Acta Veterinaria et Zootechnica Sinica

Advance in Classification and Enzymological Characteristics of Microbial Phytase in the Rumen

SHEN Shasha;YANG Hongjian;ZHANG Xiaoming

2011, 42(12): 1655-1660. doi:

Abstract ( 509 )

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Ruminants have the ability to utilize phytate phosphate by rumen microorganisms. Until now, domestic research on ruminal microbe phytase is insufficient. In this paper, phytase origin and activity in the rumen was briefly reviewed, and five kinds of rumen bacteria were capable of producing phytase. Among these bacteria, phytase activity was particularly prevalent in Selenomonas ruminantium (199.1703.6 U·mL-1). Based on their optimal pH and catalytic mechanisms, ruminal phytases are classified into acid phytase and cysteine phosphatases phytase, respectively. According to the stereospecificity of phytase, rumen microbes may exist 3phytase and 5phytase. Furthermore, kinetics of enzymatic reaction characteristics of phytases were summarized and discussed comprehensively. In a brief, phytase of S. ruminantium has a optimal temperature of 5055 ℃, an optimal pH range of 4.05.5, and exhibits the highest specificity constant for myoinositol hexakisphosphate and ATP. The optimal phytase activity of Megasphaera elsdenii is displayed at pH5.0 and 60 ℃, and it also displays strict specificity for myoinositol hexakisphosphate.

Gene Expression Analysis upon Cold Induced by Affymetrix Pig Chip

ZHANG Dongjie;LIU Di;BIE Shu;SUN Hongtao;WANG Liang;WANG Wentao;HE Xinmiao

2011, 42(12): 1661-1665. doi:

Abstract ( 455 )

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Cold stress can cause animal performance descending, induce various diseases, even result in death. Min pig is a unique anticold breed in north region of China. In order to study regulation mechanisms of gene expression under cold stress, gene profiling was performed using Affymetrix pig chip on the basis of equally mixed RNA samples from 2 treatments. Thirtyfour differentially expressed genes were detected, among them, twentyseven genes were downregulated and seven genes were upregulated. Among downregulated genes, seven genes (OAS1, IRG6, ISG20, DDX58, IFIT1, ISG15, CXCL9) were associated with interferon induction, one (FST) was associated with growth, two (MX1, PG2) were associated with antivirus, two (S100A9, Numb) were associated with cell growth and apoptosis. But the specific functions of the upregulated genes were unknown, which may have relationship to animal cold resistant. Several genes were selected for Realtime PCR analysis, and the results showed that their expression patterns were consistent with the result of gene chip hybridization. The severe cold stress could cause immunity declining and growth slowing.

Metaanalysis of the Effect of the Estrogen Receptor Gene PvuⅡ olymorphism on Pig Litter Traits

DUAN Lian;WANG Zhipeng;GAO Huijiang

2011, 42(12): 1666-1679. doi:

Abstract ( 413 )

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The aim of this study was to perform the Metaanalysis of the effect of the estrogen receptor gene PvuⅡ polymorphism on pig litter traits. The 33 papers identified from CNKI, WANFANG Data, VIP and PubMed (including 9 429 sows) were analyzed, and a Metaanalysis was performed to assess and combine the data. Under a randomeffects model, litter sizes were significantly lower in the AA genotype group compared with those in the genotype AB and BB groups. Heterogeneity within and between studies was observed. In conclusion, the Metaanalysis demonstrated that the allele B and genotype BB were beneficial for pig litter traits. Heterogeneity between studies was mainly due to the effect of pig breeds and many other factors.

Developmental Patterns and Correlation of Adiponectin Receptors，LHR，CYP11A1 and StAR mRNA Expression in Testis of Wannan Hua Pigs

SHAO Kang;ZHOU Jie;WU Xiaoxue;SHU Baoping;LUO Lianhui;SHENG Sheng;ZHANG Jia;LI Weixin;YIN Zongjun

2011, 42(12): 1680-1685. doi:

Abstract ( 463 )

