畜牧兽医学报
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2018 Vol.  49 No.  7
Published: 2018-07-23

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2018 Vol. 49 (7): 0-0 [Abstract] ( 19 ) [HTML 1KB] [PDF 452KB] ( 23 )
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2018 Vol. 49 (7): 0-0 [Abstract] ( 14 ) [HTML 1KB] [PDF 359KB] ( 14 )
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2018 Vol. 49 (7): 0-0 [Abstract] ( 3 ) [HTML 1KB] [PDF 165KB] ( 12 )
1327 GAO Xiao-long, WEI Hai-tao, SONG Yan-jun, WANG Yue, YIN Yu-qing, WANG Lin, MEI Li, FENG Xiao-yu
Progress Research of Influenza D Virus

Since the first influenza D virus(IDV) was isolated from swine in 2011, it has been reported that IDVs are widely isolated from swine, cattle and sheep in Mexico, France, Ireland, Italy, China, Japan and northwest Africa. Based on the phylogenetic analysis of whole-genome sequences of these novel influenza viruses, two genetic lineages can be distinguished, D/OK and D/660 lineages. The isolates from China belong to D/OK lineage, and the stains of D/660 lineage are mainly spread in North America. Although IDV can cause mild respiratory disease in susceptible animals, the virus may enter the blood leading to viremia, which poses a serious threat to public health. To raise the awareness about IDV, this review describes the latest researches progress in the study of IDV including its characterization, epidemiology, pathogenicity, diagnosis and prevention.

2018 Vol. 49 (7): 1327-1333 [Abstract] ( 41 ) [HTML 1KB] [PDF 1082KB] ( 53 )
1334 WANG Yan-xin, XIE Shu-yu, CHEN Dong-mei, WANG Yu-lian, PAN Yuan-hu, PENG Da-peng, TAO Yan-fei, YUAN Zong-hui
Progress in the Application of Electrochemical Immunosensor in Food Safety Detection

The electrochemical immunosensor combines traditional immunoassay with modern biosensor electrochemical analysis technology, which has become a hot research topic in recent years. The sensor exhibits high selectivity of immunoassay and high sensitivity of electrochemical analysis, as well as other advantages such as small size, convenience, low cost, simple preparation, real-time on-line detection, etc., and furthermore, it is widely used in the environmental monitoring, medical clinical trials and the field of food analysis. This paper focused on the introduction of carbon nanomaterials, and summarized the research status and application of electrochemical immunosensors in food safety testing. The aim of this study is to analyse the prospect of its future application in the area of food rapid detection through summarizing the application of the electrochemical immunosensor.

2018 Vol. 49 (7): 1334-1342 [Abstract] ( 22 ) [HTML 1KB] [PDF 934KB] ( 30 )
1343 LI Ai, ZHANG Xiu-xiu, HUANG Wan-long, XIE Ling-li, MIAO Xiang-yang
Identification and Analysis of circRNAs in Intramuscular Adipose Tissues between Large White and Laiwu Pigs

The purpose of this study was to explore circRNAs potential role in adipose tissue, which would lay a basis for further understanding the regulation role of circRNA in fat deposition and lipid metabolism. In this study, we explored the expression profile of circRNAs in intramuscular adipose tissue from Large White and Laiwu pigs using RNA-seq and bioinformatics. The target genes to the differentially expressed circRNAs were predicted by miRanda and Targetscan. The enrichment analysis of target genes was performed by GO and KEGG. The results showed that 181 differentially expressed circRNAs were identified. Among them, 102 were up-regulated expression in intramuscular adipose tissue of Laiwu pigs, and 79 were down-regulated. GO and KEGG enrichment analysis of target genes of ssc_circ_0002807 and ssc_circ_0009352 with more miRNA binding sites were peformed. The target genes were found to be enriched in the biological process involved in lipid metabolism such as phospholipid homeostasis, cholesterol homeostasis, etc. And ssc_circ_0002807, ssc_circ_0005382, ssc_circ_0009172 and ssc_circ_0004824 were also confirmed by qRT-PCR. It is infered that ssc_circ_0002807 and ssc_circ_0009352 may regulate fat deposition and metabolism.

2018 Vol. 49 (7): 1343-1353 [Abstract] ( 27 ) [HTML 1KB] [PDF 3709KB] ( 29 )
1354 ZHANG Tong-yu, FAN Hong-ying, ZHU Cai-ye, LIU Jia-xin, DENG Tian-yu, DU Li-xin, WANG Li-xian, ZHAO Fu-ping
Identification of Candidate Genes Involved in Fat Deposition in Hulun Buir Sheep Tails Using FST within the Variable Window Sizes

The aims of this study were to reveal the genetic mechanism of fat deposition in the tail of sheep, and to explore the candidate genes involved in sheep tail fat. A total of 288 individuals from two lines of Hulun Buir sheep with different tail types were used to identify candidate genes controlling different tail types of sheep. These individuals included 142 fat-tailed and 146 thin-tailed sheep. The whole-genome single locus FST values were calculated based on the Illumina Ovine 600K SNP genotype data. The cubic smoothing spline method was used to define the numbers and sizes of variable windows. A total of 23 144 variable windows were identified within the whole genome. The largest window with 775 SNPs was located at chr12:35 241 750-43 798 950 bp, with a size of 8.56 Mb. The W statistic was constructed to detect the windows with selection signature. Twenty-seven of variable windows reached extremely significant level(P<0.001). After annotation, these windows harbored 337 candidate genes, of which 22 were related to the fat metabolism. Through GO analysis, these candidate genes were mainly enriched in the intracellular components, organonitrogen compound metabolic process and small molecule metabolic process. The FST method with variable windows was used to effectively identify candidate genes associated with fat deposition in the tail of sheep. These genes can be used to breed a new sheep breed with small size. The results will provide an important reference for breeding short-tailed sheep.

