151 results about "Riemerella anatipestifer" patented technology

The invention relates to a novel veterinary antibacterial agent and in particular relates to a lauroyl arginine ethyl ester ion pair compound derivative which is prepared from lauroyl arginine ethyl ester LAE and an organic acid through reactions. As a livestock aquatic animal antibacterial agent, the derivative is capable of preventing and treating duckling riemerella anatipestifer diseases caused by riemerella anatipestifer, is capable of treating peritonitis diseases caused by escherichia coli and staphylococcus aureus, is capable of preventing and treating fish septicemia caused by aeromonas sobria, and has remarkably improved prevention and treatment effects when being compared with a conventional LAE veterinary antibacterial agent. The derivative can be completely degraded in human bodies and animal bodies and is small in toxic and side effect, environment drug resistance bacteria caused by residual antibacterial agents discharged into the environment can be avoided, and small adverse environment influence can be caused while an effective antibacterial effect is achieved.

The invention relates to a riemerella anatipestifer antibody indirect ELISA method detection kit and an application thereof; the kit comprises: a) an antibody detection plate, b) enzyme conjugate working fluid, c) a positive control, d) a negative control, e) a sample diluent, f) 10X concentrated washing liquid, g) a substrate coloured solution A, h) a substrate coloured solution B, and i) a stopping solution; the beneficial and positive effects of the invention are that: the kit is simple in operation; the requirements for apparatuses needed are not high; the kit can be operated by everyone, can meet the requirements with different levels such as epidemic disease monitoring, hygienic epidemic prevention, intensive culture, and individual culture, is suitable for large-scope popularization and application, and has wide market prospects.

The invention discloses a riemerella anatipestifer indirect coagulation antibody detection kit and application thereof. The kit comprises a riemerella anatipestifer indirect coagulation antigen, standard positive serum, standard negative serum and a diluent. The riemerella anatipestifer indirect coagulation antibody detection kit and the application thereof have the beneficial effects that the kit is simple and convenient to operate, can be widely popularized and used in related grassroots veterinarian departments and farms; the kit only needs a few simple consumable materials and incubators when being used detecting samples without any other device, and can be used even in duck farms with relatively simple and rough conditions; the result can be determined intuitively, and can be observed by eyes; professional knowledge is not required for operators, special training is not required, and the detection can be performed only referring to a specification; the kit is low in cost, is economical and practical, and can be accepted by most of users.

The invention belongs to the technical field of biology and particularly relates to a riemerella anatipestifer-escherichia coli shuttle expression vector as well as a construction method and an application thereof. The riemerella anatipestifer-escherichia coli shuttle expression vector disclosed by the invention has a nucleotide sequence of SEQ ID NO:1. The invention further discloses a construction method and the application of the riemerella anatipestifer-escherichia coli shuttle expression vector. The riemerella anatipestifer-escherichia coli shuttle expression vector pRES constructed by the construction method can stably exist and can be copied in riemerella anatipestifer and escherichia coli; an exogenous gene carried by the riemerella anatipestifer-escherichia coli shuttle expression vector can be stably expressed in the riemerella anatipestifer; the riemerella anatipestifer-escherichia coli shuttle expression vector can be used for complementary assay of riemerella anatipestifer deletion mutation strains, research of recombinant riemerella anatipestifer vaccines and the like and has a wide application prospect.

The invention relates to the field of animal medicine, in particular to a method for detecting duck plague virus antibody. The method particularly comprises the steps of the preparation of solid phase antigen, primary antibody combination, secondary antibody combination, developing, detection, judgment and the like. The method is an indirect enzyme-linked immuno sorbent assay (ELISA) method established based on purified recombinant UL51 protein, which has good specificity, and when duck hepatitis virus (DHV), Riemerella anatipestifer (RA), duck Escherichia coli (E.coli) are detected, the detection results are all positive; intra batch or batch-to-batch repeated tests show that variation coefficients are all less than 10%, and duck plague virus (DPV) vaccine immunized duck positive sera diluted at ratio of 1:3200 can be detected.

The invention provides a pair of specific primers for detecting riemerella anatipestifer, the nucleotide sequence of the specific primers comprises an upstream primer omp A-F:5minute-actcaaggaagagcggatca-3minute; a downstream primer ompA-R: 5minute-gcttcagcagaaccaactcc-3minute. The invention also provides a PCR detection kit containing the specific primers. The detection kit provided by the invention has the beneficial effects that the operation is simple, the specificity and the sensitivity are high; the result determination is intuitive, and the result can be watched by eyes after cataphoresis.

