size, surface adsorption, and lubricating properties in the temperature range of 5-85°C. The overall random coil structure of BSM showed a gradual loosening with increasing temperature as charac......Thermostability of bovine submaxillary mucin (BSM) was studied in terms of its structure, hydrodynamic size, surface adsorption, and lubricating properties in the temperature range of 5-85°C. The overall random coil structure of BSM showed a gradual loosening with increasing temperature...... as characterized by circular dichroism (CD) spectroscopy, but this change was fully reversible upon lowering temperature. Extended heating up to 120min at 80°C did not make any appreciable changes in the structure of BSM when it was cooled to room temperature. The hydrodynamic size of BSM, as studied by dynamic...

  13. First isolation of a novel thermostable antifungal peptide secreted by Aspergillus clavatus.

    Science.gov (United States)

    Skouri-Gargouri, Houda; Gargouri, Ali

    2008-11-01

    A novel antifungal peptide produced by an indigenous fungal strain (VR) of Aspergillus clavatus was purified. The antifungal peptide was enriched in the supernatant after heat treatment at 70 degrees C. The thermostable character was exploited in the first purification step, as purified peptide was obtained after ultrafiltration and reverse phase-HPLC on C18 column application. The purified peptide named "AcAFP" for A. clavatus antifungal peptide, has molecular mass of 5773Da determined by MALDI-ToF spectrometry. The N-terminal sequence showed a notable identity to the limited family of antifungal peptides produced by ascomycetes fungi. The AcAFP activity remains intact even after heat treatment at 100 degrees C for 1h confirming its thermostability. It exhibits a strong inhibitory activity against mycelial growth of several serious human and plant pathogenic fungi: Fusariuym oxysporum, Fusarium solani, Aspergillus niger, Botrytis cinerea, Alternaria solani, whereas AcAFP did not affect yeast and bacterial growth.

  14. An optimized formulation of a thermostable spray dried virus-like particles vaccine against human papillomavirus

    Science.gov (United States)

    Saboo, Sugandha; Tumban, Ebenezer; Peabody, Julianne; Wafula, Denis; Peabody, David S.; Chackerian, Bryce; Muttil, Pavan

    2016-01-01

    Existing vaccines against human papillomavirus (HPV) require continuous cold-chain storage. Previously, we developed a bacteriophage virus-like particle (VLP) based vaccine for Human Papillomavirus (HPV) infection, which elicits broadly neutralizing antibodies against diverse HPV types. Here, we formulated these VLPs into a thermostable dry powder using a multi-component excipient system and by optimizing the spray drying parameters using a half-factorial design approach. Dry powder VLPs were stable after spray drying and after long-term storage at elevated temperatures. Immunization of mice with a single dose of reconstituted dry powder VLPs that were stored at 37°C for more than a year elicited high anti-L2 IgG antibody titers. Spray dried thermostable, broadly protective L2 bacteriophage VLPs vaccine could be accessible to remote regions of the world (where ~84% of cervical cancer patients reside) by eliminating the cold-chain requirement during transportation and storage. PMID:27019231

  15. MOLECULAR CLONING AND CHARACTERIZATION OF NOVEL THERMOSTABLE LIPASE FROM SHEWANELLA PUTREFACIENS AND USING ENZYMATIC BIODIESEL PRODUCTION

    Directory of Open Access Journals (Sweden)

    Fahri Akbas

    2015-02-01

    Full Text Available A novel thermostable lipase from Shewanella putrefaciens was identified, expressed in Escherichia coli, characterized and used in biodiesel production. Enzyme characterization was carried out by enzyme assay, SDS-PAGE and other biochemical reactions. The recombinant lipase was found to have a molecular mass of 29 kDa and exhibited lipase activity when Tween 80 was used as the substrate. The purified enzyme showed maximum activity at pH 5.0 and at 80°C. The recombinant lipase was used for the transesterification of canola oil and waste oil. The enzyme retains 50% of its activity at 90°C for 30 minutes. It is also able to retain 20% of its activity even at 100 °C for 20 minutes. These properties of the obtained new recombinant thermostable lipase make it promising as a biocatalyst for industrial processes.

  16. The Newcastle connection: A software subsystem for constructing distributed UNIX systems

    International Nuclear Information System (INIS)

    Randell, B.

    1985-01-01

    The Newcastle connection is a software subsystem that can be added to each of a set of physically interconnected UNIX or UNIX look-alike systems, so as to construct a distributed system which is functionally indistinguishable at both the user and the program level from a conventional single-processor UNIX system. The techniques used are applicable to a variety and multiplicity of both local and wide area networks, and enable all issues of inter-processor communication, network protocols, etc., to be hidden. A brief account is given of experience with such distributed systems, the first of which was constructed in 1982 using a set of PDP11s running UNIX Version 7, and connected by a Cambridge Ring - since this date the Connection has been used to construct distributed systems based on various other computers and versions of UNIX, both at Newcastle and elsewhere. The final sections compare our scheme to various precursor schemes and discuss its potential relevance to other operating systems. (orig.)

  17. In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop.

    Science.gov (United States)

    Shivhare, Devendra; Mueller-Cajar, Oliver

    2017-07-01

    To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice ( Oryza sativa ) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function. © 2017 American Society of Plant Biologists. All Rights Reserved.

  18. Tyr51: Key Determinant of the Low Thermostability of the Colwellia psychrerythraea Cold-Shock Protein.

    Science.gov (United States)

    Lee, Yeongjoon; Kwak, Chulhee; Jeong, Ki-Woong; Durai, Prasannavenkatesh; Ryu, Kyoung-Seok; Kim, Eun-Hee; Cheong, Chaejoon; Ahn, Hee-Chul; Kim, Hak Jun; Kim, Yangmee

    2018-05-18

    Cold-shock proteins (Csps) are expressed at lower-than-optimum temperatures, and they function as RNA chaperones; however, no structural studies on psychrophilic Csps have been reported. Here, we aimed to investigate the structure and dynamics of the Csp of psychrophile Colwellia psychrerythraea 34H, ( Cp-Csp). Although Cp-Csp shares sequence homology, common folding patterns, and motifs, including a five β-stranded barrel, with its thermophilic counterparts, its thermostability (37 °C) was markedly lower than those of other Csps. Cp-Csp binds heptathymidine with an affinity of 10 -7 M, thereby increasing its thermostability to 50 °C. Nuclear magnetic resonance spectroscopic analysis of the Cp-Csp structure and backbone dynamics revealed a flexible structure with only one salt bridge and 10 residues in the hydrophobic cavity. Notably, Cp-Csp contains Tyr51 instead of the conserved Phe in the hydrophobic core, and its phenolic hydroxyl group projects toward the surface. The Y51F mutation increased the stability of hydrophobic packing and may have allowed for the formation of a K3-E21 salt bridge, thereby increasing its thermostability to 43 °C. Cp-Csp exhibited conformational exchanges in its ribonucleoprotein motifs 1 and 2 (754 and 642 s -1 ), and heptathymidine binding markedly decreased these motions. Cp-Csp lacks salt bridges and has longer flexible loops and a less compact hydrophobic cavity resulting from Tyr51 compared to mesophilic and thermophilic Csps. These might explain the low thermostability of Cp-Csp. The conformational flexibility of Cp-Csp facilitates its accommodation of nucleic acids at low temperatures in polar oceans and its function as an RNA chaperone for cold adaptation.

  19. Design and Testing of a Thermostable Platform for Multimerization of Single Domain Antibodies

    Science.gov (United States)

    2012-08-01

    H.J. Properties , production, and applications of camelid single domain antibody fragments. Appl. Microbiol. Biot. 2007, 77, 13‒22. 2. Goldman...Conway, J.; Sherwood, L.J.; Fech, M.; Vo, B.; Liu, J.L.; Hayhurst, A. Thermostable llama single domain antibodies for detection of Botulinum A...antiparallel coiled-coil inserted. J. Mol. Bio. 2001, 306, 25‒35. 9. Liu, J.L.; Anderson, G.P.; Goldman, E.R. Isolation of anti- toxin single domain

  20. Site-saturation mutagenesis of Glomerella cingulata cutinase gene for enhanced enzyme thermostability

    Science.gov (United States)

    Hanapi, Wan Nurhidayah Wan; Iuan-Sheau, Chin; Mahadi, Nor Muhammad; Murad, Abdul Munir Abdul; Bakar, Farah Diba Abu

    2015-09-01

    Cutinase is an important biocatalyst for various industrial applications. This enzyme which has dual functionality comparable to esterases and lipases, is efficient in the hydrolysis of soluble esters and emulsified triacylglycerols. Naturally-occurring enzymes usually have disadvantages when applied in non-natural catalysis due to Glomerella cingulata cutinase enzyme thermostability. It is postulated that by increasing the rigidity at certain amino acid positions showing high mobility based on the three-dimensional structure of G. cingulata cutinase, the improvement in thermostability will be achieved. The amino acid N82 of G. cingulata cutinase was selected based on its high B-factor value determined via the B-FITTER program. Megaprimer PCR was employed to introduce mutations at the chosen site by randomization using NNK degenerate primers. About 300 transformants were selected for screening of positive cutinase variants. The N82_V14 cutinase variant was observed to be more thermostable at an almost 2-fold increase when exposed at 50°C for 1 hr as compared to the wild-type enzyme. This study may provide valuable information regarding thermal stability of cutinases denaturation at high temperatures.

  1. Expression and characterization of thermostable glycogen branching enzyme from Geobacillus mahadia Geo-05

    Directory of Open Access Journals (Sweden)

    Nur Syazwani Mohtar

    2016-12-01

    Full Text Available The glycogen branching enzyme (EC 2.4.1.18, which catalyses the formation of α-1,6-glycosidic branch points in glycogen structure, is often used to enhance the nutritional value and quality of food and beverages. In order to be applicable in industries, enzymes that are stable and active at high temperature are much desired. Using genome mining, the nucleotide sequence of the branching enzyme gene (glgB was extracted from the Geobacillus mahadia Geo-05 genome sequence provided by the Malaysia Genome Institute. The size of the gene is 2013 bp, and the theoretical molecular weight of the protein is 78.43 kDa. The gene sequence was then used to predict the thermostability, function and the three dimensional structure of the enzyme. The gene was cloned and overexpressed in E. coli to verify the predicted result experimentally. The purified enzyme was used to study the effect of temperature and pH on enzyme activity and stability, and the inhibitory effect by metal ion on enzyme activity. This thermostable glycogen branching enzyme was found to be most active at 55 °C, and the half-life at 60 °C and 70 °C was 24 h and 5 h, respectively. From this research, a thermostable glycogen branching enzyme was successfully isolated from Geobacillus mahadia Geo-05 by genome mining together with molecular biology technique.

  2. 'THERMOST' for analysing thermo-structural behaviour of LWR fuel rods under PCI conditions

    International Nuclear Information System (INIS)

    Nuno, H.; Ogawa, S.; Kobayashi, H.

    1983-01-01

    As a method for evaluating fuel rod performance under power ramping or load following operations, the combined FROST/ THERMOST system has been developed and brought into practical use. FROST was presented at the IAEA Blackpool Meeting in 1978, and THERMOST is the subject of this paper. The major purpose of THERMOST is to analyse very detailed thermal and structural fuel behaviour in a rather localised part of the fuel rod whereas FROST deals with whole rod general performance. The code handles two-dimensional thermal and structural analyses simultaneously by using a finite element method, in axial section or in lateral section. It consists of a fundamental FEM system of generalised constitution, and a surrounding subroutine system which characterises fuel behaviour, such as temperature distribution, thermal expansion, elastoplasticity, creep, cracking, swelling, growth, etc. Thermal analysis is handled by heat conduction and heat transfer element (six kinds), and structural analysis by axisymmetric ring and lateral plane element (six kinds). Boundary problems such as contact, friction and cracking are treated by gap and crack elements. A sample calculation of PCI performance on a PWR fuel rod under ramping conditions is presented with some in-pile test data. (author)

  3. Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

    Science.gov (United States)

    Fredslund, Folmer; Otten, Harm; Gemperlein, Sabrina; Poulsen, Jens Christian N; Carius, Yvonne; Kohring, Gert Wieland; Lo Leggio, Leila

    2016-11-01

    Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the T m for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

  4. Expression of Acidothermus cellulolyticus thermostable cellulases in tobacco and rice plants

    Directory of Open Access Journals (Sweden)

    Xiran Jiang

    2017-01-01

    Full Text Available The production of cellulases in plants is an economical method for the conversion of lignocellulosic biomass into fuels. Herein we report the expressions of two thermostable Acidothermus cellulolyticus cellulases, endo-1,4-β-D-glucanase (E1 and exoglucanase (Gux1, in tobacco and rice. To evaluate the expression of these recombinant cellulases, we expressed the full-length E1, the catalytic domains of E1 (E1cd and Gux1 (Gux1cd, as well as an E1–Gux1cd fusion enzyme in various subcellular compartments. In the case of tobacco, transgenic plants that expressed apoplast-localized E1 showed the highest level of activity, about three times higher than those that expressed the cytosolic E1. In the case of rice, the level of cellulase-specific activity in the transgenic plants ranged from 11 to 20 nmol 4-methylumbelliferone min−1 mg−1 total soluble protein. The recombinant cellulases exhibited good thermostability below 70 °C. Furthermore, transgenic rice leaves that were stored at room temperature for a month lost about 20% of the initial cellulase activity. Taken together, the results suggested that heterologous expression of thermostable cellulases in plants may be a viable option for biomass conversion.

  5. Improvement in the thermostability of chitosanase from Bacillus ehimensis by introducing artificial disulfide bonds.

    Science.gov (United States)

    Sheng, Jun; Ji, Xiaofeng; Zheng, Yuan; Wang, Zhipeng; Sun, Mi

    2016-10-01

    To determine the effects of artificial disulfide bridges on the thermostability and catalytic efficiency of chitosanase EAG1. Five artificial disulfide bridges were designed based on the structural information derived from the three-dimensional (3-D) model of chitosanase EAG1. Two beneficial mutants (G113C/D116C, A207C-L286C) were located in the flexible surface loop region, whereas the similar substitutions introduced in α-helices regions had a negligible effect. Mut5, the most active mutant, had a longer half-life at 50 °C (from 10.5 to 69.3 min) and a 200 % higher catalytic efficiency (K cat/K m) than that of the original EAG1. The contribution of disulfide bridges to enzyme thermostability is mainly dependent on its location within the polypeptide chain. Strategical placement of a disulfide bridge in flexible regions provides a rigid support and creation of a protected microenvironment, which is effective in improving enzyme's thermostability and catalytic efficiency.

  6. Isolation and molecular characterization of thermostable phytase from Bacillus subtilis (BSPhyARRMK33).

    Science.gov (United States)

    Reddy, Chinreddy Subramanyam; Achary, V Mohan Murali; Manna, Mrinalini; Singh, Jitender; Kaul, Tanushri; Reddy, Malireddy K

    2015-03-01

    The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg(-1) obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74% of its original activity after incubation at 95 °C for 10 min. In the presence of Ca(2+), the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is achieved at 7.0 and 55 °C, respectively. Thermostable nature and wide range of pH are promising features of recombinant BsPhyARRMK33 protein that may be employed as an efficient alternative to commercially known phytases and thereby alleviate environmental eutrophication.

  7. Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

    Science.gov (United States)

    Pavlov, Andrey R.; Pavlova, Nadejda V.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

    2012-01-01

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases. PMID:22320201

  8. Antimicrobial activity and high thermostability of a novel BLIS secreted by Enterococcus Mundtii isolated from Lebanese cow’s milk

    Directory of Open Access Journals (Sweden)

    Imad AL Kassaa

    2016-12-01

    Full Text Available AL Kassaa, I., Safourim, N., Mostafa, N. and Hamze, M. Antimicrobial activity and high thermostability of a novel BLIS secreted by Enterococcus Mundtii isolated from lebanese cow’s milk. 2016. Lebanese Science Journal, 17(2: 166-176. Lactic acid bacteria (LAB are used in many fields such as fermentation agents, increasing nutritional value and improving organoleptic quality of food. Also they are used as probiotics and preservatives against pathogens and spoilage microbes by producing antimicrobial substances such as bacteriocins. Fifty cow’s milk samples were collected and 175 LAB isolates were isolated and identified by using biochemical method. Fifteen isolates showed an antimicrobial activity against Listeria monocytogenes ATCC® 19115™. One strain, BL4 which showed the strongest activity, was chosen to extract and characterize its antimicrobial substance in order to evaluate its potential use as a new food protective agent. This strain was identified as Enterococcus mundtii by pyrosequencing method. The active substance was extracted using solvent method. This Bacteriocin like Inhibitory Substances “BLIS” can support a high temperature (121 ˚C for a long time and resist pH variation. The BLIS BL4 can be considered as a peptide active against many food pathogen and food-spoilage microbes, such as Listeria monocytogenes and Penicillium spp. BLIS BL4 can be used in food application as bio-preservative to reduce food-spoilage and food-borne diseases in food products.

  9. Survey for Newcastle disease viruses in poultry and wild birds in ...

    African Journals Online (AJOL)

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  10. Newcastle disease B1 vaccine strain in wild rock pigeons in Atlanta, Georgia

    Science.gov (United States)

    From June to October of 2012, samples were collected from wild Rock Pigeons (Columba livia) in urban neighborhoods of Atlanta, Georgia to ascertain the prevalence of pigeon paramyxovirus serotype-1 (PPMV-1). PPMV-1 strains are a subset of avian paramyxovirus serotype-1 (APMV-1) commonly isolated fro...

  11. Survival of Newcastle disease virus (NDV) strain V4- UPM coated on ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-08-04

    Aug 4, 2008 ... Results showed that the virus coated onto the carrier foods offal without additive remained stable at ≥ MID value for ≈3 ... meat (Shane, 1984). Velogenic .... the byproducts of food processing that is expected to eliminate any ...

  12. Lung Transcriptome Data from Chickens with Newcastle Disease Virus--Impact of Gender Immune Response

    Data.gov (United States)

    US Agency for International Development — To determine the gender impact on the immune response of chickens, the mRNA was isolated and sequenced from the lungs of 48 chickens of 2 lines as three time-points...

  13. Effect of a velogenic newcastle disease virus on body and organ ...

    African Journals Online (AJOL)

    They were weighed before being sacrificed and their internal organs (liver, spleen, thymus, heart, bursa of Fabricius, brain and adrenal glands) were removed and weighed. Sections of these organs were taken and stored in Bouin's solution for 24 h and lat er sent for histology. The liver, spleen, thymus, heart and adrenal ...

  14. Expression of Hemagglutinin–Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Amir Ghaffar Shahriari

    2016-07-01

    Conclusion: Developments in genetic engineering have led to plant-based systems for recombinant vaccine production. In this research, tobacco plant was used to express F and HN epitopes of NDV. Our results indicate that for the production of recombinant vaccine, it is a novel strategy to use concatenated epitopes without their genetic fusion onto larger scaffold structure such as viral coat protein.

  15. A Good Death? Report of the Second Newcastle Meeting on Laboratory Animal Euthanasia

    Directory of Open Access Journals (Sweden)

    Penny Hawkins

    2016-08-01

    Full Text Available Millions of laboratory animals are killed each year worldwide. There is an ethical, and in many countries also a legal, imperative to ensure those deaths cause minimal suffering. However, there is a lack of consensus regarding what methods of killing are humane for many species and stages of development. In 2013, an international group of researchers and stakeholders met at Newcastle University, United Kingdom to discuss the latest research and which methods could currently be considered most humane for the most commonly used laboratory species (mice, rats and zebrafish. They also discussed factors to consider when making decisions about appropriate techniques for particular species and projects, and priorities for further research. This report summarises the research findings and discussions, with recommendations to help inform good practice for humane killing.

  16. Scientific, Engineering, and Financial Factors of the 1989 Human-Triggered Newcastle Earthquake in Australia

    Science.gov (United States)

    Klose, C. D.

    2006-12-01

    This presentation emphasizes the dualism of natural resources exploitation and economic growth versus geomechanical pollution and risks of human-triggered earthquakes. Large-scale geoengineering activities, e.g., mining, reservoir impoundment, oil/gas production, water exploitation or fluid injection, alter pre-existing lithostatic stress states in the earth's crust and are anticipated to trigger earthquakes. Such processes of in- situ stress alteration are termed geomechanical pollution. Moreover, since the 19th century more than 200 earthquakes have been documented worldwide with a seismic moment magnitude of 4.5losses of triggered earthquakes. An hazard assessment, based on a geomechanical crust model, shows that only four deep coal mines were responsible for triggering this severe earthquake. A small-scale economic risk assessment identifies that the financial loss due to earthquake damage has reduced mining profits that have been re-invested in the Newcastle region for over two centuries beginning in 1801. Furthermore, large-scale economic risk assessment reveals that the financial loss is equivalent to 26% of the Australian Gross Domestic Product (GDP) growth in 1988/89. These costs account for 13% of the total costs of all natural disasters (e.g., flooding, drought, wild fires) and 94% of the costs of all earthquakes recorded in Australia between 1967 and 1999. In conclusion, the increasing number and size of geoengineering activities, such as coal mining near Newcastle or planned carbon dioxide Geosequestration initiatives, represent a growing hazard potential, which can negatively affect socio-economic growth and sustainable development. Finally, hazard and risk degrees, based on geomechanical-mathematical models, can be forecasted in space and over time for urban planning in order to prevent economic losses of human-triggered earthquakes in the future.

