Browse by section

Content of VETERINARY SCIENCE in our journal

Published in last 1 year |

In last 2 years |

In last 3 years |

All

Please wait a minute...


For Selected: Download Citations EndNote Ris BibTeX Toggle Thumbnails

Select

Preparation of Monoclonal Antibodies Against DPV and Development of Colloidal Gold Strip for DPV Detection ZHAO Dan-dan, YANG Guo-ping, DIAO You-xiang, CHEN Hao, TI Jin-feng, ZHANG Lu, ZHANG Ying, LI Chuan-chuan Scientia Agricultura Sinica    2016, 49 (14): 2796-2804.   DOI: 10.3864/j.issn.0578-1752.2016.14.013 Abstract （428）   HTML （3）    PDF （1746KB）（891）       Knowledge map    Save 【Objective】 Duck plague（DP） is an acute, septic contagion, caused by duck plague virus（DPV）, with the characteristics of head and neck swelling, the mucosa of esophageal and cloacal bleeding and yellowish-white ulcer, head and neck skin has a yellowish-white gelatin sample. Once an outbreak of this disease manifested by with high morbidity and high mortality, it would cause serious harm to the duck industry. The aim of this assay is to establish a method of colloidal gold strip for the detection of duck plague virus (DPV) rapidly. 【Method】 The main antigenic domain of DPV was chosen and analyzed by using of the Protean Biology software to design a pair of primer to amplify the aim gene by PCR. Then the fragment was inserted into prokaryotic expression vector pET-28a to construct recombinant plasmid. Then it was transformed into Rosetta for expression. During the experiment, the authors have groped the concentration of the IPTG and the induction time. After purification, the concentration of the aim protein was tested and was also analyzed and identified by Western blotting. Hybridoma cell lines stably secreting monoclonal antibody against gB protein of DPV were generated by fusing SP2/0 myeloma cells with splenocytes from the immunized mice, which used the gB protein of DPV, expressed and purified with prokaryotic, as the antigen. The monoclonal antibody-based colloidal gold immunochromatography strip was developed for the detection of DPV. The purified DPV-gB monoclonal antibody, named H6F6, was labeled with colloidal gold, with the appropriate pH between 8.0 and 8.5 and the concentration was 15 times dilution. The purified A8D7 monoclonal antibody, with the concentration of twice dilution, and the goat anti-mouse immunoglobulin G (IgG) antibody, with the concentration of ten times dilution, were blotted on nitrocellulose membrane as test line and control line, respectively. 【Result】 Hybridoma cell lines designated as A8D9, E6C3, H11F8, H6A10, stably secreting monoclonal antibody against gB protein of DPV. The titres of ascitic fluid was1:103, 1:103, 1:105, 1:103, respectively by indirect ELISA and the immunoglobulin subtype of the monoclonal antibodies was IgG2b, IgG2a, IgG2b, IgG1,with the light chain of kappa. The result of western blot showed that the four monoclonal antibodies were able to specifically recognize gB protein of DPV. The result of IFA showed that the four monoclonal antibodies were specific to DPV. The detection results indicated that the strip was specific to DPV and had no cross reaction with DRV, EDS-76V, AIV-H9N2, and TMUV. The detection limit of DPV were 50 times dilution. 38 clinical suspected samples were simultaneously detected by immunochromatography strip and PCR while the results showed 91.6 % accuracy between them. The monoclonal antibodies-based colloidal gold strip was highly specific and sensitive and more convenient for the clinical diagnosis of DPV.【Conclusion】The colloidal gold strip was highly specific and sensitive and more convenient for the clinical diagnosis of DPV. Reference | Related Articles | Metrics Cited: Baidu(5)

Select

Construction and Characterization of Recombinant Duck Enteritis Virus Expressing the Green Fluorescent Protein SUN Ying, LI Jun-ping, HUANG Xiao-jie, LI Ling, CAO Ming-hui, LI Qi-hong, LI Hui-jiao, YANG Cheng-huai Scientia Agricultura Sinica    2016, 49 (14): 2805-2812.   DOI: 10.3864/j.issn.0578-1752.2016.14.014 Abstract （348）   HTML （2）    PDF （1808KB）（364）       Knowledge map    Save 【Objective】Compared with duck enteritis virus(DEV) virulent strain, the vaccine strain has a 528 bp deletions at the UL2, resulting to a 176 aa deletion after amino acids 65. To study the effect of UL2 gene on virus biological properties and explore the feasibility of the DEV as a carrier to express foreign gene, a recombinant DEV expressing the green fluorescent protein (GFP) were constructed;【Method】In this study, the UL2 gene of DEV was chosen as a target site and homologous arm for recombination. Two fragments of UL2 gene were amplified by polymerase chain reaction (PCR) with DNA of DEV cell-adapted strain as template, and were cloned into the pMD-18T vector. The expression cassette including GFP gene and gpt gene controlled by CMV promoter was cloned into UL2 gene as a transfer vector pT-UL2-GFP-gpt. Confluent CEF monolayers were transfected with DEV and Lipofectamine 2000 was used as the transfer vector. When the cytopathic effect (CPE) was observed, the total supernatant and cells were harvested. The infected virus was diluted and then plated on the fresh CEF, and overlaid with M199-FBS containing 1% agarose. When green fluorescent plaques were observed, plaque-purification was carried out to obtain a green fluorescent plaque population termed rDEV-△UL2-GFP-gpt, PCR and sequencing assay were used to identify the recombinant virus. CEF cells cultured in 25cm2 flasks were inoculated with recombinant virus at an MOI of 0.01. The cells and supernatants were harvested respectively every 12 hours, the titer of virus were measured and the one-step growth analyses was performed; To evaluate the genetic stability of GFP gene in the recombinant virus, the virus was passaged in primary CEF 20 times. Four-week-old specific- pathogen-free (SPF) ducks were inoculated intramuscularly with the recombinant virus, and the ducks were challenged with lethal DEV (CVCC AV1221) by intramuscular injection at 14 days post vaccination, then the ducks were observed for symptom of disease and death.【Result】The recombinant expression vector pT-UL2-GFP-gpt was correctly constructed, identified by double-enzyme digestion. After 8 hours of transfection, spindle cells with green fluorescent were appeared. After 8 rounds of plaque-purification, the purified rDEV-△UL2-GFP-gpt were obtained. The results of the PCR and sequencing indicated that the GFP expression cassette has already successfully insert into the DEV genome, which replaced 196-723 nucleotide of UL2. The recombinant virus possessed growth kinetics were similar to that of the parental virus, the cell titer peaked at 36 hours with the peak titer 106.2TCID50/0.1mL, and the supernatant titer peaked at 72 hours with the peak titer 105.5TCID50/0.1mL. The virus were passaged in CEF cells 20 times, the GFP gene was stably maintained in 1st to 5th passages, however, from the 6th passage, there was little CPE without green fluorescent, and in 15th to 20th passages, most CPE had no green fluorescent, GFP mutated during subculture. All immunized animals were protected against subsequent challenge with lethal DEV, the insertion of the GFP gene did not alter the protective efficacy of parental virus. 【Conclusion】In this research, the recombinant DEV expressing the green fluorescent protein were successfully constructed, and firstly has confirmed that the deletion of UL2 gene has no effect on virus replication in cells and the immunogenicity in ducks. This study laid a foundation for the research of the function of the DEV UL2 gene and the DEV vector vaccine. Reference | Related Articles | Metrics Cited: Baidu(1)

Select

Construction and Characterization of a Recombinant Duck Enteritis Virus Expressing VP2 Gene of Goose Parvovirus CHEN Liu, YU Bin, NI Zheng, HUA Jiong-gang, YE Wei-cheng, YUN Tao, ZHANG Cun Scientia Agricultura Sinica    2016, 49 (14): 2813-2821.   DOI: 10.3864/j.issn.0578-1752.2016.14.015 Abstract （444）   HTML （2）    PDF （1525KB）（482）       Knowledge map    Save 【Objective】Duck enteritis virus (DEV) and goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting ducklings, Muscovy ducklings and goslings. According to the most recent virus taxonomy reported in 2012 by the International Committee on Taxonomy of Viruses (ICTV), DEV (also referred to Anatid herpesvirus 1) is classified into the genus Mardivirus, the subfamily Alphaherpesvirinae of Herpesviridae. Many herpesviruses, such as Pseudorabies virus (PRV), Marek's disease virus (MDV), Herpesvirus of turkey（HVT）have been widely made as live viral vector for the expression of foreign antigens, and there were some reports about DEV as live viral vector in recent years. To control DEV and GPV infection, a recombinant vectored DEV expressing GPV VP2 was constructed in this study based on the bacterial artificial chromosome (BAC) clone pDEV-EF1 which carries DEV full-length genome (Chen L, et al. , 2015), and then the biological characteristics of the obtained recombinant virus rDEV-VP2 were analyzed to explore the possibility of rDEV-VP2 as duplex live carrier vaccine. 【Method】 The recombinant BAC clone pDEV-VP2 carrying GPV VP2 gene was generated by two-step Red/ET recombination in E. coli. pDEV-VP2 was constructed by inserting codon optimized-GPV VP2 expression cassette between DEV US7 and US8 genes on pDEV-EF1. The recombinant viruses rDEV-VP2 and rDEV-VP2-Cre without BAC sequence were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. And the growth curve in vitro, plaque size and expression of GPV VP2 in CEFs were analyzed. The antibody level of GPV VP2 in sera of rDEV-VP2-incoculated ducklings was detected by an indirect-ELISA method based on the GPV VP2 protein. 【Result】 The recombinant viruses rDEV-VP2 and rDEV-VP2-Cre were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Growth curves show that the growth kinetics of rDEV-VP2 was basically consistent with those of parental virus in vitro. And the plaque size of rDEV-VP2 was slightly increased compared to the parental virus rDEV-BAC. Immunofluorescence assay and Western blot analysis showed that GPV VP2 protein is expressed in recombinant virus-infected CEFs. And the rDEV-VP2 infection could induce 7-day-old Muscovy ducklings to produce antibody specific for GPV VP2. 【Conclusion】 In this study, the antigen gene VP2 of GPV was inserted into the genome of DEV US7 and US8, and an recombinant infectious BAC clone of DEV was successfully constructed. Then the corresponding recombinant virus rDEV-VP2 was rescued, and its cellular growth characteristics were basically consistent with those of parental virus, and rDEV-VP2 could induce Muscovy ducklings to produce VP2-specific antibody. These studies have laid a foundation for developing bivalent vaccine controlling DEV and GPV infection. Reference | Related Articles | Metrics Cited: Baidu(2)