The invention discloses a riemerella anatipestifer infection attenuated live virus vaccine and a preparation method thereof. The riemerella anatipestifer infection attenuated live virus vaccine disclosed by the invention contains a riemerella anatipestifer GD strain freeze-drying protection agent and a riemerella anatipestifer GD strain is preserved in China Center for Type Culture Collection (CCTCC) on April 1, 2016, with the preservation number of CCTCC NO: M2016161. The riemerella anatipestifer infection attenuated live virus vaccine disclosed by the invention has good safety and does not have any adverse effect on ducks; the riemerella anatipestifer infection attenuated live virus vaccine has a good immune effect, can be used for remarkably improving the resistance of young ducks on riemerella anatipestifer, can be used for clinically preventing riemerella anatipestifer infection and has important economic and social benefits.

The invention relates to a vaccine immunologic adjuvant and a vaccine, in particular to an astragalus polyose adjuvant and application thereof in an I-riemerella anatipestifer inactivated vaccine. The vaccine adjuvant is an astragalus polyose solution with the concentration of 30 milligrams per milliliter. An astragalus polyose inactivated vaccine for the I-riemerella anatipestifer is prepared from a riemerella anatipestifer RA-1 inactivated bacteria liquid as the vaccine and the astragalus polyose solution as the adjuvant through mixing. The astragalus polyose adjuvant improves the immune efficacy of the prior inactivated vaccine, effectively prevents and controls infectious serositis of duck, and provides a basis for the research of a novel immunologic adjuvant. The vaccine has good safety and immunogenicity, and can effectively prevent the infectious serositis of duck.

The invention relates to preparation and application of an egg yolk antibody containing florfenicol drug resistance gene protein (FloR). Riemerella anatipestifer (CCTCC No: M208128) resistant to florfenicol is separated from a duck dies clinically; PCR is adopted to amplify a section of the drug resistance gene (floR); the section of the drug resistance gene is cloned to an expression plasmid pET28a (+) for transfecting host bacteria BL21 (DE3). The recombination strain is subject to induction expression, and fusion protein containing the target gene section and having a HIS label is prepared; the fusion protein exists in the form of an inclusion body; the inclusion body is extracted for the preparation of an immunogen; the immunogen is used for immunizing a laying hen, so as to obtain the egg yolk antibody capable of improving the sensitivity of the drug resistance strain to florfenicol; the egg yolk antibody is used in combination with florfenicol to remarkably improve the treatment effect on a duck infected with a drug-resistant riemerella anatipestifer strain ; as other strains resistant to florfenicol are also mediated through a floR gene, the egg yolk antibody can be applied to the veterinary clinical treatment on livestock and poultry infected with strains resistant to florfenicol.

The invention discloses a riemerella anatipestifer CH1 global regulator phoP attenuated strain and a construction method thereof, and belongs to the technical field of biological engineering. The attenuated strain is constructed by knocking out a phoP gene by means of a homologous recombination method and utilizing a bacterial conjugation method. The prepared attenuated strain has a preservation number of CCTCC NO:M2014557, and is preserved on November 7, 2014. The attenuated strain has low toxicity, and a foundation is established for construction of subsequent vaccine vectors and construction of a riemerella anatipestifer gene regulatory network. The invention further provides application of the attenuated strain in vaccines.

The invention discloses a Riemerella anatipestifer OmpA / MotB truncated recombinant protein, its antibody and a preparation method and an application thereof. The recombinant protein has immunoreactivity similar to natural OmpA / MotB protein, can bind to RA antibody specifically, will not generate cross reactions with Salmonella anatum positive serum, duck colibacilosis positive serum, duck swollen head septicemia virus positive serum, avian influenza virus (H5) positive serum, duck plague virus positive serum, duck hepatitis virus positive serum and the like, and can be used as a detection antigen for detecting whole bacteria antibody. As the recombinant protein is not whole bacteria, there is no risk of spreading bacteria when the recombinant protein is applied to detection. In addition, the preparation method of the recombinant protein is simple, is suitable for large-scale industrial production, can realize standardization of products and is beneficial to result comparison between different laboratories.

The invention discloses a kit for detecting riemerella anatipestifer and a method for using the kit. At least two pairs of specific primers F3, B3, FIP and BIP for loop-mediated isothermal amplification are placed in the kit for detecting riemerella anatipestifer. For making use convenient, the kit also comprises lycine, magnesium sulfate, dNTPs, ThermoPol reaction buffer and Bst DNA polymerase; or a color developing reagent SYBR GREEN I is also added. The kit and the method have the advantages of convenient application on base layer, short detection time, low cost and simple and convenient operation.