  17. Engaging the oldest old in research: lessons from the Newcastle 85+ study

    Directory of Open Access Journals (Sweden)

    Davies Karen

    2010-09-01

    Full Text Available Abstract Background Those aged 85 and over, the oldest old, are now the fastest growing sector of the population. Information on their health is essential to inform future planning; however, there is a paucity of up-to-date information on the oldest old, who are often excluded from research. The aim of the Newcastle 85+ Study is to investigate the health of a cohort of 85-year-olds from a biological, medical and psychosocial perspective. This paper describes the methods employed for the successful recruitment, retention and evaluation of this cohort. Methods Participants were all individuals born in 1921 and registered with a participating general practice in Newcastle and North Tyneside, UK. Involvement comprised detailed health assessments, by a nurse, in their usual place of residence and/or review of their general practice medical records. Results Of the 1453 individuals eligible to participate, 72% (n = 1042 were recruited; 59% (n = 851 consented to both health assessment and review of general practice records. Key factors for successful involvement included protected time to engage with family and other key gatekeepers, minimising participant burden, through for example home based assessment, and flexibility of approach. Cognitive impairment is a significant issue; due consideration should be given to the ethical and legal issues of capacity and consent. Interim withdrawal rates at phase 2 (18 month post baseline, show 88 out of 854 participants (10% had withdrawn with approval for continued use of data and materials and a further 2 participants (0.2% had withdrawn and requested that all data be destroyed. Attrition due to death of participants within this same time frame was 135 (16%. Conclusion Our recruitment rates were good and compared favourably with other similar UK and international longitudinal studies of the oldest old. The challenges of and successful strategies for involving, recruiting and retaining the oldest old in research

  18. A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.

    Science.gov (United States)

    Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

    2018-01-01

    We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH 1 and CL domains was deleted by substitution of Cys with Ala (Fab ΔSS ). DSC measurements showed that the Tm values of Fab ΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab ΔSS . The resulting Fab (mutSS Fab ΔSS ) had the mutations H:V177C and L:Q160C in Fab ΔSS , confirming the formation of the disulfide bond between CH 1 and CL. The thermostability of mutSS Fab ΔSS was approximately 5 °C higher than that of Fab ΔSS . Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab ΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Engineering and introduction of de novo disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement.

    Science.gov (United States)

    Farnoosh, Gholamreza; Khajeh, Khosro; Latifi, Ali Mohammad; Aghamollaei, Hossein

    2016-12-01

    The organophosphorus hydrolase (OPH) has been used to degrade organophosphorus chemicals, as one of the most frequently used decontamination methods. Under chemical and thermal denaturing conditions, the enzyme has been shown to unfold. To utilize this enzyme in various applications, the thermal stability is of importance. The engineering of de novo disulphide bridges has been explored as a means to increase the thermal stability of enzymes in the rational method of protein engineering. In this study, Disulphide by Design software, homology modelling and molecular dynamics simulations were used to select appropriate amino acid pairs for the introduction of disulphide bridge to improve protein thermostability. The thermostability of the wild-type and three selected mutant enzymes were evaluated by half-life, delta G inactivation (ΔGi) and structural studies (fluorescence and far-UV CD analysis). Data analysis showed that half-life of A204C/T234C and T128C/E153C mutants were increased up to 4 and 24 min, respectively; however, for the G74C/A78C mutant, the half-life was decreased up to 9 min. For the T128C/E124C mutant, both thermal stability and Catalytic efficiency (kcat) were also increased. The half-life and ΔGi results were correlated to the obtained information from structural studies by circular dichroism (CD) spectrometry and extrinsic fluorescence experiments; as rigidity increased in A204C/T2234C and T128C/E153C mutants, half-life and ΔGi also increased. For G74C/A78C mutant, these parameters decreased due to its higher flexibility. The results were submitted a strong evidence for the possibility to improve the thermostability of OPH enzyme by introducing a disulphide bridge after bioinformatics design, even though this design would not be always successful.

  20. Protein thermostability prediction within homologous families using temperature-dependent statistical potentials.

    Directory of Open Access Journals (Sweden)

    Fabrizio Pucci

    Full Text Available The ability to rationally modify targeted physical and biological features of a protein of interest holds promise in numerous academic and industrial applications and paves the way towards de novo protein design. In particular, bioprocesses that utilize the remarkable properties of enzymes would often benefit from mutants that remain active at temperatures that are either higher or lower than the physiological temperature, while maintaining the biological activity. Many in silico methods have been developed in recent years for predicting the thermodynamic stability of mutant proteins, but very few have focused on thermostability. To bridge this gap, we developed an algorithm for predicting the best descriptor of thermostability, namely the melting temperature Tm, from the protein's sequence and structure. Our method is applicable when the Tm of proteins homologous to the target protein are known. It is based on the design of several temperature-dependent statistical potentials, derived from datasets consisting of either mesostable or thermostable proteins. Linear combinations of these potentials have been shown to yield an estimation of the protein folding free energies at low and high temperatures, and the difference of these energies, a prediction of the melting temperature. This particular construction, that distinguishes between the interactions that contribute more than others to the stability at high temperatures and those that are more stabilizing at low T, gives better performances compared to the standard approach based on T-independent potentials which predict the thermal resistance from the thermodynamic stability. Our method has been tested on 45 proteins of known Tm that belong to 11 homologous families. The standard deviation between experimental and predicted Tm's is equal to 13.6°C in cross validation, and decreases to 8.3°C if the 6 worst predicted proteins are excluded. Possible extensions of our approach are discussed.

  1. Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis.

    Science.gov (United States)

    Mohammadi, Mohsen; Sepehrizadeh, Zargham; Ebrahim-Habibi, Azadeh; Shahverdi, Ahmad Reza; Faramarzi, Mohammad Ali; Setayesh, Neda

    2016-11-01

    Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis. The recombinant mutant proteins were over-expressed in E. coli and purified by affinity chromatography on the Ni-NTA system. Circular dichroism spectroscopy, differential scanning calorimetry and kinetic parameters (Km and kcat) were determined. Our results have shown that the secondary structure of all lipases was approximately similar to one another. The MutG2P and MutG59P were more stable than wild type by approximately 2.3 and 2.9 in T 1/2 , respectively. The catalytic efficiency (kcat/Km) of MutH279K was enhanced by 2-fold as compared with the wild type (p<0.05). These results indicate that using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML and it also could be used for improving other properties of enzyme to the desired requirements as well as further mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Identification of thermostable β-xylosidase activities produced by Aspergillus brasiliensis and Aspergillus niger

    DEFF Research Database (Denmark)

    Pedersen, Mads; Lauritzen, Henrik Klitgaard; Frisvad, Jens Christian

    2007-01-01

    Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable beta......-xylosidases. The beta-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75 degrees C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60 degrees C. At 75 degrees C, these values were 38 and 44%, respectively. Whereas A. niger...

  3. Thermal Degradation Mechanism of a Thermostable Polyester Stabilized with an Open-Cage Oligomeric Silsesquioxane

    Directory of Open Access Journals (Sweden)

    Yolanda Bautista

    2017-12-01

    Full Text Available A polyester composite was prepared through the polymerization of an unsaturated ester resin with styrene and an open-cage oligomeric silsesquioxane with methacrylate groups. The effect of the open-cage oligomeric silsesquioxane on the thermal stability of the thermostable polyester was studied using both thermogravimetric analysis and differential thermal analysis. The results showed that the methacryl oligomeric silsesquioxane improved the thermal stability of the polyester. The decomposition mechanism of the polyester/oligomer silsesquioxane composite was proposed by Fourier transform infrared spectroscopy (FTIR analysis of the volatiles.

  4. Methods of hydrolyzing a cellulose using halophilic, thermostable and ionic liquids tolerant cellulases

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Tao; Datta, Supratim; Simmons, Blake A.; Rubin, Edward M.

    2018-01-09

    The present invention provides for an isolated or recombinant polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence of a Halorhabdus utahensis cellulase, such as Hu-CBH1, wherein said amino acid sequence has a halophilic thermostable and/or thermophilic cellobiohydrolase (CBH) activity. In some embodiments, the polypeptide has a CBH activity that is resistant to up to about 20% of ionic liquids. The present invention also provides for compositions comprising and methods using the isolated or recombinant polypeptide.

  5. Evaluations on power ramp data of PWR fuels by FROST and THERMOST codes

    International Nuclear Information System (INIS)

    Murai, K.; Ogawa, S.; Nuno, H.; Kondo, Y.

    1987-01-01

    An evaluation is presented of power ramp data of Mitsubishi's PWR fuel rods tested in R-2, Studsvik, which was analysed by FROST and THERMOST codes. The analyses give good predictions for measured diameter changes and on-power rod elongations. The work indicates that FROST is capable of analysing both radial and axial pellet-cladding mechanism interaction (PCMI) appropriately, and that predicted states of PCMI (i.e. stress and strain which cannot be measured directly) are considered to be reliable. The ramp data used in the present analyses were obtained in two joint programmes with five Japanese PWR utilities (KEPCO, KYEPCO, SEPCO, HEPCO, and JAPCO). (UK)

  6. Protein features as determinants of wild-type glycoside hydrolase thermostability

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus; Kiemer, Lars; Nielsen, Morten

    2017-01-01

    -silico methods guiding the discovery process would be of high value. To develop such an in-silico method and provide the data foundation of it, we determined the melting temperatures of 602 fungal glycoside hydrolases from the families GH5, 6, 7, 10, 11, 43 and AA9 (formerly GH61). We, then used sequence...... and homology modeled structure information of these enzymes to develop the ThermoP melting temperature prediction method. Futhermore, in the context of thermostability, we determined the relative importance of 160 molecular features, such as amino acid frequencies and spatial interactions, and exemplified...

  7. Spatial distribution of free-of-charge pathology submissions to the California Animal Health and Food Safety laboratories during the exotic Newcastle outbreak in 2002-2003.

    Science.gov (United States)

    Soberano, Gustavo; Carpenter, A Tim E; Cardona, Carol; Charlton, Bruce

    2009-03-01

    After the 1971-1973 outbreak of exotic Newcastle disease (END) in California, a free-of-charge diagnostic submission program was created for backyard poultry flocks. This program was implemented to improve disease surveillance in small poultry flocks. The aim of this study was to evaluate the spatial distribution of free-of-charge pathology submissions to the California Animal Health and Food Safety laboratories during the END outbreak in 2002-2003. Cases and controls were selected from within a 100-mile (161-km) radius of each of three laboratories, and their geographic distributions were evaluated. Global clustering of cases was significant around all three laboratories, with mixed results at the local clustering level and the only significant clustering at the focal level around the Davis laboratory with an observed to expected ratio of approximately 5. The area of influence for all three laboratories was about 20 miles (32 km). The significant clustering of cases around the laboratories indicates that more public information about the free-of-charge program could result in coverage of a larger portion of the population; however, the value of the information resulting from increased sampling should be considered relative to the additional cost of obtaining it.

  8. Problems of increasing of thermostability of highly permeable Ni-Zn ferrites and relative materials for telecommunications

    Energy Technology Data Exchange (ETDEWEB)

    Gonchar, A. E-mail: letyuk@mail.ru; Andreev, V.; Letyuk, L.; Shishkanov, A.; Maiorov, V

    2003-01-01

    The work considers ways of increasing of thermostability of ferrites of the basic systems NiO-ZnO-Fe{sub 2}O{sub 3} and MgO-ZnO-Fe{sub 2}O{sub 3} and relative materials for telecommunication. Sufficient results in increasing of the thermostability were achieved by doping Cu ions and controlling rejection of Fe{sub 2}O{sub 3} content from equimolar composition. These results allow to increase the Curie temperature to 130-140 deg. C for Ni-Zn ferrites with initial permeability 2000.

  9. The ''THERMOST'' for analysing thermo-structural behaviour of LWR fuel rod under PCI conditions

    International Nuclear Information System (INIS)

    Nuno, H.; Ogawa, S.; Kobayashi, H.

    1983-01-01

    As one of the methods for evaluating the fuel rod performances under power ramping or load following operations, the combined ''FROST'' and ''THERMOST'' system has been developed and being brought into practical use. The former had already been presented at Blackpool Meeting in 1978, and the latter is going to be presented in this paper. The major purpose of the THERMOST is to analyse very detailed thermal and structural fuel behaviours in a rather localized part of fuel rod whereas the FROST deals with whole-rod-wide general performances. The code handles 2-dimensional thermal and structural analyses simultaneously by using finite element method, in axial section wide or in lateral section wide. It consists of a fundamental FEM system of generalized constitution and its surrounding subroutine system which characterizes fuel behaviours such as temperature distribution, thermal expansion, elastoplasticity, creep, cracking, swelling, growth, etc. Thermal analysis is handled by heat conduction and heat transfer elements (6 kinds) and structural analysis by axisymmetric ring and lateral plane elements (6 kinds). Boundary problems such as contact, friction and cracking are treated by gap and crack elements. A sample calculation of PCI performance on a PWR fuel rod under ramping condition is presented with some inpile test data. (author)

  10. Molecular Dynamics Simulation of Barnase: Contribution of Noncovalent Intramolecular Interaction to Thermostability

    Directory of Open Access Journals (Sweden)

    Zhiguo Chen

    2013-01-01

    Full Text Available Bacillus amyloliquefaciens ribonuclease Barnase (RNase Ba is a 12 kD (kilodalton small extracellular ribonuclease. It has broad application prospects in agriculture, clinical medicine, pharmaceutical, and so forth. In this work, the thermal stability of Barnase has been studied using molecular dynamics simulation at different temperatures. The present study focuses on the contribution of noncovalent intramolecular interaction to protein stability and how they affect the thermal stability of the enzyme. Profiles of root mean square deviation and root mean square fluctuation identify thermostable and thermosensitive regions of Barnase. Analyses of trajectories in terms of secondary structure content, intramolecular hydrogen bonds and salt bridge interactions indicate distinct differences in different temperature simulations. In the simulations, Four three-member salt bridge networks (Asp8-Arg110-Asp12, Arg83-Asp75-Arg87, Lys66-Asp93-Arg69, and Asp54-Lys27-Glu73 have been identified as critical salt bridges for thermostability which are maintained stably at higher temperature enhancing stability of three hydrophobic cores. The study may help enlighten our knowledge of protein structural properties, noncovalent interactions which can stabilize secondary peptide structures or promote folding, and also help understand their actions better. Such an understanding is required for designing efficient enzymes with characteristics for particular applications at desired working temperatures.

  11. High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

    Science.gov (United States)

    Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

    2016-01-01

    Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from RNA in RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

  12. Crystallization and preliminary X-ray characterization of two thermostable DNA nucleases

    International Nuclear Information System (INIS)

    Kuettner, E. Bartholomeus; Pfeifer, Sven; Keim, Antje; Greiner-Stöffele, Thomas; Sträter, Norbert

    2006-01-01

    Two thermostable DNA nucleases from archaea were crystallized in different space groups; the crystals were suitable for X-ray analysis. Temperature-tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA-cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4-methyl pentandiol, dioxane, ethanol or 2-propanol as precipitants. The addition of a 10-mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 Å using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes

  13. Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding.

    Directory of Open Access Journals (Sweden)

    Chi-Ho Chan

    Full Text Available Most thermophilic proteins tend to have more salt bridges, and achieve higher thermostability by up-shifting and broadening their protein stability curves. While the stabilizing effect of salt-bridge has been extensively studied, experimental data on how salt-bridge influences protein stability curves are scarce. Here, we used double mutant cycles to determine the temperature-dependency of the pair-wise interaction energy and the contribution of salt-bridges to ΔC(p in a thermophilic ribosomal protein L30e. Our results showed that the pair-wise interaction energies for the salt-bridges E6/R92 and E62/K46 were stabilizing and insensitive to temperature changes from 298 to 348 K. On the other hand, the pair-wise interaction energies between the control long-range ion-pair of E90/R92 were negligible. The ΔC(p of all single and double mutants were determined by Gibbs-Helmholtz and Kirchhoff analyses. We showed that the two stabilizing salt-bridges contributed to a reduction of ΔC(p by 0.8-1.0 kJ mol⁻¹ K⁻¹. Taken together, our results suggest that the extra salt-bridges found in thermophilic proteins enhance the thermostability of proteins by reducing ΔC(p, leading to the up-shifting and broadening of the protein stability curves.

  14. Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis

    International Nuclear Information System (INIS)

    Varshney, Nishant Kumar; Suresh Kumar, R.; Ignatova, Zoya; Prabhune, Asmita; Pundle, Archana; Dodson, Eleanor; Suresh, C. G.

    2012-01-01

    A thermostable penicillin G acylase from A. faecalis has been crystallized in two space groups: C222 1 and P4 1 2 1 2. X-ray diffraction data were collected to 3.3 and 3.5 Å resolution, respectively. The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C222 1 , with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 Å, and P4 1 2 1 2, with unit-cell parameters a = b = 85.6, c = 298.8 Å. Data were collected at 293 K and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-terminal residue of the β-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G acylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme

  15. Thermostable 𝜶-Amylase Activity from Thermophilic Bacteria Isolated from Bora Hot Spring, Central Sulawesi

    Science.gov (United States)

    Gazali, F. M.; Suwastika, I. N.

    2018-03-01

    α-Amylase is one of the most important enzyme in biotechnology field, especially in industrial application. Thermostability of α-Amylase produced by thermophilic bacteria improves industrial process of starch degradation in starch industry. The present study were concerned to the characterization of α-Amylase activity from indigenous thermophilic bacteria isolated from Bora hot spring, Central Sulawesi. There were 18 isolates which had successfully isolated from 90°C sediment samples of Bora hot spring and 13 of them showed amylolytic activity. The α-Amylase activity was measured qualitatively at starch agar and quantitatively based on DNS (3,5-Dinitrosalicylic acid) methods, using maltose as standard solution. Two isolates (out of 13 amylolytic bacteria), BR 002 and BR 015 showed amylolytic index of 0.8 mm and 0.5 mm respectively, after being incubated at 55°C in the 0.002% Starch Agar Medium. The α-Amylase activity was further characterized quantitatively which includes the optimum condition of pH and temperature of α-Amylase crude enzyme from each isolate. To our knowledge, this is the first report on isolation and characterization of a thermostable α-Amylase from thermophilic bacteria isolated from Central Sulawesi particularly from Bora hot spring.

  16. Structure of the Aeropyrum pernix L7Ae multifunctional protein and insight into its extreme thermostability

    International Nuclear Information System (INIS)

    Bhuiya, Mohammad Wadud; Suryadi, Jimmy; Zhou, Zholi; Brown, Bernard Andrew II

    2013-01-01

    The crystal structure of A. pernix L7Ae is reported, providing insight into the extreme thermostability of this protein. Archaeal ribosomal protein L7Ae is a multifunctional RNA-binding protein that directs post-transcriptional modification of archaeal RNAs. The L7Ae protein from Aeropyrum pernix (Ap L7Ae), a member of the Crenarchaea, was found to have an extremely high melting temperature (>383 K). The crystal structure of Ap L7Ae has been determined to a resolution of 1.56 Å. The structure of Ap L7Ae was compared with the structures of two homologs: hyperthermophilic Methanocaldococcus jannaschii L7Ae and the mesophilic counterpart mammalian 15.5 kD protein. The primary stabilizing feature in the Ap L7Ae protein appears to be the large number of ion pairs and extensive ion-pair network that connects secondary-structural elements. To our knowledge, Ap L7Ae is among the most thermostable single-domain monomeric proteins presently observed

  17. Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design.