2018 Vol. 49 (7): 1354-1365 [Abstract] ( 8 ) [HTML 1KB] [PDF 1590KB] ( 24 )
1366 NIU Jia-qiang, WANG Yu-heng, SUOLANG Si-zhu, CHANGBA Yang-zong, XU Ye-fen, GUO Min, CHENG Ling-hua, YANG Shi-cheng
Construction of Yak Smad 4 Gene 3'UTR Dual-luciferase Reporter Vector and Its Targeting Validation to bta-miR-146a

The aim of this study was to identify the targeting relationship between Smad 4 gene and bta-miR-146a, which would provide a theoretical basis for their regulating mechanism in follicular development of yak ovary. The conservation of miR-146a in different species was analyzed by miRBase database and MEGA 6.0 software. At the same time, TargetScan software was used to predict the potential target genes of miR-146a, and the targeting validation was performed by construction of psiCHECK2 dual-luciferase reporter vector. The results of bioinformatics analysis showed that the miR-146a mature sequence was highly conserved in vertebrates, and a total of 178 potential target genes related to ovarian follicular development were predicted, then, among these genes, Smad 4 gene was selected to be further analyzed, showing that its 3'UTR had the binding site to bta-miR-146a. The wild and mutant type psiCHECK2 dual-luciferase reporter vectors of 3'UTR of yak Smad 4 gene were successfully constructed, and the dual-luciferase assay result showed that the luciferase activity of wild vector of 3'UTR of yak Smad 4 gene was distinctly down-regulated by the bta-miR-146a mimics, and was significantly lower than that of mutant vector and NC group(P<0.05), respectively. In conclusion, this research preliminarily confirmed that yak Smad 4 gene was the target gene of bta-miR-146a, suggesting that bta-miR-146a might play roles in the development of yak follicles by regulating Smad 4 gene.

2018 Vol. 49 (7): 1366-1376 [Abstract] ( 16 ) [HTML 1KB] [PDF 2039KB] ( 22 )
1377 TANG Peng, LING Xiao-xiao, GAO Ze-cheng, JIA Cong-jun, LIANG Chun-nian, WU Xiao-yun, CHU Min, YAN Ping
Skin Histological Observation of Yak with Different Coat Colors and MC1R Functional Verification

The objectives of this study were to elucidate the characteristics of skin histomorphology in yaks with different coat colors and to discover the potential molecular mechanism by which MC1R induced melanin synthesis. Skin tissues were collected from the hindquarters of 6 Datong yaks(dark brown) and 6 Tianzhu yaks(white). Toluidine blue staining was used to reveal the content and distribution of both melanin and mature melanocytes in the skin tissues of different coat colors. MC1R expression was determined by quantitative real-time polymerase chain reaction(qRT-PCR). Afterward,shRNA interference vectors for MC1R were transfected into B16 murine melanoma cells,and transfection efficiency was quantified by qRT-PCR and Western blot assay. The expressions of hair color-related genes(Agouti,MITF and TYR) in each group were analyzed and compared,and melanin production was detected with a microplate reader at different time points. The results showed that a large number of melanocytes distributed around the epidermis and hair follicles of both types of yaks. Tiny amounts of melanin were detected at the base of the epidermis and the hair roots of Tianzhu white yak skin tissues,while large amounts of melanin were discovered in the epidermis and hair follicles of Datong yak skin tissues. The expression of MC1R in Tianzhu white yaks was significantly lower than that in Datong yaks(P<0.01).In B16 cells with successful inhibition of MC1R,expression of TYR was very significantly lower(P<0.01),MITF expression was significantly lower(P<0.05),Agouti expression was not significantly different(P>0.05),and melanin content was lower than in cells that expressed MC1R. These results indicate that MC1R plays an important role in melanin synthesis and formation of coat color. To summarize,shRNA MC1R vectors were successfully transfected into B16 mouse melanoma cells;inhibition of MC1R in B16 cells was consistent with reduced expression of the downstream gene TYR,which lowered the melanin content in skin tissues. Therefore,we conclude that massive deposition of melanin and high expression of MC1R are responsible for the differences in yak coat colors.

2018 Vol. 49 (7): 1377-1386 [Abstract] ( 24 ) [HTML 1KB] [PDF 4454KB] ( 25 )
1387 LI Lan-qi, LIU Jiang-jing, CHEN Xiao-li, LI Xin, LU Yong-qiang, WANG Shui-lian, WANG Dong
Study on the Activity Alteration of Dairy Cow during Estrus

In order to improve the detection rate of estrus determination via pedometer, the activity alteration of dairy cows affected by parities, seasons and types of estrus treatment was compared before and after estrus. 157 randomly selected dairy cows(150 postpartum healthy lactating dairy cows and 7 primiparous ones) in a dairy farm were analyzed in this study. The activity of the experimental cows before and after estrus were measured by pedometer, and the estrus status were confirmed by rectal examination. The detection of pedometer showed that the activities of cows after the onset of estrus(>1 500) were significantly higher than those during diestrus(<500)(P<0.05). The maximum activities(2 179±1 073) was observed at 4.5 h after the onset of estrus and gradually decreased till the end of the estrus to the level of diestrus. The amount of increased activity and its duration of cows during estrus were markedly influenced by parities, seasons and types of estrus treatment(P<0.05). In conclusion, parities, seasons and types of estrus treatment must be taken into account comprehensively when we use pedometer to detect estrus Holstein cows automatically. And then set an estrus prediction threshold according to local specific circumstances.

2018 Vol. 49 (7): 1387-1393 [Abstract] ( 20 ) [HTML 1KB] [PDF 1103KB] ( 23 )
1394 FU Li-xia, MA Tao, DIAO Qi-yu, CHENG Shu-ru, SONG Ya-zhe
Effect of Different Concentrates on Nutrient Digestibility and Excretion of Purine Derivatives in Urine of Mutton Sheep

The aim of this study was to establish a model for estimating microbial nitrogen with purine derivatives. Ten Dorper×thin-tailed Han crossbred, non-castrated adult rams with(47.4±4.4) kg BW were assigned to 10 experimental diet treatments with different concentrate sources in a single factor design, and 10 rams in each treatment. The experimental diet included 10 pelleted TMR with a single concentrate ratio of 30%. The experiment included 10 periods in which 10 lambs were fed with one of the 10 pelleted TMR. Each period lasted for 20 d(15 d for adaptation and 5 d for trial period) and the total experiment lasted for 200 d. This experiment study the effect of 10 different concentrates on nutrient digestibility and excretion of purine derivatives in urine of mutton sheep. The results showed that:1)The digestibility of nutrients in diets was related to the content of nutrients, and the digestibility of diets with different concentrates was different. The best predictors of dietary digestible crude protein(DCP) was crude protein(CP). The urinary nitrogen, fecal nitrogen and intake nitrogen of lambs fed 10 diets with different concentrates showed significant difference(P<0.01). 2) The proportion of allantoin, uric acid, xanthine(included hypoxanthine) in total purine derivative(PD) excretion were significantly different(P<0.01) among 10 treatments, and the ratios were 85.60%-92.47%, 2.54%-7.18%, 3.20%-7.41%, respectively. Purine nitrogen indexes were significantly different(P<0.01) among 10 treatments, and range was 0.03-0.10. However, PD excretion had a strong linear relationship with intake nitrogen and microbial nitrogen(MN). Intake N had a strong relationship with apparent digestibility of N, urine nitrogen and retained N. A linear relationship was found between retained N(Y) and intake N:Y=0.244 9×intake N-1.319 9(R2=0.825). A linear relationship was found between PD excretion and intake N:PD excretion=0.119 7×intake N+10.161(R2=0.925), and a linear relationship was found between PD excretion and MN:MN=0.746 1×PD+1.785 4(R2=0.898).

