Acta Veterinaria et Zootechnica Sinica

The Population Genetic Relationships among 47 Chinese and Western Pig Populations Infered by AFLP Markers

GAO Jun;REN Jun;KEN Siggens;AI Hua-shui;GARY Evens;HUANG Lu-sheng

2009, 40(3): 285-290. doi:

Abstract ( 1355 )

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AFLP markers were detected by the two-color fluorescent detection in a sample of DNA pool of 21 Asian native pig populations, 19 European and American commercial pig populations, 5 wild pig populations, 1 Duroc×Chinese wild pig hybrid population and 1 Chinese sythetic pig breed. 312 polymorphic markers were detected by a total of 22 AFLP primer combinations. The genetic similarity coefficients of 47 pig populations were calculated from AFLP data, and molecular phylogenetic tree was constructed by UPMGA method. The results showed that Chinese native and wild pig populations had remarkable genetic differentiation with European and American commercial pig populations and wild pig populations, indicating that Chinese native pig populations may originate from Asian wild pigs, while European and American commercial pig populations were domesticated from European and American wild pigs. Although different populations of commercial pig breeds including Duroc, Landrace and Large White had close relationship with each other, they also, to some extent, displayed genetic differentiation. Nanchang White pig and Large White, L95 dam line from PIC and Meishan pig had intimate genetic relationships with each other, which were consistent with its breeding history. Moreover, the cluster results of many other Chinese native pig breeds, including Meishan, Jiaxing Black, Guanchao, Tengtian Spotted, Shangyou Spotted and Wanan Spotted, were consistent with morphology, geographical distribution and exiting classification. It was concluded that AFLP provided a valuable tool for assaying genetic diversity and population genetic relationships in farm animals.

Relationship between the Combined Genotypes of ESR and FSHβ Genes and Reproductive Traits in Eight Pig Breeds

LIU Chan-juan;ZENG Yong-qing;WEI Shu-dong;TANG Hui;FAN Xin-zhong;SUN Yan-xiao;CHEN Qi-mei;LI Hua

2009, 40(3): 291-295. doi:

Abstract ( 1447 )

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The polymorphisms of estrogen receptor (ESR) and follicular-stimulating hormone beta subunit (FSHβ) genes were detected by DNA sequencing and PCR-SSCP methods, and effects of combined genotypes of ESR and FSHβ loci on reproductive traits in pigs were analyzed by the least square analysis. Data of total number born (TNB) and number born alive (NBA) from 323 sows, including five Shandong native pig breeds and three western pig breeds, were collected. The results showed that the polymorphic sites of both ESR and FSHβ genes were found in all populations tested. The combined genotypes distribution, however, revealed great differences between Shandong native pig breeds and western pig breeds. It was demonstrated that the effects of combined genotypes on TNB and NBA were significant (P<0.05). The sows with combined genotype BBBB are the most prolific. For the first and later parities sows, BBBB combined genotype produced 2.54 TNB, 2.65 NBA and 2.29 TNB, 2.35 NBA more than AAAA combined genotype, respectively. Moreover, the genetic effects of combined genotypes on litter size traits were not the simple summation of the isolated genotypes.

Study on Genetic Variation of Inbred Families of Wuzhishan Miniature Pig Using Microsatellite DNA Loci

LI Kai;MU Yu-lian;HAN Jian-lin;YANG Shu-lin;LIU Lan;YUAN Xin-xu;GUO Yong;FENG Shu-tang

2009, 40(3): 296-302. doi:

Abstract ( 767 )

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To assess the level and pattern of genetic variation of three inbred families of Wuzhishan Miniature pig (WZSP) from the 13^th to the 18^th generations， 14 microsatellite DNA markers were used to estimate their genetic heterozygosity (H)， polymorphic information content (PIC) and number of alleles (N).The results showed that there were a total of 38 alleles at the 14 loci and the estimates of average H， PIC and N in the pooled samples were 0.42， 0.37 and 2.71， respectively. The average H and N in three families ranged from 0.41 to 0.45， and from 2.29 to 2.50， respectively. In addition， the average H and N from the 13th to the 18th generations were 0.31， 0.42， 0.36， 0.41， 0.37 and 0.20， and 2.21， 2.57， 2.43， 2.57， 2.36 and 1.64， respectively. It was noted that three microsatellites （Sw71， Sw510 and Sw2409） had been fixed in all three families at the 13^th generation， however， some loci like Sw205， Sw874 and Sw936 among others still demonstrated high levels of heterozygosity， though very low N from 2 to 4， in all three families across all generations. In conclusion， this study revealed the pattern of genetic variation at microsatellite loci in the inbred families of WZSP with one of the most important findings to be the high level of unexpected heterozygosity at some loci， e.g. Sw874 and Sw936 that could be part of the unique genetics of these families. Whether functional genes linked with such microsatellite loci were involved in the maintenance of viability of these inbred families of WZSP remains for further investigation.