    Science.gov (United States)

    Wu, Tzu-Hui; Chen, Chun-Chi; Cheng, Ya-Shan; Ko, Tzu-Ping; Lin, Cheng-Yen; Lai, Hui-Lin; Huang, Ting-Yung; Liu, Je-Ruei; Guo, Rey-Ting

    2014-04-10

    Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

    Science.gov (United States)

    Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki

    2013-01-01

    A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent Km values for the cofactors NAD(P)+ and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O–20% 2-propanol and H2O–50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols. PMID:23354700

  19. Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

    Science.gov (United States)

    Pek, Han Bin; Lim, Pei Yu; Liu, Chengcheng; Lee, Dong-Yup; Bi, Xuezhi; Wong, Fong Tian; Ow, Dave Siak-Wei

    2017-05-01

    To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively. Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

  20. Characterization of an extremely salt-tolerant and thermostable phytase from Bacillus amyloliquefaciens US573.

    Science.gov (United States)

    Boukhris, Ines; Farhat-Khemakhem, Ameny; Blibech, Monia; Bouchaala, Kameleddine; Chouayekh, Hichem

    2015-09-01

    The extracellular phytase produced by the Bacillus amyloliquefaciens US573 strain, isolated from geothermal soil located in Southern Tunisia was purified and characterized. This calcium-dependent and bile-stable enzyme (PHY US573) was optimally active at pH 7.5 and 70 °C. It showed a good stability at pH ranging from 4 to 10, and especially, an exceptional thermostability as it recovered 50 and 62% of activity after heating for 10 min at 100 and 90 °C, respectively. In addition, PHY US573 was found to be extremely salt-tolerant since it preserved 80 and 95% of activity in the presence of 20 g/l of NaCl and LiCl, respectively. The gene corresponding to PHY US573 was cloned. It encodes a 383 amino acids polypeptide exhibiting 99% identity with the highly thermostable phytases from Bacillus sp. MD2 and B. amyloliquefaciens DS11 (3 and 5 residues difference, respectively), suggesting the existence of common molecular determinants responsible for their remarkable heat stability. Overall, our findings illustrated that in addition to its high potential for application in feed industry, the salt tolerance of the PHY US573 phytase, may represent an exciting new avenue for improvement of phosphorus-use efficiency of salt-tolerant plants in soils with high salt and phytate content. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Enhanced Thermostability of Arabidopsis Rubisco Activase Improves Photosynthesis and Growth Rates under Moderate Heat Stress[OA

    Science.gov (United States)

    Kurek, Itzhak; Chang, Thom Kai; Bertain, Sean M.; Madrigal, Alfredo; Liu, Lu; Lassner, Michael W.; Zhu, Genhai

    2007-01-01

    Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Δrca) line. In a long-term growth test at either constant 26°C or daily 4-h 30°C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions. PMID:17933901

  2. Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods.

    Science.gov (United States)

    Chen, Ana; Li, Yamei; Nie, Jianqi; McNeil, Brian; Jeffrey, Laura; Yang, Yankun; Bai, Zhonghu

    2015-10-01

    Thermostability has been considered as a requirement in the starch processing industry to maintain high catalytic activity of pullulanase under high temperatures. Four data driven rational design methods (B-FITTER, proline theory, PoPMuSiC-2.1, and sequence consensus approach) were adopted to identify the key residue potential links with thermostability, and 39 residues of Bacillus acidopullulyticus pullulanase were chosen as mutagenesis targets. Single mutagenesis followed by combined mutagenesis resulted in the best mutant E518I-S662R-Q706P, which exhibited an 11-fold half-life improvement at 60 °C and a 9.5 °C increase in Tm. The optimum temperature of the mutant increased from 60 to 65 °C. Fluorescence spectroscopy results demonstrated that the tertiary structure of the mutant enzyme was more compact than that of the wild-type (WT) enzyme. Structural change analysis revealed that the increase in thermostability was most probably caused by a combination of lower stability free-energy and higher hydrophobicity of E518I, more hydrogen bonds of S662R, and higher rigidity of Q706P compared with the WT. The findings demonstrated the effectiveness of combined data-driven rational design approaches in engineering an industrial enzyme to improve thermostability. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. INCREASING THE THERMOSTABILITY OF THE NEUTRAL PROTEINASE OF BACILLUS-STEAROTHERMOPHILUS BY IMPROVEMENT OF INTERNAL HYDROGEN-BONDING

    NARCIS (Netherlands)

    EIJSINK, VGH; VRIEND, G; VANDERZEE, [No Value; VANDENBURG, B; VENEMA, G

    1992-01-01

    In an attempt to increase the thermostability of the neutral proteinase of Bacillus stearothermophilus the buried Ala-170 was replaced by serine. Molecular-dynamics simulations showed that Ser-170 stabilizes the enzyme by formation of an internal hydrogen bond. In addition, the hydroxy group of

  4. Synergistic function of four novel thermostable glycoside hydrolases from a long-term enriched thermophilic methanogenic digester

    Directory of Open Access Journals (Sweden)

    Meng eWang

    2015-05-01

    Full Text Available In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme cocktail, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60°C to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy.

  5. Structure of a thermostable serralysin from Serratia sp. FS14 at 1.1 Å resolution.

    Science.gov (United States)

    Wu, Dongxia; Ran, Tinting; Wang, Weiwu; Xu, Dongqing

    2016-01-01

    Serralysin is a well studied metalloprotease, and typical serralysins are not thermostable. The serralysin isolated from Serratia sp. FS14 was found to be thermostable, and in order to reveal the mechanism responsible for its thermostability, the crystal structure of serralysin from Serratia sp. FS14 was solved to a crystallographic R factor of 0.1619 at 1.10 Å resolution. Similar to its homologues, it mainly consists of two domains: an N-terminal catalytic domain and a `parallel β-roll' C-terminal domain. Comparative studies show that the shape of the catalytic active-site cavity is more open owing to the 189-198 loop, with a short 310-helix protruding further from the molecular surface, and that the β-sheets comprising the `parallel β-roll' are longer than those in its homologues. The formation of hydrogen bonds from one of the nonconserved residues (Asn200) to Lys27 may contribute to the thermostability.

  6. Virus-like particle nanoreactors: programmed en capsulation of the thermostable CelB glycosidase inside the P22 capsid

    NARCIS (Netherlands)

    Patterson, D.P.; Schwarz, B.; El-Boubbou, K.; Oost, van der J.; Prevelige, P.E.; Douglas, T.

    2012-01-01

    Self-assembling biological systems hold great potential for the synthetic construction of new active soft nanomaterials. Here we demonstrate the hierarchical bottom-up assembly of bacteriophage P22 virus-like particles (VLPs) that encapsulate the thermostable CelB glycosidase creating catalytically

  7. Interference of Infectious Bursal Diseases (IBD) Virus and Vaccine ...

    African Journals Online (AJOL)

    The interference of Infectious bursal disease (IBD) virus and vaccine with the immune response of the grey brested guinea fowl (Numida meleagridis galeata palas) to Newcastle desease (ND) “LaSota” vaccine was studied using hemagglutination inhibition (HI) test for detection of ND virus antibody and agar gel ...

  8. The opportunities and rewards of distance learning programs offered by the university of Newcastle

    International Nuclear Information System (INIS)

    Lyall, D.

    2002-01-01

    Full text: The University of Newcastle offers, via the distance learning mode of study, three innovative postgraduate programs designed for Nuclear Medicine Scientists and Practitioners. The Graduate Diploma in Nuclear Medicine is designed for Nuclear Medicine Scientists who have been absent from the work force for an extended period of time or have completed a qualification in medical imaging, who are currently working as a Nuclear Medicine Scientist. The Graduate Diploma fosters and encourages a greater depth of knowledge of Nuclear Medicine through the understanding and application of clinical skills. The Master of Nuclear Medicine is designed to develop within Nuclear Medicine Scientists and Practitioners a greater depth of knowledge of Nuclear Medicine through the acquisition and application of research skills. The Master of Applied Management (Health) is designed for Nuclear Medicine Scientists, practitioners, health managers and administrators who desire to acquire essential management skills while expanding their clinical skills. Graduates of the Graduate Diploma are eligible to sit the certification examination for the Nuclear Medicine Technology Certification Board in the United States of America. They may also eligible to apply to the Australian and New Zealand Society of Nuclear Medicine Accreditation Board for interim accreditation. Graduates of the Masters of Nuclear Medicine enjoy the same opportunities as per the Graduate Diploma. They also enjoy a greater success with promotion and have the skills necessary to develop a rewarding career in research. Copyright (2002) The Australian and New Zealand Society of Nuclear Medicine Inc

  9. A Good Death? Report of the Second Newcastle Meeting on Laboratory Animal Euthanasia

    Science.gov (United States)

    Hawkins, Penny; Prescott, Mark J.; Carbone, Larry; Dennison, Ngaire; Johnson, Craig; Makowska, I. Joanna; Marquardt, Nicole; Readman, Gareth; Weary, Daniel M.; Golledge, Huw D. R.

    2016-01-01

    Simple Summary Millions of laboratory animals are killed each year worldwide. However, there is a lack of consensus regarding what methods of killing are humane for many species and stages of development. This report summarises research findings and discussions from an international meeting of experts and stakeholders, with recommendations to inform good practice for humane killing of mice, rats and zebrafish. It provides additional guidance and perspectives for researchers designing projects that involve euthanasing animals, researchers studying aspects of humane killing, euthanasia device manufacturers, regulators, and institutional ethics or animal care and use committees that wish to review local practice. Abstract Millions of laboratory animals are killed each year worldwide. There is an ethical, and in many countries also a legal, imperative to ensure those deaths cause minimal suffering. However, there is a lack of consensus regarding what methods of killing are humane for many species and stages of development. In 2013, an international group of researchers and stakeholders met at Newcastle University, United Kingdom to discuss the latest research and which methods could currently be considered most humane for the most commonly used laboratory species (mice, rats and zebrafish). They also discussed factors to consider when making decisions about appropriate techniques for particular species and projects, and priorities for further research. This report summarises the research findings and discussions, with recommendations to help inform good practice for humane killing. PMID:27563926

  10. A comparison of subjective and objective measures of physical activity from the Newcastle 85+ study.

    Science.gov (United States)

    Innerd, Paul; Catt, Michael; Collerton, Joanna; Davies, Karen; Trenell, Michael; Kirkwood, Thomas B L; Jagger, Carol

    2015-07-01

    Little is known about physical activity (PA) in the very old, the fastest growing age group in the population. We aimed to examine the convergent validity of subjective and objective measures of PA in adults aged over 85 years. A total of 484 participants aged 87-89 years recruited to the Newcastle 85+ study completed a purpose-designed physical activity questionnaire (PAQ), which categorised participants as mildly active, moderately active and very active. Out of them, 337 participants wore a triaxial, raw accelerometer on the right wrist over a 5-7-day period to obtain objective measures of rest/activity, PA intensity and PA type. Data from subjective and objective measurement methods were compared. Self-reported PA was significantly associated with objective measures of the daily sedentary time, low-intensity PA and activity type classified as sedentary, activities of daily living and walking. Objective measures of PA were significantly different when low, moderate and high self-reported PA categories were compared (all P PAQ demonstrated convergent validity with objective measures of PA. Our findings suggest that this PAQ can be used in the very old to rank individuals according to their level of total PA. © The Author 2015. Published by Oxford University Press on behalf of the British Geriatrics Society.

  11. Aerial gamma ray and magnetic survey: Powder River II Project, Newcastle Quadrangle, Wyoming. Final report

    International Nuclear Information System (INIS)

    1979-04-01

    Thick Phanerozoic sediments (greater than 17,000 ft) fill the northwest trending Powder River Basin which is the dominant tectonic structure in the Newcastle quadrangle. Lower Tertiary sediments comprise more than 85% of exposed units at the surface of the Basin. A small portion of the Black Hills Uplift occupies the eastern edge of the quadrangle. Residual magnetics clearly reflect the great depth to crystalline Precambrian basement in the Basin. The Basin/Uplift boundary is not readily observed in the magnetic data. Economic uranium deposits of roll-type configuration are present in the southwest within the Monument Hill-Box Creek District in fluvial sandstones of the Paleocene Fort Union Formation. Numerous small claims and prospects are found in the Pumpkin Buttes-Turnercrest District in the northwest. Interpretation of the radiometric data resulted in 86 statistical uranium anomalies listed for this quadrangle. Most anomalies are in the eastern-central portion of the map within Tertiary Fort Union and Wasatch Formations. However, several lie in the known uranium districts in the southwest and northwest

  12. Identification and characterization of a novel thermostable pyrethroid-hydrolyzing enzyme isolated through metagenomic approach

    Directory of Open Access Journals (Sweden)

    Fan Xinjiong

    2012-03-01

    Full Text Available Abstract Background Pyrethroid pesticides are broad-spectrum pest control agents in agricultural production. Both agricultural and residential usage is continuing to grow, leading to the development of insecticide resistance in the pest and toxic effects on a number of nontarget organisms. Thus, it is necessary to hunt suitable enzymes including hydrolases for degrading pesticide residues, which is an efficient "green" solution to biodegrade polluting chemicals. Although many pyrethroid esterases have consistently been purified and characterized from various resources including metagenomes and organisms, the thermostable pyrethroid esterases have not been reported up to the present. Results In this study, we identified a novel pyrethroid-hydrolyzing enzyme Sys410 belonging to familyV esterases/lipases with activity-based functional screening from Turban Basin metagenomic library. Sys410 contained 280 amino acids with a predicted molecular mass (Mr of 30.8 kDa and was overexpressed in Escherichia coli BL21 (DE3 in soluble form. The optimum pH and temperature of the recombinant Sys410 were 6.5 and 55°C, respectively. The enzyme was stable in the pH range of 4.5-8.5 and at temperatures below 50°C. The activity of Sys410 decreased a little when stored at 4°C for 10 weeks, and the residual activity reached 94.1%. Even after incubation at 25°C for 10 weeks, it kept 68.3% of its activity. The recombinant Sys410 could hydrolyze a wide range of ρ-nitrophenyl esters, but its best substrate is ρ-nitrophenyl acetate with the highest activity (772.9 U/mg. The enzyme efficiently degraded cyhalothrin, cypermethrin, sumicidin, and deltamethrin under assay conditions of 37°C for 15 min, with exceeding 95% hydrolysis rate. Conclusion This is the first report to construct metagenomic libraries from Turban Basin to obtain the thermostable pyrethroid-hydrolyzing enzyme. The recombinant Sys410 with broad substrate specificities and high activity was the most

  13. Diseases of poultry in Akure, Nigeria: a ten year analysis of clinical ...

    African Journals Online (AJOL)

    An analysis of poultry diseases diagnosed at the avian clinic of Veterinary Hospital of the Ministry of Agriculture and Natural Resources, Akure, Ondo-State, was carried out using data from 22,927 cases reported at the clinic over a period of ten years. The commonest diseases reported were Newcastle, 9.8%, chronic ...

  14. Engineering improved thermostability of the GH11 xylanase from Neocallimastix patriciarum via computational library design.

    Science.gov (United States)

    Bu, Yifan; Cui, Yinglu; Peng, Ying; Hu, Meirong; Tian, Yu'e; Tao, Yong; Wu, Bian

    2018-04-01

    Xylanases, which cleave the β-1,4-glycosidic bond between xylose residues to release xylooligosaccharides (XOS), are widely used as food additives, animal feeds, and pulp bleaching agents. However, the thermally unstable nature of xylanases would hamper their industrial application. In this study, we used in silico design in a glycoside hydrolase family (GH) 11 xylanase to stabilize the enzyme. A combination of the best mutations increased the apparent melting temperature by 14 °C and significantly enhanced thermostability and thermoactivation. The variant also showed an upward-shifted optimal temperature for catalysis without compromising its activity at low temperatures. Moreover, a 10-fold higher XOS production yield was obtained at 70 °C, which compensated the low yield obtained with the wild-type enzyme. Collectively, the variant constructed by the computational strategy can be used as an efficient biocatalyst for XOS production at industrially viable conditions.

  15. Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus.

    Science.gov (United States)

    Raghunath, Pendru

    2014-01-01

    Vibrio parahaemolyticus is the leading cause of seafood borne bacterial gastroenteritis in the world, often associated with the consumption of raw or undercooked seafood. However, not all strains of V. parahaemolyticus are pathogenic. The thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH) encoded by tdh and trh genes, respectively, are considered major virulence factors in V. parahaemolyticus. However, about 10% of clinical strains do not contain tdh and/or trh. Environmental isolates of V. parahaemolyticus lacking tdh and/or trh are also highly cytotoxic to human gastrointestinal cells. Even in the absence of these hemolysins, V. parahaemolyticus remains pathogenic indicating other virulence factors exist. This mini review aims at discussing the possible roles of tdh and trh genes in clinical and environmental isolates of V. parahaemolyticus.

  16. Characterization of thermostable cellulase produced by Bacillus strains isolated from solid waste of carrageenan

    Science.gov (United States)

    Listyaningrum, N. P.; Sutrisno, A.; Wardani, A. K.

    2018-03-01

    Cellulase-producing bacteria was isolated from solid waste of carrageenan and identified as Bacillus licheniformis C55 by 16S rRNA sequencing. The optimum condition for cellulase production was obtained at pH and temperature of 8.0 and 50°C, respectively in a medium containing glucose as carbon source and 1.0% carboxymethyl cellulose (CMC) to stimulate the cellulase production. Most remarkably, the enzyme retained its relative activity over 50% after incubation at 50°C for 90 minutes. Substrate specificity suggested that the enzyme is an endoglucanase. The molecular mass of Bacillus licheniformis C55 crude cellulase was found about 18 kDa by SDS-PAGE analysis. This thermostable enzyme would facilitate development of more efficient and cost-effective forms of the process to convert lignocellulosic biomass into high-value products.

  17. Chemoenzymatic Synthesis of Oligo(L-cysteine) for Use as a Thermostable Bio-Based Material.

    Science.gov (United States)

    Ma, Yinan; Sato, Ryota; Li, Zhibo; Numata, Keiji

    2016-01-01

    Oligomerization of thiol-unprotected L-cysteine ethyl ester (Cys-OEt) catalyzed by proteinase K in aqueous solution has been used to synthesize oligo(L-cysteine) (OligoCys) with a well-defined chemical structure and relatively large degree of polymerization (DP) up to 16-17 (average 8.8). By using a high concentration of Cys-OEt, 78.0% free thiol content was achieved. The thermal properties of OligoCys are stable, with no glass transition until 200 °C, and the decomposition temperature could be increased by oxidation. Chemoenzymatically synthesized OligoCys has great potential for use as a thermostable bio-based material with resistance to oxidation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes.

    Science.gov (United States)

    Bhalla, Aditya; Bansal, Namita; Kumar, Sudhir; Bischoff, Kenneth M; Sani, Rajesh K

    2013-01-01

    Second-generation feedstock, especially nonfood lignocellulosic biomass is a potential source for biofuel production. Cost-intensive physical, chemical, biological pretreatment operations and slow enzymatic hydrolysis make the overall process of lignocellulosic conversion into biofuels less economical than available fossil fuels. Lignocellulose conversions carried out at ≤ 50 °C have several limitations. Therefore, this review focuses on the importance of thermophilic bacteria and thermostable enzymes to overcome the limitations of existing lignocellulosic biomass conversion processes. The influence of high temperatures on various existing lignocellulose conversion processes and those that are under development, including separate hydrolysis and fermentation, simultaneous saccharification and fermentation, and extremophilic consolidated bioprocess are also discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Thermostability and photophysical properties of mixed-ligand carboxylate/benzimidazole Zn(II)-coordination polymers

    International Nuclear Information System (INIS)

    Barros, Bráulio Silva; Chojnacki, Jaroslaw; Macêdo Soares, Antonia Alice; Kulesza, Joanna; Lourenço da Luz, Leonis; Júnior, Severino Alves

    2015-01-01

    The reaction between Zn(NO 3 ) 2 ·6H 2 O or Zn(CH 3 COO) 2 ·2H 2 O and isophthalic acid (1,3-H 2 bdc) in the presence of benzimidazole (Hbzim) in dimethylformamide (DMF)/ethanol (EtOH)/H 2 O solvent mixture at room temperature yielded two structurally different coordination polymers: [Zn 2 (1,3-bdc) 2 (Hbzim) 2 ] (1) and [Zn 2 (1,3-bdc)(bzim) 2 ] (2). (1) is a 2D-layered framework with a molecule of benzimidazole coordinated to the Zn center, whereas (2) is a 3D framework with benzimidazolate species acting as a co-ligand and bridging two Zn(II) ions. Reactions performed at 90 °C led to the formation of coordination polymers structurally similar to (2), independently of the Zn(II) source used. In the absence of benzimidazole, the reaction between ZnAc 2 .2H 2 O and 1,3-H 2 bdc at 90 °C resulted in the formation of (3), a 3D coordination polymer Zn(HCOO) 3 (Me 2 NH 2 + ). It was observed that the thermostability and the photophysical properties of (1) and (2) are strongly dependent on the coordination modes and packing of benzimidazole in the solid state. These materials present photoluminescence in the wide range of the spectrum, from UV to IR. A full understanding of a physical process occurring in these intriguing systems, including complete energy level diagrams with possible transitions were provided. - Graphical abstract: Display Omitted - Highlights: • Structurally different Zn(II)-coordination polymers were prepared. • The formation of frameworks was counter anion and temperature dependent. • Photoluminescence in the wide range of the spectrum, from UV to IR was observed. • Thermostability and luminescence depended on bzim packing in the structure

  20. Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution.

    Science.gov (United States)

    Shivange, Amol V; Roccatano, Danilo; Schwaneberg, Ulrich

    2016-01-01

    Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE,  an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The "best" combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure-function relationships of enzymes.

