In order to understand the prevalence of goose-origin Tembusu virus（TMUV） in Guangdong province, tissue samples of suspected TMUV infected geese from June 2020 to June 2021 were uesd for isolation and identification of TMUV. The results showed that three strains of TMUV which could cause significantly cytopathic effect in BHK-21 cells were identified. The nucleotide homology among the three isolates were from 99.2% to 99.7%, the viral genome homology among three isolates and cluster 2.1 reference strains were from 95.9% to 99.3%, which was considered to be generally higher than that with cluster 2.2 reference strains, which was from 95.0% to 96.4%. The results of phylogenetic analysis and E protein amino acid sequence analysis showed that the three goose-origin isolates were all clustered in cluster 2.1 branch. Comparing to the previously reported avian TMUV strains, multiple amino acid variations in E protein were noticed in Goose/GD/939/2021 and Goose/GD/1025/2021 of three isolates found in this study. It′s suggested that all of the three goose-origin strains of TMUV isolated in Guangdong located in cluster 2.1, which laid the foundation for the epidemic and evolution of TMUV and the prevention and control of associated diseases in Guangdong.

番鸭呼肠孤病毒(Muscovy duck reovirus,MDRV)近年来在雏鹅中的分离率增加,造成了一定的经济损失.本研究从临床上表现出跛行、肝脏和脾脏出现白色点状坏死的雏鹅中采集肝脏和脾脏,采用PCR方法进行MDRV鉴定,对阳性病料经番鸭胚和鹅胚进行病毒分离,并对分离株进行鉴定和σC蛋白基因测序,将分离株对雏鹅和雏番鸭进行致病性试验.流行病学调查结果显示:疑似感染MDRV雏鹅的病毒阳性率为18.7％,病毒分离率为33％;禽胚分离结果显示,成功分离获得1株鹅源MDRV,命名为846-Goose-CHN-2020株,接种禽胚后,禽胚死亡时间在2～10d;846-Goose-CHN-2020株经禽胚传至第5代时死胚率下降;遗传进化结果显示:846-Goose-CHN-2020 株与鸭源 MDRV 相似为 91％～97.2％,与鹅源 MDRV(GRV-GD2020)相似为99.8％;致病性结果显示,846-Goose-CHN-2020株对雏鹅和雏番鸭具有致病性,主要表现为脾脏出现白色坏死点.本研究完善了广东地区鹅源MDRV的流行病学和致病性信息,为鹅源MDRV的研究提供了一定的科学基础.

为构建鹅鼠伤寒沙门菌单基因缺失株并鉴定其生物学特性,本研究以广东地区鹅源鼠伤寒沙门菌流行株A29为研究对象,采用 λ-Red同源重组技术,分别构建crp、hfq基因缺失株(A29Δcrp和A29Δhfq)及相应基因回补株,并分别采用相应引物经PCR鉴定.通过细菌培养,对各菌株生物学特性进行比较;通过PCR鉴定各菌株的遗传稳定性;采用K-B纸片扩散法检测各菌株的药物敏感性;将各菌株纯培养物经腹腔注射4周龄小鼠,测定各菌株对小鼠的毒力,并观察感染后小鼠的临床症状及各组织剖检病变与组织病理变化.PCR和测序结果表明,正确构建各基因缺失株和相应回补株.生物学特性试验结果显示,与亲本株相比,A29Δcrp菌落直径极显著变小(P＜0.001),不产酸、不产生H2S,菌体极显著变短(P＜0.001);A29Δhfq菌落边缘不整齐,不产生H2S,菌体极显著变长(P＜0.001),表明crp、hfq基因影响细菌的菌落形态、生化特性和形态特征.菌株遗传稳定试验结果显示,菌株传至60代时,基因缺失株仍能够稳定缺失.药敏试验结果显示,与亲本株比较,A29Δcrp对氨基糖苷类、碳青霉烯类、环丙沙星、甲氧苄啶、阿奇霉素等药物的敏感性降低,对四环素类药物的敏感性增强,A29Δhfq对除青霉素类、头孢唑啉以外的12种药物的敏感性增强.小鼠毒力试验结果显示,与亲本株的LD50(2.479×104 cfu)相比,A29Δcrp 的 LD50(5.379×105 cfu)和 A29Δhfq 的 LD50(5.012×106 cfu)均明显上升.各菌株感染小鼠后的临床症状及各组织剖检病变基本一致;组织病理变化观察显示,与亲本株相比,A29Δcrp组小鼠肝组织可见多量大小不一的坏死灶,脾组织红髓面积增大、白髓面积减小,A29Δhfq对小鼠脾脏、肝脏及脑组织损伤均较小.本研究为探究鼠伤寒沙门菌crp 和hfq基因功能奠定了基础,同时也为进一步研发鹅沙门菌病疫苗提供了参考依据.