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The aims of this study were to observe the developmental patterns and the correlation of adiponectin receptors and testosterone synthesisrelated factors mRNA expressions in testis of Wannan Hua pig. To investigate the relationship of adiponectin with the generation of testosterone. Five Wannan Hua boars were sampled at birth, 30, 45, 90, and 180 days of age, respectively. The expression of AdpR1, AdpR2, LHR, CYP11A1 and StAR mRNA in testis were detected by realtime fluorescence quantitative RTPCR. The expression of AdpR1, CYP11A1 and StAR mRNA were significantly changed with age increasing (P<0.01). The expression of AdpR1 mRNA increased at first and then decreased with age increasing, while CYP11A1 and StAR mRNA showed an increasing at first, then decreasing, and increasing at last. All of their expression increased to the top at 45 days of age. LHR were also significantly changed with age increasing (P<0.05), which increased at first then decreased. Significant positive correlation was found between mRNA expression of AdpR1 and CYP11A1 (r=0.587, P<0.05). The mRNA expression of LHR were significantly correlated with CYP11A1 (r=0.528, P<0.05) and StAR (r=0.552, P<0.05), respectively. And significant positive correlation was found between CYP11A1 and StAR mRNA expression (r=0.709, P<0.01). The high expression of AdpR1 mRNA in testis suggested that the action of adiponectin on testis may mediated by AdpR1. The positive correlation between AdpR1 and CYP11A1 mRNA expression indicated that adiponectin contribute the generation of testosterone.

The Expression Pattern of Wnt/βcatenin Signal Pathway Related Genes and MyHCs during Porcine Skeletal Muscle Development

YANG Qiumei;SHI Xine;SHEN Qingwu;LIU Yueguang;GAO Xiaojuan;CHEN Zongzheng;YANG Gongshe

2011, 42(12): 1686-1695. doi:

Abstract ( 433 )

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The aim of this study was to investigate the mechanism of effect of Wnt/βcatenin signal pathway on the porcine skeletal muscle fiber types development. The longissimus doris from Large White pigs at the age of 1 day, 1 week, 2 weeks, 1 month, 2 months and 6 months were used as research materials. Realtime PCR, Western blot and immunohistochemical methods were used to identify the morphologic and histologic change of porcine skeletal muscle during development process, and research the expression of Wnt/βcatenin signal pathway related genes and MyHCs during longissimus doris development. Realtime PCR results showed that MyHCⅠ, GSK3β, βcatenin and Fz3 mRNA expression decreased, MyHCⅡb mRNA increased gradually, MyHCⅡa mRNA increased and then decreased from 1 day to 2 weeks old, and MyHCⅡx mRNA did not change significantly during the development process of porcine longissimus doris. The results of immunohistochemical showed that the slow mucle protein was gray; fast muscle protein was brown. The fast and slow muscle fibers were crossdispersion distribution from 1 day to 1 week old, then slow fibers were coated by fast muscle fibers after 2 weeks old and then the slow muscle fibers decreased gradually. Western bolt results showed that the slow muscle protein decreased significantly from 1 day to 2 weeks old, its change was not significant from 2 weeks to 2 months old, then decreased at 6 months old. The fast muscle protein increased with development, it was extremely increased from 2 weeks to 6 months old. The protein expression of βcatenin and GSK3β were consistent with their mRNA. The protein of pβcatenin was much higher at 2 weeks old than that at 1 day old, and then increased continuously, Tyr216pGSK3β was reduced significantly during early growth, ser9pGSK3β was decreased during later growth. These results suggest that the expression of Wnt/βcatenin signaling pathway related genes βcatenin, Fz3 and GSK3β were positively correlated with MyHCⅠ, but negatively correlated with MyHCⅡ. It played an important role on composition of muscle fiber type during skeletal muscle development.

Using the MPVA Model to Analyze the Impact of GeneGene Interaction between CXCR1 and IL8 2789/2862 on the Risk of Mastitis Susceptibility of Chinese Holstein

ZHANG Yaqin;YANG Zhangping;MAO Yongjiang;CHEN Renjin;CHEN Ying;JI Dejun

2011, 42(12): 1696-1703. doi:

Abstract ( 444 )

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The aims of this study were to identify the interaction of CXCR1 and IL8 and their impacts on the risk of mastitis susceptibility of Chinese Holstein, and to test the feasibility of using MultiLocus penetrance variance analysis (MPVA) model in analyzing genegene interactions. The polymorphisms of 7 SNP sites including the 5′ flanking region and coding region of CXCR1 and intron 3, exon 4 and exon 5 of IL8 were detected by PCRSSCP and direct sequencing for 634 Chinese Holstein, including 102 individual with mastitic, from a northern farm of China, and to identify the interaction of CXCR1 and IL8 and their impacts on the risk of mastitis susceptibility using MPVA. The interactions of CXCR118301768IL8 2789/2862 showed significant effects on the mastitis susceptibility of cow using MPVA model.The results indicate that the interactions of CXCR118301768IL8 2789/2862 is the best multigenotype model to estimate the risk of mastitis susceptibility of Chinese Holstein. MPVA can be used to estimate the risk of mastitis susceptibility of Chinese Holstein.