CRS-PCR Polymorphisms of Prolactin Gene and Its Relationship with Milk Production Traits in Chinese Holstein Cows

WANG Li-juan;LI Qiu-ling;WANG Chang-fa;WANG Hong-mei;LI Jian-bin;HOU Ming-hai;GAO Yun-dong;ZHONG Ji-feng

2009, 40(3): 303-308. doi:

Abstract ( 816 )

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Single nucleotide polymorphisms of exon 4 C8377G and intron 4 T8510C of PRL gene were detected in Chinese Holstein cows by CRS-RFLP. At 8377 site,the least square mean of milk yield of this population was higher for individuals with genotype GG than that for individuals with genotype CC (P<0.05); The least square mean of protein percentage of this population was higher for individuals with genotype GC than that for individuals with genotype CC (P<0.05).At 8510 site, the least square mean of milk yield of this population was higher for individuals with genotype CC than that for individuals with genotype TT (P<0.05).

Association between Polymorphism of STAT1 Gene and Milk Production Traits in Chinese Holstein Cattle

CHU Min;ZAN Lin-sen

2009, 40(3): 309-314. doi:

Abstract ( 778 )

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The polymorphisms in intron 11，19，25 and 3′UTR of STAT1 gene were detected in 199 Chinese Holstein cattles with PCR-SSCP and association between the polymorphisms and milk production traits of three birth orders were analyzed. There were three polymorphism sites in Chinese Holstein cattles, which were in intron11 and 3′UTR. We found that it was G/A (28739) and G/C (28873) in intron 11, and C/T (141930) in 3′UTR. The association between different genotypes of three sites and 305 days milk yield, fat percent (F%), and protein percent (P%) of three birth orders was analyzed by least square analysis. The results showed that in intron 11，there were no significant differences among three genotypes on 305 days milk yield, and fat percent(F%)of all birth orders(P>0.05), but the tendency of BB>AB>AA was showed. Cows with genotype BB had significantly higher protein percent (P%) in the first and second birth orders (P<0.05) than those with AB and AA. And in 3′UTR, there were no significant differences among three genotypes on fat percent (F%) (P>0.05), while the tendency of CC>CT>TT was showed either. And cows with genotype CC or CT had significantly higher 305 days milk yield in the first and second birth orders (P<0.05); and cows with genotype CC had significantly higher protein percent (P%) than those with genotype TT in the first and third birth orders(P<0.05).

Genetic Variations in Promoter Region of TG Gene and Its Association with Carcass and Meat Quality Traits in Cattle

ZHANG Lu-pei;REN Hong-yan;GAN Qian-fu;LI Jun-ya;LI Heng-de;XU Shang-zhong;GAO Xue;CHEN Jin-bao

2009, 40(3): 315-319. doi:

Abstract ( 754 )

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In this experiment, promoter region of TG gene from eight breeds was amplified and sequenced. Two SNPs were identified at position 82 and 422 of TG gene (X05380). The effects of these polymorphisms of TG gene on carcass and meat quality traits were analyzed. The results showed that C422T polymorphism had no association with marbling score and any other carcass and meat quality traits. G82A polymorphism significantly associated with live weight and loin muscle area(LMA)(P<0.05). Individuals with genotype GG and GA had significantly larger live weight and LMA than those with AA genotype (P<0.05). In a word, G82A SNP might be a good DNA marker to be used in MAS for carcass traits.

Study on Genetic Diversity of T128 del in Agouti Gene Intron 1 in Chinese Main Indigenous Goat Breeds

TANG Chun-juan;LI Xiang-long;ZHOU Rong-yan;LI Lan-hui;FENG Fu-jun;LI Dong-feng;WANG Jian-tao

2009, 40(3): 320-326. doi:

Abstract ( 1353 )

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Agouti gene plays an important role in the pigment synthesis of domestic animals. A SNP-T128 del was found by sequencing. The genetic diversity of T128 del in ten Chinese indigenous goat breeds was analyzed, as well as the relationship between the genotypes and the coat color by PCR-RFLP. Two alleles of A (non-del) and B (del), three genotypes of AA, AB and BB were detected. The genotype AA was superior in brown coat color goat breeds (77.41%); genotype AB existed mainly in white coat color goat breeds (76.14%) and the goat breeds with black coat color had BB genotype mostly (87.92%), so it was deduced that the T128 del might has the effect on the pigment synthesis of goat. The expected heterozygosity, the gene flow and the mean coefficient of gene differentiation of T128 del among ten goat breeds were 0.471 8, 0.319 0 and 0.439 3, respectively. The results indicated that most goat breeds investigated here were poor in genetic diversity at T128 del.