为了解广东省部分地区鹅源鸡毒支原体流行情况,试验对50例鹅慢性呼吸道病病例进行鸡毒支原体检测,结果显示:鸡毒支原体mgc2基因PCR检测阳性21份,进一步纯化培养出8个分离株,经茵体染色镜检及16 S rRNA基因系统进化树分析,确定为鸡毒支原体,且与疫苗株同源性达97.8％以上;分离株1117经卵黄囊接种6日龄鹅胚和经气管接种50日龄鸟鬃鹅,证明该分离株对鹅胚及鹅有一定的致病力.研究表明鸡毒支原体在广东部分地区鹅群中具有广泛流行性,并对鹅有致病性,该结果为广东地区鹩源鸡毒支原体流行病学监测提供参考.

为了解广东省主要养禽区域禽源巴氏杆菌的流行情况,本试验在2018～2019年期间对经临床症状与病理剖检作初步诊断为禽霍乱的临床病例,从心血和肝脏进行细菌分离并纯化出20株细菌,经培养特性与菌体形态观察、生化鉴定及种特异性PCR鉴定,确定20株分离菌株均为多杀性巴氏杆菌.采用荚膜多重PCR和脂多糖多重PCR对20株多杀性巴氏杆菌进行分型鉴定,结果荚膜型以A为主,脂多糖型以L1为主,表明目前广东家禽中的多杀性巴氏杆菌仍以A:1(NAMIOKA分型方法为A:5)为主要血清型.药敏结果显示有70％的分离菌对四环素耐受,对其他药物的耐药率均在15％以下.致病性试验结果表明,分离菌株对鹅具有强致病性,引起发病鹅的多种组织出血和坏死;经病理组织切片可见多杀性巴氏杆菌典型的病理组织学变化.试验结果为广东省部分地区禽霍乱的流行病学监测和防治提供参考依据.

以2018年分离鉴定的1株鹅星状病毒为研究对象(LHC株),采用分子克隆方法获得病毒衣壳蛋白基因序列,通过生物信息学方法分析分离株分子进化情况与纤突蛋白变异情况,构建纤突蛋白原核表达质粒对纤突蛋白进行原核表达,免疫新西兰白兔制备兔抗纤突蛋白多克隆抗体,以原核表达的衣壳蛋白验证其反应活性.结果 显示,LHC株为鹅源星状病毒2型毒株,第425～665位氨基酸残基为衣壳蛋白纤突结构,2014-2018年不同地区分离到的鹅星状病毒2型毒株的纤突蛋白享有很高的序列及结构相似性,表明2014-2018年中国不同地区间的鹅星状病毒2型毒株尚未发生明显变异.制备的兔抗纤突蛋白血清可与纤突蛋白及衣壳蛋白特异性结合,表明重组纤突蛋白具有良好的免疫原性,制备的兔抗纤突蛋白多克隆抗体具有良好的反应活性.为鹅星状病毒纤突蛋白功能研究、血清学检测方法的建立以及亚单位疫苗的制备奠定理论基础.

To understand the prevalence, coinfection with other viruses, underlying genetic evolution, recombination, and molecular biological characteristics of goose circovirus (GoCV) in Guangdong, China, from December 2019 to August 2020, 310 tissue samples of geese showing stunted growth and feather disorder syndrome were collected from this region and analyzed. GoCV, Tembusu virus, waterfowl paramyxovirus, avian influenza virus, fowl adenovirus type 4, and duck plague virus were detected with PCR or real-time PCR. Thirty-one complete GoCV viral genomes were obtained from 164 PCRverified GoCV nucleotide-positive samples and subjected to phylogenetic analysis, gene recombination analysis, and genome secondary structure prediction. The results showed that more than half of the samples were GoCV positive, and 31.1% of the GoCV- positive samples were from coinfections with at least one of the other viruses. The phylogenetic analysis showed that the GoCVs could be divided into three genome types. The genes of most main epidemic strains now circulating in Guangdong belonged to the Ia subtype, and some strains gradually formed a new Ib subtype. The secondary structure of the viral genome was similar to that of other known circoviruses. Furthermore, B cell linear epitope prediction and protein structure homology modeling of the viral capsid protein were performed based on the viral amino acid sequences. The results showed that the spatial structure of the capsid protein of the 31 sequenced strains was similar to that of duck circovirus and consisted of two b-sandwich conformations. A total of five B cell linear epitopes were predicted, and four of them were mapped on the predicted model of the capsid protein of GoCVs. This report provides a reference for the epidemiology of GoCV in Guangdong, understanding the elemental composition of the virus genes and proteins, selecting representative vaccine strains, constructing targeted immune preparations for GoCV, and strengthening prevention and control of the disease.