  1. Enzimas termoestáveis: fontes, produção e aplicação industrial Thermostable enzymes: sources, production and industrial applications

    Directory of Open Access Journals (Sweden)

    Eleni Gomes

    2007-02-01

    Full Text Available REVIEW: Living organisms encountered in hostile environments that are characterized by extreme temperatures rely on novel molecular mechanisms to enhance the thermal stability of their proteins, nucleic acids, lipids and cell membranes. Proteins isolated from thermophilic organisms usually exhibit higher intrinsic thermal stabilities than their counterparts isolated from mesophilic organisms. Although the molecular basis of protein thermostability is only partially understood, structural studies have suggested that the factors that may contribute to enhance protein thermostability mainly include hydrophobic packing, enhanced secondary structure propensity, helix dipole stabilization, absence of residues sensitive to oxidation or deamination, and increased electrostatic interactions. Thermostable enzymes such as amylases, xylanases and pectinases isolated from thermophilic organisms are potentially of interest in the optimization of industrial processes due to their enhanced stability. In the present review, an attempt is made to delineate the structural factors that increase enzyme thermostability and to document the research results in the production of these enzymes.

  2. Vaccines to Combat Livestock Diseases in Sub-Saharan Africa ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    This project is applying modern biotechnology to engineer a thermostable, single dose vaccine that protects cattle, sheep, and goats from five main diseases - lumpy skin ... In partnership with UNESCO's Organization for Women in Science for the Developing World (OWSD), IDRC is pleased to announce that the first call for ...

  3. ENFERMEDAD DE NEWCASTLE EN AVES DE TRASPATIO DEL EJE CAFETERO COLOMBIANO

    Directory of Open Access Journals (Sweden)

    Marlyn Romero P

    2009-08-01

    Full Text Available Objetivo. Evaluar la circulación del virus de la enfermedad de Newcastle (ENC en aves de traspatio de siete municipios del eje cafetero por medio de la técnica ELISA. Materiales y métodos. Fueron encuestados 465 predios para evaluar las normas de bioseguridad de las aves de traspatio de los municipios de Armenia, Circasia, Quimbaya, Montenegro, Filandia, Calarcá y La Tebaida del Departamento del Quindío. Se analizaron 662 muestras de suero para detectar anticuerpos IgG contra el virus de la ENC por ELISA. Resultados. La seroprevalencia de la ENC en la población de aves evaluada fue del 30.7%.(203/662, que corresponde al 38.5% de los predios encuestados. No se encontró asociación entre la especie y la seroreactividad (p=0.21, ni entre la distribución de los anticuerpos por grupos productivos y la edad (p>0.05. Los predios de traspatio son manejados de forma extensiva, albergan en las mismas instalaciones aves de diferentes especies y edades en el 64.3% (299/465, con edades en intervalos entre tres meses y cinco años. Fueron detectadas malas prácticas de manejo de la gallinaza y la mortalidad, sin encontrar asociación estadísticamente significativa entre las variables evaluadas y la presencia de anticuerpos anti-virus de ENC (p>0.05. Conclusiones. Es necesario el establecimiento de planes adecuados de vacunación de las aves de traspatio, la vigilancia epidemiológica activa, la observación de los casos sospechosos, sin dejar de lado la educación y capacitación, sobre el adecuado manejo de los animales, dirigida a los propietarios de las aves como medidas de control de la enfermedad.

  4. Engineering of an Extremely Thermostable Alpha/Beta Barrel Scaffold to Serve as a High Affinity Molecular Recognition Element for Use in Sensor Applications

    Science.gov (United States)

    2015-12-23

    Molecular Recognition Element For Use in Sensor Applications Report Title The overall goal of the project was to evolve a highly thermostable enzyme ( alcohol ...SECURITY CLASSIFICATION OF: The overall goal of the project was to evolve a highly thermostable enzyme ( alcohol dehydrogenase D (AdhD) from Pyrococcus...furiosus) to bind an explosive molecule, RDX. The enzyme naturally catalyzes the nicotinamide cofactor-dependent oxidation or reduction of alcohols

  5. Serologic surveillance of wild and pen-reared ring-necked pheasants (Phasianus colchicus) as a method of understanding disease reservoirs

    Science.gov (United States)

    Dwight, Ian; Coates, Peter S.; Stoute, Simone T.; Senties-Cue, C. Gabriel; Gharpure, Radhika V.; Pitesky, Maurice E.

    2018-01-01

    We investigated exposure to infectious diseases in wild (n=33) and pen-reared (n=12) Ring-necked Pheasants (Phasianus colchicus) in the Central Valley of California during 2014 and 2015. Serologic tests were positive for antibodies against hemorrhagic enteritis (HE), infectious bursal disease (IBD), and Newcastle disease (ND) viruses in both wild and pen-reared pheasants.

  6. Soroepidemiologia da doença de Newcastle em plantéis de avestruzes dos Estados da Bahia e de São Paulo

    OpenAIRE

    Fernandes,Lia Muniz Barretto; Silva,Priscila Sousa da; Ramos,Izabella; Sales,Tatiane Santana; Herval,Elen Fabiane Guimarães; Batinga,Thaís de Brito; Maia,Paulo César Costa; César,André Eduardo Rocha; Doretto Júnior,Luciano; Meyer,Roberto; Freire,Songeli Menezes

    2010-01-01

    Estudos sorológicos em Avestruzes (Struthio camelus) são ferramentas úteis para analisar os riscos relacionados à Doença de Newcastle nesses plantéis e à avicultura nacional. No presente estudo, amostras de sangue foram obtidas de avestruzes de ambos os sexos, de diferentes faixas etárias e sem apresentação de sintomatologia clínica, criadas nos Estados da Bahia e de São Paulo com o objetivo de avaliar a presença de anticorpos contra o vírus da Doença de Newcastle por meio de ELISA indireto. ...

  7. Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis.

    Science.gov (United States)

    Hesampour, Ardeshir; Siadat, Seyed Ehsan Ranaei; Malboobi, Mohammad Ali; Mohandesi, Nooshin; Arab, Seyed Shahriar; Ghahremanpour, Mohammad Mehdi

    2015-03-01

    Phytase efficiently catalyzes the hydrolysis of phytate to phosphate; it can be utilized as an animal supplement to provide animals their nutrient requirements for phosphate and to mitigate environmental pollution caused by unutilized feed phosphate. Owing to animal feed being commonly pelleted at 70 to 90 °C, phytase with a sufficiently high thermal stability is desirable. Based on the crystal structure of PhyA and bioinformatics analysis at variant heat treatments, 12 single and multiple mutants were introduced by site-directed mutagenesis in order to improve phytase thermostability. Mutated constructs were expressed in Pichia pastoris. The manipulated phytases were purified; their biochemical and kinetic investigation revealed that while the thermostability of six mutants was improved, P9 (T314S Q315R V62N) and P12 (S205N S206A T151A T314S Q315R) showed the highest heat stability (P phytase to be used as an animal feed supplement.

  8. Crystallization and preliminary X-ray diffraction studies of the precursor protein of a thermostable variant of papain

    International Nuclear Information System (INIS)

    Roy, Sumana; Choudhury, Debi; Chakrabarti, Chandana; Biswas, Sampa; Dattagupta, J. K.

    2011-01-01

    The crystallization of the precursor of a thermostable variant of papain and the collection of diffraction data to 2.6 Å resolution are reported. The crystallization of a recombinant thermostable variant of pro-papain has been carried out. The mutant pro-enzyme was expressed in Escherichia coli as inclusion bodies, refolded, purified and crystallized. The crystals belonged to space group P2 1 , with unit-cell parameters a = 42.9, b = 74.8, c = 116.5 Å, β = 93.0°, and diffracted to 2.6 Å resolution using synchrotron radiation. Assuming the presence of two molecules in the asymmetric unit, the calculated Matthews coefficient is 2.28 Å 3 Da −1 , corresponding to a solvent content of 46%. Initial attempts to solve the structure using molecular-replacement techniques were successful

  9. Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties

    Directory of Open Access Journals (Sweden)

    Koh Eunhee

    2007-07-01

    Full Text Available Abstract Background EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information. Results Here we report for the first time the 2.1-Å resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic α/β hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other α/β hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the β8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1. Conclusion Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1.

  10. Thermostability of Multidomain Proteins: Elongation Factors EF-Tu from Escherichia coli and Bacillus stearothermophilus and Their Chimeric Forms

    Czech Academy of Sciences Publication Activity Database

    Šanderová, Hana; Hůlková, Marta; Maloň, Petr; Kepková, M.; Jonák, Jiří

    2004-01-01

    Roč. 13, č. 1 (2004), s. 89-99 ISSN 0961-8368 R&D Projects: GA AV ČR IPP1050128; GA ČR GA204/98/0863; GA ČR GA303/02/0689 Institutional research plan: CEZ:AV0Z4055905; CEZ:AV0Z5052915 Keywords : elongation factor EF-Tu, thermostability, chimeric protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.116, year: 2004

  11. Bilirubin oxidase-like proteins from Podospora anserina: promising thermostable enzymes for application in transformation of plant biomass.

    Science.gov (United States)

    Xie, Ning; Ruprich-Robert, Gwenaël; Silar, Philippe; Chapeland-Leclerc, Florence

    2015-03-01

    Plant biomass degradation by fungi is a critical step for production of biofuels, and laccases are common ligninolytic enzymes envisioned for ligninolysis. Bilirubin oxidases (BODs)-like are related to laccases, but their roles during lignocellulose degradation have not yet been fully investigated. The two BODs of the ascomycete fungus Podospora anserina were characterized by targeted gene deletions. Enzymatic assay revealed that the bod1(Δ) and bod2(Δ) mutants lost partly a thermostable laccase activity. A triple mutant inactivated for bod1, bod2 and mco, a previously investigated multicopper oxidase gene distantly related to laccases, had no thermostable laccase activity. The pattern of fruiting body production in the bod1(Δ) bod2(Δ) double mutant was changed. The bod1(Δ) and bod2(Δ) mutants were reduced in their ability to grow on ligneous and cellulosic materials. Furthermore, bod1(Δ) and bod2(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and triple mutants were more affected than single mutants, evidencing redundancy of function among BODs and mco. Overall, the data show that bod1, bod2 and mco code for non-canonical thermostable laccases that participate in the degradation of lignocellulose. Thanks to their thermal stability, these enzymes may be more promising candidate for biotechnological application than canonical laccases. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide.

    Science.gov (United States)

    Liu, Yi; Cui, Wenjing; Liu, Zhongmei; Cui, Youtian; Xia, Yuanyuan; Kobayashi, Michihiko; Zhou, Zhemin

    2014-09-01

    Self-assembling amphipathic peptides (SAPs) are the peptides that can spontaneously assemble into ordered nanostructures. It has been reported that the attachment of SAPs to the N- or C-terminus of an enzyme can benefit the thermo-stability of the enzyme. Here, we discovered that the thermo-stability and product tolerance of nitrile hydratase (NHase) were enhanced by fusing with two of the SAPs (EAK16 and ELK16). When the ELK16 was fused to the N-terminus of β-subunit, the resultant NHase (SAP-NHase-2) became an active inclusion body; EAK16 fused NHase in the N-terminus of β-subunit (SAP-NHase-1) and ELK16 fused NHase in the C-terminus of β-subunit (SAP-NHase-10) did not affect NHase solubility. Compared with the deactivation of the wild-type NHase after 30 min incubation at 50°C, SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 45%, 30% and 50% activity; after treatment in the buffer containing 10% acrylamide, the wild-type retained 30% activity, while SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 52%, 42% and 55% activity. These SAP-NHases with enhanced thermo-stability and product tolerance would be helpful for further industrial applications of the NHase. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates.

    Science.gov (United States)

    Zhu, Hong; Reynolds, L Bruce; Menassa, Rima

    2017-06-19

    Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.

  14. Development of thermostable Peste des Petits Ruminants (PPR) virus vaccine and assessment of molecular changes in the F gene

    International Nuclear Information System (INIS)

    Palaniswami, K.S.; Thangavelu, A.; Velmurugan, R.

    2005-01-01

    Two Indian PPRV isolates were subjected to thermal hardening procedures to increase the proportion of temperature-resistant virions. Initial infectivity loss was compensated by titre increases on subsequent cell passages at 37 deg C. The immunogenicity of 'thermostable' viruses was assessed by virulent PPRV challenge and for safety by host animal inoculation and antibodies assessment. Vaccine viruses were not found using PCR on ocular and nasal swabs, although virus nucleic acid and antigens were demonstrated in spleen and lymph nodes by FAT and PCR. One vaccine strain (MIB187(T)) giving 100% protection (tested on only a few animals) was freeze dried and the minimum protective dose calculated. Changes in the virus genome after thermo-adaptation were examined using RT-PCR to amplify portions of the F gene, and three base changes were observed in the thermostable PPR strain (compared with the F gene sequence of the Nigerian PPRV strain). At room temperature, the titre and potency of the thermo-adapted vaccine remained constant up to one month at the10 5.5 TCID 50 level, and was 10 4.5 TCID 50 /100 μl after two months. Field trials with over 40 000 doses of the thermostable vaccine under various environmental conditions have given serum neutralization titres exceeding 2 3 and are assumed protective. (author)

  15. Capability and dependency in the Newcastle 85+ cohort study. Projections of future care needs.

    Science.gov (United States)

    Jagger, Carol; Collerton, Joanna C; Davies, Karen; Kingston, Andrew; Robinson, Louise A; Eccles, Martin P; von Zglinicki, Thomas; Martin-Ruiz, Carmen; James, Oliver F W; Kirkwood, Tom B L; Bond, John

    2011-05-04

    Little is known of the capabilities of the oldest old, the fastest growing age group in the population. We aimed to estimate capability and dependency in a cohort of 85 year olds and to project future demand for care. Structured interviews at age 85 with 841 people born in 1921 and living in Newcastle and North Tyneside, UK who were permanently registered with participating general practices. Measures of capability included were self-reported activities of daily living (ADL), timed up and go test (TUG), standardised mini-mental state examination (SMMSE), and assessment of urinary continence in order to classify interval-need dependency. To project future demand for care the proportion needing 24-hour care was applied to the 2008 England and Wales population projections of those aged 80 years and over by gender. Of participants, 62% (522/841) were women, 77% (651/841) lived in standard housing, 13% (106/841) in sheltered housing and 10% (84/841) in a care home. Overall, 20% (165/841) reported no difficulty with any of the ADLs. Men were more capable in performing ADLs and more independent than women. TUG validated self-reported ADLs. When classified by 'interval of need' 41% (332/810) were independent, 39% (317/810) required help less often than daily, 12% (94/810) required help at regular times of the day and 8% (67/810) required 24-hour care. Of care-home residents, 94% (77/82) required daily help or 24-hour care. Future need for 24-hour care for people aged 80 years or over in England and Wales is projected to increase by 82% from 2010 to 2030 with a demand for 630,000 care-home places by 2030. This analysis highlights the diversity of capability and levels of dependency in this cohort. A remarkably high proportion remain independent, particularly men. However a significant proportion of this population require 24-hour care at home or in care homes. Projections for the next 20 years suggest substantial increases in the number requiring 24-hour care due to

  16. A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability

    Directory of Open Access Journals (Sweden)

    Steven M. Swift

    2015-06-01

    Full Text Available Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

  17. Field-acclimated Gossypium hirsutum cultivars exhibit genotypic and seasonal differences in photosystem II thermostability.

    Science.gov (United States)

    Snider, John L; Oosterhuis, Derrick M; Collins, Guy D; Pilon, Cristiane; Fitzsimons, Toby R

    2013-03-15

    Previous investigations have demonstrated that photosystem II (PSII) thermostability acclimates to prior exposure to heat and drought, but contrasting results have been reported for cotton (Gossypium hirsutum). We hypothesized that PSII thermotolerance in G. hirsutum would acclimate to environmental conditions during the growing season and that there would be differences in PSII thermotolerance between commercially-available U.S. cultivars. To this end, three cotton cultivars were grown under dryland conditions in Tifton Georgia, and two under irrigated conditions in Marianna Arkansas. At Tifton, measurements included PSII thermotolerance (T15, the temperature causing a 15% decline in maximum quantum yield), leaf temperatures, air temperatures, midday (1200 to 1400h) leaf water potentials (ΨMD), leaf-air vapor pressure deficit (VPD), actual quantum yield (ΦPSII) and electron transport rate through PSII (ETR) on three sample dates. At Marianna, T15 was measured on two sample dates. Optimal air and leaf temperatures were observed on all sample dates in Tifton, but PSII thermotolerance increased with water deficit conditions (ΨMD=-3.1MPa), and ETR was either unaffected or increased under water-stress. Additionally, T15 for PHY 499 was ∼5°C higher than for the other cultivars examined (DP 0912 and DP 1050). The Marianna site experienced more extreme high temperature conditions (20-30 days Tmax≥35°C), and showed an increase in T15 with higher average Tmax. When average T15 values for each location and sample date were plotted versus average daily Tmax, strong, positive relationships (r(2) from .954 to .714) were observed between Tmax and T15. For all locations T15 was substantially higher than actual field temperature conditions. We conclude that PSII thermostability in G. hirsutum acclimates to pre-existing environmental conditions; PSII is extremely tolerant to high temperature and water-deficit stress; and differences in PSII thermotolerance exist between

  18. Carbohydrate-Based Ice Recrystallization Inhibitors Increase Infectivity and Thermostability of Viral Vectors

    Science.gov (United States)

    Ghobadloo, Shahrokh M.; Balcerzak, Anna K.; Gargaun, Ana; Muharemagic, Darija; Mironov, Gleb G.; Capicciotti, Chantelle J.; Briard, Jennie G.; Ben, Robert N.; Berezovski, Maxim V.

    2014-07-01

    The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL-1 during 40 days, and HSV-1, 2.7 Log10 PFU mL-1 during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL-1 after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors.

  19. On-Demand Production of Flow-Reactor Cartridges by 3D Printing of Thermostable Enzymes.

    Science.gov (United States)

    Maier, Manfred; Radtke, Carsten P; Hubbuch, Jürgen; Niemeyer, Christof M; Rabe, Kersten S

    2018-05-04

    The compartmentalization of chemical reactions is an essential principle of life that provides a major source of innovation for the development of novel approaches in biocatalysis. To implement spatially controlled biotransformations, rapid manufacturing methods are needed for the production of biocatalysts that can be applied in flow systems. Whereas three-dimensional (3D) printing techniques offer high-throughput manufacturing capability, they are usually not compatible with the delicate nature of enzymes, which call for physiological processing parameters. We herein demonstrate the utility of thermostable enzymes in the generation of biocatalytic agarose-based inks for a simple temperature-controlled 3D printing process. As examples we utilized an esterase and an alcohol dehydrogenase from thermophilic organisms as well as a decarboxylase that was thermostabilized by directed protein evolution. We used the resulting 3D-printed parts for a continuous, two-step sequential biotransformation in a fluidic setup. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Thermostable Bacteriocin BL8 from Bacillus licheniformis isolated from marine sediment.

    Science.gov (United States)

    Smitha, S; Bhat, S G

    2013-03-01

    To isolate and characterize bacteriocin, BL8, from the bacteria identified as Bacillus licheniformis from marine environment. One-hundred and twelve bacterial isolates from sediment and water samples collected off the coast of Cochin, India, were screened for antibacterial activity. Strain BTHT8, identified as Bacillus licheniformis, inhibited the growth of Gram-positive test organisms. The active component labelled as bacteriocin BL8 was partially purified by ammonium sulphate fractionation and was subjected to glycine SDS-PAGE. The band exhibiting antimicrobial activity was electroeluted and analysed using MALDI-TOF mass spectrometry, and the molecular mass was determined as 1.4 kDa. N-terminal amino acid sequencing of BL8 gave a 13 amino acid sequence stretch. Bacteriocin BL8 was stable even after boiling at 100 °C for 30 min and over a wide pH range of 1-12. A novel, pH-tolerant and thermostable bacteriocin BL8, active against the tested Gram-positive bacteria, was isolated from Bacillus licheniformis. This study reports a stable, low molecular weight bacteriocin from Bacillus licheniformis. This bacteriocin can be used to address two important applications: as a therapeutic agent and as a biopreservative in food processing industry. © 2012 The Society for Applied Microbiology.

  1. High yield recombinant thermostable α-amylase production using an improved Bacillus licheniformis system

    Directory of Open Access Journals (Sweden)

    Shi Gui-Yang

    2009-10-01

    Full Text Available Abstract Background Some strains of Bacillus licheniformis have been improved by target-directed screening as well as by classical genetic manipulation and used in commercial thermostable α-amylase and alkaline protease production for over 40 years. Further improvements in production of these enzymes are desirable. Results A new strain of B. licheniformis CBBD302 carrying a recombinant plasmid pHY-amyL for Bacillus licheniformis α-amylase (BLA production was constructed. The combination of target-directed screening and genetic recombination led to an approximately 26-fold improvement of BLA production and export in B. licheniformis. Furthermore, a low-cost fermentation medium containing soybean meal and cottonseed meal for BLA production in shake-flasks and in a 15 liter bioreactor was developed and a BLA concentration of up to 17.6 mg per ml growth medium was attained. Conclusion This production level of BLA by B. licheniformis CBBD302(pHY-amyL is amongst the highest levels in Gram-positive bacteria reported so far.