The Effect of Cathepsin on in vitro Developmental Capacity of Oocytes from Adult Sheep

LIU Zhitao;TIAN Shujun;SUN Shuchun;CHEN Xiaoyong;ZHANG Yanpu;HU Yuanyuan;Guo Junai

2011, 42(12): 1704-1711. doi:

Abstract ( 456 )

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This study was conducted to explore the mRNA expression of cathepsin (CTSB, CTSS) in cumulus cells during in vitro maturation(IVM) from sheep and to detect the correlation beween the mRNA expression of CTSB and CTSS in cumulus cells and in vitro developmental capacity of oocytes. The expression abundance of CTSB and CTSS in cumulus cells of sheep was detected at IVM 0 h，IVM 8 h，IVM 16 h and IVM 24 h through Realtime PCR. The expression abundance of CTSB and CTSS in cumulus cells was detected before and after IVM in the mature medium which contains cathepsin inhibitor E64 with different concentration. The effect of E64 with different concentration in mature medium on in vitro maturation, fertilization and developmental capacity of oocytes were also observed. The mRNA expression of CTSB and CTSS in cumulus cells was downregulated continuously during in vitro maturation (IVM 0 h, IVM 8 h and IVM 16 h, P<0.01), the mRNA expression of CTSB and CTSS in the groups with 5 and 10 μmol·L-1 E64 for IVM 24 h were significantly lower than that of the control and 1 μmol·L-1 E64 groups, nevertheless, there were no significant difference between the two former groups (P>0.05). The blastocysts rate in the groups of 5 and 10 μmol·L-1 E64（26.82%, 30.95%) were higher than those of the control and 1 μmol·L-1 E64 groups(15.63%, 18.23%, P<0.05), but there were no significant difference in the percentage of oocytes mature and cleavage rate among all the groups (P>0.05). The result demonstrate that the mRNA expression abundance of CTSB and CTSS in cumulus cells from sheep is downregulated continuously during IVM, there is negative correlation between the mRNA expression of CTSB and CTSS in cumulus cells and the following embryo developmental capacity；the cathepsin inhibitor E64 could improve the capacity of oocytes developing into blastocysts after fertilization by downregulating the mRNA expression abundance of CTSB and CTSS in cumulus cells.

Regulation of TYR and MITF mRNA Expression by CDK5 in Alpaca Melanocytes

ZHANG Ruina;FAN Ruiwen;CHENG Zhixue;TIAN Xue;LIU Ja;GAO Lei;MA Zheng;DONG Changsheng

2011, 42(12): 1712-1717. doi:

Abstract ( 453 )

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In order to investigate whether CDK5 could participate in the coat color formation of alpaca, the regulation of CDK5 on TYR and MITF expression in alpaca melanocytes was tested in this study. The localization of CDK5 in cultured alpaca melanocytes was examined by immunohistochemistry. Then CDK5 was transfected into cultured alpaca melanocytes by liposomes. Following the transfection, CDK5 protein was detected by Western blot and the expression of MITF and TYR was detected by quantitative realtime PCR(qRTPCR). The immunohistochemistry results showed that CDK5 was expressed in cytoplasm and nucleus of cultured alpaca melanocytes; Western blot results showed that the level of CDK5 protein was apparently higher in transfected melanocytes than that in normal melanocytes, and qRTPCR results showed that the mRNA abundance of MITF was downregulated while the mRNA abundance of TYR was upregulated by the transfection with CDK5, and the expression level of MITF and TYR mRNA in transfected melanocytes was 0.264 9 and 3.931 3 folds higher than that in controlled melanocytes respectively. The results suggest that CDK5 participate in the formation of coat color by regulating TYR and MITF expression in alpaca melanocytes.