The invention discloses a culture medium for production of a riemerella anatipestifer vaccine, which comprises a basic culture medium and an additive. The basic culture medium comprises the following components in percentage: 1.5 to 1.9 percent of tryptone, 0.2 to 0.7 percent of yeast extract, 0.2 to 0.7 percent of beef extract, 0.2 to 0.7 percent of hydrolytic albumin, 0.1 to 0.4 percent of glucose, 0.5 percent of NaCl, 0.1 to 0.7 percent of monopotassium phosphate, 0.5 to 0.9 percent of Na2HPO4.12H2O, 0.1 to 0.2 percent of (NH4)2SO4 and 0.01 to 0.03 percent of NH4Cl, wherein the percentage of each component is weight / volume percentage. The additive is sterilely extracted infected-area strong cattle blood through a jugular vein blood taking or carotid artery blood bleeding method, is subjected to sterile fiber removal, is subjected to freeze thawing at the temperature of 20 DEG C below zero and at room temperature for three times, is preserved for later use at the temperature of 20 DEG C below zero, or is directly preserved at the temperature of 20 DEG C below zero after the blood is extracted and subjected to sterile fiber removal, and is subjected to freeze thawing for three times before use. The culture medium has the advantages of capacities of reducing cost, increasing nutrition, overcoming the defects of complex operation and high labor cost during culture by using live animals or embryos, and use for large-scale fermentation production of the riemerella anatipestifer vaccines.

The invention relates to a Riemerella anatipestifer bivalent inactivated vaccine and a preparation method thereof, which belongs to the technical field of preparing livestock biological preparations and controlling poultry diseases. The antigens of the vaccine are a Riemerella anatipestifer serum I strain and a serum II strain, and the proportion of the strain antigens is proper in fit. The immune period can be as long as more than 70 days through one injection of the vaccine, and the immune protection efficiency to serum I and serum II reaches more than 90 percent. The vaccine has the advantages of simplifying immunization procedures, reducing the stress reaction of inoculate animals and saving cost for production and injection. The vaccine used for commercial production can end the reality that the control on duck infectious serositis in China excessively depends on drugs, and can provide strong technical support and material guarantee for the healthy stable development of the duck breeding industry.

The invention belongs to the technical field of biological products, and particularly relates to two dominant serotype strains of a riemerella anatipestifer disease area and its application. The strains are riemerella anatipestifer RAHB-1 and riemerella anatipestifer RAHB-2, which are deposited in the China Center for Type Culture Collection (Wuhan University Depository Center). The riemerella anatipestifer RAHB-1 and riemerella anatipestifer RAHB-2 are subjected to inoculation culture to obtain bacteria liquid, the bacteria liquid can be mixed, and a vaccine used for preventing duck scrositisis prepared by formaldehyde inactivation and addition of hydrogenated alumina gel for mixing. The vaccine has high safety, low cost, good immune effect and easy absorption, provides basic guarantee for clinical application of the vaccine, and is easy to promote.

The invention relates to the field of animal medicine, in particular to a method for detecting duck plague virus (DPV) antibodies. The method specifically comprises the steps of preparation of solid phase antigens, primary antibody combination, secondary antibody combination, color development, detection, judgment and the like. The method is a purified recombinant UL55 protein-based indirect enzyme-linked immuno sorbent assay (ELISA) method, and has good specificity; the results of detecting the positive serums of duck virus hepatitis viruses (DHV), riemerella anatipestifer (RA), duck escherichia coli (E.coli), duck-derived salmonella, duck swollen head hemorrhagic disease viruses and duck-derived influenza viruses are negative; and the repeated tests in enzyme-labeled plates or between enzyme-labeled plates display that variation coefficients are less than 10 percent, and the method can detect out the positive serums of DPV weak virus vaccine immune ducks, wherein the DPV weak virus vaccine are diluted according to a ratio of 1:6400.

The invention provides a riemerella anatipestifer lipopolysaccharide mutant strain and application thereof. The collection number of the riemerella anatipestifer lipopolysaccharide mutant strain is CGMCC No.10147. The oil emulsion deactivated vaccine prepared by the riemerella anatipestifer lipopolysaccharide mutant strain has 100% cross protection capacity on the domestically leading serum 1, 2 and 10 representative strains of the riemerella anatipestifer, and can be applied as a wide-spectrum vaccine of the riemerella anatipestifer.