Select

Selection of a Live Chicken Embryo Attenuated Duck Tembusu Virus Vaccine YU Ke-xiang, MA Xiu-li, YUAN Xiao-yuan, LIU Cun-xia, HU Feng, LING Hong-li, LI Yu-feng, HUANG Bing Scientia Agricultura Sinica    2016, 49 (14): 2822-2829.   DOI: 10.3864/j.issn.0578-1752.2016.14.016 Abstract （393）   HTML （1）    PDF （1165KB）（616）       Knowledge map    Save 【Objective】 Tembusu virus BZ-2010 strain was continuously passaged in specific-pathogen-free embryonic (SPF) eggs in order to select a live attenuated vaccine candidate of good safety and immunogenicity properties. 【Method】 Tembusu virus BZ-2010 strain was cultured for 120 passages in SPF eggs. The safety of the 120th passage viral strain was evaluated with 1-day-old SPF ducklings and 30-week-old egg-laying ducks. The property of virulent return of VC2 viral strain was evaluated with 1-day-old SPF ducklings. The neutralizing antibodies were detected after the 18-week-old breeding ducks were immunized with VC2 strain. The protective effects were evaluated after the 25-week-old breeding ducks were immunized with VC2 strain. E gene and NS4A gene of BZ_2010 and VC2 strains were amplified by RT-PCR and sequenced. 【Result】 The average death time of SPF eggs was shortened by passage virus and viral titer was increased with the escalation of passage times in SPF chicken embryonic eggs. ELD50 of the 20th virus was 10-5.3/0.1mL and ELD50 of the 120th virus was 10-5.8/0.1mL.The viral titer reached the plateau at passage 80 and remained unchanged further passages. The experimental ducks showed no clinical symptoms after 1-day-old ducklings and 30-week-old breeding ducks were immunized with VC2 strain by subcutaneous injection and by intramuscular injection, respectively. The results showed that VC2 strain had a good safety. No symptoms appeared in 1-day-old ducklings in which VC2 strain were cultured for 5 passages. 1-day-old ducklings were infected with the 5th tissue suspension and no symptoms were observed in liver pathological section by microscope. The results showed that VC2 strain had a good stability. Sequence analysis revealed that the E protein of Tembusu VC2 evolved amino acid changes in positions 86, 157, 189, 301, and 302, respectively. The NS4A protein of Tembusu VC2 only had one amino acid change in position 54 in that phenylalanine was replaced by Leucine. The level of antibodies rose very quickly, reached the plateau at the 4th week and remained a long time. Ducks were challenged by TMUV virulent strain at 2 and 50 weeks after immunization with VC2 strain in the experimental group. There was no symptom, normal stool, and regular egg production in the vaccinated group after challenge of virulent strain. The results showed that VC2 strain could provide complete protection for the challenge of TMUV virulent strain.【Conclusions】 An attenuated strain of TMUV with good immunogenicity and high safety was acquired through serial passages of SPF chicken embryos. The level of antibodies rose very quickly and remained a long time after immunization of the VC2 attenuated strain. The toxicity attack experiments showed that VC2 could provide complete protection for the challenge of TMUV virulent strain. Reference | Related Articles | Metrics

Select

Criterion of Ovarian Lesions of Duck Tembusu Virus Disease LIN Jian, YANG Zhi-yuan, HE Ping-you, DUAN Hui-juan, ZOU Li-hong, YANG Bao-shou, ZHAO Ji-cheng, PAN Jie, WANG Xiao-lei, LIU Li-xin, LIU Yue-huan Scientia Agricultura Sinica    2016, 49 (14): 2830-2836.   DOI: 10.3864/j.issn.0578-1752.2016.14.017 Abstract （364）   HTML （4）    PDF （1016KB）（452）       Knowledge map    Save 【Objective】The objective of this study is to understand the process and regularities of ovarian lesions on ducks infected with duck Tembusu virus, and to provide data for the efficiency evaluation of vaccine using laying ducks ovary pathological inspection.【Method】Seventy 260-day-old laying cherry ducks were inoculated intramuscularly with duck Tembusu virus (HB strain) at dosage of 0.5ml(100 DID50)/duck. Clinical symptoms were observed, and the egg production and feed intake were recorded daily. Serum samples were collected from all ducks for virus isolation via wing vein on 2 days post inoculation (dpi). Each serum sample was inoculated into five 6-day-old SPF chicken embryos via yolk-sac route at the inoculum of 0.1 ml per embryo. Then they were hatched at 37℃ further. The chicken embryos died in 24h were abandoned. The viral nucleic acid was detected by RT-PCR in the death chicken embryos during 24-72 h. If there were more than one (including one) death chicken embryos, and the nucleic acid testing was positive, then it was concluded that virus isolation was positive. On 4-10 dpi, 10 ducks were necropsied and the gross lesions of the reproductive system were observed every day. The pathologic rate was calculated, and the gross lesions of the reproductive system were conducted including whether there were eggs in the fallopian tube, whether the follicle was deformed, hemorrhaged or ruptured or not. According to the statistical pathologic rate, the time, the content of the examination and the criterions for determining lesion ovary were confirmed. 【Result】 (1) Feed intake and egg production decreased significantly on 3-6 dpi. The mental state of the duck on 7 dpi improved, and feed intake began to rise on 8 dpi. (2) Virus isolation of 70 ducks were all positive except one duck. The virus positive isolation rate was 98.6% (69/70) on 2 dpi. (3) On 4-10 dpi, a total of 64 laying duck reproductive organs can be determined. (4) On 4-10 dpi, the ovarian lesions rate were 66.7% (6/9), 100% (10/10), 100% (10/10), 100% (9/9), 100% (9/9), 100% (9/9) and 100% (8/8) respectively. (5) Eggs were found in fallopian tube of 2 ducks among 10 ducks that were necropsied on 4 dpi, and there was no egg found in fallopian tube of the remaining 62 ducks. The egg negative rate was 96.9(62/64). (6) On 4-10 dpi, the proportion of deformed follicular duck and hemorrhaged follicular duck were both 96.9% (62/64), and the proportion of deformed and hemorrhaged follicular duck was 95.3%（61/64）, while the proportion of ruptured follicular duck was 34.4% (22/64).【Conclusion】 (1) The time for the examination of the pathological changes of ovary was determined as 7 to 8 dpi. (2) The criterion of abnormal follicle is that one of the lesions of deformation and hemorrhage or both were found. The criterion of lesion ovary is that three abnormal follicles or more appeared and no egg in the fallopian tube. Reference | Related Articles | Metrics

Select

Dynamic Study on Maternal Antibody of Duck Tembusu Virus Disease Inactivated Vaccine (HB Strain) HAN Chun-hua, ZHAO Ji-cheng, DUAN Hui-juan, LIN Jian,YANG Zhi-yuan, XIE Jia, PAN Jie, WANG Xiao-lei, LIU Li-xin, LIU Yue-huan Scientia Agricultura Sinica    2016, 49 (14): 2837-2843.   DOI: 10.3864/j.issn.0578-1752.2016.14.018 Abstract （507）   HTML （4）    PDF （362KB）（486）       Knowledge map    Save 【Objective】The objective of this study is to evaluate the efficacy of maternal antibodies induced by Duck Tembusu Virus Disease Inactivated Vaccine and to determine the age of optimal initial immunity.【Method】Fertilized eggs were collected at random from the Cherry Valley Duck farm which was 135 days post-vaccination with Duck Tembusu Virus Disease Inactivated Vaccine (HB strain), ten progeny ducklings from the immunized breed ducks and 5 progeny ducklings from non-immunized breed ducks were randomly selected when they were 5 ,7 ,10, and 15 days old. Serum samples were collected from all ducks for the detection of maternal antibody, then the ducks were challenged with Duck Tembusu virus (HB strain) at 0.1ml（100DID50）/duck intramuscularly. Clinical symptoms of the challenged ducks were observed within 10 days, such as food intake, feces, abnormal clinical sighs and death. Serum samples were collected from all ducks for virus isolation via jugular vein on 2 days post inoculation (DPI). Each serum sample was inoculated into five 6-day-old SPF chicken embryos at the inoculum of 0.1 ml per embryo. Then they were hatched at 37℃ for 168h. The chicken embryos died within 24h were discarded. If more than one (including one) death chicken embryos were obsearved, then it was concluded that virus isolation was positive. The rate of protection of ducklings with maternal antibody and the morbidity of ducklings without maternal antibody were calculated. On 5 dpi, all ducklings were weighed respectively, and the average daily gain was calculated. The effect of maternal antibody on the weight gain of ducklings were analyzed by T test for paired samples. The efficacy of maternal antibodies was evaluated by neutralizing antibody titer, body weight changes and virus isolation.【Result】 (1) The number of positive maternal antibody titers peaked in 1 day old ducklings was 56.1%（37/66）, then fell to 40% (4/10) in ducklings on day 5, 50% (5/10) on day 7, 30% (3/10) on day 10, and 0% (0/10) on day 15. (2) On viral challenge, the control group showed signs of depression (20/20), neurologic disturbances (6/20) and death (2/20). Ducklings with positive maternal antibody titers showed mild depression. (3) On 5 dpi, the average daily gain of 5-, 7-, 10- and 15-day old ducklings with maternal antibody were 115.5, 142.8, 177.8 and 162.2g, respectively, and that of the ducklings without maternal antibody were 54.5, 91, 165 and 118.8g, respectively. (4) The rate of protection against challenge with DTMUV of 5-, 7-, 10- and 15-day old ducklings with maternal antibody were 50%(5/10), 60%(6/10), 20%(2/10) and 0%(0/10), respectively. The morbidity of 5-, 7-, 10- and 15-day old ducklings without maternal antibody were all 100%. (5) The average weight gain and efficacy reached a peak in 5-day old and 7-day old ducklings, which were 50% (5/10) and 60% (6/10), respectively. Although the maternal antibodies decreased between 10 days old and 15 days old ducklings (20% and 0%), it still has protective effect compared with the control group. 【Conclusion】(1) Duck Tembusu Virus Disease killed vaccine maternal antibodies, so it play an important role in the protection of 10-day-old ducklings against virus infection; (2) Vaccination age is optimized between 7 to 10 days of age. Reference | Related Articles | Metrics