Assessment of Genetic Diversity and Genetic Differentiation of Chinese Native Goose Using Microsatellite Markers

CHEN Kuan-wei;SONG Wei-tao;XU Wen-juan;ZHU Wen-qi;LI Hui-fang;TANG Qing-ping

2009, 40(3): 327-332. doi:

Abstract ( 769 )

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The genetic diversity and differentiation of twenty six Chinese native goose populations were investigated using 25 microsatellite markers. The total number of animals examined was 1 560. A total of 198 alleles were observed, with mean number 7.92 alleles per locus. Effective alleles number varied from 1.307 0 to 5.795 5, with an average of 3.066 2. The polymorphic information content (PIC) ranged from 0.324 to 0.538, the observed heterozygosity (Ho) ranged from 0.535 1 to 0.754 7, and the expected heterozygosity (He) ranged from 0.383 1 to 0.604 9. As a complete populations, the average Ho , He and PIC were 0.668 6, 0.457 7 and 0.564 0, respectively. Considerable genetic differentiation was observed and 20.7% of the total genetic variation originated from the differences among breeds. The largest Reynolds genetic distance and lowest gene flow were between Youjiang and Yongkang grey geeses, meanwhile, the nearest Reynolds genetic distance and highest gene flow were between Wugangtong and Huoyan geeses. Consensus tree was constructed using the Neighbour-Joining method based on the Reynolds genetic distance, and five groups were found. The results may provide basic molecular data for the research on the germplasm characteristics of native goose breeds in China and scientific basis for the conservation and utilization of those breeds.

Study on the Change of GPR54 Gene Development Expression in Hypothalamus, Pituitary and Ovaries in Pigs

ZHOU Chun-bao;;WANG Jin-neng;LU Yan-feng;DING Jia-tong

2009, 40(3): 333-337. doi:

Abstract ( 798 )

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Four female Sujiang pigs were randomly selected at different stages of birth, 60 days, 120 days, puberty and 180 days, respectively and slaughtered, tissue samples of hypothalamus, pituitary and ovaries were collected. Development expression of GPR54 mRNA which was related to puberty before and after in hypothalamus, pituitary and ovaries were determined by quantitative RT-PCR with β-actin as an internal control. The results showed that the expression abundance of GPR54 mRNA increased gradually from birth to puberty, and after puberty the expression abundance had degressive tendency. At different stages of development, the expression abundance at the stage of puberty was significantly different with that at the stages of birth and 180 days（P<0.05）. In different tissues, the expression abundance was highest in ovary and lowest in hypothalamus, the expression abundance in hypothalamus was significantly different with those in ovary and pituitary（P<0.05）.

Different Expression of LHR mRNA in the Uterus of Four Estrous Cycle Phases in Jining Gray Goat

SHEN Ying;WANG Hui;WANG Shu-ying;WANG Li-qin;HOU Yan-meng;HUANG Li-bo

2009, 40(3): 338-342. doi:

Abstract ( 1253 )

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Total RNA extracted from the uterus of Jining gray goat was used for RT-PCR amplification of goat luteinizing hormone receptor (LHR) gene. A cDNA fragment of 289 bp in size was obtained and cloned into pGEM-T vector. Positive clones were identified and sequenced. Using GAPDH as an inner control, an optimal semi-quantitative RT-PCR method was established to analyze LHR mRNA expression levels in the goat uterus of four estrous cycle phases. Sequencing analysis indicated that this fragment was a partial sequence of LHR cDNA and identical to the reported LHR mRNA (AF379199). The LHR mRNA expression was different in the uterus of Jining gray goat during the four estrous cycle phases, being lowest in the estrus (0.454±0.06), highest in the diestrus (0.705±0.09), intermediate in the proestrus (0.553±0.07).The expression in the estrus was significantly lower than that in other oestrous cycle phases (P<0.05).The results provides molecular data for further elucidating the mechanisms of reproductive endocrine secretion in goats.

Segmental Distribution and Ontogenetic Regulation of NHE3 mRNA Expression in Intestine of Pigs

ZOU Shi-geng;FENG Ding-yuan;HUANG Zhi-yi;LI Yan;WU Tong-shan;ZUO Jian-jun;ZHI Ai-min;DONG Ze-min;ZHANG Chang-ming;LIU Qing-shen

2009, 40(3): 343-348. doi:

Abstract ( 693 )

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Segmental distribution and ontogenetic regulation of sodium hydrogen exchanger (NHE3) mRNA expression were evaluated in pigs along the horizontal axis of the intestine. A total of 35 littermate purebred Lantang gilts and 35 littermate purebred Landrace gilts were divided into seven groups at the ages of day 1, 7, 26, 30, 60, 90 and 150, respectively.Samples of intestinal segments (duodenum, jejunum, ileum and colon) were collected. The NHE3 mRNA abundance was determined by realtime RT-PCR using SYBR GreenⅠRT-PCR mix Kit. Results showed that the abundance of NHE3 mRNA was highest in colon and lowest in jejunum and ileum (P<0.05). The ontogenetic expression of NHE3 mRNA in duodenum and jejunum of Lantang and Landrace pigs were similar. The highest expression abundance of NHE3 mRNA were 7 d of Lantang and 30 d of Landrace in duodenum, 7 d of Lantang and 26 d of Landrace in jejunum (P<0.05). The expression pattern of NHE3 mRNA in colon was differed from duodenum and jejunum. Abundance of NHE3 mRNA of Lantang pigs on 26, 90 and 150 d were lower than that of Landrace pigs (P<0.05) in colon. The results indicated that NHE3 mRNA expression could be regulated by developmental stages, breeds and segments of intestine in pigs.