研究从面部肿胀的成年狮头鹅眶下窦中分离到一株细菌,经生长特性、菌体染色镜检、生化试验、16 S rDNA测序比对及HMTp210基因扩增方法对其进行鉴定,确定分离菌株为血清型B型的副鸡禽杆菌,具有NAD依赖性;副鸡禽杆菌hagA基因系统进化树显示分离株与国内大部分副鸡禽杆菌分离株的亲缘性相近,与国外副鸡禽杆菌分离株的差异较大;药敏试验结果显示喹诺酮类药物和氟苯尼考的MIC较低,分别为0.25 μg/mL和4μg/mL,氨苄西林等6种药物的MIC值均大于128 μg/mL,表明菌株具有较强的耐药性;分离纯培养物人工感染7日胚龄鸡胚后24～48h内致死鸡胚,经眶下窦感染80日龄乌棕鹅,48h鹅开始出现流鼻液、鼻窦部肿胀,72 h剖检可见眶下窦有干酪样渗出物,从渗出物中又重新分离出副鸡禽杆菌;表明分离到的副鸡禽杆菌对鹅具有较强的致病力.

2019～2020年从广东肇庆地区的部份鹅场中收集到12份疑似小鹅瘟病例,对疑似小鹅瘟病例进行病原核酸鉴定,对阳性病例进行病毒分离和VP1基因的序列扩增和遗传进化分析.结果表明,12份疑似小鹅瘟病例中,共检测到7份阳性样品,阳性检测率为58.3％.从阳性病例样本中分离获得7株小鹅瘟病毒毒株,其中1株与Ⅰa亚群毒株核苷酸序列相似性高达99.1％～99.3％,另6株则聚类形成此前未报道过的新亚群,本文将其命名为Ⅰf亚群.本研究结果进一步完善了我国广东地区小鹅瘟的流行病学信息,为本地区小鹅瘟病毒的流行病学调查及该病的综合防控奠定理论基础.

为了解广东地区禽源致病性大肠杆菌（APEC）对5种β-内酰胺类抗生素的耐药情况和超广谱β-内酰胺酶（extended-spectrumβ-lactamases, ESBLs）基因（blaCTX-M、blaTEM、blaOXA）的携带情况,以更好地指导临床治疗,试验对2014—2019年从广东多地病死禽中分离鉴定得到的102株APEC采用PCR方法作系统进化分群与ESBLs基因检测,采用K-B纸片扩散法检测APEC对5种β-内酰胺类抗生素的敏感性,并分析不同禽源APEC对不同药物的耐药情况、102株APEC对5种抗生素的多重耐药性分布情况和耐药谱型,以及菌株耐药基因与耐药表型相关性。结果表明:A、B1、B2和D群比例分别为65.69%、9.80%、2.94%、21.57%。blaCTX-M、blaTEM、blaOXA基因的检出率分别是43.14%、35.29%、8.82%,组合blaCTX-M+blaTEM的阳性率为13.73%。APEC对2种青霉素类抗生素（阿莫西林、氨苄西林）和头孢拉定的耐药率均在85.71%以上,对头孢唑啉和头孢噻呋的耐药率分别为62.75%、47.06%。blaCTX-M基因与头孢唑啉和头孢噻呋的耐药率呈极显著相关（P＜0.01）,验证了blaCTX-M基因主要介导对头孢类抗生素的水解作用。说明广东地区APEC对5种β-内酰胺类抗生素的耐药情况较严重,耐药基因blaCTX-M、blaTEM的检出率要高于blaOXA基因。

为了解鸭疫里默氏杆菌在广东佛山地区养殖水禽群中的流行、耐药和对雏鸭的致病力等情况,本研究从该地区6个鹅场、9个鸭场共采集130份有疑似传染性浆膜炎临床症状的肝、脑样品,经分离、纯化和鉴定获得15株鸭疫里默氏杆菌.对新分离株的16S rRNA进行测序及遗传进化分析,并进一步检测新分离株的耐药性和对雏鸭的致病性.结果表明:所有分离株16S rRNA遗传进化上均与鸭疫里默氏杆菌血清Ⅰ型参考株TRa1处于同一进化分支;15株分离菌对阿莫西林、青霉素、链霉素等8种常用抗生素表现多重耐药,而对头孢唑啉、大观霉素和氟苯尼考敏感.选取一株鸭源菌株(Y15)进行雏鸭致病性试验,感染组雏鸭死亡率为60％,病死鸭表现典型鸭疫里默氏杆菌病临床症状.本研究结果为进一步了解鸭疫里默氏杆菌在广东地区水禽群中的流行、耐药性以及致病力情况提供参考依据.