Select

The Pathogenicity of Duck Reovirus on SPF Chicken Embryo LIU Xiao-li, LIU Ting, LIU Bo, CHENG Guo-fu, GU Chang-qin, ZHANG Wan-po, HU Xue-ying Scientia Agricultura Sinica    2016, 49 (14): 2844-2849.   DOI: 10.3864/j.issn.0578-1752.2016.14.019 Abstract （397）   HTML （3）    PDF （2757KB）（466）       Knowledge map    Save 【Objective】Previous studies showed that avian reovirus could infect vertically through egg, and avian reovirus can cause avian viral arthritis, respiratory and intestinal disease, myocarditis, hepatitis, immune suppression and so on. HP080421 strain of duck reovirus(DRV) isolated in the authors’ laboratory can cause soft duck feet as the main clinical features, and sick duck showed that a lot of white necrotic stove was on the surface of liver and spleen, and also kidney swelling and bleeding as the main pathological features. In this study, the pathogenicity of DRV to chicken embryo was investigated and whether the isolated HP080421 strain could infect chickens through the pathological changes of chicken was discussed, in order to provide a theoretical basis for the prevention and control of DRV infection.【Method】The infection model of SPF chicken to DRV was established through allantoic cavity inoculation SPF chicken embryos by using the isolated and identified HP080421 strains of DRV isolated in the lab. After chicks hatched from embryos, pathological examination methods such as clinical observation, pathological section examination, HE staining and immunohistochemical staining were used to study the pathobiology and pathogenicity of the SPF chicken embryo infected by DRV.【Result】Clinical observation found that chicken embryos were able to peck the shell after 22 - 23 days, but could not go out from the eggshell by themselves compared to the control group. At necropsy, liver and spleen were breakable and slightly swelling, many different size and yellow-white necrotic foci were consistently observed in the spleen and liver of the experimental group; the brain tissue was slightly swelling with few bleeding spots covered on it. Histopathological examination of H.E staining revealed necrotic foci in spleen and liver, which consisted of a necrotic center with lymphocytes infiltration at the periphery; in the Bursa of Fabricus, as well as in the thymus, lymphocyte depletion was apparent and cavities had developed in medulla. Besides, other organs such as lung, brain and kidney, showed different degrees of congestion and edema. Immunohistochemical detection showed that liver, lung, spleen and bursa of fabriciusa had positive signals, and were located in the cytoplasm and nucleus of epithelial cells and macrophages.【Conclusion】The results showed that the virus strain HP08421 could infect SPF chicken embryo and cause some specific pathologic changes. The pathological changes mainly focus on the liver and lymphoid organs, so DRV can be infected by vertical transmission of chickens, and can also lead to immune suppression. This study has expounded the possibility of infection of chickens by DRV, through the infection model of SPF chicken to DRV, as a matter of fact, in the actual process of production, farmers should prevent chicken embryo pollution by DRV to cut off the route of transmission, to achieve the purpose of preventing DRV infection. Reference | Related Articles | Metrics

Select

猪瘟 |猪瘟病毒 |流行病学 |地理信息系统 |数据库 WANG Qin, TU Chang-Chun, HUANG Bao-Xu, XU Lu, GAI Hua-Wu, FAN Xue-Zheng, GUO Huan-Cheng, XU He-Min, LI Jin-Hua, ZHAO Qi-Zu, NING Yi-Bao, ZHENG Ran, SHEN Qing-Chun Scientia Agricultura Sinica    2013, 46 (11): 2363-2369.   DOI: 10.3864/j.issn.0578-1752.2013.11.021 Abstract （588）      PDF （771KB）（1121）       Knowledge map    Save 【Objective】 The objective of this study is to strengthen the epidemiology information management of classical swine fever (CSF), grasp the worldwide CSF data, analyze and track CSF and predict the epidemic situation accurately. 【Method】The novel software program epidemiology information system of classical swine fever, developed by using DB, GIS and Bioinformatics technology, was developed and named as CSFinfo, which integrated with ArcGIS Desktop, ArcMapObjects, Delphi and DNAStar 6.0 etc. and based on Digital Map Database of China. 【Result】Above 1000 classical swine fever virus isolates from China, 554 E2 sequences from China and 642 E2 sequences from CSF database on EU reference laboratory for CSF (Hanover German) generate into the CSFinfo. China has become the second country which has a complete CSF database after establishing CSFinfo successfully. 【Conclusion】By using CSFinfo, information and pattern of CSF outbreak can be demanded easily and conveniently. Additionally, CSFinfo is a coupled software DNAstar6.0 software and it is a great innovation. The capability for dataset extension and phylogenetic analysis were performed by using CSFinfo. Establishment of CSFinfo can provide technical supports for CSF epidemiology detection and analysis of data, strengthen the ability to understand and handle distribution and occurrence trend of CSFV, improve the quality of analyze and reduce strategic decision risk for government of China. Reference | Related Articles | Metrics

Select

Inhibitory Activity of Chinese Herbal Aqueous Extracts to Tetracycline-Resistant E. coli of Swine HUANG Ming-Qian, KONG Xiang-Feng, XIAO Li-Chun, GUO Xiao-Quan Scientia Agricultura Sinica    2013, 46 (11): 2370-2376.   DOI: 10.3864/j.issn.0578-1752.2013.11.022 Abstract （627）      PDF （532KB）（661）       Knowledge map    Save 【Objective】 This study was conducted to determine the inhibitory activity of Chinese herbal aqueous extracts to tetracycline-resistant E. coli of swine in order to provide a basis for their application in the swine production. 【Method】 Paper disk method and PCR method were used to evaluate tetracycline sensitivity to 10 isolates of E. coli of swine and their tetracycline-resistance genes, respectively, as well as the agar diffusion and double dilution to test the inhibitory activity of Chinese herbal aqueous extracts and their mixtures with tetracycline to 10 isolates of resistant E. coli of swine. 【Result】 All of the 10 tested isolates were resistant to tetracycline, 10 of which carried the tet A, two of which carried tet B, and 9 of which carried tet C. The extracts of Fructus crataegi, Fructus forsythiae, Flos lonicerae and Fructus schisandrae had stronger antibacterial activity, while their mixtures with tetracycline presented the decreased inhibition zone diameter and increased minimal inhibitory concentration. 【Conclusion】 The tetracycline resistance of the tested strains was related to the existances of resistance genes, including tetA, tetB and tetC. The extracts of 4 Chinese herbs tested in this study had stronger inhibitory activity against the isolates of E. coli, and could not be used with the tetracycline togather. Reference | Related Articles | Metrics Cited: Baidu(3)

Select

Study on the Cloning of Coding Sequence and Ontogenetic mRNA Expression of Ovine UCP4 Gene ZHOU Sha-Sha, LIU Bao-Feng, WEI Lin-Lin, WANG Jing-Lin, LIU Jian-Hua, LIANG Chen, QIAO Li-Ying, LIU Wen-Zhong Scientia Agricultura Sinica    2013, 46 (10): 2110-2118.   DOI: 10.3864/j.issn.0578-1752.2013.10.017 Abstract （438）      PDF （735KB）（646）       Knowledge map    Save 【Objective】This study aims to clone the coding sequence (CDS) of ovine UCP4 gene, to analyze the CDS and its coding protein structures, and to profile the ontogenetic mRNA expression so that to lay a theoretical foundation for future research on the structure and function of this gene. 【Method】The CDS was amplified from ovine cerebrum tissue by reverse transcription- polymerase chain reaction (RT-PCR). Physicochemical features and structural characteristics of ovine UCP4 were analyzed by bioinformatics methods. Ninety-six animals from two sheep breeds with significant differences in tail types (Guangling Large Tailed- and Small Tailed-Han Sheep) were used to study the ontogenetic mRNA expression by real-time quantitative PCR in eight tissues (cerebrum, cerebellum, hypothalamus, pituitary, and subcutaneous fat, perirenal fat, mesenteric fat and tail fat) from 2-to 12-month old age at two-month interval. 【Result】The sequencing data showed that the length of complete CDS was 972 bp encoding a protein of 323 amino acids. The isoelectric point (pI) of the protein was 9.43, and the molecular weight was 35.73 kDa. The percentages of helix, strand and loop were 56.04%, 7.12% and 36.84%, respectively, in the secondary structure. This transmembrane protein had no signal peptide but with two predicted glycosylation sites and 15 potential phosphorylation sites. The UCP4 mRNA expressed in all these tissues, but overexpressed in brain tissue other than in adipose tissue. Generally, breed, tissue and the months of age had significant influences on the UCP4 mRNA expression. 【Conclusion】 The obtained whole CDS of ovine UCP4 gene, expression profile and its limiting factors are of scientific significance to further studies on the relationship between the gene structure and energy metabolism. Reference | Related Articles | Metrics Cited: Baidu(1)

Select

Effects of Prion Protein on the Regulation of Classical and Alternative Activation of BV2 Microglia in vitro FU Yong-Yao, SHI Fu-Shan, WANG Ji-Hong, YANG Li-Feng, ZHOU Xiang-Mei, YIN Xiao-Min, ZHAO De-Ming Scientia Agricultura Sinica    2013, 46 (9): 1932-1938.   DOI: 10.3864/j.issn.0578-1752.2013.09.021 Abstract （403）      PDF （557KB）（770）       Knowledge map    Save 【Objective】 The purpose of this study is to investigate the effects of PrPC on various forms of microglial activation. 【Method】 BV2 microglia were treated, respectively, with IFN-γ, IL-4, or IL-10, and the mRNA expression of PRNP was examined by RT-PCR. Then the effects of si-RNA-mediated disruption of PRNP on different parameters of microglial activation in IFN-γ, IL-4, or IL-10-stimulated microglia were analyzed by RT-PCR and western-blot. 【Result】PRNP mRNA expression was invariably downregulated in microglia upon exposure to IFN-γ, IL-4, or IL-10. PRNP silencing prior to cytokines treatment reduced the responsiveness of microglia to INF-γ treatment, significantly altered IL-4-induced microglial activation phenotype, and had no effect on IL-10-induced microglial activation.【Conclusion】Together, these results support a role of PrPC in the modulation of the shift of microglia from a quiescent state to an activated phenotype and in the regulation of the microglial response during classical and alternative activation. Reference | Related Articles | Metrics

Select

Expression of Ghrelin and GHS-R1a mRNA in the Bovine Oocytes During in Vitro Maturation MI Yan, ZHANG Ling-Li, LI Hai-Jun, DU Chen-Guang, CAO Gui-Fang Scientia Agricultura Sinica    2013, 46 (9): 1939-1945.   DOI: 10.3864/j.issn.0578-1752.2013.09.022 Abstract （588）      PDF （597KB）（528）       Knowledge map    Save 【Objective】 The aim of this study was to investigate the possibility of Ghrelin and GHS-R1a mRNA expression in bovine oocytes produced in vitro and the relationship between FSH and the expression of Ghrelin and GHS-R1a mRNA. 【Method】Relative real time RT-PCR technology was applied to detect the pattern of Ghrelin and GHS-R1a mRNA expression in oocytes, and the effect of different concentrations FSH and the combination of FSH and FSH binding inhibitor on the expression of Ghrelin and GHS-R1a mRNA were observed. 【Result】 Relative real time RT-PCR experiments confirmed that the Ghrelin and GHS-R1a mRNA levels in oocyte varied depending on the developmental stage, with the significant decrease of Ghrelin and GHS-R1a mRNA expression at 8 h in oocyte maturation process. And addition of FSH inhibited the expression of Ghrelin and GHS-R1a mRNA within oocytes, and this inhibition effect could be attenuated by FSH binding inhibitor. 【Conclusion】 The expression of Ghrelin and GHS-R1a mRNA could be declined with the rising of FSH concentration during in vitro maturation of bovine oocytes. Reference | Related Articles | Metrics