Optimization of the Conditions for Isolating Ruminal trans11 18:1 Hydrogenating Bacteria of Dairy in vitro

LI Changhao;JIN Di;WANG Jiaqi;ZHAO Shengguo;LI Dan;BU Dengpan;ZHOU Lingyun

2011, 42(12): 1718-1723. doi:

Abstract ( 441 )

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The objective of this study was to establish the optimal conditions for enrichment and isolating of ruminal trans11 18:1 (TVA) hydrogenating bacteria in vitro. TVA was added into anaerobic mediums with different final concentrations (0, 30, 40, 50, 60 μg·mL-1). Ruminal microbes mixed were inoculated into mediums for continuous cultivation, samples were collected every 4 h and used for detecting TVA concentration and OD values. In addition, TVA was added into the anaerobic medium with a final concentration of 50 μg·mL-1, then the cultures were transfered for six generations for enrichment. Changes of the bacterial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling. Different DGGE bands of the enrichment cultures were identified by 16S rDNA gene sequencing. The result showed that the concentration of TVA in mediums was significantly decreased at 4 h during continuous incubation and then maintained constancy at 12 h. After incubation for 12 h, degradation rates of TVA with the concentration for 50 and 60 mg·mL-1 were higher than that of other concentration. Besides, the OD value of the culture medium increased and then decreased, and reached the peak at 12 h and then decreased. The amounts and types of suspected TVA hydrogenated strains significantly increased at the 4th generation. Sequencing results for the bands showed that most of them belong to Lactobacillus gasseri and uncultured bacterium. This study determined the suitable TVA adding amount, transfer time and generations for enrichment culture of ruminal TVA hydrogenating bacteria, and laid foundation for the subsequent selective separating culture of TVA hydrogenating bacteria.

Expression of NonStructural Protein 2C of Footandmouth Disease Virus in Insect Cells and Its Application

ZHANG Xiaoli;;LU Zengjun;MA Xiaojun;CAO Yimei;FU Yuanfang;LIU Zaixin;XIE Qingge

2011, 42(12): 1724-1731. doi:

Abstract ( 447 )

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In recent years, the potential value of nonstructural protein (NSP) 2C was well documented for distinguishing footandmouth disease virus (FMDV) infected animals from vaccinated animals. In this study, 2C gene of FMDV was cloned into baculovirus transfer vector pFastbacHTB, and then the recombinant vectors were transformed into DH10Bac which contain backbone Bacmid. The white colonies were selected and Bacmid2C recombinant vectors were used for transfection of Sf9 cells. After three times amplification of the viral stocks, the NSP2C was expressed in High Five cells. The SDSPAGE showed that a 38.93 kD fusion protein 2C was successfully expressed, and the products showed a specific reactivity with serum from FMDV infected animal using Westernblot analysis and DotELISA. An indirect ELISA based on fusion protein 2C was developed, the results showed that this 2CELISA not only could be applied to distinguish FMDV infected animals from vaccinated animals, but also could be used to detect early anti2C antibodies in cattle and pigs which had been infected with FMDV. The baculovirus expressed 2C appears to be a suitable antigen for the development of a reliable diagnostic test. This study make a good foundation for development of diagnosis method for detection of antibodies against multiple NSPs to discriminate the FMDV infected animals from vaccinated ones, and this will increase our capability to check the infectious virus carrier and finally improve FMDV infection control.

The Optimum siRNA Screening of Targeting Porcine Integrin αv Subunit Gene as FMDV Receptor Against FMDV Replication

LUO Jihuai;DU Junzheng;GAO Shandian;ZHANG Guofeng;CHANG Huiyun

2011, 42(12): 1732-1737. doi:

Abstract ( 388 )

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To screen the optimum siRNA targeting porcine integrin αV subunit as FMDV receptor against FMDV replication, three siRNA sequences were selected according to porcine integrin αV mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized. After PK15 cells were transected with siRNAs by Lipofectamine 2000, qRTPCR and 100 TCID50 were used to evaluate the level of αV expression and resistance of FMDV O/CHA/99, respectively. The results showed that compared with control and mock group, the αV subunit mRNA expression were obviously suppressed in all three experimental groups (iαV480, iαV1719 and iαV2077), especially the expression rate in iαV480 group was reduced by 90.1% in 24 hours. Lower virus titers of iαV480 group by TCID50 indicated that PK15 cells increased the resistance to FMDV O/CHA/99. The optimum siRNA toward porcine integrin αV subunit gene are successfully screened in this study, it would be helpful work about studying of interference FMDV receptor gene antiFMD transgene in swine.

