CN101288774A - Construction method of mice infection model of Suis Eperythrozoonsis - Google Patents
Construction method of mice infection model of Suis Eperythrozoonsis Download PDFInfo
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- CN101288774A CN101288774A CNA2007100479529A CN200710047952A CN101288774A CN 101288774 A CN101288774 A CN 101288774A CN A2007100479529 A CNA2007100479529 A CN A2007100479529A CN 200710047952 A CN200710047952 A CN 200710047952A CN 101288774 A CN101288774 A CN 101288774A
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Abstract
The invention provides a construction method of the SPF mouse infection model of an epperythrozoon in the biological technology field, which includes the steps of (1) the separation and purification of the epperythrozoon, (2) the feeding of the SPF mouse in the isolation chamber for the fumigation and the disinfection, (3) the vaccination, (4) the detection evaluation of the animal model. The invention successively constructs the SPF mouse infection model of the epperythrozoon and is verified by three methods, including fresh blood squash detection, PCR detection and transmission electron microscope detection. The model can be applied to the deep researches in the aspects of the etiology, the pathogenic force, the pathology and the immunology of eperythrozoonosis, etc.
Description
Technical field
What the present invention relates to is a kind of construction method of animal model of technical field of bioengineering, is specifically related to a kind of construction method of mouse infection model of eperythrozoon suis.
Background technology
Eperythrozoonosis (Eperythrozoonosis) is that to parasitize cause in erythrocyte surface, blood plasma and the bone marrow a kind of by Eperythrozoon (Eperythrozoon) be the Amphixenosis of main clinical characteristics with packed cell volume reduction, hemoglobin concentration decline, leukocytosis, anemia, jaundice, heating.This disease has a very wide distribution, infection host is many, causes huge harm for people's health and Developing of Animal Industry.Domestic and international in recent years medical worker has carried out since the deep research, particularly the eighties in last century eperythrozoonosis, and the eruption and prevalence of eperythrozoonosises such as report people and poultry has more and more caused the great attention of Chinese scholars to this disease.Eperythrozoon still can not external a large amount of cultivations and propagation, for follow-up study work has brought difficulty but at present.And inapparent infection rate that should disease is higher, and normal and toxoplasmosis, colibacillosis, streptococcicosis, other diseases accompanying infections such as atypical swine fever.Therefore, set up one stable, the animal model that can reflect the swine eperythrozoonosis infection character is for this sick pathogenesis of research, immunology, epidemiology is all particularly important.
Find that through literature search the Eperythrozoon The Animal Model Study mainly is to infect the laboratory animal of cutting spleen or injection dexamethasone by the whole blood with eperythrozoon suis to obtain to existing method.As: Liu Bing etc. have described a kind of method for building up of eperythrozoon suis mouse infection model in 2007 " Chinese veterinary science " 37 phase 12-15 pages or leaves.Utilize the Kunming white mice of extracing spleen or injection dexamethasone, set up the eperythrozoon suis infection model with lumbar injection mode artificial challenge eperythrozoon suis whole blood, and verified by the method for PCR.Though this method also can be set up the animal model of eperythrozoon suis,, there are two defectives in this model: (1) adopts the whole blood counteracting toxic substances can bring impurity such as leukocyte inevitably into, and the source of infection is not single, can influence result of the test.(2) adopt mice modeling under the immunosuppressive condition, can't truly reflect the state of natural occurrence, more unilateral.Therefore, infect if an animal model can stably copy Eperythrozoon, and can overcome this two defectives, will have the value of uniqueness to the further investigation of Eperythrozoon.
Summary of the invention
The technical problem to be solved in the present invention the invention provides a kind of construction method of mouse infection model of eperythrozoon suis.The present invention adopts the eperythrozoon suis of separation and purification to obtain by intravenous injection SPF mice as the source of infection.The present invention adopts the blood tabletting to detect, and PCR detects and three kinds of methods of transmission electron microscope detection combine verifies.
The present invention is achieved by the following technical solutions:
The present invention includes following steps:
1. separation and purification goes out eperythrozoon suis from the Sanguis sus domestica of infected pigs's eperythrozoonosis;
The eperythrozoon suis of described separation and purification is meant that the eperythrozoon suis of separating is resuspended in the aseptic normal saline from the swine eperythrozoonosis Sanguis sus domestica, this suspension is the eperythrozoon suis suspension of separation and purification.