Alleviation Effect of pGRF Gene Plasmid on Growth Depression of Weaned Piglets after Immune Challenge

DONG Hai-jun;WANG Kang-ning;Li Xia

2009, 40(3): 349-355. doi:

Abstract ( 893 )

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The purpose of this study was to evaluate the effect of pGRF gene plasmid on growth performance and immunological function of immune-challenged weaned piglets, and also the potential mechanism of these effects were discussed. Eighteen weaned piglets(Duroc×Landrace×Large White) with ages of (35±2)d and initial body weight (BW) (7.86±0.59)kg were not castrated and randomly assigned into three treatments according to gender and BW by single factor design. Three treatments were pGRF gene plasmid group, pGRF gene plasmid plus lipopolysaccharide(LPS) group and LPS group. Each treatment had six replicates and one piglet in each replicate. The results were as follows:The piglets in the pGRF gene plasmid plus LPS group had a better growth performance than those in the LPS group(P<0.05). The F/G of piglets in the pGRF gene plasmid plus LPS group was lower than that in the LPS group(P>0.05). The serum IGF-1 level in the pGRF gene plasmid plus LPS group was significantly higher than that in the LPS group(P<0.05 or P<0.01). The serum IgG level in the pGRF gene plasmid plus LPS group was higher than that in the LPS group(P<0.05). The serum IL-1 and IL-6 levels in the pGRF gene plasmid plus LPS group were significantly lower than that in the LPS group(P<0.05 or P<0.01). In conclusion, compared with the LPS group, in the pGRF gene plasmid plus LPS group had lower levels of serum IL-1 and IL-6 and higher levels of serum IgG. The statuses of immune challenge induced by LPS injection was improved by pGRF gene plasmid. The characteristics of resisting diseases were increased. However, the characteristics of pGRF’s immunoregulation may closely related to the secretion of IGF-I.

Effects of Tannin Content in Sainfoin（Onobrychis viciifolia）Hay on Digestibility of Nutrients and Utilization of Nitrogen in Sheep Diets

ZHANG Xiao-qing;LI Yong;LI Fa-di;WU Qiu-jue;YE De-he;HAO Zheng-li

2009, 40(3): 356-362. doi:

Abstract ( 740 )

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Three High Mountain Finewool wethers (one and a half years old and average live weight of 24 kg) fitted with permanent rumen fistula were used to study the effects of tannin content in Sainfoin（Onobrychis viciifolia） hay on apparent digestibility of nutrients and utilization of nitrogen (N) in sheep diets. With a 3×3 Latin square design, the experiment was divided into three periods, each phase of 19 days, the whole experiment lasted 57 days. The tannin contents were 0.00 g·kg^-1 dry matter (DM) of diet A (the control), 1.70 g·kg^-1 DM of diet B, and 3.40 g·kg^-1 DM of diet C, respectively. Correspondingly, the condensed tannin contents were 0.00 g·kg^-1 DM of diet A, 1.52 g·kg^-1 DM of diet B, and 3.03 g·kg^-1 DM of diet C. The results showed that the tannin contents in Sainfoin hay among three diets have no significant (P＞0.05) effect on the digestibility of DM, organic matter (OM), phosphorus (P), neutral detergent fiber (NDF) and acid detergent fiber (ADF) of diets, but there was a decreasing tendency towards the digestibility of calcium (Ca) （P=0.087）. Compared with diet A and B, diet C showed the better effect on protein protecting, the significantly lower(P＜0.05) excretion of urinary nitrogen and the significantly higher(P＜0.01) retention of N. Therefore, under this experiment condition, the preferable effect on the protein protection was found at 3.40 g·kg^-1 DM tannin (3.03 g·kg^-1 DM condensed tannin) of diet.

Effects of Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus Type 2 Co-infection on Proinflammatory Cytokine mRNA Expression in Peripheral Blood Mononuclear Cells of Piglets

SHI Kai-chuang;YANG Han-chun;GUO Xin;XING Hua-wei;GE Xin-na;CHEN Yan-hong;ZHA Zhen-lin

2009, 40(3): 363-370. doi:

Abstract ( 1276 )

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Six-week-old healthy piglets were experimentally inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) or porcine circovirus type 2 (PCV2) alone or in combination (PRRSV and PCV2). Realtime PCR was used to quantitatively measure proinflammatory cytokines, i.e. IL-1β, IL-6, IL-8 and TNF-α, mRNA expression in peripheral blood mononuclear cells (PBMC) from piglets. After infection, the PRRSV- and PCV2-infected group tended to have increased expression of IL-1β, IL-6, IL-8 and TNF-α mRNA, showing significant upregulation of gene expression for IL-6 and IL-8 in the PRRSV group and for IL6, IL8 and TNFα in the PCV2 group, in comparison to the control group. The PRRSV and PCV2 coinfected group showed increased gene expression for IL8 and TNFα only, but both of them showed significant upregulation as compared to the control group. Furthermore, the PRRSV and PCV2 coinfected group showed decreased levels of IL1β, IL6, IL8 mRNA, while the levels of TNFα mRNA were significantly increased, in comparison to the singly infected group. These results suggest that the excessive expression of TNFα may play an important role in the synergistic pathogenesis of PRRSV and PCV2.