2018 Vol. 49 (7): 1394-1404 [Abstract] ( 19 ) [HTML 1KB] [PDF 1398KB] ( 19 )
1405 ZHOU Yan, DIAO Qi-yu, DONG Li-feng, DENG Kai-dong, XU Gui-shan, MA Tao
The Effect of Different Ratios of Dietary Non-fibrous Carbohydrate (NFC) to Neutral Detergent Fiber (NDF) on Production Performance, Nutrient Digestibility and Methane Emissions of Dorper×Thin-tailed Han Crossbred Growing Ewes

The aim of this study was to investigate the effects of ratios of dietary non-fibrous carbohydrate to neutral detergent fiber(NFC/NDF) on the growth performance, nutrient digestibility and methane(CH4) production of Dorper×Thin-tailed Han crossbred ewes with 48.0-55 kg of body weight(BW). Thirty ewes with(48.0±0.5) kg BW were selected and divided into 3 treatments with 10 sheep in each treatment:NFC/NDF=0.78(concentrate/forage=35:65, ad libitum), NFC/NDF=1.03(concentrate/forage=50:50, feed restriction) and NFC/NDF=2.17(concentrate/forage=65:35, feed restriction). The trial lasted for 32 d, including a 17-d adaptation and a 15-d experimental period. The energy and crude protein intake were same in each treatment. The average daily weight gain of the treatment of NFC/NDF=0.78 was taken as the standard for the feed restriction of the treatment of NFC/NDF=1.03 and 2.17. The BW was measured before morning feed, and feed intake was recorded daily. The CH4 production, dietary energy and nutrient digestibility were determined during experimental period. The results showed that:1)The feed conversion efficiency in the NFC/NDF=2.17 treatment was significantly higher than that in the NFC/NDF=0.78 treatment(P<0.05), whereas no significant difference was observed between NFC/NDF=1.03 treatment and other two treatments(P >0.05). 2) The apparent digestibility of dry matter, organic matter, gross energy and metabolic rate of gross energy in NFC/NDF=2.17 treatment were the highest among the 3 treatments(P<0.05). The apparent digestibility of crude protein in NFC/NDF=2.17 treatment was higher than that in NFC/NDF=0.78 treatment(P<0.05), however, there was no significant difference between NFC/NDF=2.17 and NFC/NDF=1.03 treatments, or between NFC/NDF=0.78 and NFC/NDF=1.03 treatments(P>0.05). With the increase of ratio of dietary NFC/NDF, the apparent digestibility of NDF in NFC/NDF=2.17 treatment was the highest(P<0.05), whereas no significant difference was observed between NFC/NDF=0.78 and NFC/NDF=1.03 treatments(P>0.05). 3) The CH4 emissions in NFC/NDF=2.17 treatment was 32.53 L·d-1, which was significantly lower than that in NFC/NDF=0.78(58.03 L·d-1) and NFC/NDF=1.03(63.17 L·d-1) treatments(P<0.05), whereas no significant difference was observed between NFC/NDF=0.78 and NFC/NDF=1.03 treatments(P>0.05). The similar results were found in CH4 emission as a proportion of metabolic weight in the 3 treatments. With the increase of dietary NFC/NDF, the CH4 emission as a proportion of dry matter intake, organic matter intake and digestible organic matter intake in NFC/NDF=2.17 treatment was significantly lower than that in NFC/NDF=1.03 treatment(P<0.05), whereas no significant difference were observed between NFC/NDF=0.78 and other two treatments(P>0.05). The CH4 emission as a proportion of gross energy intake, digestible energy intake, metabolic energy intake had the similar results. The CH4 emission as a proportion of NDF intake and digestible NDF intake in NFC/NDF=1.03 treatment was significantly higher than that in the other two treatments(P<0.05), whereas no significant difference was observed between NFC/NDF=0.78 and NFC/NDF=2.17 treatments(P>0.05). The CH4 emission as a proportion of average daily gain and digestible ADF intake were similar among the 3 treatments(P>0.05). According to the production performance, nutrient digestibility, energy utilization and CH4 production, it will be recommended that NFC/NDF=2.17 under restriction feeding would achieve the best carbon-cutting results for Dorper×Thin-tailed Han crossbred ewes with 48-55 kg of BW.

2018 Vol. 49 (7): 1405-1415 [Abstract] ( 10 ) [HTML 1KB] [PDF 1026KB] ( 18 )
1416 XU Yi-nan, ZHANG Hai-hua, WANG Jing, HUO Zi-shuang, XU Yuan, WEN Bin, BAI Xiu-juan, LI Guang-yu
Effects of Dietary Calcium and Vitamin D3 Levels on Growth Performance, Tibia Development and Viscera Indices of Growing-furring Blue Foxes(male)

The aim of this study was to investigate the effects of dietary Ca and vitamin D3levels on growth performance, tibia development and viscera indices of growing-furring blue foxes under the condition of a fixed 1.4/1 ratio of Ca to P. One hundred and thirty-five male blue foxes of 120-day-old were randomly allocated into 9 groups, with 15 blue foxes per group. The 9 treatment diets were supplemented with 0%, 0.4% and 0.8% Ca, and they were supplemented with 1 000, 2 000 and 4 000 IU·kg-1 VD3. The experiment was conducted with a 3×3 factorial design using a basic diet that contained 0.8% Ca and 327 IU·kg-1VD3. The treatment diets contained 0.8%Ca and 1 327 IU·kg-1VD3, 0.8%Ca and 2 327 IU·kg-1VD3, 0.8%Ca and 4 327 IU·kg-1VD3, 1.2%Ca and 1 327 IU·kg-1VD3, 1.2%Ca and 2 327 IU·kg-1VD3, 1.2%Ca and 4 327 IU·kg-1VD3, 1.6%Ca and 1 327 IU·kg-1VD3, 1.6%Ca and 2 327 IU·kg-1VD3, 1.6%Ca and 4 327 IU·kg-1VD3. The test period lasted for 87 days. The growth performance, ingredient percent of tibia and viscera weight were measured, respectively and then we calculated the tibia development parameters and viscera indices of growing-furring blue foxes. The results showed that:1) The final BW and body length of blue foxes significantly decreased with increasing Ca levels(P<0.05). The maximum final BW and body length were in 0%-Ca group. There was no significant difference in final BW, body length and pelt length of blue foxes among different VD3 levels(P>0.05). 2) The Ca dosage added had a linear effect on Ca percent of the tibia(P<0.05). The maximum Ca percent of tibia was in 0.8%-Ca group. The dietary VD3 level had a significant effect on ash percent of tibia(P<0.05). 3) The heart and liver indices of blue foxes linearly decreased with increasing Ca levels(P<0.05). In conclusion,the results of this research indicated that the diet supplementation with 0%Ca and 1 000 IU·kg-1VD3(the diet contained 0.8% Ca and 1 327 IU·kg-1 VD3) could improve the growth performance, body length, and regulate the tibia development and viscera indices of growing-furring blue foxes.