2. the raising of mice is raised in the isolation room of fumigation;
3. mouse vein is inoculated;
Described mouse vein inoculation is meant the eperythrozoon suis suspension to the mouse inoculation purification, is specially: to 7 age in week SPF mouse tail vein injection 100ul purification the eperythrozoon suis suspension, wherein the eperythrozoon suis number is more than or equal to 10
7Individual.
4. the detection of animal model, checking eperythrozoon suis mouse infection models show goes out Eperythrozoon and infects erythrocytic structure.
Described detection is to comprise blood tabletting microscopy, the comprehensive detection of PCR detection and transmission electron microscope microscopy three-step approach.
Wherein PCR detection primer is to design according to eperythrozoon suis 1.8kb open reading frame sequence, the upstream is 5 ' CTGCC GATGT GTTAA GTCCT-3 ', the downstream is 5 '-CTGGG TGTAT GAAGA GTGGT GT-3 ', can amplify the fragment that length is 233bp.
According to above-mentioned steps, by three kinds of detection method checkings, confirm eperythrozoon suis SPF mouse infection model construction success among the present invention, also can clearly show Eperythrozoon simultaneously and infect erythrocytic ultrastructure.
Described infection, with the eperythrozoon suis of separation and purification as the source of infection.Because the source of infection is single, the foundation that makes model is standard and stable more.Described inoculation is meant: intravenous injection need not to cut spleen operation or injection dexamethasone etc. to laboratory animal and also can obtain higher infection rate.
The present invention has remedied the defective that present Eperythrozoon animal model makes up fully, is a kind of ideal animal model.Meaning of the present invention is that this model can be used for the pathogenesis of eperythrozoonosis, immunology, and foundation and laboratory facilities are studied in providing of aspects such as epidemiology.
Description of drawings
Fig. 1 infects erythrocytic transmission electron microscope picture for embodiment 1 Eperythrozoon.
Fig. 2 infects erythrocytic transmission electron microscope picture for embodiment 2 Eperythrozoons.
The specific embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.
SPF mice, i.e. specific pathogen free (Specific pathogen free animal, the SPF) abbreviation of mice.A general international standard rank for laboratory mice is applicable to most of experimentation.For aseptic level mice and regular grade mice.The SPF mice comprises different cultivars, and as the most frequently used two kinds: i.e. kunming mice and BALB/c mouse, present embodiment is applicable to SPF level different cultivars mice, be not limited to that embodiment mentions two kinds.
The structure of embodiment 1 eperythrozoon suis SPF kunming mice infection model
(1) separation and purification eperythrozoon suis
At first aseptic collection suffers from the Sanguis sus domestica 200ml of swine eperythrozoonosis, and the centrifugal 10min of 2000r/min removes supernatant.To precipitate erythrocyte mud adding equal-volume normal saline resuspended is red cell suspension.Add isopyknic lymphocyte separation medium and red cell suspension in new centrifuge tube, the centrifugal 20min of 2500r/min removes the superiors and intermediary leukocytic cream, takes off a layer erythrocyte layer.With the resuspended erythrocyte layer of isopyknic normal saline, behind 50 ℃ of water-bath 15min, can be observed Eperythrozoon under the oily mirror and separate with erythrocyte, get supernatant in a centrifuge tube with the centrifugal 10min of 2000r/min this moment.Precipitation is resuspended in the normal saline the centrifugal 10min of 2000r/min again, gets supernatant.The supernatant of twice gained is collected together, and 12000r/min is centrifugal, removes supernatant.Precipitation is resuspended in an amount of normal saline, is the eperythrozoon suis suspension of purification.
(2) raising of SPF kunming mice
The mouse cage of isolation room and raising mice needs thorough disinfection before the test.With 1% lysol solution wiping isolation room ground, door, window and mouse cage, use irradiation under ultraviolet ray 12 hours earlier, then the room of raising mice is fumigated.At first measure the volume size in room, press 30 milliliters/cubic metre of formalin, potassium permanganate 15 gram/cubic meters and water and calculate consumption for 15 milliliters/cubic metre.Before the room fumigation, check the seal in room.When stifling, earlier water is poured in the ceramic vessel, the back adds potassium permanganate, stirs, and adds formalin again, and the people promptly leaves, closed room.Fumigation is more than 24 hours, and ventilation is ventilated then.