The invention belongs to the technical field of biological products for veterinary uses, in particular to a method for preparing a riemerella anatipestifer and escherichia coli bigeminal inactivated vaccine and a preparation method thereof. The vaccine is prepared by inoculating riemerella anatipestifer I type, riemerella anatipestifer II type and escherichia coli O78 type which have excellent immunogenicity into a culture medium for culture, using formaldehyde solution to inactivate the bacteria and emulsifying the resulting product by an oil adjuvant. The bigeminal inactivated vaccine is used for preventing and controlling infectious serositis and colibacillosis of a duck. The preparation method of the vaccine has a simple operation; and the prepared vaccine has stable performance and can effectively prevent and control the infectious serositis and the colibacillosis of the duck.

The invention discloses a virulent phage sensitive to riemerella anatipestifer and application thereof. The virulent phage sensitive to riemerella anatipestifer is named RAP37 and is preserved in China General Microbiological Culture Collection Center with preservation date of March-2-2009 and preservation number of CGMCC No.2908, the diameter thereof is about 1-2mm, the periphery is not provided with halo, the phage is composed of one polyhedral head and lathy tail, the face profile of the head is hexagon and the diameter is about 60nm, the tail is about 200nm long and 8nm wide and the genome thereof is double-stranded DNA. The virulent phage sensitive to riemerella anatipestifer is named RAP37, can decompose the riemerella anatipestifer, and can treat and prevent riemerella anatipestifer disease caused by leading pathogen of the riemerella anatipestifer which endangers duck breeding industry by adopting biotherapy.

The invention belongs to the veterinary biotechnical field, in particular to a riemerella anatipestifer-escherichia coli outer membrane protein bivalent vaccine and a preparation method thereof. The riemerella anatipestifer-escherichia coli outer membrane protein bivalent vaccine is prepared by emulsifying an antigen and oil adjuvant according to the volume ratio of 1:1-1:5, wherein the antigen is a riemerella anatipestifer RA1 outer membrane protein and / or a riemerella anatipestifer RA2 outer membrane protein and an avian pathogenic escherichia coli 078 outer membrane protein. As proved by an animal experiment, a vaccine immune animal prepared by adopting the method has high vaccine safety and a remarkable immune effect, a high-titer antibody can be generated inside the animal, and the attack of the same type of riemerella anatipestifer and a plurality of serum escherichia coli can be resisted.

The invention relates to duck escherichia coli with strong virulence and good immunogenicity; and the preservation number of the strain is CGMCC No.6831. The invention further provides a duplex inactivated vaccine which comprises vaccine adjuvant and antigen, wherein the antigen is the duck escherichia coli with preservation number of CGMCC No.6831 and riemerella anatipestifer with preservation number of CGMCC No.6832. The prepared duplex inactivated vaccine of the duck escherichia coli and the riemerella anatipestifer can prevent duck colibacillosis with characteristic pathological changes, such as cellulose exudative inflammation in the heart or the liver for causing duck escherichia coli septicemia, pericarditis or pleural adhesions as characteristics, and can prevent duck infectious serositis with neurological sign, fibrinous pericarditis, perihepatitis and air sacculitis as characteristics, so that excellent market promotion foreground is achieved.

The invention discloses a method for quickly determining a bacterial number in a Riemerella anatipestifer culture, comprising the following steps of: (1) sampling; (2) centrifugal cleaning: after centrifuging for 5-10 minutes by 3000-5000 turns per minute, continuously and centrifugally washing 3 times by using physiological saline, wherein each time of centrifugal cleaning spends 5-10 minutes at rotation speed of 3000-5000 turns per minutes; (3) diluting: diluting N times by using the physiological saline; (4) detecting: detecting an OD (Outside Diameter) value at a 560 nm point under an ultraviolet spectrophotometer; and (5) calculating: calculating the number of Riemerella anatipestifer according to the following formula: a total bacterial number (CFU / ml)=an OD560nm value*2*109CFU (Clonal Formation Unit) / ml.