2018 Vol. 49 (7): 1416-1422 [Abstract] ( 15 ) [HTML 1KB] [PDF 704KB] ( 26 )
1423 REN Wan-ping, SHAO Wei, LUO Cheng-long, YU Xiong
Effects of Exogenously Added Dihydroxyacetone Phosphate on the Contents of Triglycerides and Key Metabolic Enzymes in Mouse Adipocytes

The purpose of this study was to investigate the effect of exogenously added dihydroxyacetone phosphate on the contents of triglyceride and its key metabolic enzymes in mouse adipocytes. The inoculated mouse 3T3-L1 cell into mature adipocytes were the research object, the cells were cultured in dihydroxyacetone phosphate complete medium containing 0 μmol·L-1(ck group), 20 μmol·L-1(group Ⅰ), 50 μmol·L-1(group Ⅱ) and 200 μmol·L-1(group Ⅲ) on the 14th day, and the cells were collected at 0,36 and 72 h for the detection of ELISA and other substances.The contents of key enzymes in triglyceride metabolism were detected by using double antibody sandwich technique,such as acetyl-CoA carboxylase(ACC),fatty acid synthase(FAS), hormone-sensitive lipase(HSL) and other enzymes. The results showed that:1)The contents of triglyceride in the group Ⅰ,Ⅱ and Ⅲ were significantly higher than that in ck group(P<0.01) at 36 and 72 h; The contents of glucose-6-phosphate dehydrogenase(G6PD), dihydrolipoyl transacetylase(DLAT), dihydrolipoyl dehydrogenase(DLD) and FAS in the group Ⅰ,Ⅱ and Ⅲ were significantly higher than that in ck group(P<0.01) at 36 and 72 h; 2) The contents of HSL, carnitine acyltransferase1(CPT1) and free fatty acid(FFA) in the group Ⅰ,Ⅱ and Ⅲ were significantly lower than that in ck group(P<0.01) at 36 and 72 h. The dihydroxyacetone phosphate can promote the synthesis and deposition of triglyceride in mouse adipocytes;The dihydroxyacetone phosphate can also significantly increase the contents of G6PD, DLAT, DLD and FAS in triglyceride synthesis, and can significantly reduce the contents of HSL and CPT1 in triglycerides catabolism.

2018 Vol. 49 (7): 1423-1431 [Abstract] ( 14 ) [HTML 1KB] [PDF 843KB] ( 18 )
1432 MI Si-yuan, SHI Ke-rong, ZHANG Rui-qiang, ZHANG Hai-liang, XU Qing-lei, YU Ying
RT-PCR Detection of Bovine Viral Diarrhea Virus Subtypes and Analysis of Differentially Expressed Candidate Genes in Infected Calves

To explore molecular markers of anti bovine viral diarrhea virus(BVDV) infection in dairy calves, bovine SUMOs(SUMO1, SUMO2, SUMO3, UBC9) and CD4 were selected as related candidate genes, and their differential expression status between BVDV infected and uninfected Holstein calves were detected and analyzed. Blood samples of 42 Holstein calves(Within 2 months old) were collected and whether they carried BVDV or not were identified by specific nested PCR and sequencing techniques. According to the analyses of the nested PCR and sequencing, the calves were divided into two groups(BVDV-carried and un-carried groups) to identify differentially expressed candidate genes of resistance BVDV by fluorescence quantitative PCR. The results showed that 13 cattle of the 42 Holstein calves carried BVDV-1(positive infection rate is 30.9%); the homologies of these 5' UTR reach 95.80% by the analysis of DNAMAN software; compared with the un-carried group, there were significant decreases(P<0.05) in the BVDV-carried calves for the expression of SUMO1, SUMO2 and SUMO3 genes. But there was no significant difference between the two groups for the expression of UBC9 and CD4. Only BVDV-1 was detected in the Holstein calves, which is consistent with the epidemic trend of BVDV in China. The results suggest that there may have a close relationship between BVDV-1 infection and sumoylation, SUMO1, SUMO2 and SUMO3 could be served as candidate genes of bovine BVDV resistance.

2018 Vol. 49 (7): 1432-1439 [Abstract] ( 26 ) [HTML 1KB] [PDF 1972KB] ( 27 )
1440 ZHU Jian-bo, YANG Zhen-xing, XIAO Lei, GAO Lin, MIAO Hai-sheng, HE Yu-wen, MENG Jin-xin, YANG Heng, LI Hua-chun
Establishment of a Rapid Competitive ELISA for Detecting Antibodies against Epizootic Haemorrhagic Disease Virus

The objective of this research was to establish a simple and fast operation method for detecting epizootic haemorrhagic disease virus(EHDV) antibodies. In this research, a competitive ELISA(C-ELISA) method was developed, by using purified EHDV-6 coated ELISA plates and guinea pig anti-EHDV-2 antibody as a competitive antibody. The assay had a high specificity and reproducibility, with optimal dilution of the multi-clone antibody at 1:10 000 and virus antigen at 5.12 μg·mL-1(1:10 000). The critical value of the detection method was 50%, through the serum testing of cows and sheep, including negative and positive 270 copies each. Specific tests showed that the ELISA method can only detect different serotypes of EHDV antibodies, with better group specificity. Positive serum tests on known antibody titers showed that the established C-ELISA method was more sensitive than the serum neutralization test(SNT) and Agar gel immunodiffusion test(AGID). While the serum and anticoagulant samples collected from monitoring animals were detected by C-ELISA, SNT, RT-PCR and virus isolation respectively, the results of ELISA were consistent with the other two methods. These results indicate that the C-ELISA is a rapid, sensitive and stable method for the detection of EHDV antibodies.