Construction and Characterization Analysis of the Recombinant Virus SA215(C) Strain of Pseudorabies Virus-Porcine Circovirus Type 2

WANG Yin;GUO Wan-zhu;WANG Xin;XU Zhi-wen;ZHU Ling;WANG Xiao-yu

2009, 40(3): 371-375. doi:

Abstract ( 1272 )

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ORF2 gene of porcine circovirus type 2 were cloned by PCR with the specific primers designed according to genome of PCV2 in GenBank.Following extraction and digestion，PCR products were subsequently inserted into universal transfer vector pPI-2.EGFP to generate recombinant transfer plasmid pPI-2.EGFP-ORF2.The genomic DNA of PRV SA215 strain and pPI-2.EGFP-ORF2 were co-transfected into ST cells with lipofectin，and recombinant virus SA215(C) was selected by fluorescence and PCR with ORF2 gene primers respectively.The recombinant virus was analyzed with Southern blotting and Western blotting.The results indicated that ORF2 gene of PCV2 had been inserted into the genome of PRV SA215 strain and the expressed EGFP-ORF2 fusion protein could react with PCV2 positive sera.Result of virus titers detection showed that the insertion of ORF2 gene did not influence propagation of recombinant virus.

Expression of the Capsid Precursor Protein Gene of Foot-and-mouth Disease Virus and Green Fluorescent Protein Gene in

LI Jiong;LIU Yan-hong;AN Fang-lan;LIU Jun-lin;LIU Xiang-tao;SHANG You-jun;YIN Hong

2009, 40(3): 376-382. doi:

Abstract ( 799 )

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Here we constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus (FMDV). The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by inserting capsid precursor protein gene （P1） of FMDV and enhance green fluorescent protein gene(EGFP) into pBABEpuro by turns. Both the recombinant retroviral vector and the pVSV-G plasmid were cotransfected into packaging cell GP2-293 by liposomemediated transduction. And then, the pseudovirus were produced. The pseudovirus infected BHK-21 cells and resistance cells were screened by puromycin-resistant. The green fluorescent protein were observed under fluorescence microscope. The expression of capsid precursor protein gene of FMDV was detected by indirect immunofluorescence assay. The recombinant retroviral vector pBABEpuro-P1.2A-EGFP was constructed successfully. The capsid precursor protein of FMDV and green fluorescent protein could express in BHK-21 cells. The mammalian cell expression system of the capsid precursor protein of FMDV had been constructed successfully, which laid a foundation of development of FMDV subunit vaccine.

Tandem Expression and Immunogenicity of Two Copies VP1 Gene of FMDV Serotype Asia 1

ZHANG Ke-shan;XIANG Min;WU Bin;WANG Qin-gang;CHEN Huan-chun;

2009, 40(3): 383-387. doi:

Abstract ( 765 )

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VP1 gene of foot-and-mouth disease virus (FMDV) is the major immunogenic gene as it contains T cell and B cell epitopes. In this study, we constructed a recombinant expressing plasmid(KG2VP1) including two VP1 genes(2VP1) of Asia 1 FMDV, and the 2VP1 gene was expressed in E.coli BL21. The fusional protein (GST2VP1) which was purified by Sephadex-G200 sieve chromatography demonstrated the reactionogenicity by Western blot. And the mice test indicated that the GST2VP1 can induce similar levels of ELISA and neutralization antibody like inactivated vaccine. We provide foundation for researching the immunogenicity of FMDV serotype Asia 1 and further application of GST2VP1 in future.

Study on the Immune Protective Effect of the Fusion Protein Including Two Copies of T-cell and B-cell Epitopes of Food-and-mouth Disease Virus VP1 and Enterotoxins of Escherichia coli

ZHUANG Juan;YOU Yong-jin;CHEN Bo;RAO Zhong;PAN Jie

2009, 40(3): 388-393. doi:

Abstract ( 1310 )

