Table of Content

25 April 2018, Volume 38 Issue 2

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Up-regulated Expression and Preliminary Mechanism of Interferon γin Duck Embryonic Fibroblasts Induced by Duck Enteritis Virus

LI Si-qi, YIN Hai-chang, ZHAO Li-li, WANG Yi-ping, JIN Jian-li, CHEN Hong-yan

2018, 38(2):  86-90.  DOI: 10.3969/j.issn.1674-5817.2018.02.002

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Objective In order to investigate the mechanism of interferon γ(IFNγ) up-regulation in duck embryo fibroblasts (DEF) induced by duck enteritis virus(DEV) infection.Methods The total RNA was extracted from DEF at 24 h,36 h,48 h and 60 h after DEV infection,and reverse transcripted into cDNA.The expression of IFNγand related interferon stimulated genes (ISGs) was detected by fluorescence quantitative PCR using specific primers.Results The expression level of IFNγwas significantly up-regulated at 24 h,36 h,48 h and 60 h,and the mRNA expression levels of Myxovirus resistance (Mx)、DEAD-box protein 50(DDX50)、2’-5’oligoadenylates synthesis L (OASL) were also up-regulated in DEV-infected cells compared with those of uninfected cells.Conclusions DEV-infected DEF cells were able to induce IFNγproduction and up-regulation of related ISGs.



Establishment of SYBR Green Ⅱ Based Real-time PCR Method for Detection of Helicobacter Rodentium

SUN Jing-yu, XIE Ling-zhi, FENG Jie, YU Ning, QIAN Miao, CAO Shu-yang, ZHANG Quan

2018, 38(2):  91-97.  DOI: 10.3969/j.issn.1674-5817.2018.02.003

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Objective To establish a rapid detection method for helicobacter rodentium.Method A pair of specific primers was designed according to the gene sequence of Helicobacter rodentium 16S rRNA.The method of SYBR Green Ⅱ real-time PCR was established.Results The standard curve cycle threshold had a good linear relationship with the template concentration in the range of 10² copies /µL to 10⁷ copies /µL concentration.This method has good sensitivity,only specific amplification of Helicobacter rodentium,could detect 30 copies /µL,the coefficient of variation (CV) was less than 2% within and between groups with good reproducibility.Conclusion The method of SYBR Green Ⅱ real-time PCR was established for the identification of Helicobacter rodentium.



Establishment and Preliminary Application of RT-PCR Method for Detection of Bovine Parainfluenza Virus Type3 in Bovine Origin Samples

WANG Ji, FU Rui, LI Xiao-bo, WANG Shu-jing, WANG Sha-sha, LI Wei, QIN Xiao, GONG Wei, YUE Bing-fei, HE Zheng-ming

2018, 38(2):  98-104.  DOI: 10.3969/j.issn.1674-5817.2018.02.004

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Objective To establish a RT-PCR method for detection of bovine parainfluenza virus type3 (BPIV3) in bovine origin samples.Methods The primers were designed and synthesized according to the published BPIV3 specific sequences of neuraminidase (HN) gene.RT-PCR method was established,and the specificity,sensitivity,repeatability and stability of this method were tested.Then the method was used to detect 137 bovine origin samples.Results The developed RT-PCR method was no cross reaction with bovine viral diarrhea virus (BVDV),bovine herpesvirus type1(BHV-1),Sendai virus(SV).The lowest detection virus titer is 10^-6/mL.BPIV3 cDNA maintained at -30 ℃ for 12 months,still can enlarge the fragment of about 610 bp visible purpose belt .The nucleic acid positive rate of 137 bovine origin samples were 14.6% about BPIV3 detected by RT-PCR.Conclusion The developed RT-PCR method is good in specificity,sensitivity,stability,and can be used for detecting the BPIV3 in bovine origin samples.



Effects of Azelaic Acid Adjuvant on Humoral Immune Response Induced by Hepatitis A Virus Attenuated Live Vaccine in Mice

LI Xiao-hong, JIN Xiao, JIA Sen-quan, HU Ning-zhu, HU Yun-zhan, LI Jian-fang

2018, 38(2):  105-110.  DOI: 10.3969/j.issn.1674-5817.2018.02.005

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Objective To investigate the effect of azelaic acid as an adjuvant on humoral immune response induced by hepatitis A virus (HAV) attenuated live vaccine (HepA-1) in mice to evaluate its adjuvant effect.Methods The ICR mice were injected respectively with HepA-1(18EU) and Al(OH)₃(300 mg) and four different doses(0.5 mg,1 mg,2 mg,3 mg) of azelaic acid as adjuvant group,300 µL for each and one immunization.At the same time,set up the blank group,pure vaccine group,aluminum adjuvant control group.The sera from tail vein blood of mice were collected at the end of 4,8,12 and 16 weeks after immunization and the specific IgG antibody against HAV were detected by indirect ELISA method.Results In addition to the blank group,mice in various groups generated Anti-HAV IgG at 4,8,12,16 weeks after the immunization.The antibody levels increased as time went on and reached peak at the 8th and 12th week,then decreased gradually.At 4 weeks,the level of anti-hepatitis A virus (HAV) IgG produced by adjuvant group 4 (azelaic acid 3 mg) was significantly higher than aluminum adjuvant control group (P<0.05,t = 2.640).At the same time,the level of anti-hepatitis A virus (HAV) IgG in adjuvant group 4 was significantly higher at 4th week,8th week and 12th week than pure antigen group (P<0.05,t = 3.134,2.751,2.743).Conclusion Appropriate dose of azelaic acid can obviously enhance the HepA-1 induced humoral immune response in mice.Azelaic acid is expected to become a new adjuvant for human vaccines.