为了解广东地区圆环病毒在鹅群中的流行情况.研究针对鹅圆环病毒全基因组保守序列设计特异性检测引物,采用PCR方法对送检的232份鹅疑似鹅圆环病毒感染病例进行检测.并针对其中一份阳性样品病毒全基因组进行测序,获得该毒株全基因组序列信息.对该毒株全基因进行遗传进化分析、Cap蛋白相似性分析和B细胞抗原表位预测.结果 显示,232份样品中阳性检出率为44.4％,表明鹅圆环病毒在广东地区鹅群中普遍存在,鹅圆环病毒毒株与其他已报道的鹅源毒株处于同一遗传进化分支,鹅源毒株间Cap蛋白相似性为98.0％～100％,但与鸭圆环病毒相似性低(46.9％～48.2％);Cap蛋白B细胞线性表位预测结果提示,鹅源和鸭源毒株表位位置相近,但氨基酸序列差异较大,提示两种毒株之间交叉保护力可能存在差异.研究为了解广东地区鹅源圆环病毒流行、进化情况,并进一步做好相关防控工作提供理论参考.

为了解当前广东地区鹅群新城疫病毒(NDV)遗传进化情况,本研究从广东养殖病死鹅组织样品中分离鉴定获得8株NDV,对分离株的F蛋白基因进行核苷酸及氨基酸序列分析.8株分离病毒之间F蛋白基因同源性较高,为96.3％～99.9％,但与当前国内NDV优势流行株及疫苗株同源性均较低.遗传进化分析结果显示所有新分离株均属于Ⅻ型NDVⅫb亚型分支,而当前国内NDV的优势流行株及常用疫苗株均属于Ⅶd亚型分支.氨基酸序列分析结果显示,本研究分离株F蛋白裂解位点氨基酸序列均为112K/RRQKR/F117,符合强毒株分子特征;与基因Ⅶ型代表株NDV/ZJ1相比,新分离株F蛋白B细胞中和抗原表位均出现D170N变异;而与2010年～2011年报道的广东Ⅻb亚型分离株相比,新分离株F蛋白疏水区、七肽重复序列等区域均出现氨基酸的变异.综上所述,本研究分离鉴定的8株鹅源NDV的F基因均属于Ⅻb亚型,与Ⅶd亚型流行株及常用疫苗株F基因同源性较低,且其多个氨基酸关键位点存在变异.本研究在国内首次从病死鹅体内分离到基因Ⅻ型NDV,为进一步了解基因Ⅻ型NDV在广东地区水禽中的流行、病原进化以及疫苗株更新提供参考.

有效防治猪流感需要借助于准确而快速的检测方法,为了建立一种高效、快速的多重检测方法,本研究应用H1和H3亚型猪流感病毒(SIV)的血凝素(HA)蛋白抗原偶联不同编码的荧光微球,应用间接法检测模式,建立了H1、H3亚型猪流感病毒IgG抗体荧光微球免疫学检测方法,并将该方法与血凝抑制试验(HI)、病毒中和试验(VN)和酶联免疫吸附试验(ELISA)进行比较分析.结果 表明,该方法的抗体检测灵敏度比ELISA方法高100～1 000倍;与猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪瘟病毒、伪狂犬病病毒及日本乙型脑炎病毒的阳性血清均无交叉反应,且两种亚型血清之间也不存在交叉反应.重复性试验表明,批内和批间精密度测试结果分别为10.2％和12.8％.与HI、VN和ELISA方法比较,总符合率分别为97.82％、98.27％和97.83％.综上,本研究建立的检测方法具有双重检测特性,特异性好、灵敏性高、重复性强,可用于H1、H3亚型SIV的鉴别诊断.

Four new H9N2 avian influenza viruses (AIVs) were isolated from domestic birds in Guangdong between December 2015 and April 2016. Nucleotide sequence comparisons indicated that most of the internal genes of these four strains were highly similar to those of human H7N9 viruses. Amino acid substitutions and deletions found in the HA and NA proteins indicated that all four of these new isolates may have an enhanced ability to infect humans and other mammals. A cross-hemagglutinin-inhibition assay, conducted with two vaccine strains that are broadly used in China, suggested that antisera against vaccine candidates could not provide complete inhibition of the new isolates.

