[Objective]This study was to determine molecular biology information of HA gene of H1N1 swine influenza virus isolated from Guangxi strains to provide references for monitoring swine flu in Guangxi. [Method]To analyze HA gene of H1N1 from Genbank, the primer was designed, and then HA genes of A / swine/Guangxi/1/2011 (H1N1) influenza virus were cloned, sequenced and compared via DNASTAR software. A phylogenetic tree was made using MEGA 4.0. [Result]The results indicated that the length of HA gene was 1701 bp and coded for 566 amino acids. Comparing with classical swine influenza H1N1, human -like H1N1 and Eurasian avian-like H1N1, the nucleotide homologies of HA genes were from 88.0% to 99.6%, from 76.3% to 77.3% and from 72.9% to 75.4%,respectively. Phylogenetic analysis revealed that the strain of A / swine/Guangxi/1/2011(H1N1) belonged to classical swine influenza H1N1 lineage and was similar to the strain A/California/04/2009 (H1N1), while it did not resemble human-like H1N1 and bird-like H1N1 at all. Amino acid sequence at the HA cleavage site was IPSIQSR↓G, which had characteristics of low-pathogenic influenza viruses. Among the eight glycosylation sites, six sites were located in HAl section and the rest in HA2. Amino acid in protein RBS of Guangxi strains possessed characteristics of both human and swine influenza virus. [Conclusion]This strain was pandemic(H1N1) 2009 virus and first isolated in Guangxi.

We reported for the first time the isolation of H9N2 influenza virus from dogs in southern China. Genetic analysis of an isolate revealed that it was a novel genotype closely related to avian H9N2 virus. Epidemiologic studies demonstrated that the new H9N2-subtype virus was the causative agent of a disease in canine. Therefore, an appropriate countermeasure is urgently needed.

【Objective】Epidemic survey was carried out to find out the prevalence of bovine tuberculosis in Guangxi.【Method】The tuberculin skin test (TST) and interferon gamma (IFN-g) ELISA assay were used to detect the bovine tuber-culosis in Guangxi from 2008 to 2011. Totally 5478 cows and 1580 buffaloes had been quarantined for 4 years. 【Result】Dur-ing 2008-2011, 63 dairy cows (accounted for 1.15% of the total ) and 17 buffaloes(accounted for 1.08% of the total)were detected as positive in Nanning, Liuzhou, Beihai, Qinzhou and Guilin. Overall, the positive rate of tuberculosis in cow and buffalo decreased year by year. Through self-made box of interferon gamma assay and BovigamTMkit, it was found that the positive rate of bovine tuberculosis detected by interferon gamma assay which had high accuracy was lower than that by TST.【Conclusion】For better detection and purification of bovine tuberculosis, the herd should be quarantined by tuberculin skin test, and then tested by interferon gamma assay.

文章采用案例分析方法,以广西灵川县东田村辇田尾自然村实施实施参与式整村推进扶贫项目为依托,探讨参与式方法在贫困村扶贫开发项目制定和实施过程中,如何将贫困村长期积累的经验与参与式扶贫开发的积极性充分发挥出来,分析广西参与式整村推进扶贫开发工作的现实意义。

随着宠物热的兴起，宠物诊疗业蓬勃发展，但该行业依然存在专业人员严重缺乏，行业标准和规范不完善、管理落后等问题，严重阻碍了发展，本文就广西宠物诊疗业存在的问题及应对措施进行了深入的探讨，以期为促进广西宠物诊疗业的良性发展提供借鉴。