  2. Geometric Simulation Approach for Grading and Assessing the Thermostability of CALBs

    Directory of Open Access Journals (Sweden)

    B. Senthilkumar

    2016-01-01

    Full Text Available Candida antarctica lipase B (CALB is a known stable and highly active enzyme used widely in biodiesel synthesis. In this work, the stability of native (4K6G and mutant (4K5Q CALB was studied through various structural parameters using conformational sampling approach. The contours of polar surface area and surface area of mutant CALB were 11357.67 Å2 and 30007.4 Å2, respectively, showing an enhanced stability compared to native CALB with a statistically significant P value of < 0.0001. Moreover, simulated thermal denaturation of CALB, a process involving dilution of hydrogen bond, significantly shielded against different intervals of energy application in mutant CALB revealing its augmentation of structural rigidity against native CALB. Finally, computational docking analysis showed an increase in the binding affinity of CALB and its substrate (triglyceride in mutant CALB with Atomic Contact Energy (ACE of −91.23 kcal/mol compared to native CALB (ACE of −70.3 kcal/mol. The computational observations proposed that the use of mutant CALB (4K5Q could serve as a best template for production of biodiesel in the future. Additionally, it can also be used as a template to identify efficient thermostable lipases through further mutations.

  3. Construction and engineering of a thermostable self-sufficient cytochrome P450

    Energy Technology Data Exchange (ETDEWEB)

    Mandai, Takao; Fujiwara, Shinsuke [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan); Imaoka, Susumu, E-mail: imaoka@kwansei.ac.jp [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan)

    2009-06-19

    CYP175A1 is a thermophilic cytochrome P450 and hydroxylates {beta}-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP{sup +} reductase (FNR): H{sub 2}N-CYP175A1-Fdx-FNR-COOH (175FR) and H{sub 2}N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V{sub max} value for {beta}-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k{sub m} values of these enzymes were similar. 175RF retained 50% residual activity even at 80 {sup o}C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

  4. FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

    Science.gov (United States)

    Bednar, David; Beerens, Koen; Sebestova, Eva; Bendl, Jaroslav; Khare, Sagar; Chaloupkova, Radka; Prokop, Zbynek; Brezovsky, Jan; Baker, David; Damborsky, Jiri

    2015-11-01

    There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

  5. FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

    Directory of Open Access Journals (Sweden)

    David Bednar

    2015-11-01

    Full Text Available There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

  6. Thermostable, haloalkaline cellulase from Bacillus halodurans CAS 1 by conversion of lignocellulosic wastes.

    Science.gov (United States)

    Annamalai, Neelamegam; Rajeswari, Mayavan Veeramuthu; Elayaraja, Sivaramasamy; Balasubramanian, Thangavel

    2013-04-15

    An extracellular thermostable, haloalkaline cellulase by bioconversion of lignocellulosic wastes from Bacillus halodurans CAS 1 was purified to homogeneity with recovery of 12.54% and purity fold 7.96 with the molecular weight of 44 kDa. The optimum temperature, pH and NaCl for enzyme activity was determined as 60°C, 9.0 and 30% and it retained 80% of activity even at 80°C, 12 and 35% respectively. The activity was greatly inhibited by EDTA, indicating that it was a metalloenzyme and significant inhibition by PMSF revealed that serine residue was essential for catalytic activity. The purified cellulase hydrolyzed CMC, cellobiose and xylan, but not avicel, cellulose and PNPG. Furthermore, the cellulase was highly stable in the presence of detergents and organic solvents such as acetone, n-hexane and acetonitrile. Thus, the purified cellulase from B. halodurans utilizing lignocellulosic biomass could be greatly useful to develop industrial processes. Published by Elsevier Ltd.

  7. Thermostable phycocyanin from the red microalga Cyanidioschyzon merolae, a new natural blue food colorant.

    Science.gov (United States)

    Rahman, D Y; Sarian, F D; van Wijk, A; Martinez-Garcia, M; van der Maarel, M J E C

    2017-01-01

    The demand for natural food colorants is growing as consumers question the use of artificial colorants more and more. The phycobiliprotein C-phycocyanin of Arthospira platensis is used as a natural blue colorant in certain food products. The thermoacidophilic red microalga Cyanidioschyzon merolae might provide an alternative source of phycocyanin. Cyanidioschyzon merolae belongs to the order Cyanidiophyceae of the phylum Rhodophyta. Its natural habitat are sulfuric hot springs and geysers found near volcanic areas in, e.g., Yellowstone National Park in the USA and in Java, Indonesia. It grows optimally at a pH between 0.5 and 3.0 and at temperatures up to 56 °C. The low pH at which C . merolae grows minimizes the risk of microbial contamination and could limit production loss. As C . merolae lacks a cell wall, phycocyanin with a high purity number of 9.9 could be extracted by an osmotic shock using a simple ultrapure water extraction followed by centrifugation. The denaturation midpoint at pH 5 was 83 °C, being considerably higher than the A . platensis phycocyanin (65 °C). The C . merolae phycocyanin was relatively stable at pH 4 and 5 up to 80 °C. The high thermostability at slightly acidic pH makes the C . merolae phycocyanin an interesting alternative to A . platensis phycocyanin as a natural blue food colorant.

  8. Reconstitution of β-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

    International Nuclear Information System (INIS)

    Momoi, Kyoko; Hofmann, Ute; Schmid, Rolf D.; Urlacher, Vlada B.

    2006-01-01

    CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards β-carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude Escherichia coli cell extract at 37 o C. In this system, β-carotene was hydroxylated to β-cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low β-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated β-carotene at 3- and also 3'-positions, resulting in β-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol β-cryptoxanthin produced per nmol P450 per min

  9. Construction and engineering of a thermostable self-sufficient cytochrome P450

    International Nuclear Information System (INIS)

    Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu

    2009-01-01

    CYP175A1 is a thermophilic cytochrome P450 and hydroxylates β-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP + reductase (FNR): H 2 N-CYP175A1-Fdx-FNR-COOH (175FR) and H 2 N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V max value for β-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k m values of these enzymes were similar. 175RF retained 50% residual activity even at 80 o C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

  10. Not so monofunctional--a case of thermostable Thermobifida fusca catalase with peroxidase activity.

    Science.gov (United States)

    Lončar, Nikola; Fraaije, Marco W

    2015-03-01

    Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and overexpressed in Escherichia coli with a yield of 400 mg/L. Heat treatment of disrupted cells at 60 °C for 1 h resulted in enzyme preparation of high purity; hence, no chromatography steps are needed for large-scale production. Except for catalyzing the dismutation of hydrogen peroxide, TfuCat was also found to catalyze oxidations of phenolic compounds. The catalase activity was comparable to other described catalases while peroxidase activity was quite remarkable with a k obs of nearly 1000 s(-1) for catechol. Site directed mutagenesis was used to alter the ratio of peroxidase/catalase activity. Resistance to inhibition by classic catalase inhibitors and an apparent melting temperature of 74 °C classifies this enzyme as a robust biocatalyst. As such, it could compete with other commercially available catalases while the relatively high peroxidase activity also offers new biocatalytic possibilities.

  11. Hepatitis B surface antigen incorporated in dissolvable microneedle array patch is antigenic and thermostable.

    Science.gov (United States)

    Poirier, Danielle; Renaud, Frédéric; Dewar, Vincent; Strodiot, Laurent; Wauters, Florence; Janimak, Jim; Shimada, Toshio; Nomura, Tatsuya; Kabata, Koki; Kuruma, Koji; Kusano, Takayuki; Sakai, Masaki; Nagasaki, Hideo; Oyamada, Takayoshi

    2017-11-01

    Alternatives to syringe-based administration are considered for vaccines. Intradermal vaccination with dissolvable microneedle arrays (MNA) appears promising in this respect, as an easy-to-use and painless method. In this work, we have developed an MNA patch (MNAP) made of hydroxyethyl starch (HES) and chondroitin sulphate (CS). In swines, hepatitis B surface antigen (HBsAg) formulated with the saponin QS-21 as adjuvant, both incorporated in HES-based MNAP, demonstrated the same level of immunogenicity as a commercially available aluminum-adjuvanted HBsAg vaccine, after two immunizations 28 days apart. MNAP application was associated with transient skin reactions (erythema, lump, scab), particularly evident when the antigen was delivered with the adjuvant. The thermostability of the adjuvanted antigen when incorporated in the HES-based matrix was also assessed by storing MNAP at 37, 45 or 50 °C for up to 6 months. We could demonstrate that antigenicity was retained at 37 and 45 °C and only a 10% loss was observed after 6 months at 50 °C. Our results are supportive of MNAP as an attractive alternative to classical syringe-based vaccination. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Heterologous Expression and Characterization of a Thermostable Exo-β-D-Glucosaminidase from Aspergillus oryzae.

    Science.gov (United States)

    Wu, Dingxin; Wang, Linchun; Li, Yuwei; Zhao, Shumiao; Peng, Nan; Liang, Yunxiang

    2016-02-01

    An exo-β-D-glucosaminidase (AorCsxA) from Aspergillus oryzae FL402 was heterologously expressed and purified. The deduced amino acid sequence indicated that AorCsxA belonged to glycoside hydrolase family 2. AorCsxA digested colloid chitosan into glucosamine but not into chitosan oligosaccharides, demonstrating exo-β-D-glucosaminidase (CsxA) activity. AorCsxA exhibited optimal activity at pH 5.5 and 50°C; however, the enzyme expressed in Pichia pastoris (PpAorCsxA) showed much stronger thermostability at 50°C than that expressed in Escherichia coli (EcAorCsxA), which may be related to glycosylation. AorCsxA activity was inhibited by EDTA and most of the tested metal ions. A single amino acid mutation (F769W) in AorCsxA significantly enhanced the specific activity and hydrolysis velocity as revealed by comparison of Vmax and kcat values with those of the wild-type enzyme. The three-dimensional structure suggested the tightened pocket at the active site of F769W enabled efficient substrate binding. The AorCsxA gene was heterologously expressed in P. pastoris, and one transformant was found to produce 222 U/ml activity during the high-cell-density fermentation. This AorCsxA-overexpressing P. pastoris strain is feasible for large-scale production of AorCsxA.

  13. The effect of different stabilizers on the thermostability of electron beam crosslinked polyethylene in hot water

    International Nuclear Information System (INIS)

    Hassanpour, S.; Khoylou, F.

    2003-01-01

    Plastic pipes owing to their flexibility, great lengths, easier handling and absence of corrosion have been used for hot-water installations. Crosslinked high-density polyethylene is one of the best materials, being used for this purpose. The useful lifetime of unstabilized polyethylene is predicted to vary from a few months in hot water (30-40 deg. C) to almost two years in cool water (0-10 deg. C). Polyethylene was mixed with different types of stabilizers, in order to increase its durability. The samples were irradiated at 100-150 kGy. The amount of gel fraction and the changes in mechanical properties were measured. Irradiated samples were immersed in hot water for 1000 h. The thermostability of the specimens and the existence of antioxidants were measured by the induction time technique using differential scanning calorimetry at different time intervals. Furthermore, the changes in chemical structure and mechanical properties of the samples during their immersion in hot water were determined

  14. Retrospective study on cattle and poultry diseases in Uganda

    Directory of Open Access Journals (Sweden)

    Joseph Byaruhanga

    2017-12-01

    Full Text Available Cattle and poultry enterprises are among the major contributors to food security and socioeconomic empowerment of households in Uganda. However, various diseases constrain their productivity. A two-year retrospective study between April 2012 and March 2014 was conducted using records for cattle and poultry diseases diagnosed at the Central Diagnostic Laboratory (CDL to determine prevalent diseases in Uganda. The laboratory received 836 samples from poultry (36.3% and cattle (63.7%. Of the 836 samples, 47.5% had a definitive diagnosis of disease causation. Most of the cattle and poultry diseases diagnosed were protozoan diseases (39.3% followed by bacterial (21.4%, viral (17.1%, helminthiasis (11.1%, nutritional diseases (4% and others (7.1%. For poultry, viral diseases (29.5% and protozoan diseases (27.1% especially newcastle disease (44.3% and coccidiosis (100% respectively, were the most diagnosed. While for cattle, hemo-protozoan parasites (52.1% were the most prevalent, of which 92.9% were east coast fever infection. Bacterial infection (20.5% in cattle were the second most diagnosed diseases and mastitis was the most diagnosed (46.2%. In summary, coccidioisis, collibacillosis, newcastle disease, gumboro disease, and avian helminthiasis were the most prevalent poultry diseases while in cattle, east coast fever, helminthiasis, mastitis, brucellosis and rabies were the most frequently diagnosed diseases. This study has identified the major diseases that hinder poultry and cattle production in Uganda. The data generated by CDL could be used for surveillance, monitoring and designing strategic interventions for control of poultry and cattle diseases in Uganda. Keywords: Coccidiosis, Collibacillosis, East coast fever, Mastitis, Newcastle disease, Rabies

  15. Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis.

    Directory of Open Access Journals (Sweden)

    Chengtuo Niu

    Full Text Available 1,3-1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the thermostability of a mesophilic β-glucanase from Bacillus terquilensis was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (Tm compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation.

  16. Optimization of Thermostable Alpha-Amylase Production Via Mix Agricultural-Residues and Bacillus amyloliquefaciens

    Directory of Open Access Journals (Sweden)

    Shalini RAI

    2014-03-01

    Full Text Available This study reports utilization of mixture of wheat and barley bran (1:1 for the production of thermostable alpha-amylase enzyme through a spore former, heat tolerant strain of Bacillus amyloliquefaciens in solid state fermentation. Maximum yield of alpha-amylase (252.77 U mL-1 was obtained in following optimized conditions, inoculums size 2 mL (2 × 106 CFU/mL, moisture 80%, pH 7±0.02, NaCl (3%, temperature 38±1°C, incubation for 72 h, maltose (1% and tryptone (1%. After SSF crude enzyme was purified via ammonium sulfate precipitation, ion exchange and column chromatography by DEAE Cellulose. Purified protein showed a molecular weight of 42 kDa by SDS-PAGE electrophoresis. After purification, purified enzyme was characterized against several enzymes inhibitors such as temperature, NaCl, pH, metal and surfactants. Pure enzyme was highly active over broad temperature (50-70°C, NaCl concentration (0.5-4 M, and pH (6-10 ranges, indicating it’s a thermoactive and alkali-stable nature. Moreover, CaCl2, MnCl2, =-mercaptoethanol were found to stimulate the amylase activity, whereas FeCl3, sodium dodecyl sulfate (SDS, CuCl3 and ethylenediaminetetraacetic acid (EDTA strongly inhibited the enzyme. Moreover, enzyme specificity and thermal stability conformed by degradation of different soluble starch up to 55°C. Therefore, the present study proved that the extracellular alpha-amylase extracted through wheat flour residues by organism B. amyloliquefaciens MCCB0075, both have considerable potential for industrial application owing to its properties.

  17. Evaluation of enhanced thermostability and operational stability of carbonic anhydrase from Micrococcus species.

    Science.gov (United States)

    Bhattacharya, Abhishek; Shrivastava, Ankita; Sharma, Anjana

    2013-06-01

    Carbonic anhydrase (CA) was purified from Micrococcus lylae and Micrococcus luteus with 49.90 and 53.8 % yield, respectively, isolated from calcium carbonate kilns. CA from M. lylae retained 80 % stability in the pH and temperature range of 6.0-8.0 and 35-45 °C, respectively. However, CA from M. luteus was stable in the pH and temperature range of 7.5-10.0 and 35-55 °C, respectively. Cross-linked enzyme aggregates (CLEAs) raised the transition temperature of M. lylae and M. luteus CA up to 67.5 and 74.0 °C, while the operational stability (T(1/20) of CA at 55 °C was calculated to be 7.7 and 12.0 h, respectively. CA from both the strains was found to be monomeric in nature with subunit molecular weight and molecular mass of 29 kDa. Ethoxozolamide was identified as the most potent inhibitor based on both IC(50) values and inhibitory constant measurement (K(i)). The K(m) and V(max) for M. lylae CA (2.31 mM; 769.23 μmol/mg/min) and M. luteus CA (2.0 mM; 1,000 μmol/mg/min) were calculated from Lineweaver-Burk plots in terms of esterase activity. Enhanced thermostability of CLEAs alleviates its role in operational stability for application at an on-site scrubber. The characteristic profile of purified CA from Micrococcus spp. advocates its effective application in biomimetic CO(2) sequestration.

  18. Production, Purification, and Characterization of Thermostable α-Amylase Produced by Bacillus licheniformis Isolate AI20

    Directory of Open Access Journals (Sweden)

    Yasser R. Abdel-Fattah

    2013-01-01

    Full Text Available An optimization strategy, based on statistical experimental design, is employed to enhance the production of thermostable α-amylase by a thermotolerant B. licheniformis AI20 isolate. Using one variant at time (OVAT method, starch, yeast extract, and CaCl2 were observed to influence the enzyme production significantly. Thereafter, the response surface methodology (RSM was adopted to acquire the best process conditions among the selected variables, where a three-level Box-Behnken design was employed to create a polynomial quadratic model correlating the relationship between the three variables and α-amylase activity. The optimal combination of the major constituents of media for α-amylase production was 1.0% starch, 0.75% yeast extract, and 0.02% CaCl2. The predicted optimum α-amylase activity was 384 U/mL/min, which is two folds more than the basal medium conditions. The produced α-amylase was purified through various chromatographic techniques. The estimated enzyme molecular mass was 55 kDa and the α-amylase had an optimal temperature and pH of 60–80°C and 6–7.5, respectively. Values of Vmax and Km for the purified enzyme were 454 mU/mg and 0.709 mg/mL. The α-amylase enzyme showed great stability against different solvents. Additionally, the enzyme activity was slightly inhibited by detergents, sodium dodecyl sulphate (SDS, or chelating agents such as EDTA and EGTA. On the other hand, great enzyme stability against different divalent metal ions was observed at 0.1 mM concentration, but 10 mM of Cu2+ or Zn2+ reduced the enzyme activity by 25 and 55%, respectively.

  19. Studies for methods to improve thermostability of the functionalized butadiene styrene rubbers

    Directory of Open Access Journals (Sweden)

    A. L. Rumyantseva

    2018-01-01

    Full Text Available It is well known that the tire performance properties can deteriorate in the processes of production, processing, storage and operation. One of the reasons for that is a series of processes occurring in the polymer under the influence of different factors: thermal, mechanical or chemical. This problem is particularly relevant for functionalized polymers, as functional groups can interact with each other, causing side cross linking reactions that lead to a deterioration of consumer properties of the products. The main purpose of this work was to study influence of several key factors on the thermostability of functionalized rubbers in order to find a solution: different types of antioxidants, rubber polymerizate stripping conditions and rubber processing. In accordance with the problem, solutions were found and work was carried out in several directions: changing the pH of the medium in the rubber stripping and using antioxidants containing carbonyl groups located in ?-positions to methylene groups, namely Irganox 1520 and Irganox 1076. As an evaluation factor, thermal treatment was selected in two modes: at 100 °C for 48 hours and after extruder at 130 °C for 5 minutes + 100 °C for 48 hours. At the same time, the following parameters were determined: molecular weight characteristics and Mooney viscosity of the starting polymers and after thermal aging. During the experiments, it was found that the acidity of the medium in the water degasser does not affect the crosslinking of the functionalized rubber during storage. In addition, a study was made of the effect of the type of antioxidant and its quantity on the thermal stability of functionalized styrene butadiene rubbers, as well as the study of the effect of the content of the modifying agent on the thermal stability of the product. It has been found that the use, as antioxidants, of carbonyl compounds containing a methylene group at the ?-position, leads to inhibition of the cross

  20. Cloning, purification and characterization of a thermostable β-galactosidase from Bacillus licheniformis strain KG9.

    Science.gov (United States)

    Matpan Bekler, F; Stougaard, P; Güven, K; Gül Güven, R; Acer, Ö

    2015-06-28

    A thermo— and alkalitolerant Bacillus licheniformis KG9 isolated from Taşlıdere hot water spring in Batman/Turkey was found to produce a thermostable β—galactosidase. Phylogenetic analysis showed that the 16S rRNA gene from B. licheniformis strain KG9 was 99.9% identical to that of the genome sequenced B. licheniformis strain DSM 13. Analysis of the B. licheniformis DSM 13 genomic sequence revealed four putative β—galactosidase genes. PCR primers based on the genome sequence of strain DSM 13 were used to isolate the corresponding β—galactosidase genes from B. licheniformis strain KG9. The calculated molecular weights of the β—galactosidases I, II, III, and IV using sequencing data were 30, 79, 74, and 79 kDa, respectively. The genes were inserted into an expression vector and recombinant β—galactosidase was produced in Escherichia coli. Of the four β—galactosidase genes identified in strain KG9, three of them were expressed as active, intracellular enzymes in E. coli. One of the recombinant enzymes, β—galactosidase III, was purified and characterized. Optimal temperature and pH was determined to be at 60 ºC and pH 6.0, respectively. Km was determined to be 1.3 mM and 13.3 mM with oNPG (ortho—nitrophenyl—β—D—galactopyranoside) and lactose as substrates, respectively, and Vmax was measured to 1.96 μmol/min and 1.55 μmol/min with oNPG and lactose, respectively.

  1. Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil.