Effect of Kappa-selenocarrageenan on Humoral Immune Response Induced by Hepatitis a Virus Attenuated Live Vaccine in Mice

JIA Sen-quan, JIN Xiao, LI Xiao-hong, WANG Hai-xuan, HU Ning-zhu, HU Yun-zhang

2018, 38(2):  111-116.  DOI: 10.3969/j.issn.1674-5817.2018.02.006

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Objective To investigate the effect of kappa-selenocarrageenan (KSC) on humoral immune response induced by hepatitis A virus attenuated live vaccine (HepA-1) in mice.Methods Seven experiment groups were set up,with 7 ICR mouse per group randomly,negative control group (saline),pure antigen group (18EU HepA-1),Aluminium adjuvant group [Al(OH)₃ 0.3 mg+18 EU HepA-1],KSC experimental groups (KSC 1 mg+18 EU HepA-1,KSC 0.5 mg+18 EU HepA-1,and KSC 0.25 mg +18 EU HepA-1,KSC 0.1 mg+18 EU HepA-1),in which mice were injected subcutaneously with 300 µL,immunizing one time.The anti-HAV specific IgG antibody levels were tested by indirect ELISA method in the serum of mice after the first 4th,8th,12th,16th week of injection.The healthy status of mice were observed during the study.The kidney,spleen,liver,lung and heart of mice immunized with KSC+HepA-l at the optimal dosage and those in negative control group were taken out at 16th week after immunization,and prepared into sections for pathological observation.Results HAV-specific IgG antibody of all mice were detected at four setting time point except negative control group.The antibody level of all groups increased first and then decreased.The peak of Aluminium adjuvant group at the 12th week and others at the 8th week.The level of antibody in KSC 0.5 mg+18EU HepA-1 group at 8th,12th week was higher than that in simple antigen group (t=3.103,t=2.573,P<0.05).The level of antibody in KSC 0.25 mg +18EU HepA-1 group at week 12 was higher than that in pure antigen group(t=2.602,P<0.05).The optimal dosage of mice in this test was 0.5 mg.No abnormality was observed in the physiological indexes of mice in various groups,while there was no pathological changes in the kidney,spleen,liver,lung and heart of mice immunized with HepA-l +0.5 mg KSC.Conclusion KSC can be used as an adjuvant to enhance humoral immune response induced by HepA-1 in mice.



Comparison of Peripheral Nerve Regeneration Ability in B6-St mice and C57BL/6 Mice

WU Liu-cheng, YANG Wen-jie, FAN Hai-hua, YI Jian-feng, TIAN Jia-wei, SHI Wei-min, JIANG Mao-rong

2018, 38(2):  122-125.  DOI: 10.3969/j.issn.1674-5817.2018.02.008

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Objective To explore the difference of peripheral nerve regeneration between C57BL/6 mice and B6-St mice (short tail mice) comparing sciatic nerve injury mice model.Methods Healthy C57BL/6 mice and B6-St mice were selected for study.Each group was half the male and female.All the mice were treated with left sciatic nerve injury to induce the model mice which were followed up for 8,12 and 16 days after surgery to calculate the sciatic function index (SFI).All mice were subjected to electrophysiological examination 21 days after the operation.The composite muscle motion potentials (CMAPs) were recorded.The bilateral gastrocnemius muscle of each group was used to calculate the wet weight ratio of gastrocnemius muscle.The frozen gastrectomy was performed on the gastrocnemius muscle.The cross-sectional area (CSA) of the muscle fibers was measured by HE staining.Results The SFI values of the two groups were not significantly different on 8,12 and 16 days after operation (P>0.05).There was no significant difference in the composite muscle between the two groups (P>0.05).There was no significant difference between the two groups in terms of the wet weight of the gastrocnemius muscle.There was no significant difference in the CSA between the two groups.Conclusion The peripheral nerve regeneration ability of B6-St mice is similar to that of C57BL/6 mice.