Select

Recent Advances in Research on Bovine Oocyte Maturation Technology in vitro JIA Zhen-Wei, TIAN Jian-Hui, AN Lei, ZHANG Jia-Xin Scientia Agricultura Sinica    2013, 46 (8): 1716-1724.   DOI: 10.3864/j.issn.0578-1752.2013.08.022 Abstract （521）      PDF （483KB）（1088）       Knowledge map    Save Bovine oocyte in vitro maturation (IVM) is an important reproductive technology, however, as these oocytes were maturated in vitro that is deficient in growing process and some crucial event in vivo during antral follicular development prior to the LH surge, which interfere cytoplasmic maturation, thus resulting in reduced developmental capacity compared to in vivo maturated counterparts. Oocyte maturation is a complex process that involves events of nuclear, cytoplasmic and molecular maturation. Bovine oocyte maturation during in vitro culture is affected by many factors including source of oocyte, bidirectional communication between the oocyte and its surrounding cumulus cells and maturation environment. Up to date, to improve the developmental competence of bovine oocytes in vitro, some new technologies, for example, attenuating spontaneous oocyte maturation, improving oocyte maturation using exogenous oocyte-secreted growth factors and simulated physiological oocyte maturation have been developed for enhancing bovine oocytes maturation in vitro by simulating in vivo oocytes growing environment. This paper reviewed the maturation processes of bovine oocytes, the factors affecting oocyte maturation in vitro and the latest approach to improve the maturation rate of bovine oocytes in vitro. Reference | Related Articles | Metrics

Select

Cloning and Bioinformatics Analysis of Full-Length cDNA Sequence of YAP1 Gene in Sheep SUN Wei, LI Da, SU Rui, MA Yue-Hui, GUAN Wei-Jun, ZHANG You-Fa, CHEN Ling, WU Wen-Zhong, ZHOU Hong Scientia Agricultura Sinica    2013, 46 (8): 1725-1735.   DOI: 10.3864/j.issn.0578-1752.2013.08.023 Abstract （547）      PDF （945KB）（942）       Knowledge map    Save 【Objective】 There was no report on YAP1 gene and its protein in the past days, this study was carried out in order to clone the YAP1 cDNA in sheep and provide a basic foundation for the study of YAP1 genetic characters , as well as analyze its tissue different level expression patterns. 【Method】 Hu sheep was regarded as the research object, and the full-length cDNA sequence of the YAP1 gene was acquired from the longissimus dorsi muscle by RT-PCR and RACE, and the sequence was analyzed in depth by bioinformatics method. 【Result】 The full-length cDNA sequence of the YAP1 gene was successfully obtained from Hu sheep longissimus dorsi muscle. Bioinformatics technology was successfully used to analyze the YAP1 homology among different species, transmembrane region of YAP1 sequence, subcellular localization, hydrophilic, potential signal peptide cleavage sites, glycosylation sites, phosphorylationlocus, product function prediction analyisis, YAP1 secondary structure and teriary structure.【Conclusion】The full-length of YAP1 gene was 1 712bp, including 1 212bp coding sequence encoding 403 amino acids. The homology analysis found that the nucleotide sequence and amino acid sequence of sheep YAP1 gene share the highest 78.77% and 92.51% identity with the Cricetulus YAP1 while there was no large difference between Human and Chimpanzee. The amino acid sequence analysis revealed that the YAP1 gene of sheep encoded water-soluble protein and its relative molecular weight was 44079.0 Da, isoelectric point was 4.91, was a kind of hydrophilic non-transmembrane protein. Subcellular localization of YAP1 was in the mucleus, and it did not belong to the secreted protein. The YAP1 protein contained 33 phosphorylation sites, 7 glycosylation sites and 2 WW domain. The secondary structure of YAP1 was mainly composed of random coil. The tertiary structure of domain area of YAP1 protein showed a forniciform helix structure. Forecast YAP1 had a key role in regulatory functions process. The above studies have laid an information foundation for further study of the YAP1 gene function in the sheep muscle development process. Reference | Related Articles | Metrics Cited: Baidu(8)

Select

Molecular Characteristics of Extended-Spectrum β-Lactamases in Clinical Isolates of Proteus mirabilis from Poultry PAN Yu-Shan, YUAN Li, WU Hua, LIU Jian-Hua, LIU Zhen-Zhen, ZHAO Yi-Shuang, HU Gong-Zheng Scientia Agricultura Sinica    2013, 46 (7): 1463-1469.   DOI: 10.3864/j.issn.0578-1752.2013.07.017 Abstract （569）      PDF （703KB）（764）       Knowledge map    Save 【Objective】This experiment was conducted to explore the genotpye of extended-spectrum β-lactamases (ESBLs) gene and the genetic environment of blaCTX-M carried by Proteus mirabilis. 【Method】The isolates were characterized by isolation and identification, antimicrobial susceptibility testing, confirmation of ESBLs, resistant genes screening, and conjugation experiments. PCR mapping was performed to investigate the genetic environment of blaCTX-M. 【Result】 Among the 21 P. mirabilis clinical isolates from poultry, 10 (47.6%) isolates exhibited extended-spectrum β-lactamase (ESBL) phenotypes. The most common types of ESBL gene screened by PCR and sequencing experiments were blaCTX-M-14 (n=6) , blaOXA-1 (n=6), followed by blaCTX-M-65 (n=4). The blaOXA-10 (n=1) was also detected, while blaSHV was not detected. Nucleotide sequence analysis revealed that insertion sequence ISEcp1B elements present in the upstream region of blaCTX-M genes in all ESBL-producing P. mirabilis, and IS903D was also detected in downstream of CTX-M-9 group genes.【Conclusion】This is the first report the blaCTX-M-65 gene was found in P. mirabilis in China, and the results showed that CTX-M-tpye ESBL in P. mirabilis is no longer an unusual phenomenon in poultry origins. Reference | Related Articles | Metrics

Select

Effects of Vitamin D3 on Growth Performance, Slaughter Performance, Carcass Quality and Tibia Quality of Geese WANG Di, WANG Bao-Wei, GE Wen-Hua, ZHANG Ming-Ai, YUE Bin, CHEN Miao-Lu, WANG Jiao, MENG Ling-Feng Scientia Agricultura Sinica    2013, 46 (7): 1470-1480.   DOI: 10.3864/j.issn.0578-1752.2013.07.018 Abstract （766）      PDF （928KB）（854）       Knowledge map    Save 【Objective】This experiment was conducted to study the effects of dietary vitamin D3 level on growth performance, carcass quality and tibia quality of geese aged 0 to 15 weeks.【Method】Three hundred and sixty 1-day-old Qingnonghui liver goose were selected and randomly assigned into 6 treatment groups with 6 replicates for each treatment and 10 in each replicate．The geese were fed with basal diet supplemented with 0, 200, 400, 800, 1 600, and 3 200 IU•kg-1 vitamin D3, respectively. Geese were raised for fifteen weeks and their feed intake and weight were recorded to get the growth performance. One or two geese were selected and slaughtered from each replicate to measure slaughter performance, carcass quality and tibia development at the end of 4 and 15 weeks.【Result】Osteomalacia rate of the controlled group was 36.67% between weeks 0 to 4 and 26.67% between weeks 5 to 15, which was remarkably higher than that of treatment groups. There was no clinical poisoning symptoms when addition of vitamin D3 reached 3 200 IU•kg-1. Compared with the controlled group, the daily gain and daily feed intake of geese aged 0 to 4 weeks were significantly increased by supplementation of 400 IU•kg-1 vitamin D3 (P＜0.05)．Adding vitamin D3 could enhance the daily gain of geese aged 5 to 15 weeks significantly (P＜0.05), but there was no significant difference among the treatment groups. Supplementation of 800 IU•kg-1 vitamin D3 could significantly improve the slaughter rate (P＜0.01) and significantly increase the half net carcass rate of geese aged 15 weeks (P＜0.05). Adding vitamin D3 significantly affected the color of pectoral muscle (P＜0.05). BMD, bone weight, tibia calcium and tibia phosphorous of geese aged 4 weeks and BMD, bone weight and bone ash of geese aged 15 weeks were significantly improved by supplementation of vitamin D3(P＜0.05). 【Conclusion】 Vitamin D3 plays a regulatory role in the growth performance, slaughter performance, carcass quality and tibia quality of geese. Osteomalacia rate of geese increased notably without the addition of vitamin D3. The optimal vitamin D3 level in geese diet should be 471.70 IU•kg-1 between 0 and 4 weeks, and 548.63 IU•kg-1 between 5 and 15 weeks. Reference | Related Articles | Metrics

Select

Study on Induction of Porcine Putative Embryonic Germ Cells into Nerve Cells in vitro CONG Yi-Mei, MA Jing, SUN Rui-Zhen, WANG Jian-Yu, XUE Bing-Hua, HU Kui, YIN Zhi, LIU Zhong-Hua Scientia Agricultura Sinica    2013, 46 (6): 1272-1279.   DOI: 10.3864/j.issn.0578-1752.2013.06.022 Abstract （659）      PDF （894KB）（770）       Knowledge map    Save 【Objective】The objective of the study is to detect the potential of porcine putative embryonic germ cells differentiating into nerve cells. 【Method】Co-culture of Gonad-mesonephros (GM) region stromal cells and primordial germ cells was used to get porcine putative embryonic germ cells. Directional differentiation method was adopted for neural differentiation. 【Result】 Compared with MEF , GM region stromal cells as feeder could support the growth of porcine putative embryonic germ cells with no significant difference in the proliferation of embryonic germ cells. Porcine putative embryonic germ cells had strong AP activity, Q-PCR results indicated the expression of Oct4, Sox2 and Nanog genes. Proliferating ability of porcine putative embryonic germ cells showed a "S" type, namely the growth of latency, logarithmic phase and plateau phase. Embryoid body could be derived after suspending culture of porcine putative embryonic germ cells in vitro. After induction, porcine putative embryonic germ cells could differentiate into a variety of neural cell types with expression of neural stem cells, neurons and glial cells markers.【Conclusion】These results indicate that porcine putative embryonic germ cells can be derived from porcine early gonadal and have the ability of differentiating into a variety of neural lineage cells in vitro. Reference | Related Articles | Metrics

Select

The Research and Control Situation of Ecology and Animal Poisoning of Locoweed in Western Natural Grassland of China ZHOU Qi-Wu, ZHAO Bao-Yu, LU Hao, WANG Shan-Shan, ZHANG Liang, WEN Wei-Li, YANG Xiao-Wen Scientia Agricultura Sinica    2013, 46 (6): 1280-1296.   DOI: 10.3864/j.issn.0578-1752.2013.06.023 Abstract （791）      PDF （1054KB）（991）       Knowledge map    Save Natural grassland is an important production base of grassland and animal husbandry, and it is also a very important ecological barrier in China, so it is a great strategic importance for China to maintain ecological and food security and protect survival environment of human beings and fulfill permanent utilization of grassland. For a long time, some natural and human factors lead to grassland desertification and contagious poisonous weeds in the rangeland, such as grassland drought, overgrazing, blind reclamation, shortages of fund and lagging management. Especially in the last few decades, the rapid spreading of poisonous-weeds have caused some ecological problems such as decrease of vegetation community and biodiversity, single vegetation and desertification, and it seriously affects the grassland ecological balance and the sustainable development of local animal husbandry. “Locoweeds” has been a primary poisonous weed in the western natural grassland, and it brings about a billion RMB in economical losses every year. The research history, species and ecological distribution and the situation of the toxicity disaster, the toxic ingredients and mechanism of locoweeds in grassland are summarized in this paper, and the problems of locoweed research in the future are given, it is of great significance for understanding the ecological function of locoweeds and comprehensive control of the locoweed disaster in western natural grassland of China. Reference | Related Articles | Metrics Cited: Baidu(21)