[Objective]The present experiment was conducted to further understand the epidemic features, toxicity levels, and genotypes of porcine origin avian paramyxovirus serotype 1 in Guangxi in order to provide the relevant data for carrying out future researches on porcine paramyxovirus, and to provide references for the prevention and control of the disease. [Method]The virus was isolated from suspected samples of pigs using chicken embryos inoculation. The characteristics of the virus were determined with hemagglutination (HA), hemagglutination-inhibition (HI) test, mean death time of chicken embryos (MDT), and intracerebral phthogenicity index (ICPI). The virus strain was identified and F gene fragments were amplified using RT-PCR and sequenced. [Result]The pig source avian paramyxovirus serotypeⅠisolate was isolated successfully from suspected samples of pigs. Its titer of HA and HI was 26 and chick embryo minimal lethal dose was 10-8/0.1 mL. The MDT and ICPI of this isolate were 60 h and 1.45, respectively. Amino acid composition was R-R-Q-R-R-F of F gene 112-117 splitting sites, which was in accordance with those of NDV standard virulent strain HER33 and F48E9. The nucleotide homologues were 86.5 and 85.7%, respectively, and the amino acid homologues were 90.1 and 90.8% after comparing the F gene of strain with standard virulent strain F48E9 and moderate virulence strain Beaudette C. [Conclusion]The isolate was virulent strain belonging to A PMV-1 gene typeⅡand traditional strains. It signified that hosts of APMV-1 were spreading and expanding constantly in Guangxi area.

Double antibody sandwich ELISA(DAS-ELISA) for IFN-γ in water buffalo was developed.In this method,two mice ascites McAbs against water buffalo interferon-γ were purified by ammonium sulfate precipitation(AS),and McAbs 1E4 was used as a capture antibody and HRP-labeled McAbs 4A11 as a detection antibody.The optimal reaction conditions for the assay were determined.The result indicated that the optimal concentration of capture antibody was 625ng/mL,the working concentration of HRP-labeled 4A11 was 1∶100,and the detection limit of the sandwich ELISA for IFN-γ was 25ng/mL.Compared with tubculin skin test(TST),the diagnosis specificity,sensitivity and coincidence test of DAS-ELISA were 62%,87% and 75%,respectively.Therefore,this DAS-ELISA assay will be a useful tool in the bovine tuberculosis diagnosis in water buffalo.

本研究采用RT-PCR方法从培养的鹅外周血淋巴细胞总RNA中成功扩增到鹅IFN-γ基因。将克隆在pMD18-T载体中的鹅IFN-γ基因插入原核表达载体pGEX-6P-1,得到重组质粒pGEX-6P-1-IFN-γ。重组质粒转化大肠杆菌BL21,经IPTG诱导后作SDS-PAGE分析,得到约43 ku融合表达蛋白特异条带。用GST亲和纯化柱对原核表达产物中目的蛋白进行纯化,得到1.2 mg/L的纯化蛋白。用100TCID50病毒量在鹅副黏病毒(GPMV)/鹅胚成纤维细胞系统上测定表达蛋白的抗病毒活性,观察不同处理组的细胞形态并用RT-PCR方法鉴定,发现表达蛋白稀释倍数小于104可抑制GPMV复制,按照Reed-Muench法计算表达的鹅IFN-γ抗病毒效价为2.0×103U/mL,表明表达蛋白具有良好的抗病毒活性。