A Simple gRNA in vitro Screening Method

CHENG Da-xin, XU Li-ran, YU Qing-qing, GAO Shou-cui, WANG Xiao-jing, LIU Yi, LIU En-qi, ZHAO Si-hai

2018, 38(2):  126-129.  DOI: 10.3969/j.issn.1674-5817.2018.02.009

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Objective To explore a simple guide ribonucleic acid (gRNA) in vitro screening method.Methods gRNAs target rabbits torsin family 2 member A (TOR2A) gene were used as an example and screened out two efficiency gRNAs.First,design the gRNAs by online software.Three or more gRNA sequences were choose and inserted behind T7 promoter.After in vitro transcription,gRNAs were purified and then quantified by NanoDrop.Secondly,a pair of primers were designed to amplify about 300~1000 bp genomic sequence which included the gRNAs target sequences.The last step,PCR products,gRNA candidate and Cas9 nuclease were incubated together and then were checked by agarose gel electrophoresis.Results In this study,three gRNAs that target rabbits TOR2A gene were screened.The gRNA1 was failed to guide Cas9 to cleave the PCR products that contain the target sequences,while gRNA2 and gRNA3 were identified to successfully guide the Cas9 cleavage.Conclusion The gRNA in vitro screening method that used in this experiment was simple and efficient.



A Survey of Eimeria Infections in Experimental Rabbits in Fujian Province

WEN Fu-li

2018, 38(2):  130-134.  DOI: 10.3969/j.issn.1674-5817.2018.02.010

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Objective To investigate the infection of coccidian in experimental rabbits in Fujian province.Methods Feces were collected from 2 experimental rabbit producers and 12 experimental units in 6 prefecture level cities of Fujian province.The oocysts were collected by the method of saturated brine floating.The oocysts per gram of faeces (OPG) was determined and the sporulated oocysts were identified.Results A total of 195 samples were collected and analyzed in this survey,with 28 positive samples,the total infection rate was 14.36%,and the average OPG (represent infection intensity) was 2 333.33.The infection rate of 2 experimental rabbit producers was 22.22%,and the infection rate of 12 experimental units was 13.10%.A total of 10 kinds of rabbit coccidia were detected,all of them were mixed infection.Eimeria tenella was the dominant species.The infection rate of coccidian in the ordinary experimental rabbits was 15.30%,and coccidian can not be found in the cleanliness experimental rabbits.The positive rate of coccidian in weanling rabbits was the highest,the infection rate was 17.50%,the growing rabbits was the second.The breeding rabbits had the lowest infection rate and the highest infection intensity.In our province,bio-pharmaceutical enterprises had the highest infection rate of rabbit coccidia,the average OPG was 5 678.39,and the infection rate was also higher.In hospitals,universities and other research institutes,there are varying degrees of experimental coccidia infection.Conclusions The infection rate of coccidian in experimental rabbits in Fujian province is lower than other provinces.In daily work,we should further strengthen the experimental rabbit coccidian monitoring,prevention and control.It is suggested that rabbit coccidian should be included in the national standard of conventional experimental rabbits as necessary test items.



Characterization of Growth and Reproductive Performance in Microtus Fortis

BAI Xiong, LIN jin-xing, WANG Xiao-dong, FENG Jie, GAO Jie, XIE Jian-yun

2018, 38(2):  135-140.  DOI: 10.3969/j.issn.1674-5817.2018.02.011

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Objective To characterize and to compare the growth and reproductive performance of Microtus fortis of Dongtinghu stock (M.f.calamorum) and Qingtongxia stock (M.f.fortis).Methods Females and males of each stock,aged about 10 weeks,were long-term monogamously mated,the reproductive characters were analyzed.Body weight of 60 Microtus fortis (30 females and 30 males) of each stock were measured from born to 98 days of age,and the growth curves were fitted by 3 nonlinear growth models.Results For Microtus fortis of dongtinghu stock,average litter size was 4.70 ±1.15,litter interval was (30.78 ±10.21) d,weight at birth and weaning was (4.09±0.13) g and (27.31±1.10) g respectively.For Microtus fortis of qingtongxia stock,average litter size was 3.58±1.00,litter interval was (35.60±12.48) d,weight at birth and weaning was (4.37±0.09) g and (28.22±0.46) g respectively.Growth curve showed that the sexual dimorphism of Dongtinghu Microtus fortis and Qingtongxia Microtus fortis occurred at 14 d and 28 d respectively.The fitting results showed that the females had earlier time of inflection than that of males.Conclusion The reproductive capacity of Microtus fortis in Dongtinghu stock is higher than that of Qingtongxia stock.The body weight of male and female has significant dimorphic in both stocks.Femals reached sexual maturity earlier than males.



Research Progress on Infection Mechanism and Diagnostic Methods of Ectromelia Virus

ZHOU Jie, ZHAO Li-juan, TAO Ling-yun, HU Jian-hua, GAO Cheng

2018, 38(2):  160-164.  DOI: 10.3969/j.issn.1674-5817.2018.02.016

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The paper mainly reviewed the infection and spread mechanisms of Ectromelia virus (ECTV) in animals,including natural disease resistance mechanism of the mice,the body's immune response and pathogenic mechanism of virus infection.Furthermore,we described the animal experiments,pathological diagnosis,serological methods,nucleic acid methods of ECTV and their advantages and disadvantages.This article can help to understand infection mechanism and different diagnostic methods of ECTV,which may contribute to control the spread of ECTV.