Etiology and Serology Investigation of Avian Leukosis form the Different Regions of Shandong Province of China in 20092010

NI Wei;SUN Honglei;QU Yue;YANG Feng;LIU Sidang

2011, 42(12): 1738-1742. doi:

Abstract ( 394 )

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In order to clarify the prevalence about etiology and serology of the AL in Shandong Province, 1 827 samples of serum samples were collected from four regions, Tai’an, Liaocheng, Qingdao and Linyi, in Shandong Province, including 597 samples of grandparent layer serum, 710 samples of parent layer serum and 520 samples of commercial layer serum；at the same time, 1 209 samples of cotton swabs were collected from these regions above, including 597 samples of grandparent layer swabs, 460 samples of parent layer swabs and 172 samples of commercial layer swabs. Serums were tested for ALVJ and ALVAB antibody and cotton swabs were tested for P27 antigen through ELISA. Result were as follows: The antibody of ALVJ, ALVAB and antigen of p27 of grandparent layer is 0, 0.84%, 17.93% respectively, the diversity is big in different regions of P27 and the rate of antigen of Tai’an, Liaocheng, Qingdao is 52.5%, 0.58%, 0 respectively. The antibody of ALVJ, ALVAB and antigen of P27 of parent layer is 8.45%, 3.10%, 30.48% respectively, and the diversity is big in different regions among the rate of antibody of ALVJ and ALVAB and antigen of P27. The antibody of ALVJ, ALVAB and antigen of p27 of commercial layer is 13.46%, 8.65%, 18.42% respectively, and the diversity is also big in different regions among the rate of antibody of ALVJ and ALVAB and antigen of P27. The evidence relative of antibody positive rate and antigen positive rate in Grandparent, Parent and Commercial chickens reveals that the possibility about existence of vertical infection. The ALVJ antibody in Shandong Province of Granparent chickens weren’t detected, and ALVAB antibody is very low (0.84%), but the positive rate of antigen of P27 is great in some grandparent layer, we conjectured that there probably existed immunological tolerance infection; the positive rate of antibody and antigen of Parent and Commercial chickens were all higher than that of Granparent chickens, which illuminated that there has been ALVJ and ALVAB infection in them; at the same time the ALVJ antibody is higher than ALVAB antibody in Parent and Commercial chickens, and it shows that the ALV infection is mainly ALVJ, we should research the route of transmission of ALV and intensify quarantine and purification.

Expression and Immunogenicity of Transferrinbinding Protein A Gene of Haemophilus parasuis

XIN Wei;LI Yu;HUANG Xiaohui;WEI Jianzhong

2011, 42(12): 1743-1749. doi:

Abstract ( 440 )

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An experiment was conducted to express transferrinbinding protein A (TbpA) of Haemophilus parasuis (HPS) and investigate its immunogenicity. Three pairs of specific primers were designed according to the tbpA gene sequence of HPS SH0165 strain from GenBank, three fragments of tbpA1, tbpA2 and tbpA3 containing multiple antigen epitopes were accquired by PCR and cloned into pET32a(+) vector respectively. It was confirmed that three fragments were inserted into the prokaryotic expression vector correctly by using Restriction endonuclease digestion and sequencing. The plasmids were transformed into E. coli BL21(DE3) and expressed by induction of IPTG. The specific proteins were determined by SDSPAGE and their immune activity was analyzed by Western blotting. The immunogenicity of the proteins was analyzed by vaccination efficacy assessment in mice and bctericidal assays. The results showed that three fragments of 1 140, 897 and 666 bp were amplified by PCR and expressed proteins with the sizes of 62, 54 and 44 kD. The rTbpA1 can partly protect mice from the challenge of H. parasuis LJ3 strain and a significant bactericidal activity was also detected. These results indicated that the successfully expressed rTbpA1, rTbpA2 and rTbpA3 proteins were of effective reactogenicity, and the rTbpA1 could induces an immune response and might be useful as an antigen for vaccine and serological detection against Glsser's disease.