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In this study, the fusion proteins including two copies of T-cell epitope(aa21-40) and B-cell epitope(aa141-160) of VP1 of food-and-mouth disease virus typed O and labile enterotoxin B subunit (LTB) and/not stable enterotoxinⅠ (STI) of Escherichia coli were analyzed as candidate vaccine against FMDV and E.coli. The proteins 2020-2020VP1,2020-B-2020 and 2020-B-2020-STI were used to vaccinate guinea pigs, rabbits and 6-8 weeks old BALB/c mice, then the immune responses were observed. The fusion proteins could induce proliferation of spleen T cells in vaccinated guinea pigs, and could elicit FMDV neutralizing antibody. It could be concluded that the three fusion proteins could activate FMDVspecial cellular and humoral immune-response simultaneously. At the same time, the fusion proteins 2020-B-2020 and 2020-B-2020-STI could protect mice from challenge of enterotoxigenic E. coli C83902 and the protecting-rate were 60%/1.5 MLD and 100%/1.5 MLD respectively. The fusion protein including STI enterotoxin could induce neutralizing antibody for STI toxin in rabbits, moreover, the fusion protein had no STI toxicity. The fusion proteins 2020-B-2020 and 2020-B-2020-STI could be recognized specially by CTB antibody in ELISA. So that, it can be concluded that the fusion protein 2020-B-2020-STI is potent to be efficient FMDV and ETEC vaccine.

Tendency of Genetic Variation on the F and HN Genes of Newcastle Disease Viruses Isolated from Waterfowls in China

XU Huai-ying;QIN Zhuo-ming;WANG You-ling;OUYANG Wen-jun;TAN Lei-tao;HE Ye-feng;JIN Wei-jiang;YUAN Xiao-yuan

2009, 40(3): 394-401. doi:

Abstract ( 802 )

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Eight Newcastle disease virus (NDV) isolates from waterfowl such as geese and duck were obtained in China during 19962005, and then the fusion (F) protein gene and haemagglutinin-neuraminidase (HN) gene of those isolates were amplified and sequenced for further phylogenetic analysis. Phylogenetic trees were constructed separately based on variable region fragment (47420 nt) of the F gene and full-length deduced amino acid sequence of the HN gene of 40 NDV strains, including 16 NDV reference strains in the world and the 24 prevalent waterfowl NDV isolates. The results showed that 16 NDV reference strains were identical in the two different phylogenetic trees. While, only 15 in 24 NDV isolates from waterfowl were similar, the others were different. In addition, 24 waterfowl-derived NDV strains were all classified as genotype Ⅶ on the basis of deduced amino acid sequence of the HN gene, which were different from that by F gene. Furthermore, there had feeble correlation between the complete F and HN protein sequences, and highly close correlation between full-length F protein sequences and variable region fragments. The results showed that evolutionary rates of the F and HN genes were respectively independent. All those data showed that waterfowls might be the major reservoir hosts of NDV and we should pay more attention to those NDV strains isolated from waterfowls.

Molecular Identification and Evolvement Analysis of Shigella Boydii Strain hn03 Isolated from Chicken

YANG Xia;CHEN Lu;XU Lan-ju;LIU Hong-ying;CHANG Hong-tao;WANG Chuan-qing

2009, 40(3): 402-408. doi:

Abstract ( 1280 )

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Molecular identification and evolvement analysis of Shigella boydii strain hn03 isolated from chicken were researched in this study. Eight pairs of specific primers for housekeeping genes, e.g. thrB/ thrC, trpC/trpB, purM/purN and mdh/argR, were designed according to the related sequences of shigella deposited in the GenBank database, and were used to amplify corresponding genes of hn03. The PCR products were cloned into pMD18-T vector and sequenced, the sequences reported in this paper have been submitted to GenBank. By using the BLAST and ClustalX program, the sequencing results were analyzed and the phylogenetic trees for 8 housekeeping genes in 4 regions of chromosome were established. Based on the phylogenetic analysis, 3 clusters of shigella strains were identified and nonetheless, were clearly within Escherichia coli. In this study， the locations are distributed identically for the shigella boydii isolated from chicken in four regions of chromosome, which are all contained in cluster 1. Shigella boydii isolated from chicken is probably originated from shigella boydii isolated from human on the level of whole gene.

Effects of Codon Optimization in Tsol18 on Its Expression and Antigenicity of the Recombinant Protein

DING Jun-tao;LUO Xue-nong;WANG Ying;ZHANG Shao-hua;CHEN Xiao-yu;ZHENG Ya-dong;JING Zhi-zhong;CAI Xue-peng

2009, 40(3): 409-415. doi:

Abstract ( 1492 )

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The expressive vector of pGEX-Tsol18-sp was constructed using the genetic modified Tsol18 gene of Taenia solium onchospere by the site-directed mutation of 4 bases in its coding sequence and removing of the sequence fragment encoding its N-terminal signal peptide. After induced with IPTG, the expressed proteins of TSOl18 and TSOl18-sp were probed by SDS-PAGE, Western blot and its stability was evaluated by storing at 4 ℃. The effects of immuno-protection of these two recombinant proteins were compared by ELISA and counting of Cysticercus in the muscle tissue of experimental pigs. The results revealed that the modified Tsol18-sp, in which 7 bases were synonymously mutated, could be expressed in the recombinant pGEX-Tsol18-sp vector. The soluble recombinant protein with a molecular weight of 38.7 ku can be probed by the specific serum against Taenia solium oncosphere, and only 20.1% was degradated in storage at 4 ℃ for 2 months. Both of the recombinant proteins can induce high titre of antibodies in the vaccinated pigs, but the recombinant protein TSOL18-sp showed a better protection rate（100%） against the challenge of Cysticercus cellulose than TSOL18 revealed（20%） by the reduced counts of Cysticercus in the muscle tissue of vaccinated pigs. It was suggested that the TSOL18-SP is a effective and stabile antigen, and could be used as a potential antigen for the immunoprophylaxis of Cysticercosis cellulosae.