To the Editor: A novel swine-originated influenza A virus known as pandemic (H1N1) 2009 was first isolated from humans in Mexico in April 2009 (1), and a worldwide pandemic followed, which affected >214 countries and resulted in >18,000 deaths (2). In August 2010, the World Health Organization stated that the pandemic caused by this virus had ended. As this virus emerged, animals, including swine, turkeys, ferrets, cats, and cheetahs, were found to have been infected (3). In addition, transmission from humans to pigs in porcine herds has been reported (4). Swine influenza A virus (SIV) belongs to the family Orthomyxoviridae and is a causative agent of respiratory disease in pigs (5). Currently, 3 subtypes of influenza viruses are circulating in the swine population globally: H1N1, H3N2, and H1N2 (6,7). Although pigs can be simultaneously infected with avian influenza viruses and human influenza viruses, the viruses can exchange genes and produce new variants, which suggests that pigs have become mixing vessels for influenza viruses (8). Pandemic (H1N1) 2009, caused by a virus usually circulating in pigs in Europe and Asia, is a triple hybrid that contains swine, human, and avian virus gene segments, which further emphasizes that SIVs pose a serious threat to public health. We describe an outbreak of pandemic (H1N1) 2009 virus, which was isolated from a pig farm in Guangxi Province, People’s Republic of China, and report the consequences of subsequent epidemiologic studies. In January 2011, an outbreak of severe respiratory problems occurred in pigs on a pig farm. Nine hundred growing and fattening pigs exhibited clinical signs of influenza, including fever, cough, runny nose, loss of appetite, lethargy, edema, watery eyes, conjunctivitis, diarrhea, and vomiting. The incidence rate was ≈80%, and the death rate was 22%. The outbreak lasted ≈2 weeks. However, no outbreak of respiratory disease occurred in other pig farms in the same area simultaneously, and no evidence of human-to-pig transmission was found. We collected lung samples from 3 dead pigs with underlying illness for reverse transcription PCR and virus isolation in 10-day-old specific pathogen–free embryonated chicken eggs. Viral RNA was extracted from the tissue suspension and allantoic fluids. Virus isolation was assessed by hemagglutination inhibition (HI) assay and neuraminidase inhibition assay by using a panel of reference serum samples (National Reference Laboratory for Avian Influenza, Harbin Veterinary Research Institute, Harbin City, China). Later, 3 viruses were isolated. All of them had hemagglutination (HA) activity, and the HA titers ranged from 128–256. The HA-positive isolates were further identified as subtype H1N1. Subsequently, nucleotide sequences of the 8 viral genes were amplified and sequenced (GenBank accession nos. {type:entrez-nucleotide-range,attrs:{text:JN222372-JN222379,start_term:JN222372,end_term:JN222379,start_term_id:339782164,end_term_id:339782180}}JN222372-JN222379). This analysis showed high identities to a pandemic strains A/California/04/2009 (H1N1), hemagglutinin (99.2%), neuraminidase (99.1%), matrix (99.3%), nucleoprotein (99.5%), nonstructural protein (98.5%), polymerase acidic protein (98.5%), polymerase basic protein 1 (99.7%), and polymerase basic protein 2 (99.6%) genes. Bacteria were cultured from spleen, liver, and heart-blood samples from 5 pigs. Four pigs were infected with porcine streptococci, and 1 pig with mild symptoms was negative for the bacterium. We sought to gain more insight into the epidemiology of pandemic (H1N1) 2009 virus in Guangxi Province and collected 600 bronchial swab samples and 200 blood serum samples when pigs were slaughtered every month from February through June 2011. The samples were used to isolate virus in 10-day-old specific pathogen–free embryonated chicken eggs, and then the virus isolates were subjected to sequencing of a partial genome of the HA gene. Overall, we obtained 10 strains of subtype H1N1 influenza virus, including 5 strains of classic swine H1N1, 3 strains of Eurasian avianlike H1N1, and 2 strains of pandemic (H1N1) 2009 virus, which were derived from 3,000 bronchial swab samples. In addition, a serologic survey was implemented by using HI testing with pandemic (H1N1) 2009 virus and SIV (H1N1) antigens. Serologic studies showed that 251 of the 1,000 samples tested had positive HI titers for pandemic (H1N1) 2009 virus, and 248 of these samples had positive HI titers for SIV (H1N1). Notably, cross-reactivity of pandemic (H1N1) 2009 virus between H1 subtype viruses has been reported recently in pigs (9). However, the higher rate of positive test results indicated that swine serum samples contained antibodies against pandemic (H1N1) 2009 virus. Our findings strengthened previous data by showing that growing and fattening pigs are susceptible to infection of pandemic (H1N1) 2009 virus (4). Analysis of the complete genome sequence of the subtype H1N1 isolates suggests that no gene reassortment occurred. The results of serologic studies demonstrated that uninfected pig farms are also susceptible to pandemic (H1N1) 2009 virus infection. Our results suggest that the pandemic virus is currently circulating in swine populations and posing a challenge to pigs in southern China. Increasing serologic surveillance of pigs for prevention and better control of pandemic influenza is urgently needed in China.