Cytotoxicity of the Cytolethal Distending Toxin of Haemophilus parasuis

CHEN Xi;WANG Xiangru;XU Xiaojuan;GUO Fengjuan;CHEN Huanchun;CAI Xuwang

2011, 42(12): 1750-1755. doi:

Abstract ( 406 )

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The study was aimed at prokaryotic expressing cytolethal distending toxin of Haemophilus parasuis, and acted on Pig iliac endothelial cells (PIEC) to investigate toxin effects. According to the complete genome sequence of Haemophilus parasuis SH0165 (serovar 5), which finished by our laboratory, we designed a set of specific primer, the cdtA, cdtB and cdtC gene was amplified by PCR, respectively. The 681, 834 and 531 bp amplified DNA fragment was cloned into pET28a and the expression were induced in E. coli BL21 (DE3) by IPTG for 3 hours. Results of Sodium docecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) and Western blot assay showed that the cdtA, cdtB and cdtC gene was expressed in the form of inclusion body, and the recombinant protein was about 36, 34 and 28 kDa. Recombinant toxin acted on PIEC for 3 hours in vitro, and observed the morphological change after another culture of 72 hours. These results showed that CdtABC holotoxin induced PIEC distension, vacuolization and apoptosis, while the other test groups changed unobviously. The study indicated that CDT may play an important role in the infection and pathopoiesis of bacterial.

Cloning and Preliminary Characterization of a SmadA Gene from Echinococcus granulosus

LI Jing;;ZHANG Chuanshan;LV Guodong;WANG Junhua; WEI Xufa;LIN Renyong;YAN Genqiang

2011, 42(12): 1756-1762. doi:

Abstract ( 418 )

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The objectives of this study were to clone the EgSmadA gene of Smad family from Echinococcus granulosus, identify its structure and expression profile of the protein, and construct its yeast twohybrid system. The EgSmadA gene was cloned and analyzed by bioinformatics, and detected its expression profile by immunohistochemisty. The results showed that EgSmadA genomic DNA is 4 069 bp, contains 4 exons, 3 introns and a complete open reading frame of 957 bp in length, which encodes for a peptide of 318 amino acids. A conserved MH2 (Mad homology) domain of Smad family was detected in the Cterminal of EgSmadA, and the yeast twohybrid system of EgSmadA and EgSmadAMH2 were successfully constructed. Immunolocalization results indicated that EgSmadA was expressed in the tegument of protoscolex and cellular germinal layer of metacestode. The results provided some new clues for further studying the biological functions of EgSmadA in the growth and development of Echinococcus.

Sequence and Phylogenetic Analysis of Mitochondrial nad1 and nad4 Genes for Coenurus cerebralis in Goats in Hunan Province

HE Desi;XU Pingyuan;PENG Yunchao

2011, 42(12): 1763-1767. doi:

Abstract ( 379 )

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The objectives of the present study were to examine sequence variation in the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene and NADH dehydrogenase subunit 4 (nad4) gene among Coenurus cerebralis isolates in Hunan Province，and to reconstruct their phylogenetic relationship using nad1 and nad4 sequences. The partial nad1 (pnad1) and nad4 (pnad4) were amplified from each C. cerebralis， and pnad1 and pnad4 sequences were aligned using the ClustalX 1.81. MP, and NJ trees of pnad1 and pnad4 were constructed using the software Phylip 3.67 version 4.0 and Mage version 4.0, and ML tree was also constructed using Puzzle version 5.2. Sequence homology analysis was performed using the Megalign program of the software DNAStar version 5.0. The results showed that the lengths of pnad1 and pnad4 sequences were 666 bp and 887 bp, respectively. The constructed phylogenetic tree revealed that the Hunan isolates and the Taenia multiceps available in GenBank were clustered in the same clade. There is no significant variation in pnad1 and pnad4 sequences within C. cerebralis, while interspecies difference is obvious. It is concluded that pnad1 and pnad4 sequences can be used as genetic marker for population genetic studies of cestodes. The results of the present study provided foundation for further studies of molecular epidemiology of C. cerebralis，and for diagnosis of the resultant disease.

Detection of Anaplasma marginale, Anaplasma centrale, and Anaplasma ovis by Nested Polymerase Chain Reaction Assay

LI Shuqing;WANG Guiqiang;CHEN Zhifei;ZHAO Junlong;WANG Qiaoquan;ZHANG Ziqun;YOU Xuan;HU Yongqiang;DU Kai;YAO Baoan

2011, 42(12): 1768-1775. doi:

Abstract ( 424 )

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To detect and differentiate A. marginale, A.centrale, and A. ovis, an msp4 nested PCR assay was developed by two universal and 3 specific pairs of primers. Sensitivity test revealed that this method was able to detect as little as 0.2 pg of DNA (approximately 6 infected erythrocytes). No crossreaction with Babesia bovis, B. bigemina, Mycoplasma wenyonii, and DNA from other organisms was observed in the specificity test. 1 119 clinical blood samples of cows, beef, buffalos and sheep from six different areas were tested by this method and 106 of them were positive for Anaplasma spp. Of the 106 samples, 46 were positive for A. marginale, 15 for A. centrale, and 35 for A. ovis, and 4 showed mixed infections with A. centrale and A. ovis, 3 with A. marginale and A. centrale, and 3 with A. marginale and A. ovis. The results had 98.5% (835/848) coincidence with the OIE recommended msp5 nested PCR method. The results also provided molecular evidence for the existence of A. centrale in China. The study reveals that cattle can be simultaneously infected by more than one Anaplasma spp. This study indicated that the msp4 nested PCR assay is specific and sensitive for the detection and differentiation of A. marginale, A. centrale, and A. ovis.