2018 Vol. 49 (7): 1440-1450 [Abstract] ( 22 ) [HTML 1KB] [PDF 1018KB] ( 18 )
1451 HE Yan-chun, YANG Wen-ping, FU Shao-zu, ZHENG Hai-xue, YANG Xiao-pu
The Research on Molecular Mechanism of PCBP2 Promoting the Multiplication of Foot-and-mouth Disease Virus

Poly(rC) binding protein 2(PCBP2) belongs to a class of proteins that bind to poly(C) stretches of both RNA and DNA. PCBP2 has roles in maintaining mRNA stability and regulating translation. Meanwhile PCBP2 is a negative regulator in VISA-mediated antiviral signaling to reduce production of IFN-β. Previous study have shown that PCBP2 can affect the multiplication of foot-and-mouth disease virus(FMDV), but the specific mechanism is unclear. Here, we investigated the molecular mechanisms about PCBP2 promoting the multiplication of FMDV. Results:1) We found that FMDV structural protein VP0, 2B, VP2, 2C and 3D can interact with PCBP2 via Co-IP and GST pull-down tests. 2) Further study revealed that PCBP2 is a negative regulator in VISA-mediated antiviral signaling, and 2C, 3D, VP0 and 2B can promote the negative regulation of PCBP2 on VISA-mediated antiviral signaling via the Dual-Luciferase Reporter Assay. 3) Meanwhile we found that FMDV VP0 can increase specific degradation of VISA mediated by PCBP2 through Western blot. In a word, FMDV VP0 can increase degradation of VISA mediated by PCBP2 via VP0-PCBP2 interaction to reduce the production of IFN-β, and enhance the FMDV activity in cell.

2018 Vol. 49 (7): 1451-1459 [Abstract] ( 17 ) [HTML 1KB] [PDF 2673KB] ( 14 )
1460 LIANG Wen-hua, JIA Yun-hui, XU Cheng-zhi, WU Yun-pu, CHEN Yan, YANG Huan-liang, QIAO Chuan-ling, CHEN Hua-lan
Preparation and Antiviral Activity of Two Neutralizing Monoclonal Antibodies against HA Protein of H1N1 Swine Influenza Virus

This study aimed to prepare monoclonal antibodies(MAbs) against HA protein of Eurasian avian-like H1N1(EA H1N1) virus and analyze their biological activities. After viral proliferation in chicken embryos, the purified whole-virus protein of A/swine/Henan/11/2005(HN11) was used to immunize BALB/c mice. The splenic cells of the mice were fused with the SP2/0 cells after the third immunization. After screening by indirect ELISA and hemagglutination inhibition test and three circles of sub-clone, two hybridoma cell strains secreting MAbs against HA protein of the HN11 were obtained, which was designated as 2B6 and 4C7, respectively. The ELISA titers of two MAbs were higher than 1:107, and HI titer were higher than 5 log2. These two MAbs could specifically recognize HA protein expressed by plasmid pCAGGS-H1HA in 293T cells by IFA, but not in Western-blot assay. The results of neutralization test indicated that two MAbs have specific neutralization activities for EA H1N1. The protective efficacies of two MAbs were further verified in BALB/c mice, which suggested that MAbs 2B6 and 4C7 can effectively provide protection against the infection with homologous and heterologous H1N1 influenza viruses. Two MAbs, with high viral neutralization activity and strong anti-viral infection ability, were prepared, could be used for further research on anti-viral infection and the molecular basis of antigenic variation of EA H1N1 SIVs.

2018 Vol. 49 (7): 1460-1466 [Abstract] ( 18 ) [HTML 1KB] [PDF 1311KB] ( 16 )
1467 GUO Hai-yong, YUAN Hong-xing, SUN Tai-ran, CAO Jing, SONG Qin-ye, LI Tan-qing, LU Ji-cheng
Preparation and Immunological Activities of Transfer Factor from the Spleen of Pig Immunized with Porcine Reproductive and Respiratory Syndrome Attenuated-live Virus Vaccine

The objective of this study was to prepare transfer factor(TF) from the spleen of pig immunized with porcine reproductive and respiratory syndrome(PRRS) attenuated-live virus vaccine, then detect its immune activities and expect to provide a scientific basis for developing a new immunopotentiator and prevention and control of PRRS. TF was extracted from spleen collected from healthy grown pigs which had been vaccinated with PRRS attenuated-live virus vaccine. The main components of TF were detected using sulfonyl salicylic acid, ninhydrin and ammonium molybdate-ammonium nitrate reactions, and kinds and contents of amino acids were measured by high performance liquid chromatography. The regulating effects on phagocytosis of peritoneal macrophage and lymphocytic proliferation of mice were checked by chicken erythrocyte phagocytosis test and MTT assay, respectively. Furthermore, nine piglets were divided into group A, B and C of 3 each. The piglets in the group A were injected intramuscularly with one dose of PRRS attenuated-live virus vaccine in left side opisthotic part and 1 mL of TF in the right side, respectively. Each one in the group B was injected intramuscularly the mixed liquor of one dose of PRRS attenuated-live virus vaccine with 1 mL of TF, and in the group C was did the mixed liquor of one dose of PRRS attenuated-live virus vaccine with 1 mL of normal saline. The proliferative activity of porcine peripheral blood lymphocyte(PBLC) from each group was measured by MTT on day 20 post-inoculation. There were nucleic acid and 18 kinds of amino acids in TF prepared in this study, containing 21.5 μg of nucleic acid and 3.479 mg total amino acids per mL of TF. Phagocytic ability of peritoneal macrophage to chicken erythrocytes and proliferative activity of PBLC from mice were enhanced significantly following stimulation of TF. Moreover, the proliferation capacity of porcine PBLC was also increased remarkably when the four-week-old piglets were injected with PRRS attenuated-live virus vaccine and TF simultaneously in different parts or their mixture. The transfer factor was prepared from the spleen of pig immunized with PRRS attenuated-live virus vaccine and it could increase significantly phagocytosis of macrophage and proliferative activity of PBLC.