Select

Continuous Observation of Subgroup J Avian Leukosis for Three Groups of Commercial Layer Chicken BIAN Xiao-Ming, LI De-Qing, ZHAO Peng, CUI Zhi-Zhong Scientia Agricultura Sinica    2013, 46 (2): 409-416.   DOI: 10.3864/j.issn.0578-1752.2013.02.020 Abstract （484）      PDF （860KB）（539）       Knowledge map    Save 【Objective】 The objective of the study is to understand the occurrence and development of subgroup J avian leukosis in the commercial layer chickens as well as the relationship with the isolation of the virus and antibodies.【Method】Thirty-one chickens suspectable of ALV-J at the age of 39 to 43 weeks from 3 groups were observed and compared continuously in clinical status, pathology, viremia and development of neutralizing antibodies. 【Result】 It indicated that the infection rate with ALV-J in these three groups was very high. Twenty-eight chickens were infected with ALV-J or had persistent viremia, of which 14 were detected as immunotolerized persistent viremia without neutralizing antibodies. Indicative myelocytomatosis of ALV-J was found in chickens of these 3 groups, and could be detected in almost all of the organs and tissues. Of these 31 chickens, 13 had tumor/hemangioma in three or more organs and 1 chicken had in at least 5 organs.【Conclusion】Result of this observation indicates that ALV-J is still epidemical in Chinese commercial layer chickens, and the virus is getting stronger during the epidemic. Reference | Related Articles | Metrics Cited: Baidu(4)

Select

Establishment of Hybridoma Cell Lines Secreting Anti-Kasugamycin Monoclonal Antibody and Identification of Their Immunological Properties GONG Fang, ZHI Ai-Min, LI Qing-Mei, HU Xiao-Fei, ZHAO Qi-Fa, ZHANG Xiao-Hui, LU Qi-Yu, ZHANG Gai-Ping Scientia Agricultura Sinica    2013, 46 (2): 417-423.   DOI: 10.3864/j.issn.0578-1752.2013.02.021 Abstract （662）      PDF （531KB）（787）       Knowledge map    Save 【Objective】 The aim of the study is to generate high sensitivity and high specificity monoclonal antibody against kasugamycin and identify its immunological characteristics.【Method】KSM was coupled with the carrier protein BSA and OVA by using EDC method and the immunogen KSM-BSA and coating antigen KSM-OVA were synthesized respectively, and SDS-PAGE was used to identify KSM-BSA. Balb/c mouse was immunized by using KSM-BSA. Immunization interval was 4 weeks. The titer of polyclonal antibody was detected by indirect ELISA and blocking ELISA after four times immunization. The high titer and high sensitivity mouse was selected for cell fusion. High sensitivity and high specificity monoclonal antibody was prepared after several times subcloning,and the immunological characteristics were characterized. The preparation of ascites was carried out using in vivo induction method. 【Result】 SDS-PAGE results showed that KSM-BSA artificial antigen was synthesized successfully.Indirect ELISA showed a high titer above 1:104 of the antiserum of all the six Balb/c mouse. The sensibility of antiserum of No.3 mice was the best, which IC50 was 73.9ng•mL-1. Hybridoma cell lines of 1G7 and 2C8 were screened after cell fusion, all the isotypes of the mAb were IgG1.Titers of the mAb were 1:5.12×103 and 1:1.28×103 in supernatant,1:5.12×105 and 1:2.56×105 in ascites. The IC50 of KSMmAb was 8.902 ng•mL-1 to kasugamycin. The rate of cross reaction of the mAb with other compounds was less than 0.03%.【Conclusion】KSM artificial antigens were synthesized successfully and the monoclonal antibody with high sensitivity and specificity was obtained. This study has laid a foundation for a KSM immunological fast detection method. Reference | Related Articles | Metrics Cited: Baidu(4)

Select

Fluorescent Quantitative PCR as an Alternative Method for Efficacy Testing of Lapinized Hog Cholera Virus CHEN Kai, YAO Hua-Wei, WANG Chang-Jiang, XU Lu, FAN Xue-Zheng, ZHAO Qi-Zu, ZOU Xing-Qi, ZHU Yuan-Yuan, ZHAO Yan, YANG Guang-You, WANG Qin Scientia Agricultura Sinica    2013, 46 (1): 162-169.   DOI: 10.3864/j.issn.0578-1752.2013.01.019 Abstract （747）      PDF （609KB）（636）       Knowledge map    Save 【Objective】 A rapid, sensitive and specific one-step fluorescent quantitative PCR method as a substitute for rabbit fever testing for hog cholera lapinized virus (HCLV) vaccine efficacy was established. 【Method】 A pair of primers and a HCLV specific MGB probe were designed on the 3’UTR region of the HCLV genome for fluorescent quantitative PCR (FQ-PCR). The method was tested for specificity, sensitivity and conformity after optimization. 【Result】 The FQ-PCR sensitivity was 4.35 cDNA copies. The correlation coefficient between CT value and cDNA copies was 0.9998. Amplification efficiency was 101.14%. The FQ-PCR method was specific for HCLV and did not show amplifications for CSFV, BVDV, BDV, PRRSV, FMDV and other pathogens. A total of 34 samples from 17 batches of the four vaccine manufacturers were tested after serial dilutions using the rabbit fever test and FQ-PCR. Eleven samples were disqualified for lack of fever in two rabbits with the Ct values falling between 21.15 and 27.30 and viral content of 8.80×102copy/μL-6.52×104copy/μL. Twelve samples induced fever in one rabbit and no fever in the others with the Ct values between 17.47 and 23.70 and viral content of 1.10×104copy/μL-8.55×105copy/μL. The Ct values of 11 positive samples with both rabbits showing typical fever were from 17.10 to 20.81 with the viral content of 8.27×104copy/μL-1.11×106copy/μL.【Conclusion】The FQ-PCR kits established in this study is specific, sensitive and positively correlated with rabbit fever testing, and thus could be used for quantitative examination of semi-finished HCLV vaccine. Reference | Related Articles | Metrics Cited: Baidu(15)

Select

Establishment of Cell Lines Transcribing shRNA Targeted to Jiv Gene and Constuction of CSFV Resistant Transgenic Piglet GUO Kang-Kang, LEI An-Min, NING Peng-Bo, CHENG Min, HE Lei, LIU Wei, TAN Xue-Chao, XU Lei, CAO Wei-Wei, ZHANG Yan-Ming Scientia Agricultura Sinica    2013, 46 (1): 170-178.   DOI: 10.3864/j.issn.0578-1752.2013.01.020 Abstract （558）      PDF （782KB）（615）       Knowledge map    Save 【Objective】To construct postive cell strans inserted the shRNA fragments targeting to porcine J-Domain protein interacting with virus gene, the interference effects were evaluated by comparing the proliferation of classical swine fever virus (CSFV) in constructed cell strains and the cell strain with significant CSFV resistance would be provided as the basic materials for constructing the CSFV-resistant transgenic pig. 【Method】 Four shRNA interfering fragments were designed tagerting to pig Jiv gene and 4 lentivirus inserted the designed shRNA fragment（named P1, P2, P3 and P4）. Four positive PK-15 cell strains transfected with lentivirus were established individually and the interference effects of CSFV were evaluated by determining the RNA of CSFV in positive cell strains after 72 h infected with CSFV by real-time RT-PCR . The porcine fetus fibroblasts were isolated and infected with lentivirus P2 which interfered the proliferation of CSFV remarkerably in cell. A positive porcine fetus fibroblast strain stable expressing the shRNA interfering fragment targeting to Jiv gene was established which also was the donor cells for constructing the transgenic pig. The nuclear of positive pircine fetus fibroblast was transferred into matured enucleated porcine oocytes, and somatic embryos were obtained and transplanted into the receptor sows for constructing transgenic pig. The foreign gene was identified in obtained transgenic pig.【Result】 Four PK-15 cell strains inserted into the shRNA interfering fragments targeting to the Jiv gene of porcine were established, the P2 cell strain (inserted into P2 interfering fragment) was significantly CSFV-resistant. The nuclear of porcine fetus fibrlblast inserted P2 interfering fragment was donor cells for constructing transgenic pig by somatic cell cloning and embryo transplantation, and a transgenic piglet was obtained.【Conclusion】 The expression of Jiv gene can influence the proliferation of CSFV in cells. A PK-15 cell strain with stable expressing shRNA P2 was established which could resist CSFV proliferation remarkablly. A trnasgenic piglet was constructed by somatic cell cloning and embryo transplantation which carries the P2 interfering fragment targeting to porcine Jiv gene. Reference | Related Articles | Metrics Cited: Baidu(1)

Select

Construction of a Chimeric Classical Swine Fever Virus C-strain Containing E2 of Group Ⅱ Isolate Remains and Its Biological Characteristics TONG Chao, CHEN Ning, LIAO Xun, YUAN Xue-Mei, LI Xiao-Liang, FANG Wei-Huan Scientia Agricultura Sinica    2013, 46 (1): 179-186.   DOI: 10.3864/j.issn.0578-1752.2013.01.021 Abstract （533）      PDF （589KB）（752）       Knowledge map    Save 【Objective】 The classical swine fever (CSF) C-strain vaccine has proven to be safe and highly efficacious. However, classical swine fever virus (CSFV) genotype Ⅱisolates became dominant in recent years, making it more challenging to bring the disease into total control. This study was attempted to engineer, by reverse genetic approach, a novel C-strain-based vaccine candidate that matches molecular variations of field isolates, and to characterize its biological features. Those results could provide foundations for further development of a marker vaccine for differential diagnostic purpose.【Method】An infectious recombinant CSF virus was generated by exchanging the 5’-end 870-bp of E2 gene in C-strain with the same fragment from group 2 strain HZ1-08. The resulting recombinant virus RecCHZE2 was rescued and its pathogenicity and immunogenicity were characterized in rabbits and pigs. 【Result】 Rabbits inoculated with RecCHZE2 exhibited typical temperature profiles similar to the C-strain with spleen enlargement. Pigs inoculated with the recombinant chimeric virus did not show fever and significant decline of the number of white blood cells. Mild transient viremia was detected at day 12 post-inoculation. The antibody responses specific to the field strain HZ1-08 reached the peak 20 days post-inoculation. The neutralizing antibody titers from pigs immunized with RecCHZE2 were similar to both HZ1-08 and C strains while the titer from pigs immunized with RecC were lower for group 2 strain HZ1-08.【Conclusion】The recombinant virus RecCHZE2 remains avirulent but differs in immunogenic properties from the C-strain. Reference | Related Articles | Metrics