Study on the Structure and Quantitative Changes of Mucosal Immunityassociated Cells in Small Intestine of the Adult Yak

ZUO Yuzhen;GAO Shijie;SHAO Jianhua;FANG Mei;JIA Ning

2011, 42(12): 1776-1781. doi:

Abstract ( 481 )

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In order to reveal the structure and the distribution and number changes of small intestinal immunity in the adult yak, the morphological structure and intraepithelial lymphocytes, goblet cells, plasma cells and mast cells of small intestine of adult yak were investigated by histochemical technique, the scion image software analysis techniques and the electron microscope technique. The results showed that there were mucousmembrane, submucosa, tunica muscularis and serosa in small intestine of the yak. The ratio of high/crypt depth and the thickness of tunica muscularis of duodenum is the highest; the high of villi and the crypt depth of jejunum are the longest and highest. They are significantly differed that the high of villi, the crypt depth, the ratio of high/crypt depth and the thickness of tunica muscularis in each segment of small intestine (P<0.01). The number of intraepithelial lymphocytes, goblet cells and mast cells reduced gradually from duodenum, jejunum to ileum, but the number of plasma cells exhibits an increasing trend, which are significantly differed(P <0.01). In the electron microscope, there are tight junction, gap junction and hemidesmosome, a lot of microvillus in small intestine of the yak. The nucleus of intraepithelial lymphocytes is larger and cytoplasms is less. Goblet cells are typical goblet shape, its top are larger because of a number of secretory granules; Plasma cells are circular or elliptical, the chromatin is distributed along the nuclear membrane, cytoplasms are richer in endoplasmic reticulum; Mast cells are elliptical, there are strong electron density particles in its cytoplasm. Digestion and absorption of arcticalpine pasture are enhancing greatly by the structure of small intestine in the yak; there are regular distributions of mucosal immunityassociated cells in each segment of small intestine, showing strong mucosal immunity.

The cDNA Cloning and Prokaryotic Expression of Mongolia Sheep Ghrelin Gene

LIANG Jianrong;CAO Guifang;ZHAO Pengwei;WEN Shiyong;CHENG Lanling;TU Yong

2011, 42(12): 1787-1794. doi:

Abstract ( 453 )

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To construct Mongolia sheep Ghrelin prokaryotic expression vector, and observe its expression in host Escherichia coli strain BL2l(DE3), the cDNA of Ghrelin gene was amplified from abomasum fundic gland mRNA of Mongolia sheep by RTPCR. PCR product was cloned into the T vector pMD19T to construct pMD19TGhrelin for sequencing. Then the cDNA was subcloned into the prokaryotic expressing plasmid vector pET32a(+) and transformed into host Escherichia coli strain BL2l(DE3)for expression. The clone was induced by IPTG and was identified by SDSPAGE and Westernblot. Comparing with the Ghrelin sequence of sheep reported in GenBank, there were two nucleotide differences, but it did not affect the amino acid sequence; the expression product was observed with soluble protein; The result of Westernblot showed that the recombinant protein was recognized by Hisantibody specifically. The nucleotide sequence isolated from the recombinant plasmid pET32aGhrelin was the same as expected; the Ghrelin protein was expressed successfully in Escherichia coli, which provided a basis for further investigation of Ghrelin function.