The invention discloses a loop-mediated isothermal amplification-based nucleic acid test method for Riemerella anatipestifer. The method includes the following steps: a specific primer sequence is designed according to the gyrB gene of the Riemerella anatipestifer, the DNA (deoxyribonucleic acid) of a sample to be tested is extracted as a template, and LAMP (loop-mediated isothermal amplification) reaction is carried out; an LAMP result is analyzed, and if the LAMP amplification result is positive, then the sample to be tested contains the Riemerella anatipestifer. The test method aimed at the Riemerella anatipestifer, which is provided by the invention, is simple, fast, specific, sensitive and economic, the result judgement method is simple, and the test method is suitable for use in clinic or grass-roots labs, and has a broad application prospect.

The invention discloses a rapid differential diagnosis method of riemerella anatipestifer disease and colibacillosis. The method includes that riemerella anatipestifer 42-KD outer membrane protein gene and Escherichia coli phoA gene are selected to synthesize two pair of specificity primers used for gene amplification of the two germs, the amplified segments are respectively 809bp and 622bp, difference of the two is 187bp, and the established RA and E.coli double PCR method can rapidly, specifically and sensitively can detect and differentiate the two bacteria. The invention has the advantage that the method of the invention has lower required expense, higher efficiency and stronger specificity compared with the traditional differential diagnosis method.

The invention belongs to the field of biotechnology, specifically relates to a monoclonal antibody against riemerella anatipestifer (RA). The preservation number of the monoclonal antibody 5G7 is CGMCC No. 5165. With adopting the monoclonal antibody to prepare the immune colloidal gold strip for detecting the RA, the pathogen of the RA can be directly detected, and the RA strains such as serotypes of 1, 2, 3 and 11 can be synchronously detected, such that the monoclonal antibody against the RA has a wide application value.

The invention discloses a shuttle plasmid and a construction method and application thereof. The shuttle plasmid includes a starting replicator shown as SEQ ID NO.1, can be replicated in riemerella anatipestifer and can stably exist in escherichia coli and the riemerella anatipestifer. Therefore, the shuttle plasmid can be used for anaplerosis of gene knockout mutants of the riemerella anatipestifer and provide a powerful tool for identification of gene functions of the riemerella anatipestifer.

The invention discloses a riemerella anatipestifer OmpH intercepted recombinant protein, and a preparation method and application thereof, and belongs to the field of biological engineering. The amino acid sequence of the riemerella anatipestifer OmpH intercepted recombinant protein is shown as SEQ ID NO:1; then, a gene engineering technology is used; on the clone basis, the connection with a prokaryotic expression vector pET-32a (+) is performed; a gene engineering product induced and expressed by a colon bacillus BL21(DE3) expression system is successfully converted; the expression form of the protein is OmpHM / His fusion protein; the molecular weight of the expressed fusion protein is 28kDa; the product is designed into fusion expression, and the effect of being convenient to purify is mainly considered; meanwhile, the expression protein maintains the natural activity of the protein. The product can be applied to the detection of detection antigen of a riemerella anatipestifer antibody by an indirect ELISA (enzyme-linked immuno sorbent assay) method, and can also be used as a gene engineering sigmasubunit vaccine for preventing the riemerella anatipestifer.

The invention belongs to the field of biotechnology, and discloses a riemerella anatipestifer tervalent inactivated vaccine. The inactivated vaccine comprises a riemerella anatipestifer serotype I inactivated whole bacterium, a serotype II inactivated whole bacterium, a serotype X inactivated whole bacterium and an adjuvant. The immune protection ratio of the inactivated vaccine against the toxicity attack from the serotype I, II and X type riemerella anatipestifer virulent strains reaches up to 100%. The inactivated vaccine provided by the invention has a good immune protection effect on all serotype strain infections.

The invention discloses a method for mixed culture and re-separation of riemerella anatipestifer and escherichia coli. The method for mixed culture and re-separation of riemerella anatipestifer and escherichia coli comprises the following steps of (1) performing strain recovery; (2) screening riemerella anatipestifer and escherichia coli with antibiotics sensitivity differences; (3) inoculating the riemerella anatipestifer and the escherichia coli into a culture medium for mixed culture at a certain time interval or different diluting concentration; (4) measuring the minimum inhibitory concentration of antibiotics in the second step on the screened riemerella anatipestifer and escherichia coli; (5) preparing an antibiotic plate culture medium; (6) separating mixed culture bacterium liquid. The method for mixed culture and re-separation of riemerella anatipestifer and escherichia coli has the advantages that two kinds of bacteria are used outside duck poultry bodies and can jointly grow for more than 20 generations; conditions are provided for studying drug resistance transfer and relevant mechanisms of the two kinds of bacteria under the symbiosis condition; important significance is realized on disease prevention and control of duck breeding industry.