2018 Vol. 49 (7): 1467-1474 [Abstract] ( 17 ) [HTML 1KB] [PDF 1514KB] ( 33 )
1475 ZHANG Hui-yong, WU Jing-sheng, LI Guo-hui, YU Yan, YANG Jian-bo, SU Yi-jun, YIN Jian-mei, HAN Wei
Characteristic Analysis on Vertical Transmission of Avian Leukosis Virus in Huiyang Chicken and Wenshang Barred Chicken

This experiment was conducted to find the vertical transmission rule of avian leukosis virus(ALV) in indigenous chickens. Huiyang chicken and Wenshang Barred chicken were selected as materials,two experimental groups were set up based on virus isolation detection results of plasma and semen at the age of 43 weeks and pedigree analysis:group Ⅰ,male and female parent were ALV-positive Huiyang chicken and ALV-negative Wenshang Barred chicken which was divided into three groups and eight chicken in each group;group Ⅱ,male and female parent were ALV-negative Wenshang Barred chicken and ALV-positive Huiyang chicken.The hybrid offsprings were selected and raised separately,the meconium,cloacal swabs and plasma were collected at 1, 42, 42 days of age for p27 antigen detection and virus isolation detection. The subtype of ALV-positive chicken through virus isolation were verified by PCR,sequencing and phylogenetic analysis. The results showed as follows:Positive rates of p27 antigen from meconium, cloacal swabs and virus isolation in offsprings of ALV-negative roosters were significantly higher than those in offsprings of ALV-positive hens(P<0.05). The virus isolation rate in offsprings of ALV-negative roosters and ALV-positive hens were 8.54% and 0%,respectively. There was no significant correlation between amount of ALV carried or shed by ALV-positive roosters and hens and positive rate of p27 antigen in their offsprings(P>0.05). The false positive rate of p27 antigen from meconium and cloacal swabs was high, and misdetection rate of p27 antigen from meconium was 15% compared to virus isolation. Sequencing and phylogenetic analysis showed that ALV-positive chicken through virus isolation were all ALV-J. This study suggest that roosters would play an important role in vertical transmission of ALV,and it would be necessary to strengthen the surveillance of roosters which were infected with ALV in eradication process of avian leuiosis.

2018 Vol. 49 (7): 1475-1481 [Abstract] ( 14 ) [HTML 1KB] [PDF 1057KB] ( 10 )
1482 ZHOU Zhong-wen, LIN Gui-fang, WANG Hao-hao, SU Shuai, CUI Zhi-zhong, SUN Shu-hong
Biocharacteristics of Recombinant Marek's Disease Virus Expressing the env Gene of Reticuloendotheliosis Virus

The study aimed to construct a Marek's disease virus(MDV) recombinant expressing the env gene of Reticuloendotheliosis virus(REV), and explore its biocharacteristics preliminarily. Firstly, env gene was amplified from the DNA of REV SNV strain and cloned into eukaryotic expression vector pcDNA3.1(+). The env gene expression cassette and the kanamycin resistance gene(kanr) amplified from the recombinant plasmid were then cloned into pUC18 vector, respectively. Using the primers containing homologous arms on both sides of the meq gene of MDV strain SC9-1, the REV env gene expression cassette and the kanr gene were amplified and integrated into the site of meq gene in the genome via Red E/T homologous recombination system. The kanr gene was further knocked out by the Arab sugar induced flg recombinant enzyme to obtain the recombinant BAC plasmid, naming SC9-env BAC. The recombinant MDV strain SC9-env was rescued after transfecting the SC9-env BAC plasmid into CEF cells, and identified by indirect immunofluorescence(IFA) using the MDV monoclonal antibody H19 and the REV monoclonal antibody 11B118. Replication of SC9-env on CEF cells, the pathogenicity of SC9-env, and the protective efficacy of SC9-env against challenge of MDV and REV in SPF chickens were further evaluated. The results showed that the REV env protein was stably expressed by the recombinant virus SC9-env. The recombinant virus SC9-env showed similar replication capacity to the parental virus SC9-1, but had no pathogenicity to SPF chickens. In addition, SC9-env protected 92% of SPF chickens against challenge of MDV strain rMd5, which showed no significant difference with SC9-1, and eased the weight loss and the decreased inactivated antibody levels caused by REV infection. In conclusion, the constructed MDV recombinant expressing the env gene of REV possesses good protective efficacy against infection of MDV and REV in SPF chickens.

2018 Vol. 49 (7): 1482-1491 [Abstract] ( 10 ) [HTML 1KB] [PDF 2714KB] ( 17 )
1492 LIU Lu, GUO Jie, ZHU Ke-meng, WANG Ning, XIN Jiu-qing, LIU Yu-fen, LIU Heng-gui
Screening and Identification of a B-cell Epitope on P97 Protein of Mycoplasma hyopneumoniae Using Yeast Random Display Library Technology

The experiment was conducted to construct a yeast random display library of P97 antigen from Mycoplasma hyopneumoniae(Mhp), and the specific B cell epitopes on P97 protein were screened and identified by using the library. The p97 gene was amplified from Mhp genome using specific primers. All TGA codons within the p97 gene were mutated into TGG by polymerase chain reaction, Mutated p97 gene was randomly digested into about 100-250 bp in the length with DNaseⅠ. After adding adenine at the 3' terminus, the digested fragments were ligated into yeast expression vector pCT-XcmⅠ treated with XcmⅠ. The ligation product was transformed into Saccharomyces cerevisiae EBY100 to obtain a random yeast display library of P97 protein.The positive yeast clones in the library that react with the sera from Mhp-infected porcine were screened by flow cytometry and verified the minimum peptide sequences of epitopes. Results:The size of the constructed library was 2.86×106 clones. The inserted fragments were randomly distributed and covered the full length of p97 mutant gene. Upon induction and staining with the sera from Mhp-infected porcine, the positive clones were able to be detected from the library using flow cytometry. Then the sorting of the library was carried out using FACS and a positive peptide, named as 184, was identified. After one by one deletion from the C-terminal amino acid of 184, an epitope 184D(DEKTSSQKDPSTLRA) with 15 amino acids in length was determined. This epitope was able to react with many Mhp-positive porcine sera. These results indicate that the library can be used for the epitope screening from the P97 protein, and the 184D peptide on the P97 protein is a B-cell epitope which is capable of inducing antibody production in many hosts. These results will provide the base for the exploring epitope vaccine of Mhp and establishing a specific diagnostic method in the future.

2018 Vol. 49 (7): 1492-1499 [Abstract] ( 16 ) [HTML 1KB] [PDF 1528KB] ( 15 )
1500 CAI Jun-jun, YUAN Tian-qing, YANG Yin-ping, GAO Shuang, GONG Xing-wen
Virtual Screening and Activity Evaluation of Multi-target Antibacterial Lead Compounds

Pseudomonas aeruginosa and Escherichia coli are two kinds of common pathogenic bacteria existed extensively in hospitals and veterinary clinics. They can infect both human and animals and brought great challenges to disease treatment and economic development. In order to obtain novel lead compounds targeting to penicillin-binding proteins(PBPs) of P. aerμginosa and E. coli, molecular docking was performed using UCSF DOCK 6.5 against ZINC database. PBP3 of Pseudomonas aeruginosa and PBP1b, PBP4, and PBP5 of Escherichia coli were chosen as the docking targets. Lead compounds with high scores and novel structures were selected for organic synthesis. Their antibacterial activities were evaluated according to their minimum inhibitory concentrations(MIC). Grid score was used for the first round of virtual screening, while lead compounds with grid scores lower than -125.6 kJ·mol-1 were subjected to second round of screening. Amber score was used for the second round of virtual screening. Finally, approximately 200 compounds with amber score lower than -83.7 kJ·mol-1 were screened out. In view of the structural novelty, lead compound ZINC00053282 was selected for synthesis. The result of antibacterial assay indicated that it could inhibit both Gram-positive and Gram-negative bacteria, with an activity better than sulfadiazine. These results mean that this lead compound is an potential antibacterial drug and might be used to solve the drug resistance problem.