During the experiment beginning, before the staff enters isolation room, all must carry out arm part sterilization (bromo geramine of dilution in 1: 1000), change aseptic working clothing and mask then.7 age in week the SPF kunming mice available from experimental animal center, Chinese Academy of Sciences Shanghai, feed sterilization Mus grain and aquesterilisa.The SPF kunming mice is divided into each 30 of matched group and infected group, raises respectively at different isolation rooms.
(3) inoculation
Take the tail vein injection inocalation method, every mice of infected group all inoculates the eperythrozoon suis suspension of 100ul purification, and (the eperythrozoon suis number is no less than 10 in the 100ul suspension
7Individual).Every mouse inoculation 100ul of matched group physiological saline solution.
(4) the blood tabletting detects
Infect after 3 days, respectively infected group and control group mice are carried out tail vein blood, with the blood of gathering
Tabletting is observed under 1000 times of oily mirrors, can see that obvious distortion all appears in the infected group mouse red blood cell.And the mice in control group erythrocyte is normal concave-concave shape.
(5) PCR detects
According to eperythrozoon suis 1.8kb open reading frame sequence design Auele Specific Primer, the upstream is 5 '-CTGCC GATGT GTTAA GTCCT-3 ', the downstream is 5 '-CTGGG TGTAT GAAGA GTGGT GT-3 '.Experimental group and control group mice be heart blood sampling 1ml respectively, presses genome DNA extracting reagent kit (TIANGEN) the extraction template DNA is described.The PCR reaction system is: 1 * PCR buffer, 1.5mM MgCl2,250u M dNTPs, 12.5pmole primer, 1.25U Taq archaeal dna polymerase and 1ul template DNA.Reaction condition is: behind 94 ℃ of 5min, and 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 30s, carry out 36 circulations and extend 10min for back 72 ℃, the sample standard deviation of experimental group can amplify the fragment that length is 233bp, and this fragment of proof is an eperythrozoon suis after the order-checking of worker biotech firm is given birth in Shanghai.And the matched group sample can not amplify respective strap.
(6) transmission electron microscope detects
With the fixing 2h of 2.5% glutaraldehyde, with 0.1mol/L phosphoric acid rinsing liquid rinsing three times, 1% osmic acid fixative is 2-3h fixedly, reuse 0.1mol/L phosphoric acid rinsing liquid rinsing three times with the experimental mice tail vein.Use 50% ethanol respectively, 70% ethanol, 90% ethanol, 90% ethanol: 90% acetone (1: 1), 90% acetone and 100% acetone carry out the gradient dehydration.Acetone adds embedding liquid and soaks into embedding step by step.
With Leica (come card)-ULTRACUT ultramicrotome microtome slice thickness is 60nm, after the two dyeing of 3% acetic acid uranium-lead citrate, and with Hitachi (Hitachi)-7650 transmission electron microscope observation, film making (* 10000).From picture, can observe Eperythrozoon and can loosely be combined in erythrocyte surface, and not run through erythrocyte membrane.Eperythrozoon causes erythrocyte membrane to cave in attached to erythrocyte surface, and Eperythrozoon then is embedded in the erythrocyte membrane that caves in, and certain interval (as Fig. 1) is arranged between Eperythrozoon and the erythrocyte membrane.
Present embodiment has successfully made up swine eperythrozoonosis SPF kunming mice infection model, and this model can be used for this sick pathogenesis, immunology, the research of aspects such as epidemiology.
The structure of embodiment 2 eperythrozoon suis SPF BALB/c mouse infection models
(1) separation and purification eperythrozoon suis
At first aseptic collection suffers from the Sanguis sus domestica 200ml of swine eperythrozoonosis, and the centrifugal 10min of 2000r/min removes supernatant.To precipitate erythrocyte mud adding equal-volume normal saline resuspended is red cell suspension.Add isopyknic lymphocyte separation medium and red cell suspension in new centrifuge tube, the centrifugal 20min of 2500r/min removes the superiors and intermediary leukocytic cream, takes off a layer erythrocyte layer.With the resuspended erythrocyte layer of isopyknic normal saline, behind 50 ℃ of water-bath 15min, can be observed Eperythrozoon under the oily mirror and separate with erythrocyte, get supernatant in a centrifuge tube with the centrifugal 10min of 2000r/min this moment.Precipitation is resuspended in the normal saline the centrifugal 10min of 2000r/min again, gets supernatant.The supernatant of twice gained is collected together, and 12000r/min is centrifugal, removes supernatant.Precipitation is resuspended in an amount of normal saline, is the eperythrozoon suis suspension of purification.