Select

Construction and Rescue of Recombinant Classical Swine Fever Virus with Shimen Structure Protein and Flag Marker ZHU Yuan-Yuan, HAN Tao, ZOU Xing-Qi, FAN Xue-Zheng, XU Lu, WANG Qin, ZHAO Qi-Zu Scientia Agricultura Sinica    2013, 46 (1): 187-194.   DOI: 10.3864/j.issn.0578-1752.2013.01.022 Abstract （523）      PDF （605KB）（587）       Knowledge map    Save 【Objective】Vaccination is still the main strategy for CSFV control in China. CSFV HCLV(Hog Cholera Lapinized Virus, HCLV) strain could protect effectively, but it couldn’t be serologically differentiated between infected pigs and vaccinated pigs (DIVA principle), which could been effectively solved by marker vaccine and marker diagnostics.【Method】Based on infectious cDNA clone of CSFV T strain, which is a temperature-sensitive mutation strain, the complete structural protein-coding region of T strain had been replaced by that of ShiMen strain , and the recombinant plasmid SMT was constructed. Additionally, the Flag gene was inserted into C end of E1 protein of the recombinant virus as a positive marker, and the pSMT-Flag was constructed successfully. After electroporation, the recombinant viruses vSMT and vSMT-Flag were passaged by SK6 cell. 【Result】Two recombinant plasmids were identified by specific restriction enzymes，the rescued viruses could be passaged successfully and confirmed by RT-PCR, sequencing and immunoperoxidase monolayer assay(IPMA). 【Conclusion】 This marker virus could react with the Flag specific monoclonal antibody while could not influence the combination between E2 protein and its specific monoclonal antibody, which could be a potential CSF novel marker vaccine. Reference | Related Articles | Metrics Cited: Baidu(2)

Select

The Regulation of Autoinducer-2 in Avian Pathogenic Escherichia coli BAI Hao, HAN Xian-Gan, LIU Lei, DAN Xue-Qin, SONG Jun, LIU Rui, DONG Hong-Liang, LIU Hai-Wen, DING Chan, YU Sheng-Qing Scientia Agricultura Sinica    2012, 45 (24): 5110-5116.   DOI: 10.3864/j.issn.0578-1752.2012.24.017 Abstract （512）      PDF （599KB）（604）       Knowledge map    Save 【Objective】 The aim of the study was to investigate the modulation of AI-2 on the biofilm forming ability and virulence in Avian Pathogenic Escherichia coli (APEC). 【Method】 The improved crystal violet seme-quantitative method and fluorescence staining method were used to study the effects of AI-2 on the biofilm forming ability in APEC. The effects of AI-2 on the mRNA levels of the virulence genes were analyzed using real-time PCR. Viable bacteria counting method was used to evaluate the effects of AI-2 on the capability of APEC to adhere and invade DF-1cell. 【Result】The results showed that the biofilm forming ability decreased at the concentration of 0.185 mmol•L-1 AI-2,while the biofilm forming ability had no significant change at the concentration of 0.037mmol•L-1 and 0.278 mmol•L-1 AI-2. Real-time PCR showed that Al-2 decreased the transcription of pfs, vat, lux, tsh, fuyA, iucD genes, while increased the transcription of ompA and iss genes. The adherence and invasion was decreased to 57.35% and 36.64% by supplementation with AI-2 in APEC. 【Conclusion】The biofilm forming ability increased at an optimal concentration of AI-2. The transcription of the virulence genes, the adherence and invasion to DF-1 cells of the bacteria were decreased by supplementation with AI-2 in the medium to culture the APEC. These findings will be of benefit to future studies of the role of AI-2 in APEC. Reference | Related Articles | Metrics Cited: Baidu(34)

Select

Research on the Combined Forecast Model Method Based on BP Neural Network Improved by Genetic Algorithm LIANG Yi, LIU Shi-Hong Scientia Agricultura Sinica    2012, 45 (23): 4924-4930.   DOI: 10.3864/j.issn.0578-1752.2012.23.020 Abstract （551）      PDF （644KB）（881）       Knowledge map    Save 【Objective】 The combined forecasting model for studying the classic swine fever morbidity was proposed.【Method】The data was processed by ARIMA and GM(1,1) initially, then the results were used as the inputs of the majorizing BP neural network.【Result】The combined model was used to analyze the monthly data from 2000/01 to 2008/05, and the accuracy of the forecasting results from 2008/06 to 2009/06 was 97.379%. The prediction accuracy of the combined model increased by 5.469%, 3.499%, and 1.188%, respectively, compared with BP neural network, ARIMA, GM(1,1), which suggest that the combined model is more steady than traditional methods.【Conclusion】This research has supplied an efficient analytical tool for animals diseases forecasting work, verified the feasibility of the combined model in animal diseases forecasting research, and also has provided references to other animal diseases. Reference | Related Articles | Metrics Cited: Baidu(1)

Select

Construction and Animal Experiment of a Recombinant Adenovirus Expressing NcSAG1 Protein of Bovine Neospora caninum JIA Li-Jun, ZHANG Shou-Fa Scientia Agricultura Sinica    2012, 45 (22): 4705-4712.   DOI: 10.3864/j.issn.0578-1752.2012.22.017 Abstract （564）      PDF （586KB）（525）       Knowledge map    Save 【Objective】A recombinant adenovirus expressing NcSAG1 protein of bovine N. caninum was constructed. Balb/c mice were immunized with the recombinant adenovirus to evaluate the levels of humoral and cellular immune responses. 【Method】NcSAG1 gene of bovine N. caninum was amplified by PCR. pMD18-T-NcSAG1 and pCR259- NcSAG1 were constructed. The correct pCR259-NcSAG1 was transformed into HighQ-1 Transpose-Ad™ 294 and HighQ-1™ competent cells to construct transpose-Ad-NcSAG1 recombinant adenovirus. Coated with liposome, Transpose-Ad-NcSAG1, linearized by PacI, was transfected into QBI- HEK293 cells to package recombinant adenovirus Ad5-NcSAG1. The recombinant adenovirus Ad5-NcSAG1 was detected by PCR. The expression of NcSAG1 gene in QBI-HEK293 cells was detected by Western blotting. After the virus titer was determined, the virus fluid was collected to inoculate Balb/c mice and the levels of IgG antibody and cytokines IFN-γ, IL-4 in the sera were measured to evaluate the recombinant adenovirus effect. 【Result】The size of NcSAG1 gene was 982 bp. The nucleotide sequence of the gene shared 99.2% homology with that in GenBank (AF132217). The recombinant adenovirus Ad5-NcSAG1 was successfully packaged in 293 cells. The protein expressed by Ad5-NcSAG1 was 33kD and had good reactogenicity. The titer of the recombinant adenovirus Ad5- NcSAG1 was 1010TCID50/mL. The Ad5-NcSAG1 recombinant adenovirus induced Balb/c mice to produce strong humoral and cellular immune responses. 【Conclusion】 The recombinant adenovirus Ad5-NcSAG1 was successfully constructed. It could induce Balb/c mice to produce strong humoral and cellular immune responses. This study has laid a solid foundation for the clinical experiment of the novel vaccine against N. caninum. Reference | Related Articles | Metrics Cited: Baidu(1)

Select

Analysis of Immunological Characteristics of Echinococcus Granulosus Adult Worm Surface Membrane -Associated Glycoantigen GU Nu-尔?Tu-尔Xun, MI Xiao-Yun, ZHANG Zhuang-Zhi, SHI Bao-Xin, TU 尔Hong-?Yi-Mi-Ti, JIN Ying-Hong, ZHANG Mei, CHENG Xiao-Bo, ZHANG Xu, ZHAO Li, ZHANG Wen-Bao Scientia Agricultura Sinica    2012, 45 (22): 4713-4719.   DOI: 10.3864/j.issn.0578-1752.2012.22.018 Abstract （550）      PDF （547KB）（434）       Knowledge map    Save 【Objective】 The immunological characteristics of the carbohydrate antigen (glycoprotein) of E. granulosus adult surface membrane were explored in this study . 【Method】 The freez-thaw E. granulosus adult worm lysis was digested by protease K, and centrifuged to collect supernatant as crude glycoantigen materials, which was identified by SDS-PAGE, quantified by sulfuric acid anthrone method. Then, the Balb/c mice were injected hypodermically by the material six times for anti-serum, which was collected to measure immunoglobulin (IgG, IgM and IgA) by ELISA, and to determine the specificity by western- blot method. To localize the site of the antigen composition in the E. granulosus adult worm by IFA. 【Result】 The SDS-PAGE showed that there was no visible band on the gel. This suggested that the proteins of the parasite surface membrane were degraded completely. The concentration of glycoantige was 2.54mg•mL-1 in the preparation. ELISA showed that the specific antibodies (IgG, IgM, and IgA) were rised gradually with the increase of the immunization times, and the positive/negative ration (P/N) of IgG, IgM and IgA equals to 4.9, 3.2 and 6.4, respectively. Comparatively, the specific IgE was not detected. Western blotting showed that the E. granulosus adult worm surface membrane glycoantigen and native proteins could be recognized by the anti-serum. IFA proved that the glycoantigen was located on the surface membrane of E. granulosus adult worm. 【Conclusion】 The glycoantigen of E. granulosus adult worms digested proteinase K has good immunogenicity and reactivity, and could be used to develop vaccine candidates and diagnosis material. Reference | Related Articles | Metrics

Select

Fluorescence Quantitative RT-PCR Assay for Detection of Tembusu Virus YU Chun-Mei, DIAO You-Xiang, TANG Yi, CUI Jing-Teng, GAO Xu-Hui, ZHANG Ying, JU Xiao-Jun, WU Li-Li Scientia Agricultura Sinica    2012, 45 (21): 4492-4500.   DOI: 10.3864/j.issn.0578-1752.2012.21.018 Abstract （851）      PDF （1405KB）（901）       Knowledge map    Save 【Objective】The objective of the study is to establish a method for detecting Tembusu virus by SYBR GreenⅠ relative fluorescence quantitative RT-PCR. 【Method】 Special primers based on Tembusu virus NS5 and E gene were designed and a pair of primers of house-keeping gene β-actin was chosen. Then these amplified fragments were cloned into pMD18-T. Using the plasmids NS5-pMD18-T, E-pMD18-T and β-actin-pMD18-T as standard products, a real-time quantitative reverse transcription-polymerase chain reaction ( RT-PCR) was performed to construct the standard curves of NS5, E and β-actin gene and detect the sensitivity, specificity and repeatability. 【Result】 The results showed a precise linear relationship with a correlation coefficient of R2＞0.99. The detection limits was 10 copies of DNA plasmid reaction. The amplification curve showing a single peak could only been detected for Tembusu virus. The variation coefficient was less than 0.5% by within and between the group of repeatability tests. The clinical samples were detected 3 times by this method, and all results were positive.【Conclusion】 The developed real-time PCR assay was highly specific, sensitive, and reproducible and could be an available tool for diagnosis and monitoring of Tembusu virus in duck farms. Reference | Related Articles | Metrics Cited: Baidu(13)