2018 Vol. 49 (7): 1500-1510 [Abstract] ( 11 ) [HTML 1KB] [PDF 2881KB] ( 13 )
1511 DIAO Hong-xiu, LI Yi, LIN De-gui, ZHANG Di
Expression of MUC1 and Its Clinical Value in Canine Mammary Tumors

This study was conducted to assess the influences of MUC1 in cancer invasion, metastasis, classification and grade in mammary tumors. This work was designed to investigate the expression of MUC1 mRNA and MUC1 in canine mammary tissues. Forty-seven canine mammary gland tumors(20 benign and 27 malignant mammary tumors) and mammary gland tissues from 3 healthy dogs were investigated for MUC1 mRNA and MUC1 by Real-Time PCR and immunohistochemistry(IHC), respectively. The results showed that the expression levels of MUC1 gene and MUC1 in canine mammary tumors were significant higher than that in healthy mammary gland(P<0.05), and a significant correlation was observed between the expression levels of MUC1 gene and MUC1 and malignance grades. In conclusion, the expression levels of MUC1 gene and MUC1 may become new and valuable biomarkers for diagnosis and malignance grade in canine mammary tumors.

2018 Vol. 49 (7): 1511-1516 [Abstract] ( 20 ) [HTML 1KB] [PDF 2873KB] ( 17 )
1517 TIAN Wei-jun, QIN Jia-ling, YUE Chan-juan, HOU Ran-ran, LIU Kuan-hui, HU Yuan-liang
Effects of Three Kinds of Polysaccharides on Antioxidative Ability of Chicken Embryo Liver Cells

In order to develop the antioxidative polysaccharide preparations, the effects of Lycum barbarum polysaccharide(LBP), Atractylodes macrocephala polysaccharide(AMP) and Chinese Angelica polysaccharide(CAP), on resisting oxidative damage ability of Chicken embryo liver cells(CEL) were compared, vitamin C(VC) were used as control. The safe concentrations of four drugs on CEL were determined. Then they at three concentrations within safe concentrations were respectively added into cultivation system of CEL. After cultivation of 24 hours, the cells were attacked by H2O2 for an hour, the cellular vitality(A570 nm value), intra-cellular ROS content, GSH-Px and SOD activities were assayed. The result showed that the A570 nm values in H2O2 group were the lowest, ROS contents were the highest and the activities of GSH-Px and SOD were the lowest, which there were significant differences as compared with cell control group(P<0.05). The A570 nm values in 3 polysaccharides and VC groups were higher or significantly higher, ROS contents were lower or significantly lower and the activities of GSH-Px and SOD were higher or significantly higher than those in H2O2 group, especially in high concentrations(P<0.05), the actions were most significant. The actions of LBP at 3 concentrations were stronger than those of AMP and CAP at same concentration, and equivalent even superior to VC. The results indicate that three kinds of polysaccharides all possess stronger antioxidant activity, can resist the CEL vitality depress, intra-cellular ROS contents elevation and antioxidase activities decline caused by H2O2 attack, thus strengthen CEL ability of oxidation resistance, LBP has the strongest activity and can be considered as polysaccharid antioxidant.

2018 Vol. 49 (7): 1517-1523 [Abstract] ( 12 ) [HTML 1KB] [PDF 680KB] ( 19 )
1524 HU Hui-yan, JIA Qing, HOU Sheng-kui, LIU Jin, ZHANG Jing, ZHANG Wei-feng, XI Jian-zhong
Cloning and Transcriptional Activity of Swine StAR Gene Promoter

The research was designed to analyze the activity region in the promoter of StAR gene, and to explore the mechanism of expression regulation of StAR gene, thus provide new ideas for improving the fertility of the pigs from the perspective of breeding. The sequence of the promoter activity region was analyzed by online tools based on the 5'-flanking sequence of swine StAR gene published by Ensembl database. The specific primer was designed and the PCR was used to amplify the gene promoter sequence based on the reference genomic sequence of the pigs, pGL3-StAR promoter luciferase reporter gene vectors were constructed and transfected into 293T cells, the relative luciferase activity was measured by dual luciferase assay system. The results showed that the 5'-flanking sequence of swine StAR gene didn't contain the typical TATA-box and CpG island. Ten promoter fragments with different lengths were obtained and luciferase reporter gene vectors were constructed, and then to analyze their transcriptional activity through transfected into 293T cells, respectively. The core promoter region of StAR gene was located in the 5'-flanking sequence of swine StAR gene, among them, -196-+127 bp region had the highest activity value, which was significantly higher than other deletion fragments(P<0.01),suggested that the region of +127——196 bp existed an important positive regulatory element. The exon 1 played a critical role in regulating the activity of the promoter. -41——196 bp as the core promoter region, contained a primary positive regulatory element, in which there were a number of transcription factor binding sites, including GATA2, GATA4, SP1, ZNF263, Hoxa9, KLF16 and ZNF740. Bioinformatics analysis of StAR gene and detection of dual reporter gene activity with different length promoter fragments confirmed that 5'-flanking sequence of swine StAR had promoter transcriptional activity. The promoter region of the gene had been preliminarily identified, the promoter region and the main regulatory region were found, which provided a theoretical basis for further study of StAR gene transcriptional regulation mechanism.

2018 Vol. 49 (7): 1524-1532 [Abstract] ( 9 ) [HTML 1KB] [PDF 1619KB] ( 23 )
1533 ZHANG Ran-ran, WANG Lei, LI Yang, XU Jia-ping, XING Xiu-mei
Distinguishing for the Deer Blood and Antler Blood by Metabolomics Approach

The purpose of this experiment was to study the differences of the metabolite components between deer blood and antler blood, and to provide a theoretical basis for the further development of related products. The metabolites were analyzed and quantified by UPLC-QTOF-MS combined with multivariate statistical analysis(PCA, OPLS-DA). Multivariate statistical analysis showed that the deer blood and antler blood were clearly distinguished in the direction of the variable X, indicating a significant difference in the small metabolite composition. Twenty-eight kinds of significantly differentially expressed metabolites were screened. The contents of lipids, nucleotides and ergothioneine-a natural antioxidant, were higher in antler blood. Metabolomics analysis of deer blood and antler blood can provide theoretical basis for the development of deer blood related products.