(2) raising of SPF BALB/c mouse
The mouse cage of isolation room and raising mice needs thorough disinfection before the test.With 1% lysol solution wiping isolation room ground, door, window and mouse cage, use irradiation under ultraviolet ray 12 hours earlier, then the room of raising mice is fumigated.At first measure the volume size in room, press 30 milliliters/cubic metre of formalin, potassium permanganate 15 gram/cubic meters and water and calculate consumption for 15 milliliters/cubic metre.Before the room fumigation, check the seal in room.When stifling, earlier water is poured in the ceramic vessel, the back adds potassium permanganate, stirs, and adds formalin again, and the people promptly leaves, closed room.Fumigation is more than 24 hours, and ventilation is ventilated then.
During the experiment beginning, before the staff enters isolation room, all must carry out arm part sterilization (bromo geramine of dilution in 1: 1000), change aseptic working clothing and mask then.7 age in week the SPF BALB/c mouse available from experimental animal center, Chinese Academy of Sciences Shanghai, feed sterilization Mus grain and aquesterilisa.The SPF mice is divided into each 30 of matched group and infected group, raises respectively at different isolation rooms.
(3) inoculation
Take the tail vein injection inocalation method, every mice of infected group all inoculates the eperythrozoon suis suspension of 100ul purification, and (the eperythrozoon suis number is no less than 10 in the 100ul suspension
7Individual).Every mouse inoculation 100ul of matched group physiological saline solution.
(4) the blood tabletting detects
Infect after 3 days, respectively infected group and control group mice are carried out tail vein blood,, under 1000 times of oily mirrors, observe, can see that obvious distortion all appears in the infected group mouse red blood cell the blood tabletting of gathering.And the mice in control group erythrocyte is normal concave-concave shape.
(5) PCR detects
According to eperythrozoon suis 1.8kb open reading frame sequence design Auele Specific Primer, the upstream is 5 '-CTGCC GATGT GTTAA GTCCT-3 ', the downstream is 5 '-CTGGG TGTAT GAAGA GTGGT GT-3 '.Experimental group and control group mice be heart blood sampling 1ml respectively, presses genome DNA extracting reagent kit (TIANGEN) the extraction template DNA is described.The PCR reaction system is: 1 * PCR buffer, 1.5mM MgCl2,250u MdNTPs, 12.5pmole primer, 1.25U Taq archaeal dna polymerase and 1ul template DNA.Reaction condition is: behind 94 ℃ of 5min, and 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 30s, carry out 36 circulations and extend 10min for back 72 ℃, the sample standard deviation of experimental group can amplify the fragment that length is 233bp, and this fragment of proof is an eperythrozoon suis after the order-checking of worker biotech firm is given birth in Shanghai.And the matched group sample can not amplify respective strap.
(6) transmission electron microscope detects
With the fixing 2h of 2.5% glutaraldehyde, with 0.1mol/L phosphoric acid rinsing liquid rinsing three times, 1% osmic acid fixative is 2-3h fixedly, reuse 0.1mol/L phosphoric acid rinsing liquid rinsing three times with the experimental mice tail vein.Use 50% ethanol respectively, 70% ethanol, 90% ethanol, 90% ethanol: 90% acetone (1: 1), 90% acetone and 100% acetone carry out the gradient dehydration.Acetone adds embedding liquid and soaks into embedding step by step.
With Leica (come card)-ULTRACUT ultramicrotome microtome slice thickness is 60nm, after the two dyeing of 3% acetic acid uranium-lead citrate, and with Hitachi (Hitachi)-7650 transmission electron microscope observation, film making (* 10000).From picture, can observe Eperythrozoon and can loosely be combined in erythrocyte surface, and not run through erythrocyte membrane.Eperythrozoon causes erythrocyte membrane to cave in attached to erythrocyte surface, and Eperythrozoon then is embedded in the erythrocyte membrane that caves in, and certain interval (as Fig. 2) is arranged between Eperythrozoon and the erythrocyte membrane.
Present embodiment has successfully made up swine eperythrozoonosis SPF BALB/c mouse infection model, and this model can be used for this sick pathogenesis, immunology, the research of aspects such as epidemiology.