Select

Molecular Epidemiology Study on Porcine Reproductive and Respiratory Syndrome Virus in South Xinjiang ZHAO Li, LIU Yong-Hong, JIAO Hai-Hong, ZHANG Zhi-Feng, LIU Jun-Feng, CHEN Yan-Zhou, WEN Ya-Qin, LIU Bo, LI Jiang-Tao, CUI Hao-Ran, CHENG Bo, ZHANG Chun-Guang Scientia Agricultura Sinica    2012, 45 (20): 4288-4299.   DOI: 10.3864/j.issn.0578-1752.2012.20.019 Abstract （549）      PDF （835KB）（527）       Knowledge map    Save 【Objective】The objective of the study is to determine whether the porcine reproductive and respiratory syndrome virus infection is present in pig farms of South Xinjiang, and to determine the types and gene characteristics of PRRSV strain, thus pertinency effective prevention of PRRS can be made. 【Method】Seven pairs of primers were designed, and by using RT-PCR, the cloning, sequencing and sequence analysis on suspected PRRS pig tissue samples were conducted. 【Result】 There were 35 PRRSV positive in 47 test samples. The positive rate was as high as 74.47% (35/47). The 35 strains were all American type strain. There were 10 strains belong to the highly pathogenic PRRSV, accounting for 21.28% of the total tested samples (10/47), accounted for 28.57% of the positive samples(10/35); According to the comprehensive analysis, no Europe type of strains were found in the samples collected from these pig farms in this study. 【Conclusion】The American type of PRRSV strain infection and highly pathogenic strains exist in South Xinjiang. Reference | Related Articles | Metrics Cited: Baidu(4)

Select

Polymorphism Assays of Amino Acid and Establishment of a Two-Temperature PCR for Theileria Annulata Based on Tams1 Gene LUO Jin, LIU Guang-Yuan, TIAN Zhan-Cheng, XIE Jun-Ren Scientia Agricultura Sinica    2012, 45 (20): 4300-4309.   DOI: 10.3864/j.issn.0578-1752.2012.20.020 Abstract （524）      PDF （529KB）（856）       Knowledge map    Save 【Objective】 It has been widespread concern for gene polymorphism assays in species classification, pathogen detection and vaccine screening. T. annulata is an important blood protozoa in bovine and causes major hazards. In present study the Tams1 amino acid polymorphism of T. annulata was analyzed and a two-temperature PCR detection method was established for T. annulata. 【Method】The specific primers were designed of Tams1 gene of T. annulata. A nucleotide fragment of 846 bp in length was obtained by PCR amplification. The gene amino acid sequence was compared and analyzed with 12 known species in different regions of the isolates in GenBank. And other primers were designed in conserved region of Tams1 gene and the detection method of T. annulata was established by two-temperature PCR. The method was used to detect theileriosis in field.【Result】The fragment encoded 281 amino acids, including 48 basic amino acids, 42 acidic amino acids and 100 hydrophobic amino acids. Identity analysis showed that Gansu strain of T. annulata had closest relationship with Ankara (Z48739.1), Turkey (AF214911), Bahrain (AF214794) and had little relationship with Xinjiang strain (No. accession number) while had closest relationship with Italy (AF214862) and Spain (AF214815) strains. The results can be testified by sequences alignment. The two-temperature PCR detection method can detect in 0.31 fg•μL-1 blood infection. Specificity results showed that only T. annulata genome DNA test was positive, other control parasite genome template were shown to be negative, and no cross reaction with other infected bovine Piroplasmorida. The two-temperature PCR method of T. annulata was used to test 335 field samples, the positive rate was 16.33%, and the microscopic detection results of 2.25% coincidence rate was 100%. Compared with common PCR, the method had no significant difference in sensitivity. But, two-temperature PCR was higher than common PCR in specificity, and the method took a short time with repeated heating and cooling.【Conclusion】T. annulata Tams1 genes from different strains exist obvious differences. Its amino acid sequence polymorphism feature is significant. Therefore, when the gene is used as a potential candidate antigen attention should be paid to the design of peptide vaccines. Two-temperature PCR method has good sensitivity and specificity. It has significance for early detection, early prevention of T. annulata. Reference | Related Articles | Metrics

Select

Rescue and Identification of the Recombinant Bovine Rotavirus with Mutational NSP4 Gene YANG Shao-Hua, HE Hong-Bin, YANG Hong-Jun, CHEN Fang-Yuan, GAO Yun-Dong, ZHONG Ji-Feng Scientia Agricultura Sinica    2012, 45 (19): 4102-4108.   DOI: 10.3864/j.issn.0578-1752.2012.19.023 Abstract （821）      PDF （548KB）（731）       Knowledge map    Save 【Objective】The objective of the study is to rescue the attenuated rotavirus with mutational NSP4 gene by reverse genetic method and to purify further by RNAi technique and plaque clone method. 【Method】NSP4 gene of a wild type RV strain CHLY was cloned and five silent mutation nucleotides were introduced at 93 nt-109 nt and five missense mutations at 444 nt-453 nt, which resulted in M135L, I136T, A138P amino acid mutation. A recombinant plasmid △pT7-NSP4/89/D that contains T7 promoter and T7 terminator at 5′ and 3′ end of the manipulated NSP4 cDNA was constructed. △pT7-NSP4/89/D plasmid and expression vector pcDNA3.1/T7-RNAP carrying RNA polymerase in vivo was co-transfected MA104 cell layer that had been infected 24 h earlier with wild type RV as helper virus, and the transfected cell was cultured for further 24 h until harvest. The culture fluid colleted was subjected to passage on MA104 cell in the presence of Lentivirus-RNAi-H1-89, which could inhibit the amplification of helper virus specifically. The rescued virus was biologically cloned by five successive plaque purifications in MA104 cells.【Result】A RV strain with mutational NSP4 gene was successfully rescued. Compared to wild type RV, the rescued virus showed attenuated virulence to MA104 cell and mouse pups. 【Conclusion】A attenuated virulence RV was rescued by reverse genetic method and RNAi technique. The mutations of 135aa, 136aa and 138aa of NSP4 have effect on virus virulence. Reference | Related Articles | Metrics

Select

Studies on Synergetic Pathogenicity of Co-infection with Highly Pathogenic PRRSV and PCV2 FAN Pei-Hu, WEI Yan-Wu, GUO Long-Jun, WU Hong-Li, HUANG Li-Ping, LIU Chang-Ming Scientia Agricultura Sinica    2012, 45 (18): 3859-3872.   DOI: 10.3864/j.issn.0578-1752.2012.18.019 Abstract （690）      PDF （18990KB）（262）       Knowledge map    Save 【Objective】 The purpose is to study the synergetic pathogenic effect between highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and porcine cirovirus type 2 (PCV2), and to clarify the relation of ordinal co-infection and synergetic pathogenicity. 【Method】 Thirty 35-day-old piglets were randomly selected and separated into 6 groups which designated PCV2/HP-PRRSV ordinal co-infection group, HP-PRRSV/PCV2 ordinal co-infection group, HP-PRRSV+PCV2 simultaneous co-infection group, HP-PRRSV infection group, PCV2 infection group and control group, respectively. Clinical symptom and temperature were recorded everyday post-inoculation, meanwhile, weighing body weight and collecting blood samples. Variations of CD3+CD4+CD8-, CD3+CD4-CD8+, γδT, NK, granulocyte and mononuclear cell were determined by flow cytometry. Two kinds of antibodies against HP-PRRSV and PCV2 were quantified by immunoperoxidase monolayer assay (IPMA). Concentrations of IFN-γ, TNF-α, IL-10, IL-2, GM-CSF in serum were determined by ELISA. Pathological changes were observed through making pathological section for different organs. 【Result】 All the findings suggest that the pathogenicity of HP-PRRSV/PCV2 ordinal co-infection is conspicuously more severe than other testing groups. Mortality of this group is 60% surprisingly. Virus load in body is the highest while antibodies level is the lowest. Variation of immunocyte subgroups and cytokines (especially TNF-α) is the most conspicuous. 【Conclusion】 There is a synergetic pathogenicity of co-infection of pigs with HP-PRRSV and PCV2. If HP-PRRSV infecting precedes PCV2, the morbility and mortality will be conspicuously enhanced. This study has provided a scientific proof to control the two viruses. Reference | Related Articles | Metrics Cited: Baidu(5)

Select

Isolation, Characterization and Mechanism of Anti-viral Activity of Duck Avian Beta-defensin 16 ZHANG Ke-Xin, ZHANG Ming-Yue, XIN Sheng-Nan, HAN Zong-Xi, SHAO Yu-Hao, LIU Sheng-Wang, MA De-Ying Scientia Agricultura Sinica    2012, 45 (18): 3873-3882.   DOI: 10.3864/j.issn.0578-1752.2012.18.020 Abstract （790）      PDF （433KB）（461）       Knowledge map    Save 【Objective】The objective of the present study was to clone, express, and characterize the antimicrobial activity of duck avian beta-defensin (AvBD16). AvBD16 and TLR-7 dynamic changes were evaluated in different duck organizations after duck hepatitis virus infection. 【Method】The specific cDNA was obtained by PCR from bone marrow of ducks. The cDNA of duck AvBD16 was cloned into pGEX-6p-1 vector to construct recombinant plasmid, which were transformed into E. coli BL21 and the bacteria were induced with IPTG. Furthermore, the recombinant protein was purified. Peptides were synthesized according to the gene sequence. The antimicrobial activity of both recombinant and synthetic AvBDs was investigated in vitro. In addition, the mRNA expressions of AvBD16 and TLR-7 in tissues after duck hepatitis virus (DHV) infection were examined.【Result】Duck AvBD16 consisted of 155 bp encoding 50 amino acids and shared an amino acid homology (62%) with chicken AvBD3. Both recombinant and synthetic duck AvBD16 showed similar antibacterial activities. In high salt ions conditions, antibacterial activity of duck AvBD16 protein was decreased. In addition, the hemolysis activity of the peptide was extremely low. The duck AvBD16 exhibited significant antiviral activity against DHV in vitro. The mRNA expression of AvBD16 and TLR-7 in most tissues, including immune organs and liver, was upregulated or induced in response to DHV infection at different time points.【Conclusion】The duck AvBD16 gene from duck was successfully cloned, expressed in E. coli, and recombinant and synthetic AvBD16 protein showed antimicrobial activity and has no hemolytic properties. Duck AvBD16 exhibits significant antiviral activity against DHV in vitro. This result suggests that the antiviral activity against DHV of the AvBD16 is partially via a TLR7-dependent mechanism. Reference | Related Articles | Metrics Cited: Baidu(4)