We report the full-length sequence of two chicken source influenza A (H7N9) viruses found in Guangdong live poultry market (LPM) during the most recent wave of human infections (from October 2016 to the present time). These viruses carry insertion of poly-basic amino acids (KGKRTAR/G) at the protease cleavage site of the HA protein, which were previously found in the highly pathogenic (HP) human influenza A (H7N9) [IAV(H7N9)] strains. Phylogenetic analysis of these two novel avian influenza viruses (AIVs) suggested that their genomes reassorted between the Yangtze River Delta (YRD) and Pearl River Delta (PRD) clades. Molecular clock analysis indicated that they emerged several months before the HP human strains. Collectively, our results suggest that IAV(H7N9) viruses evolve in chickens through antigenic drift to include a signature HP sequence in the HA gene, which highlights challenges in risk assessment and public health management of IAV(H7N9) infections at the human-animal interface.

构建了ORF2蛋白原核表达体系Rosetta-pMAL-c5X-ORF2,经IPTG诱导表达后,对重组蛋白进行SDS-PAGE和Western blot分析,并采用镍柱亲和层析法进行纯化,以该蛋白为抗原建立液相蛋白芯片检测方法.结果显示:本试验成功表达了可溶性ORF2蛋白,具有良好的抗原性.液相蛋白芯片检测方法对猪常见的其他疾病阳性血清无交叉反应,其批内、批间变异系数分别为5％和6.6％.对102份临床血清样本检测结果显示,该方法与商品化ELISA试剂盒符合率为93.1％,关联性卡方检验显示2个方法具有一致性(P＜0.01).本试验为临床HEV血清抗体的检测提供了一种特异、灵敏的新型快速检测技术,为建立猪病多重检测方法提供了基础.

Guangdong Province is recognized for dense populations of humans, pigs, poultry and pets. In order to evaluate the threat of viral infection faced by those working with animals, a cross-sectional, sero-epidemiological study was conducted in Guangdong between December 2013 and January 2014. Individuals working with swine, at poultry farms, or live poultry markets (LPM), and veterinarians, and controls not exposed to animals were enrolled in this study and 11 (4 human, 3 swine, 3 avian, and 1 canine) influenza A viruses were used in hemagglutination inhibition (HI) assays (7 strains) and the cross-reactivity test (9 strains) in which 5 strains were used in both tests. Univariate analysis was performed to identify which variables were significantly associated with seropositivity. Odds ratios (OR) revealed that swine workers had a significantly higher risk of elevated antibodies against A/swine/Guangdong/L6/2009(H1N1), a classical swine virus, and A/swine/Guangdong/SS1/2012(H1N1), a Eurasian avian-like swine virus than non-exposed controls. Poultry farm workers were at a higher risk of infection with avian influenza H7N9 and H9N2. LPM workers were at a higher risk of infection with 3 subtypes of avian influenza, H5N1, H7N9, and H9N2. Interestingly, the OR also indicated that LPM workers were at risk of H1N1 swine influenza virus infection, perhaps due to the presence of pigs in the LPM. While partial confounding by cross-reactive antibodies against human viruses or vaccines cannot be ruled out, our data suggests that animal exposed people as are more likely to have antibodies against animal influenza viruses.

In parts of southern China, some large‐scale swine farms are adjacent to lakes and ponds that are home to many types of birds. Some swine farms will also raise poultry for consumption and sale. Swine farms in rural China may be the source of the AIV outbreak. A seroepidemiological study was conducted among swine farm residents to understand the prevalence of antibodies against avian influenza virus (AIV) H9N2 in southern China. A total of 2,006 swine farm residents were sampled. Serum samples were tested for the presence of antibodies against H9N2 AIV by hemagglutination inhibition (HI) and microneutralization assays. A total of 37 serum samples from swine farm residents were HI positive for A/chicken/Guangdong/V/2008(H9N2), and 24 serum samples (all of which were also HI positive) were microneutralization assays positive for A/chicken/Guangdong/V/2008(H9N2). Due to the special pig farming model in southern China, the residents are in close contact with different kinds of birds. Thus, controlling bird‐to‐human transmission of AIV in swine farms with poultry may be an important means of preventing widespread AIV infection in humans. J. Med. Virol. 86:597–600, 2014 . © 2014 Wiley Periodicals, Inc.