Claims (8)
1, a kind of construction method of mouse infection model of eperythrozoon suis is characterized in that, may further comprise the steps:
1. separation and purification goes out eperythrozoon suis from the Sanguis sus domestica of infected pigs's eperythrozoonosis, and the eperythrozoon suis of separating from the swine eperythrozoonosis Sanguis sus domestica is resuspended in the aseptic normal saline, and this suspension is the eperythrozoon suis suspension of separation and purification;
2. the raising of mice is raised in the isolation room of fumigation;
3. to mouse vein inoculation, the eperythrozoon suis suspension to the mouse inoculation purification is specially: to 7 age in week SPF mouse tail vein injection 100ul purification the eperythrozoon suis suspension;
4. the detection of animal model, checking eperythrozoon suis mouse infection models show goes out Eperythrozoon and infects erythrocytic structure.
2, the construction method of the mouse infection model of eperythrozoon suis as claimed in claim 1 is characterized in that, described eperythrozoon suis suspension, and wherein the eperythrozoon suis number is more than or equal to 10
7Individual.
3, the construction method of the mouse infection model of eperythrozoon suis as claimed in claim 1 is characterized in that, described infection, with the eperythrozoon suis of separation and purification as the source of infection.
4, the construction method of the mouse infection model of eperythrozoon suis as claimed in claim 1 is characterized in that, described inoculation is meant: intravenous injection.
5, the construction method of the mouse infection model of eperythrozoon suis as claimed in claim 1 is characterized in that, described detection comprises: blood tabletting microscopy, the comprehensive detection of PCR detection and transmission electron microscope microscopy three-step approach.
6, the construction method of the mouse infection model of eperythrozoon suis as claimed in claim 1 is characterized in that, described blood tabletting detects, be meant: infect after 3 days, respectively infected group and control group mice are carried out tail vein blood,, observe the blood tabletting of gathering.
7, the construction method of the mouse infection model of eperythrozoon suis as claimed in claim 1, it is characterized in that, described PCR detects, its primer is to design according to eperythrozoon suis 1.8kb open reading frame sequence, the upstream is 5 '-CTGCC GATGT GTTAA GTCCT-3 ', the downstream is 5 '-CTGGG TGTAT GAAGA GTGGT GT-3 ', can amplify the fragment that length is 233bp.
8, the construction method of the mouse infection model of eperythrozoon suis as claimed in claim 1, it is characterized in that described transmission electron microscope detects, be meant the experimental mice tail vein with the fixing 2h of 2.5% glutaraldehyde, with 0.1mol/L phosphoric acid rinsing liquid rinsing three times, 1% osmic acid fixative is 2-3h fixedly, reuse 0.1mol/L phosphoric acid rinsing liquid rinsing three times, gradient dehydration, soak into step by step, embedding, section is observed.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789430A (en) * | 2014-01-24 | 2014-05-14 | 浙江大学 | Kit for rapid detection of eperythrozoon, leptospira and toxoplasma in blood and application of kit |
| CN107164230A (en) * | 2017-04-28 | 2017-09-15 | 内蒙古医科大学 | A kind of construction method of SD rat infections people eperytozoa body Model |
| CN107367407A (en) * | 2017-05-25 | 2017-11-21 | 长沙金域医学检验所有限公司 | A kind of Pathologic specimen is fixed and dehydration treatment method |
-
2007
- 2007-11-08 CN CNA2007100479529A patent/CN101288774A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789430A (en) * | 2014-01-24 | 2014-05-14 | 浙江大学 | Kit for rapid detection of eperythrozoon, leptospira and toxoplasma in blood and application of kit |
| CN103789430B (en) * | 2014-01-24 | 2015-05-20 | 浙江大学 | Kit for rapid detection of eperythrozoon, leptospira and toxoplasma in blood and application of kit |
| CN107164230A (en) * | 2017-04-28 | 2017-09-15 | 内蒙古医科大学 | A kind of construction method of SD rat infections people eperytozoa body Model |
| CN107367407A (en) * | 2017-05-25 | 2017-11-21 | 长沙金域医学检验所有限公司 | A kind of Pathologic specimen is fixed and dehydration treatment method |
| CN107367407B (en) * | 2017-05-25 | 2020-04-24 | 长沙金域医学检验所有限公司 | Pathological specimen fixing and dehydrating treatment method |
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Open date: 20081022 |