Select

Antimicrobial Resistance Analysis and Detection of Methicillin- Resistant Staphylococcus aureus (MRSA) Among Staphylococcus aureus Strains Isolated from Bovine Mastitis SU Yang-., PU Wan-Xia, CHEN Zhi-Hua, DENG Hai-Ping Scientia Agricultura Sinica    2012, 45 (17): 3602-3607.   DOI: 10.3864/j.issn.0578-1752.2012.17.017 Abstract （648）      PDF （405KB）（574）       Knowledge map    Save 【Objective】The aim of the study is to investigate the antimicrobial resistance of S. aureus (Science) and to detect the presence of methicillin-resistant S.aureus (MRSA) among S.aureus strains isolated from bovine mastitis in Inner Mongolia, and to provide credible theory evidence for prevention and treatment of bovine mastitis. 【Method】 K-B disk diffusion method was used to test drug sensitivity of 38 total strains of S.aureus to 17 commonly used antibiotics. Meanwhile, agar screen was performed to identify the minimum inhibitory concentration of oxacillin and vancomycin to all strains. Cefoxitin, oxacillin disk diffusion and oxacillin agar screen were used to detect the phenotype of MRSA, and PCR assay was generated the genotype of MRSA as well. 【Result】 The isolates had different degrees of antimicrobial resistance to each antibiotic, the frequency of resistance to ampicillin, cefradine, penicillin, cotrimoxazole, novobiocin and streptomycin was more than 45%, yet keeping sensitive to ofloxacin, vancomycin, amikacin, ciprofloxacin, gentamicin and cefazolin was over 90%. Two of the strains with vancomycin MIC were ≥16 μg•mL-1. The MICs of oxacillin for eight of total strains were ≥ 8 μg•mL-1, others with oxacillin MICs were ≤2 μg•mL-1. The multidrug resistance was severe, 84.21% of the strains were resistant to at least 3 kinds of antimicrobial agent, four of the total strains can survive in the presence of night various antibiotics. 16(42.11%) S.aureus strains carried mecA gene detected by PCR assay. However, there were only seven of them have the minimum inhibitory concentration over 4 μg•mL-1. When cefoxitin, oxacillin disk diffusion and oxacillin agar screen methods were generated the phenotype of MRSA, only 7, 10 and 7 strains of each were identified. 【Conclusion】 The antimicrobial resistance and multidrug resistance of S aureus were serious. High incidence of MRSA and OS-MRSA has been associated with bovine mastitis in the surveyed region. Reference | Related Articles | Metrics Cited: Baidu(1)

Select

Antibiotic Resistance and Coagulase Typing of Staphylococcus aureus Isolates from Pigs and Cows in Part of China LIU Yang, LIANG Yao-Feng, JIAO Xin-An, SONG Li, ZHANG Chun-Ping, NING Yi-Bao, XU Shi-Xin Scientia Agricultura Sinica    2012, 45 (17): 3608-3616.   DOI: 10.3864/j.issn.0578-1752.2012.17.018 Abstract （694）      PDF （469KB）（664）       Knowledge map    Save 【Objective】The objective of the experiment is to study the status of antibiotics resistance and coagulase typing distribution of S.aureus in China, and provide a scientific basis for effective control of transmission and prevalence of S.aureus from animals.【Method】Antibiotics resistance was tested by broth dilution method, coagulase serotypes were identified by coagulase grouping serum, coa genes were amplified by PCR method and then sequenced for further analysis by mega software. 【Result】 A total of 124 S.aureus isolates were identified, and the result of resistance detection revealed that the resistant rate of ampicillin and penicillin was near 100%, erythromycin reached 75%, and other 10 antibiotics was below 50%. The positive rate of MRSA was 27%. No vancomycin resistant isolates was found. Six kinds of serotypes were observed and the rate of coagulase serotyping reached 91%: serotypes VII,VI and combined type were prevalent, whereas serotypes Ⅲ and Ⅴwere not found. The phylogenetic tree of coagulase gene was constructed. 【Conclusion】 The status of antibiotics resistance in China was relatively severe, swine isolates were severer than cattle ones. Coagulase serotypes were abundant and the distribution was comparative intensive, and possessed unique prevalence feature. The evolutional characteristic of coa gene had no direct relationship with coagulase serotypes, and this study has provided a theoretical basis for further veterinary research about molecular epidemiology and evolutionary tendency. Reference | Related Articles | Metrics Cited: Baidu(4)

Select

Study on Cytotoxicity of Outer Membrane Protein BP26 of Brucella CHEN Rui-Hua, ZHANG Hui, TANG Li-Yan, MENG Ru, ZHANG Yu, WANG Zhen, LI Zhi-Qiang, ZHANG Jun-Bo, CHEN Chuang-Fu Scientia Agricultura Sinica    2012, 45 (16): 3406-3413.   DOI: 10.3864/j.issn.0578-1752.2012.16.021 Abstract （722）      PDF （642KB）（764）       Knowledge map    Save 【Objective】 Outer membrane protein BP26 of Brucella is an important Brucella virulence factor. In order to reveal the biological functions of Brucella bp26 gene, HPT-8 cells were infected by bp26 gene deletion strains and treated by BP26 protein respectively. 【Method】 The BP26 protein was purified by Ni affinity chromatography and bp26 gene deletion strains was constructed by overlap technology. After the infection of bp26 gene deletion strains and the treatment of BP26 protein, HPT-8 cells were observed by electron microscope to examine the cells morphology. ELISA was used to detect cytokines in the supernatant.【Results】bp26 gene was cloned from Brucella vaccine strain M5-90 and successfully expressed in E. coli BL21(DE3).Purified protein was proved correct by SDS-PAGE, and the immunogenicity of the obtained BP26 was confirmed by Western-blot. The upper and lower arms of bp26 gene were inserted into the suicide vector PGEM-7zf, transferred to Brucella vaccine strains M5-90 and the bp26 deletion strains with genetic stability were selected successfully. The treatment of BP26 protein changed the cells morphology and significantly decreased the adhesion ability to the wall. Compared with the PBS group, the release level of IL-6,TNF-α and LDH significantly increased（P＜0.01）, but the release level of IL-10 significantly decreased (P＜0.05).  After the infection of bp26 gene deletion strains, the release level of IL-6, TNF-α were higher than M5-90 group. The release level of LDH and IL-10 were significantly lower than M5-90 group（P＜0.05）.【Conclusion】 BP26 protein and Brucella M5-90Δbp26 deletion strains were successfully obtained. BP26 protein could lead to inflammatory response and had some toxic effects on the HPT-8 cells. The bp26 gene plays an important role in Brucella infection of HPT-8 cells. Reference | Related Articles | Metrics Cited: Baidu(3)

Select

Construction of miR-34c Lentiviral Expression Vector and Its Infection in Dairy Goat Germ Line Stem Cells LIU Chao, YU Meng, ZHU Hai-Jing, LI Ming-Zhao, HUA Jin-Lian Scientia Agricultura Sinica    2012, 45 (16): 3414-3421.   DOI: 10.3864/j.issn.0578-1752.2012.16.022 Abstract （644）      PDF （1261KB）（917）       Knowledge map    Save 【Objective】In order to over-express mouse miR-34c economically and efficiently, mouse miR-34c lentiviral expression vector was constructed using two different methods.【Methods】The pri-miR-34c was amplified and inserted into downstream of CMV and U6 promotors in pLL3.7, respectively. The recombinant lentiviral vector together with lentivirus package plasmid mixtures were transfected into 293T cells to package virus. The titres of the viruses were examined and then the viruses were infected with 293T cells and dairy goat male germ line stem cells and 293T cells. The transfection efficiency was measured by the percentage of GFP expression cells, and the expression level of miR-34c was determined by qPCR.【Results】The recombinant pLL3.7-CMV-34c vector was successfully constructed, confirmed by endonuclease digestion analysis and DNA sequencing. The expression level of miR-34c in cells infected with the packaged pseudoviriius was distinctively increased. 【Conclusion】Using both CMV and U6 promotor can over-express miR-34c vector. And the virus packaged with recombined vector using U6 promoter can infect dairy goat male germ line stem cells more efficiently. Reference | Related Articles | Metrics

Select

Epidemiological Survey and Identification of Theileria Parasite Infection for Small Ruminants in Some Parts of China LI You-Quan, PENG Yu-率, LIU Zhi-Jie, GUAN Gui-Quan, YANG Ji-Fei, CHEN Ze, NIU Qing-Li, LUO Jian-Xun, YIN Hong Scientia Agricultura Sinica    2012, 45 (16): 3422-3429.   DOI: 10.3864/j.issn.0578-1752.2012.16.023 Abstract （684）      PDF （628KB）（885）       Knowledge map    Save  【Objective】The objective of this experiment is to investigate the current epidemiological status of ovine and caprine theileriosis and identified its pathogens in China. 【Method】The genomic DNA of the blood samples of sheep and goats and ticks were amplified using specific primers to T. uilenbergi, T. luwenshuni and T. ovis, respectively. The 18S rRNA gene sequences from the positive samples were obtained and used for phylogenetical analysis.【Result】On the basis of the PCR results, wide spreading of the theileriosis and significant difference in terms of pathogen species and prevalence in the investigated sites were observed. Co-infection of T. uilenbergi and T. luwenshuni was found only in Gansu Provinces. T. ovis infection was found in Kashi, Xinjiang, but T. luwenshuni infection was detected in Hubei Province but no clinical cases were found. None Theileria infection in Yunnan Province was found. 【Conclusion】The prevalence and infection situation of ovine and caprine theileriosis varied markedly in the studied sites. The data will be useful for guiding the comprehensive prevention and control of ovine and caprine theileriosis in these regions. Reference | Related Articles | Metrics Cited: Baidu(15)

Select

Establishment and Optimization of Immunohistochemistry Assay for Detection of Chronic Wasting Disease ZHANG Xin-Xin, LIU Huan-Qi, LIU Yu-Tian, SUN Cheng-You, CHI Tian-Ying, YU Xiao-Jing, DENG Ming-Yi, WANG Zhi-Liang Scientia Agricultura Sinica    2012, 45 (15): 3176-3181.   DOI: 10.3864/j.issn.0578-1752.2012.15.020 Abstract （567）      PDF （703KB）（611）       Knowledge map    Save 【Objective】The purpose of this study is to establish and optimize immunohistochemical assay for detection of chronic wasting disease (CWD) based on the monoclonal antibodies (McAbs) against Elk cellular prion protein which were independently developed by China Animal Health and Epidemiology Center. 【Method】Negative and positive slices were made by conventional paraffin method,and ABC method of IHC was used to detect the 5 McAbs.【Result】The results indicated that only two (No. 5E3 and 3B2) from 5 McAbs (No. 5A5, 3B2, 6D12, 5E3 and 1F5, respectively ) could be used for the immunohistochemical  detection of positive spinal cord section of Elk. It was showed strong specific immunoreactivity with cervid abnormal prion protein by 5E3 and weak specific immunoreactivity by 3B2. There was no any immunoreactivity by other McAbs with various experimental concentrations. Additionally, the best immunoreactivity was present when McAb 5E3 was used at 1/10000 dilution. 【Conclusion】The study demonstrated that 5E3 can be used in national surveillance of CWD. Reference | Related Articles | Metrics

First page | Prev page | Next page | Last page

Page 1 of 8, 285 records