    Directory of Open Access Journals (Sweden)

    Sandeep Kaur Saggu

    Full Text Available Proteases are one of the largest groups of hydrolytic enzymes constituting about 60% of total worldwide sales of industrial enzymes due to their wide applications in detergent, leather, textile, food and pharmaceutical industry. Microbial proteases have been preferred over animal and plant proteases because of their fundamental features and ease in production. Bacillus infantis SKS1, an alkaline protease producing bacteria has been isolated from garden soil of north India and identified using morphological, biochemical and molecular methods. 16S rDNA sequence amplified using universal primers has 99% sequence identity with corresponding gene sequence of Bacillus infantis strain FM 34 and Bacillus sp. Beige. The bacterial culture and its 16S rDNA gene sequence have been deposited to Microbial Culture Collection (Pune, India with accession number MCC 3035 and GenBank with accession number KR092197 respectively. The partially purified extract of Bacillus infantis SKS1 was thermostable and active in presence of Mg2+, acetyl acetone and laundry detergents implicating its application in industry. Production of these enzymes using this strain was maximized by optimization of various parameters including temperature, pH, media components and other growth conditions. Our results show that fructose and dextrose serve as the best carbon sources for production of these enzymes, highlighting the use of this strain for enzyme production utilizing relatively inexpensive substrates like beet molasses and corn steep liquor. Additionally, this strain showed maximum production of enzymes at 40°C similar to bacterial species used for commercial production of alkaline proteases. Characterization of alkaline proteases from this strain of Bacillus infantis and optimization of parameters for its production would help in understanding its industrial application and large-scale production.

  2. Crystal structure of a thermostable Old Yellow Enzyme from Thermus scotoductus SA-01

    International Nuclear Information System (INIS)

    Opperman, Diederik J.; Sewell, Bryan T.; Litthauer, Derek; Isupov, Mikhail N.; Littlechild, Jennifer A.; Heerden, Esta van

    2010-01-01

    Recent characterization of the chromate reductase (CrS) from the thermophile Thermus scotoductus SA-01 revealed this enzyme to be related to the Old Yellow Enzyme (OYE) family. Here, we report the structure of a thermostable OYE homolog in its holoform at 2.2 A as well as its complex with p-hydroxybenzaldehyde (pHBA). The enzyme crystallized as octamers with the monomers showing a classical TIM barrel fold which upon dimerization yields the biologically active form of the protein. A sulfate ion is bound above the si-side of the non-covalently bound FMN cofactor in the oxidized solved structure but is displaced upon pHBA binding. The active-site architecture is highly conserved as with other members of this enzyme family. The pHBA in the CrS complex is positioned by hydrogen bonding to the two conserved catalytic-site histidines. The most prominent structural difference between CrS and other OYE homologs is the size of the 'capping domain'. Thermostabilization of the enzyme is achieved in part through increased proline content within loops and turns as well as increased intersubunit interactions through hydrogen bonding and complex salt bridge networks. CrS is able to reduce the C=C bonds of α,β-unsaturated carbonyl compounds with a preference towards cyclic substrates however no activity was observed towards β-substituted substrates. Mutational studies have confirmed the role of Tyr177 as the proposed proton donor although reduction could still occur at a reduced rate when this residue was mutated to phenylalanine.

  3. Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance

    Directory of Open Access Journals (Sweden)

    Paulo S. Monteiro

    2015-03-01

    Full Text Available An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The KM for sodium phytate hydrolysis was 30.9 mM, while the kcat and kcat/KM were 1.46 ×105 s−1 and 4.7 × 106s−1.M−1, respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg2+, Cd2+, K+ and Ca2+, and it was drastically inhibited by F−. The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment.

  4. Partial Purification and Characterization of a Thermostable β-Mannanase from Aspergillus foetidus

    Directory of Open Access Journals (Sweden)

    Juliana da Conceição Infante de Marco

    2015-10-01

    Full Text Available An extracellular β-mannanase was isolated from samples of crude extract of the mesophilic fungus Aspergillus foetidus grown on soybean husk as a carbon source. The induction profile showed that β-mannanase reached a maximum activity level (2.0 IU/mL on the 15th day of cultivation. The enzyme was partially purified by ultrafiltration and gel filtration chromatography procedures and was named Man 58. Sodium dodecyl sulfate-polyacrilamide electrophoresis and zymogram analysis of Man 58 showed two bands of approximately 43 and 45 kDa with β-mannanase activity. Ultrafiltration showed that β-mannanase activity was only detected in the concentrated sample. Man 58 was most active at 60 °C and at pH 4.0. It was thermostable in the temperature range of 40–60 °C for eleven days, and the half-life at 70 °C was ten days. Man 58 showed Km and Vmax values of 3.29 mg/mL and 1.76 IU/mL respectively, with locust bean gum as a substrate. It was mostly activated by FeSO4 and CoCl2 and inhibited by MgSO4, FeCl3, CuSO4, MgCl2, ZnCl2, ZnSO4, CaCl2, CuCl2, KCl and ethylenediaminetetraacetic acid (EDTA. Phenolic compounds did not inhibit the enzyme. On the other hand, auto-hydrolysis liquor showed an inhibitory effect on Man 58 activity.

  5. Experimental study on the thermostable property of aramid fiber reinforced PE-RT pipes

    Directory of Open Access Journals (Sweden)

    Guoquan Qi

    2015-11-01

    Full Text Available Flexible composite pipes are advantageous in ultra high strength, high modulus, pH and corrosion resistance and light weight, but there are still some hidden safety troubles because they are poorer in thermostable capacity. Therefore, test samples of flexible composite pipes were prepared with high-temperature polythene (PE-RT as the neck bush and aramid fiber as the reinforcement layer. Experimental study was conducted by using HPHT vessel and differential thermal scanner for different working conditions, different temperatures, whole-pipe pressure-bearing capacity and 1000 h viability. It is shown by the environmental compatibility test that high temperature has little effect on the weight, Vicat softening temperature, mechanical properties and structures of neck bush PE-RT, but exerts an obvious effect on the tensility and whole-pipe water pressure blasting of the reinforcement aramid fiber. Besides, the drop of whole-pipe pressure-bearing capacity is caused by deformation and breaking of aramid fibers when the reinforcement layer is under the force of internal pressure. Finally, disorientation and crystallization of molecular thermal motion occur with the rise of temperature, so amorphous orientation reduces, crystallinity factor and crystalline orientation factor increase gradually, thus, disorientation of macromolecular chains increases and tensile strength decreases. It is concluded that this type of flexible composite pipe can smoothly pass 1000 h viability test. And it is recommended that it be used in the situations with temperature not higher than 95 °C and internal pressure not higher than 4 MPa.

  6. Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance

    Science.gov (United States)

    Monteiro, Paulo S.; Guimarães, Valéria M.; de Melo, Ricardo R.; de Rezende, Sebastião T.

    2015-01-01

    An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The K M for sodium phytate hydrolysis was 30.9 mM, while the k cat and k cat / K M were 1.46 ×10 5 s −1 and 4.7 × 10 6 s −1 .M −1 , respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg 2+ , Cd 2+ , K + and Ca 2+ , and it was drastically inhibited by F − . The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t 1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment. PMID:26221114

  7. Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

    DEFF Research Database (Denmark)

    da Silva, Ines Isabel Cardoso Rodrigues; Jers, Carsten; Otten, Harm

    2014-01-01

    Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM......, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino...

  8. Draft genome sequence of a thermostable, alkaliphilic α-amylase and protease producing Bacillus amyloliquefaciens strain KCP2.

    Science.gov (United States)

    Prajapati, Vimalkumar S; Ray, Sanket; Narayan, Jitendra; Joshi, Chaitanya C; Patel, Kamlesh C; Trivedi, Ujjval B; Patel, R M

    2017-12-01

    Bacillus amyloliquefaciens strain KCP2 was isolated from municipal food waste samples collected in Vallabh Vidyanagar, Gujarat, India. Strain KCP2 is noteworthy due to its ability to produce a thermostable, alkaliphilic α-amylase and a protease. These enzymes have importance in several industrial processes including bread making, brewing, starch processing, pharmacy, and textile industries. Whole genome sequencing of strain KCP2 showed that the estimated genome size was 3.9 Mb, the G + C content was 46%, and it coded for 4113 genes.

  9. Soroepidemiologia da doença de Newcastle em plantéis de avestruzes dos Estados da Bahia e de São Paulo

    OpenAIRE

    Fernandes, Lia Muniz Barretto; Silva, Priscila Sousa da; Ramos, Izabella; Sales, Tatiane Santana; Herval, Elen Fabiane Guimarães; Batinga, Thaís de Brito; Maia, Paulo César Costa; César, André Eduardo Rocha; Doretto Júnior, Luciano; Nascimento, Roberto José Meyer; Freire, Songeli Menezes

    2010-01-01

    p.135-140 Estudos sorológicos em Avestruzes (Struthio camelus) são ferramentas úteis para analisar os riscos relacionados à Doença de Newcastle nesses plantéis e à avicultura nacional. No presente estudo, amostras de sangue foram obtidas de avestruzes de ambos os sexos, de diferentes faixas etárias e sem apresentação de sintomatologia clínica, criadas nos Estados da Bahia e de São Paulo com o objetivo de avaliar a presença de anticorpos contra o vírus da Doença de Newcastle por meio de ELI...

  10. Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Covalent Immobilization

    Directory of Open Access Journals (Sweden)

    Roberta V. Branco

    2015-01-01

    Full Text Available A recombinant thermostable lipase (Pf2001Δ60 from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment (glyoxyl-DTT PFUL and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment (glyoxyl PFUL. The enzyme’s properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity, whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity. Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity E=22 and enantiomeric excess (ee (% = 91.

  11. Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii.

    Science.gov (United States)

    Cheng, Kun; Zhang, Fei; Sun, Fangfang; Chen, Hongge; Percival Zhang, Y-H

    2015-08-20

    Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin - the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 (o)C for 30 min. This enzyme was characterized and required Mg(2+) as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

  12. Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability

    Directory of Open Access Journals (Sweden)

    Jiivittha Veno

    2017-11-01

    Full Text Available In the industrial processes, lipases are expected to operate at temperatures above 45 °C and could retain activity in organic solvents. Hence, a C-terminal truncated lipase from Staphylococcus epidermis AT2 (rT-M386 was engineered by directed evolution. A mutant with glycine-to-cysteine substitution (G210C demonstrated a remarkable improvement of thermostability, whereby the mutation enhanced the activity five-fold when compared to the rT-M386 at 50 °C. The rT-M386 and G210C lipases were purified concurrently using GST-affinity chromatography. The biochemical and biophysical properties of both enzymes were investigated. The G210C lipase showed a higher optimum temperature (45 °C and displayed a more prolonged half-life in the range of 40–60 °C as compared to rT-M386. Both lipases exhibited optimal activity and stability at pH 8. The G210C showed the highest stability in the presence of polar organic solvents at 50 °C compared to the rT-M386. Denatured protein analysis presented a significant change in the molecular ellipticity value above 60 °C, which verified the experimental result on the temperature and thermostability profile of G210C.

  13. Glycine in the conserved motif III modulates the thermostability and oxidative stress resistance of peptide deformylase in Mycobacterium tuberculosis.

    Science.gov (United States)

    Narayanan, Sai Shyam; Sokkar, Pandian; Ramachandran, Murugesan; Nampoothiri, Kesavan Madhavan

    2011-07-01

    Peptide deformylase (PDF) catalyses the removal of the N-formyl group from the nascent polypeptide during protein maturation. The PDF of Mycobacterium tuberculosis H37Rv (MtbPDF), overexpressed and purified from Escherichia coli, was characterized as an iron-containing enzyme with stability towards H(2) O(2) and moderate thermostability. Substitution of two conserved residues (G49 and L107) from MtbPDF with the corresponding residues found in human PDF affected its deformylase activity. Among characterized PDFs, glycine (G151) in motif III instead of conserved aspartate is characteristic of M. tuberculosis. Although the G151D mutation in MtbPDF increased its deformylase activity and thermostability, it also affected enzyme stability towards H(2) O(2) . Molecular dynamics and docking results confirmed improved substrate binding and catalysis for the G151D mutant and the study provides another possible molecular basis for the stability of MtbPDF against oxidizing agents. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  14. Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II–acetazolamide complex

    International Nuclear Information System (INIS)

    Aggarwal, Mayank; Boone, Christopher D.; Kondeti, Bhargav; Tu, Chingkuang; Silverman, David N.; McKenna, Robert

    2013-01-01

    Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site

  15. Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II–acetazolamide complex

    Energy Technology Data Exchange (ETDEWEB)

    Aggarwal, Mayank; Boone, Christopher D.; Kondeti, Bhargav; Tu, Chingkuang; Silverman, David N.; McKenna, Robert, E-mail: rmckenna@ufl.edu [University of Florida, 1600 SW Archer Road, PO Box 100245, Gainesville, FL 32610 (United States)

    2013-05-01

    Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.

  16. Efecto de la infección de Newcastle Disease Virus en distintas fases del ciclo celular

    OpenAIRE

    Martín-Doncel, Elena

    2014-01-01

    Trabajo Fin de Máster presentado por la licenciada en Biotecnología Elena Martín Doncel para optar al título de Máster en Biología y Clínica del Cáncer por la Universidad de Salamanca, que ha sido realizado en el Instituto de Biología Molecular y Celular del Cáncer (Curso 2013/2014).

  17. Quantification and phenotypic characterisation of peripheral IFN-γ producing leucocytes in chickens vaccinated against Newcastle disease

    DEFF Research Database (Denmark)

    Andersen, Stig Henrik; Vervelde, Lonneke; Sutton, Kate

    2017-01-01

    controls, one group was vaccinated intramuscularly twice with a commercial inactivated ND virus (NDV) vaccine, and the last group was vaccinated orally twice with a commercial live attenuated NDV vaccine. PBMC were ex vivo stimulated with ConA or with NDV antigen. The ICS assay was used to determine...... the phenotype and frequency of IFN-γ positive cells. ConA stimulation induced extensive IFN-γ production in both CD3+TCRγδ+ (γδ T cells) cells and CD3+TCRγδ− cells (αβ T cells), but no significant differences were observed between the experimental groups. Furthermore, a large proportion of the IFN-γ producing...... cells were CD3− indicating that other cells than classic T cells, secreted this cytokine. NDV antigen stimulation induced IFN-γ production but to a lower extent than ConA and with a large variation between individuals. The CD3+TCR1γδ−CD8α+ (CTL) population produced the highest NDV specific IFN...

  18. A Safety and Environmental Assessment of the Biological Simulants Bacillus subtilis and Newcastle Disease Virus. Volume 1: Discussion

    Science.gov (United States)

    1993-01-01

    1988) Bacillus keratitis associated with contaminated contact lens care systems. Am J Ophthalmol105:195-197 * Doyle RJ, Keller KF, Ezzell JW (1985...cities lie within 250 miles and could easily be reached by a viral aerosol generated on the Suffield range. The distances could be covered in a

  19. Flow cytometric assessment of chicken T cell-mediated immune responses after Newcastle disease virus vaccination and challenge

    DEFF Research Database (Denmark)

    Dalgaard, T. S.; Norup, L. R.; Pedersen, A.R.

    2010-01-01

    . Despite a delayed NDV-specific antibody response to vaccination, L133 appeared to be better protected than L130 in the subsequent infection challenge as determined by the presence of viral genomes. Peripheral blood was analyzed by flow cytometry and responses in vaccinated/challenged birds were studied...... by 5-color immunophenotyping as well as by measuring the proliferative capacity of NDV-specific T cells after recall stimulation. Immunophenotyping identified L133 as having a significantly lower CD4/CD8 ratio and a lower frequency of γδ T cells than L130 in the peripheral T cell compartment...

  20. Newcastle disease outbreaks in recent years in Western Europe were caused by an old (VI) and a novel genotype (VII)

    DEFF Research Database (Denmark)

    Lomniczi, B.; Wehmann, E.; Herczeg, J.

    1998-01-01

    and Greece in the late 1960's and in Hungary in the early 1980's. In contrast, viruses that caused epizootics in Germany, Belgium, The Netherlands, Spain and Italy could be classified into a novel genotype (provisionally termed VII), hitherto undetected in Europe. It is possible that the genotype VII viruses...

  1. Inactivation of low pathogenicity notifiable avian influenza virus and lentogenic Newcastle disease virus following pasteurization in liquid egg products

    Science.gov (United States)

    Sixty seven million cases of shell eggs produced per year in the U.S. are processed as liquid egg product. The U.S. also exports a large amount of egg products. Although the U.S. is normally free of avian influenza, concern about contamination of egg product with these viruses has in the past result...

  2. Improving the thermostability by introduction of arginines on the surface of α-L-rhamnosidase (r-Rha1) from Aspergillus niger.

    Science.gov (United States)

    Li, Lijun; Liao, Hui; Yang, Yan; Gong, Jianye; Liu, Jianan; Jiang, Zedong; Zhu, Yanbing; Xiao, Anfeng; Ni, Hui

    2018-06-01

    To improve the thermostability of α-L-rhamnosidase (r-Rha1), an enzyme previously identified from Aspergillus niger JMU-TS528, multiple arginine (Arg) residues were introduced into the r-Rha1 sequence to replace several lysine (Lys) residues that located on the surface of the folded r-Rha1. Hinted by in silico analysis, five surface Lys residues (K134, K228, K406, K440, K573) were targeted to produce a list of 5 single-residue mutants and 4 multiple-residue mutants using site-directed mutagenesis. Among these mutants, a double Lys to Arg mutant, i.e. K406R/K573R, showed the best thermostability improvement. The half-life of this mutant's enzyme activity increased 3 h at 60 °C, 23 min at 65 °C, and 3.5 min at 70 °C, when compared with the wild type. The simulated protein structure based interaction analysis and molecular dynamics calculation indicate that the thermostability improvement of the mutant K406R-K573R was possibly due to the extra hydrogen bonds, the additional cation-π interactions, and the relatively compact conformation. With the enhanced thermostability, the α-L-rhamnosidase mutant, K406R-K573R, has potentially broadened the r-Rha1 applications in food processing industry. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

    Science.gov (United States)

    Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

    2017-12-01

    We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

  4. Characterization of a cold-active esterase from Serratia sp. and improvement of thermostability by directed evolution.

    Science.gov (United States)

    Jiang, Huang; Zhang, Shaowei; Gao, Haofeng; Hu, Nan

    2016-01-22

    In recent years, cold-active esterases have received increased attention due to their attractive properties for some industrial applications such as high catalytic activity at low temperatures. An esterase-encoding gene (estS, 909 bp) from Serratia sp. was identified, cloned and expressed in Escherichia coli DE3 (BL21). The estS encoded a protein (EstS) of 302 amino acids with a predicted molecular weight of 32.5 kDa. It showed the highest activity at 10 °C and pH 8.5. EstS was cold active and retained ~92 % of its original activity at 0 °C. Thermal inactivation analysis showed that the T1/2 value of EstS was 50 min at 50 °C (residual activity 41.23 %) after 1 h incubation. EstS is also quite stable in high salt conditions and displayed better catalytic activity in the presence of 4 M NaCl. To improve the thermo-stability of EstS, variants of estS gene were created by error-prone PCR. A mutant 1-D5 (A43V, R116W, D147N) that showed higher thermo-stability than its wild type predecessor was selected. 1-D5 showed enhanced T1/2 of 70 min at 50 °C and retained 63.29 % of activity after incubation at 50 °C for 60 min, which were about 22 % higher than the wild type (WT). CD spectrum showed that the secondary structure of WT and 1-D5 are more or less similar, but an increase in β-sheets was recorded, which enhanced the thermostability of mutant protein. EstS was a novel cold-active and salt-tolerant esterase and half-life of mutant 1-D5 was enhanced by 1.4 times compared with WT. The features of EstS are interesting and can be exploited for commercial applications. The results have also provided useful information about the structure and function of Est protein.

  5. Effect of Dietary Patterns on Muscle Strength and Physical Performance in the Very Old: Findings from the Newcastle 85+ Study.

    Science.gov (United States)

    Granic, Antoneta; Jagger, Carol; Davies, Karen; Adamson, Ashley; Kirkwood, Thomas; Hill, Tom R; Siervo, Mario; Mathers, John C; Sayer, Avan Aihie

    2016-01-01

    Healthy diet has been associated with better muscle strength and physical performance in cross-sectional studies of older adults but the effect of dietary patterns (DP) on subsequent decline, particularly in the very old (aged 85+), has not been determined. We investigated the association between previously established DP and decline in muscle strength and physical performance in the very old. 791 participants (61.8% women) from the Newcastle 85+ Study were followed-up for change in hand grip strength (HGS) and Timed Up-and Go (TUG) test over 5 years (four waves 1.5 years apart). Mixed models were used to determine the effects of DP on muscle strength and physical performance in the entire cohort and separately by sex. Previously we have established three DP that varied in intake of red meats, potato, gravy and butter and differed with key health and social factors. HGS declined linearly by 1.59 kgF in men and 1.08 kgF in women (both pgravy (DP1), or butter (DP3) may adversely affect muscle strength and physical performance in later life, independently of important covariates and cognitive status.