2018 Vol. 49 (7): 1533-1539 [Abstract] ( 26 ) [HTML 1KB] [PDF 1517KB] ( 17 )
1540 LIU Yan-yan, FAN Yang-yang, BU Xun, HU Yue, DONG YE Sheng-jin, TAN Qing-qing, ZHANG Quan-fang
The study on Identification of Wool Fiber of 8 Animals Using Multiplex Real-time Fluorescence PCR

In this study, the multi-real-time fluorescent PCR was used to identify and distinguish the wool fibers from 8 animal species:mink, fox, camel, rabbit, goat, sheep, duck and goose. Rapid DNA extraction from the animal wool fibers by alkaline lysis methold was further optimized by adding dithiothreitol and using silicon beads. Based on the 16S rDNA sequences of the 8 animal species, the universal PCR primers and specific TaqMan fluorescent probes were designed. Then,the multi-real-time fluorescent PCR detection system was established by optimizing the parameters of reaction conditions, such as primers, probe concentrations and annealing temperatures. Finally, the specificity, sensitivity, reproducibility and suitability of the multi-real-time fluorescent PCR system were methodologically evaluated. The results showed that the use of SDS-dithiothreitol DTT-protease K combined with Chelex-100 method and the use of silica beads for the purification of animal hair fiber DNA was the best, meeting the requirements of real-time fluorescent PCR detection. The DNA sensitivity was 0.01 ng and the detection limit was 1.0%. Therefore, the DNAs extracted from wool fibers were high quality, and this multi-real-time fluorescent PCR detection system had high specificity and sensitivity, and good reproducibility. This method could be applied for the identification of the 8 different animal species and their specific wool fibers. Furthermore, this method could extend the usage of animal wool fibers in the fields of species identification, molecular evolution study, molecular marker assisted breeding and genetic resources evaluation.

2018 Vol. 49 (7): 1540-1549 [Abstract] ( 12 ) [HTML 1KB] [PDF 3080KB] ( 17 )
1550 MA Jun-xing, HE Jun-feng, CUI Yan, ZHANG Qian, LIU Peng-gang
The Distribution of LRP6 and VEGFR2 in Yak Lungs

This experiment was conducted to investigate the expression and localization of LRP6 and VEGFR2 in yak lungs of different ages and their roles in yak lung hypoxia adaptive structure formation and the possible regulation. Newborn(1-7 days old, n=5), juvenile(5-6 months old, n=5), adult(3-6 years old, n=5) and old(7-10 years old, n=5) yaks were selected as the subjects, the expression and percise location of LRP6 and VEGFR2 in yak lung tissue of different age groups were detected by ELISA, Western-blot and immunohistochemistry. The results showed that the expression of LRP6 and VEGFR2 between the newborn and juvenile yak groups was not significant different(P>0.05); the expression between newborn, juvenile and old yak groups was significant different(P<0.05); the expression between the adult and old yak groups was significant different(P<0.05). The LRP6 was mainly distributed in the lungs of branches and their branches epithelial cells and alveolar type Ⅱ cells and the expression was stronger; the expression in pulmonary vascular endothelial cells and vascular wall smooth muscle cells was weak. The VEGFR2 was mainly distributed in the epithelial cells of the bronchial lung and its branches, wall smooth muscle cells and alveolar septum and alveolar typeⅠcells and the expression was stronger; the expression in pulmonary vascular endothelial cells and smooth muscle cells was weak. The results suggested that LRP6 and VEGFR2 not only had different expression levels in yak lung tissues of different ages, but also had important implications for the regulation of the formation and maintenance of hypoxic adaptative structures in the lungs of yak in the plateau environment.

2018 Vol. 49 (7): 1550-1557 [Abstract] ( 19 ) [HTML 1KB] [PDF 5256KB] ( 18 )
1558 WAN Chun-he, LIU Rong-chang, CHENG Long-fei, FU Guang-hua, SHI Shao-hua, CHEN Hong-mei, FU Qiu-ling, HUANG Yu
Establishment of a EvaGreen Real-Time Fluorescent Quantitative PCR Method for Vaccine Strain of Duck Enteritis Virus

A live attenuated duck enteritis virus(DEV) vaccine has been used routinely to control lethal DEV in ducks since the 1960s, which is safety, stable, efficacious, and cost effective to produce. Recently, DEV vaccine strain has been developed as a vector for expressing foreign antigens for vaccine purposes, which offered the advantage of efficiently generating both humoral and cellular immune responses and overcomed pre-existing antibodies. To the best of our knowledge, no real time fluorescent quantitative PCR(qPCR) method was reported for only detection attenuated duck enteritis virus(DEV) vaccine strain. In this study, nucleotide comparison analysis showed the UL2 had significant characteristics with 528 bp continuous gene deletion between the virulent DEVs and attenuated vaccine strains. Based on the UL2 gene characterization, an EvaGreen real time qPCR method was developed with conditional optimization. The results demonstrated that the established qPCR had good linear correlation when UL2 gene content was 5.25×101-5.25×106 copies·μL-1 with a linear correlation(R2) of 0.999 and efficiency of 100% between the cycle threshold value and the logarithm of the plasmids copy number. The axial intercept of standard curve equation was 34.95 and the slope was -3.218. The lowest limit of detection concentration was 52.5 copies·μL-1. The melting curve analysis showed one specific peaked with a melting temperature(Tm) was(90.62±0.28)℃,with no primer-dimers peak represent. No cross amplification was detected from other common duck pathogens(such as wild duck enter virus, Muscovy duck parvovirus, duck circovirus, Muscovy duck origin goose parvovirus, novel recombinant duck parvovirus, duck adenovirus A, goose hemorrhagic polyomavirus, Escherichia coli, Rimerella anatipstifer and Pasteurella multocida). Reproducibility test showed that the intra-and inter-assay were ranged from 0.55%-1.72% and 0.92%-2.49%, respectively. In conclusion, this study provided an EvaGreen real time qPCR method for DEV vaccine strain, which lays good foundation for mechanism research with live attenuated DEV vector based genetic engineering vaccines.

2018 Vol. 49 (7): 1558-1566 [Abstract] ( 15 ) [HTML 1KB] [PDF 1828KB] ( 18 )
畜牧兽医学报
 

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