The IFN-γ gene was inserted into the eukaryotic expression vector pcDNA3.1(+),and the eukaryotic expression plasmid pcDNA3.1(+)-IFN-γ was constructed and identified by sequencing.Electroporation instrument was set to 425 V of voltage and 25 μF of capacitor,then pulsed 2 times consecutively under room temperature.20 μg/mL plasmid pcDNA3.1(+)-IFN-γ was transformed into 1×106 cells/mL BHK-21 in 400 μL of volume.After consecutive selection by 600 μg/mL G418 for 14 days,resistant cell clones were selected,and the cell supernatants were identified by Western-blot test.The results indicated that goose IFN-γ was expressed in BHK-21 cells.

A H9N2 subtype strain of swine influenza virus(SIV) was isolated from swine in Guangxi by SPF chicken embryo.The SIV strain had a stable haemagglutiain(HA) values for 27 after there generations,and can be inhibited by H9 positive serum.The HA and NA genetic amplification showed that this strain had the height homology with the subtype H9N2 SIV,which named A/Swine/Guangxi/01/2009.

对具有流感临床症状的病马组织经处理后接种SPF鸡胚,分离到1株流感病毒.该病毒经鸡胚接种7代后,出现稳定的对鸡红细胞凝集效价,效价为25,能被H3亚型血清中和,与H1、H5、H7、H9亚型阳性血清无交叉反应；对HA基因及NA基因进行序列分析,结果显示:HA基因与马流感病毒H3亚型同源性最高,NA基因与马流感病毒N8亚型同源性最高,判断该病毒属于H3N8亚型马流感病毒,命名A/EQ/Guangxi/01/09.

Full-length cDNAs of polymerase genes PB2 and PB1 of avian influenza virus strain GS/GX/01/06 were amplified by RT-PCR.The cDNAs were cloned into pMD18-T vectors and sequenced.Sequence analysis showed that open reading frames of the PB2 and PB1 cDNAs contain 2 280 nt and 2 277 nt encoding 759 and 758 amino acids,respectively.Nucleotide sequence and amino acid identity rates of the polymerase genes PB2 and PB1 were over 95% between avian influenza virus strain QA/HK/G1/97 that belong to the subtype H9N2 AIV,there were in closer relation between them.

A pair of primers and a TaqMan probe were designed according to the CSFV 5′UTR sequences of CSFV taken from GenBank,for performing TaqMan fluorescent quantitative PCR.RNA of classical swine fever virus(CSFV) was used as template,and the reaction conditions of TaqMan fluorescence PCR were optimized based on conventional PCR.The TaqMan fluorescent quantitative PCR method for CSFV was developed,which showed high specificity and high sensitivity to CSFV(detection sensitivity of 5.03×10-2 μg/μL),as revealed by the sensitivity and specificity tests.Therefore,this method can be used for rapid detection of CSFV.

将原核表达的重组水牛(Bubalus bubalis)γ-干扰素成熟蛋(rGST-WBIFN-γ)以100 μg/只剂量免疫8周龄Balb/c小鼠(Mus musculus),经5次免疫后,取脾细胞与SP2/0骨髓瘤细胞进行融合,以rHIS-WB-IFN-γ和rGST-WBIFN-γ蛋白共同作为检测抗原进行间接ELISA检测,筛选并最终得到了2株能稳定分泌抗体的单细胞克隆株(分别命名为IE4和4A11),分泌抗体亚型均为IgG1型,轻链均为κ链.Western-blotting检测显示:2株单克隆抗体与原核表达的rWBIFN-γ蛋白均能特异性结合,具有良好的免疫反应原性.

2818 cattle were detected for Mycobacterium tuberculosis by tuberculin skin test(TST),and the interferon gamma(IFN-γ) assay was used to retest the positive,suspicious and some negative individuals.Compared with TST,the diagnosis sensitivity,specificity and coincidence of IFN-γ assay were 53.33%,70.49% and 67.11%,respectively.This study demonstrated that the use of TST alone in the diagnosis of bovine tuberculosis has non-specific defects and the retest with antigen-specific IFN-γ assay for the positive and suspicious individuals by TST can improve the specificity.The IFN-γ assay would be a useful tool in the diagnosis of bovine tuberculosis.