Altered Expression of Neurotrophin Receptor p75^NTR in PrP106-126-induced Neurotoxicity of N2a cells

BAI Yu;LI Yu-rong;ZHOU Xiang-mei;YIN Xiao-min;ZHAO De-ming

2009, 40(3): 416-420. doi:

Abstract ( 749 )

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Neuronal death is a pathological hallmark of prion diseases. Synthetic prion peptide PrP106-126 can cause neurotoxicity and induce apoptosis in neuronal cells. Extracellular region of neurotrophin receptor p75NTR, a cell surface protein, could bind to PrP106-126 and has apoptotic effect on neurons. Using quantitative RT-PCR， Western Blot technique, DNA Ladder assay, and flow cytometry (FCM) assay with Annexin V-FITC/PI doublestained, we investigated the p75NTRmediated neurotoxicity by PrP106126 in mouse neuroblastoma cells N2a. The results showed that in PrP106126-induced apoptosis of N2a cells, p75^NTR expression was significantly upregulated at mRNA and protein levels, and blocking the interaction of p75NTR with PrP 106-126 by p75^NTR polyclone antibody sc-6189 attenuated the apoptotic effect induced by PrP106-126. This study showed that mRNA and protein expression of receptor p75^NTR were altered in PrP106-126-induced neurotoxicity of N2a cells, and this may partially account for the pathogenesis of prion disease.

Effect of Dietary High Copper on the Cellular Apoptosis of Brain Tissue in Chickens

LI Min;PENG Xi;CUI Wei;BAI Cai-min;CUI Heng-min

2009, 40(3): 421-427. doi:

Abstract ( 779 )

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The experiment was conducted with the objective of examining the effect of dietary high copper on the cellular apoptosis of brain tissue in the chicken by the methods of flow cytometry (FCM) and immune histochemical staining.360 one-day-old Avian chickens were randomly divided into six groups，and feed on diets as follows：Control (Cu 10.89 mg·kg^-1) and high copper (Cu 100 mg·kg^-1，high copper groupⅠ；Cu 200 mg·kg^-1，high copper groupⅡ；Cu 400 mg·kg^-1，high copper group Ⅲ；Cu 600 mg·kg^-1，high copper group Ⅳ；Cu 800 mg·kg^-1，high copper groupⅤ) for six weeks. By FCM，the percentage of apoptotic brain cells was significantly increased in high copper groups Ⅲ, Ⅳ and Ⅴcompared with that of control group （P＜0.05 or P＜0.01）. By the method of immune histochemical staining，the percentage of positive cells of Bax was significantly increased in high copper group Ⅲ, Ⅳ and Ⅴ (P＜0.01)，and the percentage of positive cells of Bcl-2 decreased (P＜0.05 or P＜0.01) in high copper group Ⅳ and Ⅴin comparison with those of control group. Also, copper-zinc-superoxidase （Cu-Zn-SOD）activity was obviously decreased and malondialdehyde （MDA）content markedly increased in high copper groups Ⅲ, Ⅳ and Ⅴ than that in control group. These results showed that dietary copper in excess of 400 mg·kg-1 induced the cellular apoptosis of brain tissue in chickens.

Changes of Collogen Type I and Heat Shock Protein 90 at the Different Stages of Broiler Tibial Dyschondroplasia

TIAN Wen-xia;QIN Ping;ZHANG Yan-hong;LI Jia-kui;BI Ding-ren;PAN Si-yi;GUO Ding-zong

2009, 40(3): 428-432. doi:

Abstract ( 1348 )

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The experiment was conducted to identify the changes of collogen type I (Col I) and heat shock protein 90 (Hsp90) at the different stages of broiler tibial dyschondroplasia. 80 one day-old AA broiler chickens were randomly divided into two groups after being raised seven days. The control group was given a regular diet, whereas the experimental group was given the same diet containing thiram 100 mg·kg^-1 for four days, then both of the groups was given the same regular diet. The feeding experiment lasted 23 days. Immunohistochemistry was adopted to detect protein synthesis of Col I and Hsp90 in tibia growth plate at the fourth day, the ninth day, the sixteenth day, the twenty-third day respectively by using Col I and Hsp 90 polyclonal antibody. The results showed that synthesis of Col I and Hsp90 was elevated remarkably in prehypertrophic and hypertrophic chondrocytes at the fourth day and the ninth day after being fed thiram diet, but the synthesis of Col I and Hsp90 was identified in prehypertrophic chondrocytes around TD lesion at the sixteenth day and the twenty-third day respectively. It was deduced that the non calcification feedback regulation might have resulted in upregulated Col I in TD and the increased Hsp90 might play an important role in regulating cartilage cell cycle. As a result, the research provides new insights into understanding the pathogenic process of TD in chickens.