Effects of Mastitis on Testday Milk Performance and the Variation of SCC of Chinese Holstein

MAO Yongjiang;CHEN Ying;CHEN Renjin;CHANG Lingling;SHI Xuekui;YANG Zhangping;LIANG Xianghuan;YIN Shaohua

2011, 42(12): 1787-1794. doi:

Abstract ( 372 )

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The objective of this study was to investigate the effects of clinical mastitis and pathogenspecific mastitis on the testday milk performance and the variation of SCC for Chinese Holstein. Clinical mastitis and subclinical mastitis were indentified using CMT method from 140 milking cows in a dairy farm, Jiangsu Province. In September, 2009, and the pathogens were indentified for the quarters of subclinical mastitis cow. The effects of clinical mastitis and pathogenspecific subclinical mastitis on the testday milk performance and the variation of SCC were estimated by ANOVA. The proportion of clinical mastitis and subclinical mastitis cows were 10.00% and 51.10%，respectively. In the subclinical mastitis cows, the most events was the mixed infected by Escherichia coli and Streptococcus spp (26.00%），following was Streptococcus spp (23.30%). The proportion of cows infected by only one pathogen was 36.11%，and the proportion of cows infected by more than two pathogens was 61.11%. The effects of mastitis on testday milk yield, lactose percentage, SCC and SCS were significant at P<0.01 level. The testday milk yield and lactose percentage for clinical mastitis and subclinical mastitis cows were less than that for healthy cows, and the SCC were higher than that in healthy cows. The pathogen types significantly affected SCC and SCS of cow. The testday milk yield for subclinical mastitis cows infected by more than two pathogens were significant less than that in cows infected by only one pathogen, and the SCC were significant higher than that in cows infected by only one pathogen. The effect of mastitis on the variation of SCC was significant at P<0.01 level. The healthy cows had higher ability to maintain low SCC in milk, and the possibility having more SCC for subclinical mastitis cows was increased .The pathogens types had no significant effect on the variation of SCC. The results indicate that the subclinical mastitis infected by more than two pathogens have more serious effects than subclinical mastitis infected by only one pathogen on the testday milk yield and SCC in milk. These findings provide dairy producers with more information in which pathogenspecific subclinical mastitis cases should receive treatment and how to manage these cows.

Pathologic Research of Lymphocytic Subgroup JAvian Leukosis in Qingyuan Local Chicken

DENG Hua;WU Yunfei;LU Yukui;WANG Zhengfu;YANG Hong;MA Chunquan

2011, 42(12): 1795-1799. doi:

Abstract ( 411 )

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This experiment was conducted to explore the complicated tumor manifestation of avian leucosis subgroup J. On the basis of epidemiology survey of four Qingyuan local layer chicken farms in Guangdong province, then the avian leucosis virus was isolated and detected by PCR test, and avian leucosis virus subgroup J (ALVJ) was definite diagnosed as the pathogen. The histopathologic study showed that the most tumorous manifestation was lymphocytic leucosis (82.9%), and then was hemangioma (11.4%), myeloid leukosis was 5.7%. Abnormal proliferation of lymphocytic leukosis was occurred mainly in parenchymatous organs, including heavy swollen of liver, spleen, kidney, lung, proventriculus and pancreas. There were many ivorywhite tumors and nodules occurred in the parenchyma, and the sections were homogeneous and soft. The solid components of tumors were typical lymphoblast and neoplastic lymphoid cells, and many phanerous pathologic nuclear mitotic figures were observed. Those results confirmed that there appeared a novel tumorous manifestation in Qingyuan local chicken, lymphocytic subgroup Javian leukosis.

The Study on Immunne Response of Muscovy Duck Reovirusis Alive Vaccine

LIN Fengqiang;CHEN Shilong;CHEN Shaoying;ZHU Xiaoli;CHENG Xiaoxia;HU Qilin;WANG Shao;

2011, 42(12): 1800-1804. doi:

Abstract ( 458 )

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The experiment was conducted to access the effect of muscovy duck reovirusis alive vaccine on immune response and to explore the immune mechanism. After 1dayold muscovy ducks were immunized, the growth and development were observed, neutralization antibody in the sera, proliferation responses of the lymphocytes in the peripheral blood, thymus and bursa cells to ConA and LPS, Cytotoxic activity of cytotoxic T lymphocytes (CTL) were detected. The results showed that the growth and development of the immunized muscovy ducks were more than that of control group. The neutralization antibody began to increase until 35 days. Lymphocyte proliferation response to ConA in blood and thymus was enhanced differently in the immunized muscovy ducks, but response to LPS in blood and bursa weren’t changed differently. The cytotoxic activity of CTL in the immunized muscovy ducks was improved. So the muscovy duck reovirusis alive vaccine can improve the resistant ability of muscovy ducks to muscovy duck reovirus mainly by increasing cellular immunity．It laid theoretical basis on clinical application with muscovy duck alive vaccine.

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