A pair of primer was designed using the nucleotide sequence of Northeast white goose IFN-α gene obtained from GenBank(Accession No.AY524422).Total RNA was extracted from cultures of goose peripheral blood lymphocytes under stimulation of GPMV,and the cDNA was amplified using reverse transcription-polymerase chain reaction(RT-PCR),which was subsequently cloned into pMD18-T vector and sequenced.The results showed that the open reading frame(ORF) of Guangxi goose interferon-α(GoIFN-α) gene consisted of 576 bp,encoding 192 amino acid residues.Among them,there were seven cysteine residues and two potential glycosyl binding site(NDT and NHT).Compared with the sequences of other goose IFN-α gene in GenBank,the homologies of nucleotide and amino acid between Guangxi goose and white Northeast goose(AY524422) was found the highest(98.3 and 96.4%,respectively).However,these two indices among Guangxi goose and chicken,cow,swine,rat,dog and human,etc.were lower.These results indicated that the IFN had species specificity,the homologies of interferon among different animal species depend on their genetic relationships.

A pair of primer was designed using the nucleotide sequence of Northeast white goose IFN-α gene obtained from GenBank(Accession No.AY524422).Total RNA was extracted from cultures of goose peripheral blood lymphocytes under stimulation of GPMV,and the cDNA was amplified using reverse transcription-polymerase chain reaction(RT-PCR),which was subsequently cloned into pMD18-T vector and sequenced.The results showed that the open reading frame(ORF) of Guangxi goose interferon-α(GoIFN-α) gene consisted of 576 bp,encoding 192 amino acid residues.Among them,there were seven cysteine residues and two potential glycosyl binding site(NDT and NHT).Compared with the sequences of other goose IFN-α gene in GenBank,the homologies of nucleotide and amino acid between Guangxi goose and white Northeast goose(AY524422) was found the highest(98.3 and 96.4%,respectively).However,these two indices among Guangxi goose and chicken,cow,swine,rat,dog and human,etc.were lower.These results indicated that the IFN had species specificity,the homologies of interferon among different animal species depend on their genetic relationships.

The gene encoding protein CFP10-ESAT6 was amplified from Mycobacterium tuberculosis strain AN5 chromosomal DNA by recombinant PCR and cloned into the expression vector pET-32a to generate the recombinant plasmid pET-CFP10/ESAT6.After being identified by BamHⅠ+HindⅢ digestion and sequencing,the recombinant expression plasmid was transformed into Escherichia coli BL21(DE3).The fused protein rHIS-CFP10/ESAT6 was expressed with induction by IPTG.SDS-PAGE analysis showed that the fusion protein rHIS-CFP10/ESAT6 was 42 ku in molecular weight,and made up 40% of whole bacterial proteins.The soluble protein was purified with HIS affinity chromatography column.Western-blotting analysis indicated that the purified recombinant protein possessed good immunological activity,and could induce to generate high level of IFN-γ in whole blood,and the IFN-γ response to rHIS-CFP10/ESAT-6 was better than that to PPDa and PPDb.The results showed that the recombinant fusion protein rHIS-CFP10/ESAT6 provided a basis for the diagnosis of tuberculosis.

[Objective] The study was to clone HN and F genes from GX1 strain of goose paramyxovirus and analyze their sequences. [Method] According to the full nucleotide sequence of GPV-SF02 strain of goose paramyxovirus,two pairs of primers were designed to amplify the HN and F genes from GX1 strain of goose paramyxovirus isolated from diseased goose in Guangxi Zhuang Autonomous Region;the amplified products were ligated into pMD18-T vector and sequenced. [Result] HN and F genes of this strain tested were 1 716 and 1 662 bp in full nucleotide length,respectively;both showed the homologues of about 97.3% with GPV-SF02 strain,of 80.3%-97.5% with strains LaSota,F48E9 and JS,of just 84.8% with Miyadera strain. [Conclusion] The results show that isolated strain BX1 matches to virulent APMV-1 strain,belonging to genotype Ⅶ of APMV-1 strain.