Study on Influences of the Effect of Intracytoplasmic Sperm Injection(ICSI) in Goat

ZHANG Wen-long;ZHAO Zhu-jun;TIAN Shu-jun;SANG Rui-zi;

2009, 40(3): 433-437. doi:

Abstract ( 718 )

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Three factors affecting the effect of Intracytoplasmic Sperm Injection (ICSI) technique in goat were studied, and we found that the fertilization rate of frozen sperm was significantly higher than that of the fresh sperm(52.21% VS 26.44%,P<0.05). As for activation method, it was found that the method of single Ionomycin treatment would be more safe and convenient compared with the method of the combination of Ionomycin and 6-DMAP. What is more, the blastocyst rate of embryos cultured under the condition of 5% O₂ was significantly higher than that of embryos cultured under the condition of 20% O₂ (19.59% VS 7.81%，P<0.05).

Isolation, Identification and Characteristic Analysis of Non-structural Protein Nsp2 Gene of High Pathogenicity Porcine Reproductive and Respiratory Syndrome GS/LZh/07 Strain

WU Jin-yan;TIAN Hong;SHANG You-jun;LIU Xiang-tao;ZHENG Hai-xue;JIN Ye;YIN Shuang-hui;MAN Zi-ping;ZHAO Na;CAI Cheng-ru;LIN Fang;XIE Qing-ge

2009, 40(3): 438-443. doi:

Abstract ( 806 )

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PRRS suspect patho-material was inoculated in Marc145 and CPE was found, a strain of PRRSV was detected from cell cultures by PRRSV kit and named as GS/LZh/07. Meanwhile nonstructural protein Nsp2 gene was obtained by RT-PCR,Nsp2 90 nucleotide deletion in Nsp2 gene was detected by sequencing. Sequence analysis indicated that homology between GS/LZh/07 and classical strain was 83.0% in nucleotide sequence and 72.7% in amino acids sequence; homology between GS/LZh/07 and HP-PRRSV variation NX and SD was 95.5% in nucleotide sequence, and 90.9% in amino acids sequence. It was indicated that GS/ LZh/07 originated from popular strain 2006-2007. The results illustrated that HP-PRRSV variation strain exists in Gansu province. Succeed isolation of GS/LZh/07 can provide data for epidemiology of HP-PRRSV, at the same time it can accumulate document for preservation and manipulation of the disease, characteristic analysis of Nsp2 may offer information for HPPRRSV popular feature in Gansu province.

Development of Withdrawal Time of Eprinomectin Residue in Bovine Tissues after Subcutaneous Administration

JIANG Hai-yang;DING Shuang-yang;LIU Jin-feng;ZHAO Si-jun;HE Ji-hong;WU Cong-ming;LI Jian-cheng;SHEN Jian-zhong

2009, 40(3): 444-450. doi:

Abstract ( 1488 )

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In this study, a high performance liquid chromatography method with fluorescence detector (HPLC-FLD) was developed for determination of eprinomectin residue in bovine muscle, liver, kidney, and fat tissues. The tissue samples were extracted with acetonitrile, and cleaned up using basic SPE cartridge. Fortified at levels of 2-100 ng·g^-1, mean recoveries were 70.5%-91.2%, with coefficient of variation of 2.3%-11.5%. The limit of detection and limit of quantitation were 1 and 2 ng·g^-1, respectively. Eighteen parasite-free cross cattle were treated with 1% eprinomectin injectable oil formulation at a dose rate of 0.2 mg·kg-1body weight. Six batches of three treated animals (two males and one female) each were selected randomly at 1, 3, 7, 14, 21, and 28 days withdrawal after injection and the tissue (muscle, liver, kidney, and fat) samples were collected and analyzed by the above HPLC-FLD method. The results indicated that the residue concentration in muscle (2.1-9.3 ng·g^-1) was below the limit (10 ng·g^-1) approved by USA and the concentration in liver (29.8-625.1 ng·g^-1) was below the limits (4 800，1 500 and 2 000 ng·g^-1) recommended by USA, EU, and CAC, respectively. The levels of eprinomectin residues in kidney and fat (4.1-112.5 ng·g^-1 and 2.1-96.4 ng·g^-1) were less than the limits (300 and 250 ng·g^-1) regulated by EU and CAC. The withdrawal periods for all bovine tissues were zero days after the residue concentrationtime data were analyzed using a statistical method recommended by Committee for Veterinary Medicinal Products (CVMP) of The European Agency for the Evaluation of Medicinal Products.

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