  6. Continuous D-tagatose production by immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor.

    Science.gov (United States)

    Ryu, Se-Ah; Kim, Chang Sup; Kim, Hye-Jung; Baek, Dae Heoun; Oh, Deok-Kun

    2003-01-01

    D-Tagatose was continuously produced using thermostable L-arabinose isomerase immobilized in alginate with D-galactose solution in a packed-bed bioreactor. Bead size, L/D (length/diameter) of reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h(-1), 5.65 units, and 300 g/L, respectively. Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L x h and an average conversion yield of 48% (w/w). Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half-life of 24 days.

  7. Discovery, cloning and characterisation of proline specific prolyl endopeptidase, a gluten degrading thermo-stable enzyme from Sphaerobacter thermophiles

    DEFF Research Database (Denmark)

    Shetty, Radhakrishna; Vestergaard, Mike; Jessen, Flemming

    2017-01-01

    processes occur at elevated temperature. We present in this paper, the discovery, cloning and characterisation of a novel recombinant thermostable gluten degrading enzyme, a proline specific prolyl endoprotease (PEP) from Sphaerobacter thermophiles. The molecular mass of the prolyl endopeptidase......Gluten free products have emerged during the last decades, as a result of a growing public concern and technological advancements allowing gluten reduction in food products. One approach is to use gluten degrading enzymes, typically at low or ambient temperatures, whereas many food production...... was estimated to be 77 kDa by using SDS-PAGE. Enzyme activity assays with a synthetic dipeptide Z-Gly-Pro-p-nitroanilide as the substrate revealed that the enzyme had optimal activity at pH 6.6 and was most active from pH 5.0-8.0. The optimum temperature was 63 °C and residual activity after one hour incubation...

  8. Preparation of lactose-free pasteurized milk with a recombinant thermostable β-glucosidase from Pyrococcus furiosus

    Science.gov (United States)

    2013-01-01

    Background Lactose intolerance is a common health concern causing gastrointestinal symptoms and avoidance of dairy products by afflicted individuals. Since milk is a primary source of calcium and vitamin D, lactose intolerant individuals often obtain insufficient amounts of these nutrients which may lead to adverse health outcomes. Production of lactose-free milk can provide a solution to this problem, although it requires use of lactase from microbial sources and increases potential for contamination. Use of thermostable lactase enzymes can overcome this issue by functioning under pasteurization conditions. Results A thermostable β-glucosidase gene from Pyrococcus furiosus was cloned in frame with the Saccharomyces cerecisiae a-factor secretory signal and expressed in Pichia pastoris strain X-33. The recombinant enzyme was purified by a one-step method of weak anion exchange chromatography. The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively. The enzyme activity was not significantly inhibited by Ca2+. We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose. Conclusions The thermostablity of this recombinant β-glucosidase, combined with its neutral pH activity and favorable temperature activity optima, suggest that this enzyme is an ideal candidate for the hydrolysis of lactose in milk, and it would be suitable for application in low-lactose milk production during pasteurization. PMID:24053641

  9. Effect of thermostable α-amylase injection on mechanical and physiochemical properties for saccharification of extruded corn starch.

    Science.gov (United States)

    Myat, Lin; Ryu, Gi-Hyung

    2014-01-30

    In industry, a jet cooker is used to gelatinize starch by mixing the starch slurry with steam under pressure at 100-175 °C. A higher degree of starch hydrolysis in an extruder is possible with glucoamylase. Unfortunately, it is difficult to carry out liquefaction and saccharification in parallel, because the temperature of gelatinization will be too high and will inactivate glucoamylase. Since the temperature for liquefaction and saccharification is different, it is hard to change the temperature from high (required for liquefaction) to low (required for saccharification). The industrial gelatinization process is usually carried out with 30-35% (w/w) dry solids starch slurry. Conventional jet cookers cannot be used any more at high substrate concentrations owing to higher viscosity. In this study, therefore, corn starch was extruded at different melt temperatures to overcome these limitations and to produce the highest enzyme-accessible starch extrudates. Significant effects on physical properties (water solubility index, water absorption index and color) and chemical properties (reducing sugar and % increase in reducing sugar after saccharification) were achieved by addition of thermostable α-amylase at melt temperatures of 115 and 135 °C. However, there was no significant effect on % increase in reducing sugar of extruded corn starch at 95 °C. The results show the great potential of extrusion with thermostable α-amylase injection at 115 and 135 °C as an effective pretreatment for breaking down starch granules, because of the significant increase (P < 0.05) in % reducing sugar and enzyme-accessible extrudates for saccharification yield. © 2013 Society of Chemical Industry.

  10. DISEASES

    DEFF Research Database (Denmark)

    Pletscher-Frankild, Sune; Pallejà, Albert; Tsafou, Kalliopi

    2015-01-01

    Text mining is a flexible technology that can be applied to numerous different tasks in biology and medicine. We present a system for extracting disease-gene associations from biomedical abstracts. The system consists of a highly efficient dictionary-based tagger for named entity recognition...... of human genes and diseases, which we combine with a scoring scheme that takes into account co-occurrences both within and between sentences. We show that this approach is able to extract half of all manually curated associations with a false positive rate of only 0.16%. Nonetheless, text mining should...... not stand alone, but be combined with other types of evidence. For this reason, we have developed the DISEASES resource, which integrates the results from text mining with manually curated disease-gene associations, cancer mutation data, and genome-wide association studies from existing databases...

  11. Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand

    Directory of Open Access Journals (Sweden)

    Salleh Abu

    2009-04-01

    Full Text Available Abstract Background Many researchers have reported on the optimization of protease production; nevertheless, only a few have reported on the optimization of the production of organic solvent-tolerant proteases. Ironically, none has reported on thermostable organic solvent-tolerant protease to date. The aim of this study was to isolate the thermostable organic solvent-tolerant protease and identify the culture conditions which support its production. The bacteria of genus Bacillus are active producers of extra-cellular proteases, and the thermostability of enzyme production by Bacillus species has been well-studied by a number of researchers. In the present study, the Bacillus subtilis strain Rand was isolated from the contaminated soil found in Port Dickson, Malaysia. Results A thermostable organic solvent-tolerant protease producer had been identified as Bacillus subtilis strain Rand, based on the 16S rRNA analysis conducted, as well as the morphological characteristics and biochemical properties. The production of the thermostable organic solvent-tolerant protease was optimized by varying various physical culture conditions. Inoculation with 5.0% (v/v of (AB600 = 0.5 inoculum size, in a culture medium (pH 7.0 and incubated for 24 h at 37°C with 200 rpm shaking, was the best culture condition which resulted in the maximum growth and production of protease (444.7 U/ml; 4042.4 U/mg. The Rand protease was not only stable in the presence of organic solvents, but it also exhibited a higher activity than in the absence of organic solvent, except for pyridine which inhibited the protease activity. The enzyme retained 100, 99 and 80% of its initial activity, after the heat treatment for 30 min at 50, 55, and 60°C, respectively. Conclusion Strain Rand has been found to be able to secrete extra-cellular thermostable organic solvent-tolerant protease into the culture medium. The protease exhibited a remarkable stability towards temperature and organic

  12. Anticuerpos séricos contra la enfermedad de Newcastle e Influenza Aviar en aves rapaces de Chile.

    Directory of Open Access Journals (Sweden)

    Daniel González-Acuña

    2012-12-01

    Full Text Available Objetivo. Detectar la presencia de anticuerpos séricos sanguíneos contra los virus de la Enfermedad de Newcastle (ENC e Influenza aviar (IA, para comprender la contribución de las aves silvestres en la transmisión de estos virus en Chile. Materiales y métodos. Se analizaron 63 aves pertenecientes a los órdenes Falconiformes y Strigiformes desde centros de rehabilitación de aves de las zonas central y sur de Chile. Se realizaron las pruebas de inhibición de la hemoaglutinación (IHA para detectar anticuerpos contra el virus ENC e inmunodifusión en gel agar (IDGA y ELISA para IA. Resultados. Se detectaron 14 aves positivas (22.2% para anticuerpos séricos contra el virus de la ENC. En cambio, no se registraron anticuerpos séricos sanguíneos para el virus de la IA. Conclusiones. La presencia de aves rapaces positivas en los centros de rescate a los anticuerpos séricos contra el virus de la ENC puede ser explicada por el consumo de carne de pollos que han sido vacunados contra ENC o consumo de aves que han adquirido directamente el virus vacunal a través de los distintos procedimientos de administración (aerosoles, bebederos de la vacuna o por el ingreso a los centros de rescate de aves rapaces migratorias, las que podrían facilitar la diseminación de la infección desde los países de origen, hecho que debe ser investigado.

  13. Improved thermostability and enzyme activity of a recombinant phyA mutant phytase from Aspergillus niger N25 by directed evolution and site-directed mutagenesis.

    Science.gov (United States)

    Tang, Zizhong; Jin, Weiqiong; Sun, Rong; Liao, Yan; Zhen, Tianrun; Chen, Hui; Wu, Qi; Gou, Lin; Li, Chenlei

    2018-01-01

    We previously constructed three recombinant phyA mutant strains (PP-NP m -8, PP-NP ep -6A and I44E/T252R-PhyA), showing improved catalytic efficiency or thermostability of Aspergillus niger N25 phytase, by error-prone PCR or site-directed mutagenesis. In this study, directed evolution and site-directed mutagenesis were further applied to improve the modified phytase properties. After one-round error-prone PCR for phytase gene of PP-NP ep -6A, a single transformant, T195L/Q368E/F376Y, was obtained with the significant improvements in catalytic efficiency and thermostability. The phytase gene of T195L/Q368E/F376Y, combined with the previous mutant phytase genes of PP-NP ep -6A, PP-NP m -8 and I44E/T252R-PhyA, was then sequentially modified by DNA shuffling. Three genetically engineered strains with desirable properties were then obtained, namedQ172R, Q172R/K432R andQ368E/K432R. Among them, Q172R/K432R showed the highest thermostability with the longest half-life and the greatest remaining phytase activity after heat treatment, while Q368E/K432R showed the highest catalytic activity. Five substitutions (Q172R, T195L, Q368E, F376Y, K432R) identified from random mutagenesis were added sequentially to the phytase gene of PP-NP ep -6A to investigate how the mutant sites influence the properties of phytase. Characterization and structural analysis demonstrated that these mutations could produce cumulative or synergistic improvements in thermostability or catalytic efficiency of phytase. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Resolution Mechanism and Characterization of an Ammonium Chloride-Tolerant, High-Thermostable, and Salt-Tolerant Phenylalanine Dehydrogenase from Bacillus halodurans.

    Science.gov (United States)

    Jiang, Wei; Wang, Ya-Li; Fang, Bai-Shan

    2018-05-09

    As phenylalanine dehydrogenase (PheDH) plays an important role in the synthesis of chiral drug intermediates and detection of phenylketonuria, it is significant to obtain a PheDH with specific and high activity. Here, a PheDH gene, pdh, encoding a novel BhPheDH with 61.0% similarity to the known PheDH from Microbacterium sp., was obtained. The BhPheDH showed optimal activity at 60 °C and pH 7.0, and it showed better stability in hot environment (40-70 °C) than the PheDH from Nocardia sp. And its activity and thermostability could be significantly increased by sodium salt. After incubation for 2 h in 3 M NaCl at 60 °C, the residual activity of the BhPheDH was found to be 1.8-fold higher than that of the control group (without NaCl). The BhPheDH could tolerate high concentration of ammonium chloride and its activity could be also enhanced by the high concentration of ammonium salts. These characteristics indicate that the BhPheDH possesses better thermostability, ammonium chloride tolerance, halophilic mechanism, and high salt activation. The mechanism of thermostability and high salt tolerance of the BhPheDH was analyzed by molecular dynamics simulation. These results provide useful information about the enzyme with high-temperature activity, thermostability, halophilic mechanism, tolerance to high concentration of ammonium chloride, higher salt activation and enantio-selectivity, and the application of molecular dynamics simulation in analyzing the mechanism of these distinctive characteristics.

  15. Development of soluble and immobilized biocatalysts based on a recombinant thermostable ß-fructosidase enabling complete sucrose inversion at pasteurization temperatures

    OpenAIRE

    Menéndez, Carmen; Martínez, Duniesky; Trujillo, Luis E; Ramírez, Ricardo; Sobrino, Alina; Cutiño-Ávila, Bessy V; Basabe, Liliana; del Monte-Martínez, Alberto; Pérez, Enrique R; Hernández, Lázaro

    2014-01-01

    Biocatalysts for the industrial production of invert sugar are preferred to stably operate at high sucrose concentrations and pasteurization temperatures. Thermotoga maritima ß-fructosidase (BfrA) is more thermostable and less susceptible to substrate inhibition than the current commercial invertase from Saccharomyces cerevisiae. In this research, the non-saccharolytic host Pichia pastoris was engineered for BfrA production. Fed-batch fermentation of the recombinant yeast for 72 h using cane ...

  16. Thermo-stable carbon nanotube-TiO_2 nanocompsite as electron highways in dye-sensitized solar cell produced by bio-nano-process

    International Nuclear Information System (INIS)

    Inoue, Ippei; Yasueda, Hisashi; Yamauchi, Hirofumi; Okamoto, Naofumi; Toyoda, Kenichi; Horita, Masahiro; Ishikawa, Yasuaki; Uraoka, Yukiharu; Yamashita, Ichiro

    2015-01-01

    We produced a thermostable TiO_2-(anatase)-coated multi-walled-carbon-nanotube (MWNT) nanocomposite for use in dye-sensitized solar cells (DSSCs) using biological supuramolecules as catalysts. We synthesized two different sizes of iron oxide nanoparticles (NPs) and arrayed the NPs on a silicon substrate utilizing two kinds of genetically modified cage-shaped proteins with silicon-binding peptide aptamers on their outer surfaces. Chemical vapor deposition (CVD) with the vapor–liquid-solid phase (VLS) method was applied to the substrate, and thermostable MWNTs with a diameter of 6 ± 1 nm were produced. Using a genetically modified cage-shaped protein with carbon-nanomaterials binding and Ti-mineralizing peptides as a catalyst, we were able to mineralize a titanium compound around the surface of the MWNT. The products were sintered, and thin TiO_2-layer-coated MWNTs nanocomoposites were successfully produced. Addition of a 0.2 wt% TiO_2-coated MWNT nanocomposite to a DSSC photoelectrode improved current density by 11% and decreased electric resistance by 20% compared to MWNT-free reference DSSCs. These results indicate that a nanoscale TiO_2-layer-coated thermostable MWNT structure produced by our mutant proteins works as a superior electron transfer highway within TiO_2 photoelectrodes. (paper)

  17. Enzymatic liquefaction of agarose above the sol-gel transition temperature using a thermostable endo-type β-agarase, Aga16B.

    Science.gov (United States)

    Kim, Jung Hyun; Yun, Eun Ju; Seo, Nari; Yu, Sora; Kim, Dong Hyun; Cho, Kyung Mun; An, Hyun Joo; Kim, Jae-Han; Choi, In-Geol; Kim, Kyoung Heon

    2017-02-01

    The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol-gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol-gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol-gel transition temperature of agarose. In this study, a thermostable endo-type β-agarase, Aga16B, originating from Saccharophagus degradans 2-40 T , was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 °C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 °C, which was above the sol-gel transition temperature of 1 % (w/v) agarose (∼35 °C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol-gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type β-agarase.

  18. Combined exposure of Japanese quails to cyanotoxins, Newcastle virus and lead: oxidative stress responses.

    Science.gov (United States)

    Paskova, Veronika; Veronika, Paskova; Paskerova, Hana; Hana, Paskerova; Pikula, Jiri; Jiri, Pikula; Bandouchova, Hana; Hana, Bandouchova; Sedlackova, Jana; Jana, Sedlackova; Hilscherova, Klara; Klara, Hilscherova

    2011-10-01

    Wild birds are continually exposed to many anthropogenic and natural stressors in their habitats. Over the last decades, mass mortalities of wild birds constitute a serious problem and may possibly have more causations such as natural toxins including cyanotoxins, parasitic diseases, industrial chemicals and other anthropogenic contaminants. This study brings new knowledge on the effects of controlled exposure to multiple stressors in birds. The aim was to test the hypothesis that influence of cyanobacterial biomass, lead and antigenic load may combine to enhance the effects on birds, including modulation of antioxidative and detoxification responses. Eight treatment groups of model species Japanese quail (Coturnix coturnix japonica) were exposed to various combinations of these stressors. The parameters of detoxification and oxidative stress were studied in liver and heart after 30 days of exposure. The antioxidative enzymatic defense in birds seems to be activated quite efficiently, which was documented by the elevated levels and activities of antioxidative and detoxification compounds and by the low incidence of damage to lipid membranes. The greatest modulations of glutathione level and activities of glutathione-S-transferase, glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase and lipid peroxidation were shown mostly in the groups with combined multiple exposures. The results indicate that the antioxidative system plays an important role in the protective response of the tissues to applied stressors and that its greater induction helps to protect the birds from more serious damage. Most significant changes of these "defense" parameters in case of multiple stressors suggest activation of this universal mechanism in situation with complex exposure and its crucial role in protection of the bird health in the environment. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Tradução e adaptação cultural do Newcastle Satisfaction with Nursing Scales para a cultura brasileira

    Directory of Open Access Journals (Sweden)

    Gisele Hespanhol Dorigan

    2013-06-01

    Full Text Available Este estudo teve por objetivos traduzir e adaptar o Newcastle Satisfaction with Nursing Scales para a cultura brasileira, bem como verificar seu uso prático. O instrumento contém duas escalas e tem como objetivo avaliar as experiências vivenciadas pelo paciente e a satisfação com o cuidado de enfermagem. Para o procedimento metodológico de adaptação cultural foram seguidas as etapas de tradução, síntese, retro-tradução, avaliação pelo comitê de especialistas e pré-teste. O processo de tradução e adaptação cultural foi considerado apropriado. A avaliação pelo comitê de especialistas resultou em modificações gramaticais simples para a maioria dos itens e, no pré-teste, participaram 40 sujeitos. A versão brasileira do Newcastle Satisfaction with Nursing Scales demonstrou adequada validade de conteúdo e facilidade de compreensão pelos sujeitos. Contudo, este é um estudo que antecede o processo de avaliação das propriedades psicométricas do instrumento, cujos resultados serão apresentados em publicação posterior.

  20. Effect of Dietary Patterns on Muscle Strength and Physical Performance in the Very Old: Findings from the Newcastle 85+ Study.

    Directory of Open Access Journals (Sweden)

    Antoneta Granic

    Full Text Available Healthy diet has been associated with better muscle strength and physical performance in cross-sectional studies of older adults but the effect of dietary patterns (DP on subsequent decline, particularly in the very old (aged 85+, has not been determined.We investigated the association between previously established DP and decline in muscle strength and physical performance in the very old.791 participants (61.8% women from the Newcastle 85+ Study were followed-up for change in hand grip strength (HGS and Timed Up-and Go (TUG test over 5 years (four waves 1.5 years apart. Mixed models were used to determine the effects of DP on muscle strength and physical performance in the entire cohort and separately by sex.Previously we have established three DP that varied in intake of red meats, potato, gravy and butter and differed with key health and social factors. HGS declined linearly by 1.59 kgF in men and 1.08 kgF in women (both p<0.001, and TUG slowed by 0.13 log10-transformed seconds (log10-s in men and 0.11 log10-s in women per wave after adjusting for important covariates (both p<0.001, and also showed a nonlinear change (p<0.001. Men in DP1 ('High Red Meat' had worse overall HGS (β = -1.70, p = 0.05, but men in DP3 ('High Butter' had a steeper decline (β = -0.63, p = 0.05 than men in DP2 ('Low Meat'. Men in DP1 and women in DP3 also had overall slower TUG than those in DP2 (β = 0.08, p = 0.001 and β = 0.06, p = 0.01, respectively, but similar rate of decline after adjusting for sociodemographic, lifestyle, health, and functioning factors. The results for HGS and TUG were not affected by participants' cognitive status.DP high in red meats, potato and gravy (DP1, or butter (DP3 may adversely affect muscle strength and physical performance in later life, independently of important covariates and cognitive status.

  1. Improving the Efficiency of New Automatic Dishwashing Detergent Formulation by Addition of Thermostable Lipase, Protease and Amylase

    Directory of Open Access Journals (Sweden)

    Ashwini Naganthran

    2017-09-01

    Full Text Available The use of T1 lipase in automatic dishwashing detergent (ADD is well established, but efficiency in hard water is very low. A new enzymatic environmentally-friendly dishwashing was formulated to be efficient in both soft and hard water. Thermostable enzymes such as T1 lipase from Geobacillus strain T1, Rand protease from Bacillus subtilis strain Rand, and Maltogenic amylase from Geobacillus sp. SK70 were produced and evaluated for an automatic dishwashing detergent formulation. The components of the new ADD were optimized for compatibility with these three enzymes. In compatibility tests of the enzymes with different components, several criteria were considered. The enzymes were mostly stable in non-ionic surfactants, especially polyhydric alcohols, Glucopon UP 600, and in a mixture of sodium carbonate and glycine (30:70 buffer at a pH of 9.25. Sodium polyacrylate and sodium citrate were used in the ADD formulation as a dispersing agent and a builder, respectively. Dishwashing performance of the formulated ADDs was evaluated in terms of percent of soil removed using the Leenert‘s Improved Detergency Tester. The results showed that the combination of different hydrolysis enzymes could improve the washing efficiency of formulated ADD compared to the commercial ADD “Finish” at 40 and 50 C.

  2. Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion

    Science.gov (United States)

    Pečová, M.; Šebela, M.; Marková, Z.; Poláková, K.; Čuda, J.; Šafářová, K.; Zbořil, R.

    2013-03-01

    In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, β- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

  3. Thermostable L-arabinose isomerase from Bacillus stearothermophilus IAM 11001 for D-tagatose production: gene cloning, purification and characterisation.

    Science.gov (United States)

    Cheng, Lifang; Mu, Wanmeng; Jiang, Bo

    2010-06-01

    D-Tagatose, as one of the rare sugars, has been found to be a natural and safe low-calorie sweetener in food products and is classified as a GRAS substance. L-Arabinose isomerase (L-AI, EC 5.3.1.4), catalysing the isomerisations of L-arabinose and D-galactose to L-ribulose and D-tagatose respectively, is considered to be the most promising enzyme for the production of D-tagatose. The araA gene encoding an L-AI from Bacillus stearothermophilus IAM 11001 was cloned, sequenced and overexpressed in Escherichia coli. The gene is composed of 1491 bp nucleotides and codes for a protein of 496 amino acid residues. The recombinant L-AI was purified to electrophoretical homogeneity by affinity chromatography. The purified enzyme was optimally active at 65 degrees C and pH 7.5 and had an absolute requirement for the divalent metal ion Mn(2+) for both catalytic activity and thermostability. The enzyme was relatively active and stable at acidic pH of 6. The bioconversion yield of D-galactose to D-tagatose by the purified L-AI after 12 h at 65 degrees C reached 36%. The purified L-AI from B. stearothermophilus IAM 11001 was characterised and shown to be a good candidate for potential application in D-tagatose production. Copyright (c) 2010 Society of Chemical Industry.

  4. Directed divergent evolution of a thermostable D-tagatose epimerase towards improved activity for two hexose substrates.

    Science.gov (United States)

    Bosshart, Andreas; Hee, Chee Seng; Bechtold, Matthias; Schirmer, Tilman; Panke, Sven

    2015-03-02

    Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D-tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shells around the substrate-binding site of Var8 yielded the eight-site mutant IDF8 (ninefold improved kcat for the epimerization of D-fructose) and the six-site mutant ILS6 (14-fold improved epimerization of L-sorbose), compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site; this presumably facilitates entry of the polar substrate. The improvement in catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for further directed evolution of the ketohexose epimerase. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Thermostable gel polymer electrolyte based on succinonitrile and ionic liquid for high-performance solid-state supercapacitors

    Science.gov (United States)

    Pandey, Gaind P.; Liu, Tao; Hancock, Cody; Li, Yonghui; Sun, Xiuzhi Susan; Li, Jun

    2016-10-01

    A flexible, free-standing, thermostable gel polymer electrolyte based on plastic crystalline succinonitrile (SN) and ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate (BMImBF4) entrapped in copolymer poly(vinylidene fluoride-co-hexafluoropropylene) (PVdF-HFP) is prepared and optimized for application in solvent-free solid-state supercapacitors. The synthesized gel polymer electrolyte exhibits a high ionic conductivity over a wide temperature range (from ∼5 × 10-4 S cm-1 at -30 °C up to ∼1.5 × 10-2 S cm-1 at 80 °C) with good electrochemical stability window (-2.9 to 2.5 V). Thermal studies confirm that the SN containing gel polymer electrolyte remains stable in the same gel phase over a wide temperature range from -30 to 90 °C. The electric double layer capacitors (EDLCs) have been fabricated using activated carbon as active materials and new gel polymer electrolytes. Electrochemical performance of the EDLCs is assessed through cyclic voltammetry, galvanostatic charge-discharge cycling and impedance spectroscopy. The EDLC cells with the proper SN-containing gel polymer electrolyte has been found to give high specific capacitance 176 F g-1 at 0.18 A g-1 and 138 F g-1 at 8 A g-1. These solid-state EDLC cells show good cycling stability and the capability to retain ∼80% of the initial capacitance after 10,000 cycles.

  6. Thermostable hemicellulases of a bacterium, Geobacillus sp. DC3, isolated from the former Homestake gold mine in Lead, South Dakota.

    Science.gov (United States)

    Bergdale, Terran E; Hughes, Stephen R; Bang, Sookie S

    2014-04-01

    A thermophilic strain, Geobacillus sp. DC3, capable of producing hemicellulolytic enzymes was isolated from the 1.5-km depth of the former Homestake gold mine in Lead, South Dakota. The DC3 strain expressed a high level of extracellular endoxylanase at 39.5 U/mg protein with additional hemicellulases including β-xylosidase (0.209 U/mg) and arabinofuranosidase (0.230 U/mg), after the bacterium was grown in xylan for 24 h. Partially purified DC3 endoxylanase exhibited a molecular mass of approximately 43 kDa according to zymography with an optimal pH of 7 and optimal temperature of 70 °C. The kinetic constants, K m and V max, were 13.8 mg/mL and 77.5 μmol xylose/min·mg xylan, respectively. The endoxylanase was highly stable and maintained 70 % of its original activity after 16 h incubation at 70 °C. The thermostable properties and presence of three different hemicellulases of Geobacillus sp. DC3 strain support its potential application for industrial hydrolysis of renewable biomass such as lignocelluloses.

  7. CNA web server: rigidity theory-based thermal unfolding simulations of proteins for linking structure, (thermo-)stability, and function.

    Science.gov (United States)

    Krüger, Dennis M; Rathi, Prakash Chandra; Pfleger, Christopher; Gohlke, Holger

    2013-07-01

    The Constraint Network Analysis (CNA) web server provides a user-friendly interface to the CNA approach developed in our laboratory for linking results from rigidity analyses to biologically relevant characteristics of a biomolecular structure. The CNA web server provides a refined modeling of thermal unfolding simulations that considers the temperature dependence of hydrophobic tethers and computes a set of global and local indices for quantifying biomacromolecular stability. From the global indices, phase transition points are identified where the structure switches from a rigid to a floppy state; these phase transition points can be related to a protein's (thermo-)stability. Structural weak spots (unfolding nuclei) are automatically identified, too; this knowledge can be exploited in data-driven protein engineering. The local indices are useful in linking flexibility and function and to understand the impact of ligand binding on protein flexibility. The CNA web server robustly handles small-molecule ligands in general. To overcome issues of sensitivity with respect to the input structure, the CNA web server allows performing two ensemble-based variants of thermal unfolding simulations. The web server output is provided as raw data, plots and/or Jmol representations. The CNA web server, accessible at http://cpclab.uni-duesseldorf.de/cna or http://www.cnanalysis.de, is free and open to all users with no login requirement.

  8. Thermostability and surface morphology of nano- and micro-filled NBR/CSM and CR/CSM rubber blends

    Directory of Open Access Journals (Sweden)

    M. MARINOVIC-CINCOVIC

    2004-02-01

    Full Text Available Acrylonitrile-butadiene rubber (NBR, polychloroprene rubber (CR, chlorosulphonated polyethylene rubber (CSM and their blends were cross-linked with sulphur, ethylene-thiourea, magnesium oxide or their combination. The effect of nano- and micro- particle sized of 35 pphr SiO2 on the thermostability and surface morphology of all the crosslinked systems was investigated. Identification of the structure of nano- and micro- particle sized SiO2 filled NBR/CSM and CR/CSM crosslinked systems was carried out by Fourier transform infrared spectroscopy (FTIR with an attenuated total reflectance (ATR extension. The thermal stability of the nano- and micro- particle sized SiO2 filled NBR/CSM and CR/CSM crosslinked systems were carried out by thermogravimetric analysis (TGA. The glass transition temperature (Tg of the samples was determined by differential scanning calorimetry (DSC. The morphology of the fracture surface of the crosslinked systems was carried out by scanning electron microscope (SEM. The results show when filled with nano-particle sized of SiO2 NBR/CSM and CR/CSM polymer matrix have a strong peak from SiO–C at 1079 cm-1. This suggests the an interaction between the SiO2, which should lead to an increased thermal stability, higher values of Tg, better dispersion the nano-SiO2 and more polish, without cracks than micro-filled NBR/CSM and CR/CSM crosslinked systems.

  9. Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation.

    Science.gov (United States)

    Gutarra, Melissa L E; Godoy, Mateus G; Maugeri, Francisco; Rodrigues, Maria Isabel; Freire, Denise M G; Castilho, Leda R

    2009-11-01

    The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35-60 degrees C and pH 4.0-6.0, with a maximum activity at 50 degrees C and pH 4.0-5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 degrees C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0-C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.

  10. Effect of chemical stabilizers on the thermostability and infectivity of a representative panel of freeze dried viruses.

    Directory of Open Access Journals (Sweden)

    Boris Pastorino

    Full Text Available As a partner of the European Virus Archive (EVA FP7 project, our laboratory maintains a large collection of freeze-dried viruses. The distribution of these viruses to academic researchers, public health organizations and industry is one major aim of the EVA consortium. It is known that lyophilization requires appropriate stabilizers to prevent inactivation of the virus. However, few studies have investigated the influence of different stabilizers and lyophilization protocols on the thermostability of different viruses. In order to identify optimal lyophilization conditions that will deliver maximum retention of viral infectivity titre, different stabilizer formulations containing trehalose, sorbitol, sucrose or foetal bovine serum were evaluated for their efficacy in stabilizing a representative panel of freeze dried viruses at different storage temperatures (-20°C, +4°C and +20°C for one week, the two latter mimicking suboptimal shipping conditions. The Tissue Culture Infectious Dose 50% (TCID50 assay was used to compare the titres of infectious virus. The results obtained using four relevant and model viruses (enveloped/non enveloped RNA/DNA viruses still serve to improve the freeze drying conditions needed for the development and the distribution of a large virus collection.

  11. Novel thermostable clostridial strains through protoplast fusion for enhanced biobutanol production at higher temperature—preliminary study

    Directory of Open Access Journals (Sweden)

    Muhammad Ferhan

    2016-01-01

    Full Text Available The objective of this study is to improve the thermal stability of clostridium strains for enhanced biobutanol production. Thermostable clostridia species were developed through protoplast fusion between mesophilic clostridial species (i.e., Clostridium beijerinckii and Clostridium acetobutylicum and thermophilic clostridial species (i.e., Clostridium thermocellum. Production of biobutanol was examined in the present preliminary study using the clostridium strains and their protoplast fusants using sugar mixture with composition identical to that of wheat straw acid hydrolysate. Maximum biobutanol production of 9.4 g/L was achieved by a fused strain at 45 °C with total sugar consumption of 66% compared to that at 35 °C (i.e., 8.4 g/L production and 64% total sugar consumption. Glucose and xylose uptake rates were generally higher compared to all other individual sugars in the feedstock. In general, average cell concentrations were in close proximity for all parenting and fused strains at 35 °C; i.e., in the range of 5.12 × 107 to 5.49 × 107 cells/mL. Average cell concentration of fusants between the mesophilic clostridial species and the thermophilic clostridial species slightly increased to ~ 5.62 × 107 cells/mL at a higher temperature of 45 °C. These results, in addition to the ones obtained for the butanol production, demonstrate enhanced thermal stability of both fusants at a higher temperature (45 °C.

  12. Immobilization of anaerobic thermophilic bacteria for the production of cell-free thermostable. alpha. -amylases and pullulanases

    Energy Technology Data Exchange (ETDEWEB)

    Klingeberg, M [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Vorlop, K D [Technische Univ. Braunschweig (Germany, F.R.). Inst. fuer Technische Chemie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F. R.). Arbeitsbereich Biotechnologie 1

    1990-08-01

    For the production of cell-free thermostable {alpha}-amylases and pullulanases various anaerobic thermophilic bacteria that belong to the genera Clostridium and Thermoanaerobacter were immobilized in calcium alginate gel beads. The entrapment of bacteria was performed in full was well as in hollow spheres. An optimal limited medium, which avoided bacterial outgrowth, was developed for the cultivation of immobilized organisms at 60deg C using 0.4% starch as substrate. Compared to non-immobilized cells these techniques allowed a significant increase (up to 5.6-fold) in the specific activities of the extracellular enzymes formed. An increase in the productivity of extracellular enzymes was observed after immobilization of bacteria in full spheres. In the case of C. thermosaccharolyticum, for instance, the productivity was raised from 90 units (U)/10{sup 12} cells up to 700 U/10{sup 12} cells. Electrophoretic analysis of the secreted proteins showed that in all cases most of the amylolytic enzymes formed were released into the culture medium. Proteins that had a molecular mass of less than 450 000 daltons could easily diffuse through the gel matrix. Cultivation of immobilized bacteria in semi-continuous and fed-batch cultures was also accompanied by an elevation in the concentration of cell-free enzymes. (orig.).

  13. Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming β-cyano-L-alanine

    International Nuclear Information System (INIS)

    Omura, Hironori; Yoshida, Toyokazu; Nagasawa, Toru; Kobayashi, Michihiko; Shimizu, Sakayu

    2003-01-01

    A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable β-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of β-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical sub-units. It was stable in the pH range of 6.0 to 10.0 and up to 70degC. The enzyme also catalyzes the synthesis of various β-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the β-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the β-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed β-cyano-L-alanine synthase. Heat stable β-cyano-L-alanine synthase can be applied to the synthesis of [4- 11 C]L-2,4-diaminobutyric acid as a tracer for positron emission tomography. (author)

  14. Dynamic fluorescence studies of beta-glycosidase mutants from Sulfolobus solfataricus: effects of single mutations on protein thermostability.

    Science.gov (United States)

    Bismuto, Ettore; Febbraio, Ferdinando; Limongelli, Simona; Briante, Raffaella; Nucci, Roberto

    2003-04-01

    Multiple sequence alignment on 73 proteins belonging to glycosyl hydrolase family 1 reveals the occurrence of a segment (83-124) in the enzyme sequences from hyperthermophilic archaea bacteria, which is absent in all the mesophilic members of the family. The alignment of the known three-dimensional structures of hyperthermophilic glycosidases with the known ones from mesophilic organisms shows a similar spatial organizations of beta-glycosidases except for this sequence segment whose structure is located on the external surface of each of four identical subunits, where it overlaps two alpha-helices. Site-directed mutagenesis substituting N97 or S101 with a cysteine residue in the sequence of beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus caused some changes in the structural and dynamic properties as observed by circular dichroism in far- and near-UV light, as well as by frequency domain fluorometry, with a simultaneous loss of thermostability. The results led us to hypothesize an important role of the sequence segment present only in hyperthermophilic beta-glycosidases, in the thermal adaptation of archaea beta-glycosidases. The thermostabilization mechanism could occur as a consequence of numerous favorable ionic interactions of the 83-124 sequence with the other part of protein matrix that becomes more rigid and less accessible to the insult of thermal-activated solvent molecules. Copyright 2003 Wiley-Liss, Inc.

  15. Thermostable, salt tolerant, wide pH range novel chitobiase from Vibrio parahemolyticus: isolation, characterization, molecular cloning, and expression.

    Science.gov (United States)

    Zhu, B C; Lo, J Y; Li, Y T; Li, S C; Jaynes, J M; Gildemeister, O S; Laine, R A; Ou, C Y

    1992-07-01

    A chitobiase gene from Vibrio parahemolyticus was cloned into plasmid pUC18 in Escherichia coli strain DH5 alpha. The plasmid construct, pC120, contained a 6.4 kb Vibrio DNA insert. The recombinant gene expressed chitobiase [EC 3.2.1.30] activity similar to that found in the native Vibrio. The enzyme was purified by ion exchange, hydroxylapatite and gel permeation chromatographies, and exhibited an apparent molecular weight of 80 kDa on SDS-polyacrylamide gel electrophoresis. Chitobiose and 6 more substrates, including beta-N-acetyl galactosamine glycosides, were hydrolyzed by the recombinant chitobiase, indicating its putative classification as an hexosaminidase [EC 3.2.1.52]. The enzyme was resistant to denaturation by 2 M NaCl, thermostable at 45 degrees C and active over a very unusual (for glycosyl hydrolases) pH range, from 4 to 10. The purified cloned chitobiase gave 4 closely focussed bands on an isoelectric focusing gel, at pH 4 to 6.5. The N-terminal 43 amino acid sequence shows no homology with other proteins in commercial databanks or in the literature, and from its N-terminal sequence, appears to be a novel protein, unrelated in sequence to chitobiases from other Vibrios reported and unrelated to hexosaminidases from other organisms.

  16. Production and Characterization of Thermostable Bioplastic (Poly-beta-Hydroxybutyrate) from Bacillus Cereus NRRL-B-3711

    International Nuclear Information System (INIS)

    Asad-ur-Rehman, M.; Aslam, A.; Masood, R.; Aftab, M. N.; Ajmal, R.; Haq, I. U.

    2016-01-01

    The poly-beta-hydroxybutyrate (PHB) is a thermostable and biocompatible polyester produced by several bacteria under unbalanced nutritional conditions. Among Gram-positive bacteria, which are not well exploited for PHB production on industrial scale, Bacillus appears to be a prospective candidate due to its excellent biopolymer yield and less rigorous fermentation conditions. Batch culture fermentation was carried out for PHB production. The Bacillus cereus NRRL-B-3711 was the most efficient producer of PHB out of five Bacillus species. The optimized production was achieved with 2% glucose, 37 degree C, pH value of 8 and ammonium sulfate (2.5 g/L) as a nitrogen source. Carbon to nitrogen ratio of 10 significantly affects the PHB accumulation. The selected specie was able to accumulate PHB up to 56% (14.2) 0.07 g/L) on dry cell weight basis after optimization. This corresponds to a 1.87 folds increase in the production. The optical microscopy showed extremely flat surface of bioplastic thin film indicating the brittle failure of PHB under tensile loading. The FTIR analysis revealed C=O and -CH groups, with thermal properties e.g. Tg; 2 degree C, Tc; 54 degree C, Tm;162 degree C and percent crystallinity of 51.3 by differential scanning calorimeter (DSC), thus confirming the presence of PHB bioplastic and enhancing its industrial applications.The results surpassed those reported in the literature for PHB production. (author)

  17. Enhanced thermostability of silica-immobilized lipase from Bacillus coagulans BTS-3 and synthesis of ethyl propionate.

    Science.gov (United States)

    Kumar, Satyendra; Pahujani, Shweta; Ola, R P; Kanwar, S S; Gupta, Reena

    2006-06-01

    A lipase from the thermophilic isolate Bacillus coagulans BTS-3 was produced and purified. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The purified lipase was immobilized on silica and its binding efficiency was found to be 60%. The enzyme took 60 min to bind maximally onto the support. The pH and temperature optima of immobilized lipase were same as those of the free enzyme, i.e. 8.5 and 55 degrees C, respectively. The immobilized enzyme had shown marked thermostability on the elevated temperatures of 55, 60, 65 and 70 degrees C. The immobilized enzyme was reused for eigth cycles as it retained almost 80% of its activity. The catalytic activity of immobilized enzyme was enhanced in n-hexane and ethanol. The immobilized enzyme when used for esterification of ethanol and propionic acid showed 96% conversion in n-hexane in 12 h at 55 degrees C.

  18. A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake Mushroom Lentinus edodes

    Directory of Open Access Journals (Sweden)

    Guo-Qing Zhang

    2013-01-01

    Full Text Available A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a tested concentration of 5 mM.

  19. Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming {beta}-cyano-L-alanine

    Energy Technology Data Exchange (ETDEWEB)

    Omura, Hironori; Yoshida, Toyokazu; Nagasawa, Toru [Gifu Univ. (Japan). Dept. of Biomolecular Science; Kuroda, Masako [Ikeda Food Research Co., Ltd., Fukuyama, Hiroshima (Japan); Kobayashi, Michihiko; Shimizu, Sakayu [Kyoto Univ. (Japan). Agricultural Sciences

    2003-10-01

    A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable {beta}-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of {beta}-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical sub-units. It was stable in the pH range of 6.0 to 10.0 and up to 70degC. The enzyme also catalyzes the synthesis of various {beta}-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the {beta}-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the {beta}-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed {beta}-cyano-L-alanine synthase. Heat stable {beta}-cyano-L-alanine synthase can be applied to the synthesis of [4-{sup 11}C]L-2,4-diaminobutyric acid as a tracer for positron emission tomography. (author)

  20. A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake Mushroom Lentinus edodes

    Science.gov (United States)

    Zhang, Guo-Qing; Wu, Ying-Ying; Ng, Tzi-Bun; Chen, Qing-Jun; Wang, He-Xiang

    2013-01-01

    A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a tested concentration of 5 mM. PMID:23586045