广东地区鸭疫里默氏杆菌的分离鉴定及双重PCR检测方法的建立

对来自广东地区疑似鸭疫里默氏杆菌病的病鸭组织进行了病原菌的分离培养和生化鉴定,确定得到20株鸭疫里默氏杆菌,动物回归试验结果表明,分离的鸭疫里默氏杆菌具有很强的致病性.对这20株鸭疫里默氏杆菌进行的药敏试验表明,分离株已广泛产生耐药性.参照已经发表的2对引物以细菌全菌体为模板建立双重PCR方法,结果均能扩增出2条目的片段,经测序证实为鸭疫里默氏杆菌,而对鸭源大肠杆菌、鸭源禽多杀性巴氏杆菌、鸭源沙门菌和鸭源葡萄球菌的扩增结果均为阴性.说明建立的双重PCR方法能快速、准确的检测出鸭疫里默氏杆菌,并具有高度特异性,可用于鸭疫里默氏杆菌的快速鉴定和快速诊断.     

鸭疫里默氏杆菌明胶酶对雏鸭免疫功能影响的研究

为研究鸭疫里默氏杆菌明胶酶对雏鸭免疫功能的影响,本研究将2000 u明胶酶经颈静脉注射10日龄健康鸭建立动物模型,测定了模型动物的部分免疫指标。结果显示,注射明胶酶的雏鸭外周血T淋巴细胞非特异性酯酶阳性率、白细胞杀菌指数、红细胞C3bR花环率、血清溶血和杀菌活性、脾淋巴细胞的增殖和分泌IgG能力等显著低于阴性和空白对照组(p0.05或p0.01);明胶酶可导致雏鸭脾脏严重损害,脾中央动脉内皮细胞肿胀、脱落,淋巴细胞变性、坏死及细胞核膜不规则与染色质周边浓集。结果表明,鸭疫里默氏杆菌明胶酶对雏鸭的免疫功能具有损害作用。     

鸭疫里默氏杆菌的鉴定及药敏试验

从四川某鸭场送检的病料中分离到1株疑似鸭疫里默氏杆菌(Riemeralla anatipestifer,RA)。经细菌形态学、培养特性、生化试验、PCR扩增与动物回归试验鉴定后,证实为鸭疫里默氏杆菌。该菌尿素酶试验阳性,不能发酵多种糖类,不能液化明胶,不产生H2S,不还原硝酸盐,吲哚试验、MR试验、VP试验均为阴性;菌株对左氟沙星、氧哌嗪青霉素、庆大霉素等极为敏感,而对青霉素、头孢拉定、链霉素等耐药。     

鸭疫里默氏杆菌的分离鉴定与生物学特性研究

为了解鸭疫里默氏杆菌的流行情况和药物敏感性,本研究对广东省、山东省、江苏省等地区疑似鸭疫里默氏杆菌病的病料进行病原分离和鉴定,并测定分离株对抗菌药物的敏感性。结果显示,共鉴定到46株鸭疫里默氏杆菌,其中血清1型、2型、10型和15型菌株依次为9株、25株、1株和1株,未定血清型菌株10株;RA分离株普遍对林可霉素、多粘菌素和诺氟沙星耐药,表明RA分离株对临床常用药出现了不同程度的耐受;易感鸭的动物回归试验能够复制出典型的原始病例,且可从死亡雏鸭中分离到同源菌株。该研究结果为鸭疫里默氏杆菌病疫苗选用和临床用药提供了科学的参考依据。     

鸭疫里默氏杆菌的分离鉴定

从广州市郊区几个鸭场发病或死亡鸭的脑、心血、肝、脾等组织中分离到6株革兰氏阴性杆菌，经形态学检查、生化试验、动物实验等鉴定均为鸭疫里默氏杆菌。通过凝集试验和琼脂扩散试验鉴定：2株为鸭疫里默氏杆菌Ⅰ型，4株为鸭疫里默氏杆菌Ⅱ型。     

广东地区鸭疫里默氏杆菌RAPD分型研究

为了解广东地区鸭疫里默氏杆菌的流行情况,研究采用随机引物初步建立了鸭疫里默氏杆菌RAPD扩增多态性方法,对分离自广东地区的20株鸭疫里默氏杆菌进行鉴定。结果显示:20株鸭疫里默氏杆菌产生16种不同的指纹图谱;有些鸭场存在两种或两种以上基因型的菌株并且不同鸭场流行基因型不一样。该结果可为该地区鸭疫里默氏杆菌病的防治提供参考。鸭疫里默氏杆菌RAPD指纹图谱存在丰富的多样性,可用于鸭疫里默氏杆菌的快速基因分型及分子流行病学调查。     

鹅源鸭疫里默氏杆菌的分离和鉴定

为研究鹅源鸭疫里默氏杆菌的生化特性、药敏情况、血清型和致病性,从扬州郊区发病鹅场分离到1株细菌,经分离培养、染色镜检、生化特性鉴定、玻片凝集试验,确定为Ⅱ型鸭疫里默氏杆菌。对病料进行病毒分离试验,经过血凝试验和琼脂扩散试验,没有发现鹅新城疫病毒、禽流感病毒和小鹅瘟病毒。动物致病性试验结果表明,鸭疫里默氏杆菌分离菌株可以通过静脉注射、皮下注射和滴鼻3种途径感染鹅,出现100%的死亡率,说明分离株对扬州鹅具有很强的致病性,同时提示在鹅的免疫计划中也应该充分考虑到鸭疫里默氏杆菌的致病和传播。     

鸭疫里默氏杆菌GroEL蛋白的原核表达和单克隆抗体制备

本试验旨在研制鸭疫里默氏杆菌GroEL蛋白的单克隆抗体。将鸭疫里默氏杆菌WJ4菌株的groEL基因进行原核表达,重组蛋白经过纯化后作为免疫原免疫BALB/c小鼠。经过3次免疫后,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,用间接ELISA筛选阳性细胞株并进行3次亚克隆后制备小鼠腹水单克隆抗体。共获得2株稳定分泌鸭疫里默氏杆菌GroEL单克隆抗体的杂交瘤细胞株,分别命名为1G2B6和1G2F10。2株单克隆抗体均为IgG1亚类。腹水单克隆抗体ELISA效价达到1:102 400。Western blot检测结果表明2株单克隆抗体均能与鸭疫里默氏杆菌血清1、2和10型菌株发生特异性结合,而与禽致病性大肠杆菌、沙门氏菌和禽巴氏杆菌菌株无反应性。本研究成功制备了稳定性好、特异性强的鸭疫里默氏杆菌GroEL单克隆抗体,为进一步研制基于抗体检测鸭疫里默氏杆菌抗原的试剂盒奠定了基础。     

鸭疫里默氏杆菌PCR快速检测方法的建立

在鸭疫里默氏杆菌外膜蛋白保守区设计了一对特异性引物,建立PCR方法,对5株已知血清型的鸭疫里默氏杆菌纯培养菌和6株临床分离未定型的里默氏杆菌菌株以及病死鸭病料组织进行检测,结果表明不同样品都可扩增出592bp的特异目的条带,而对鸭源大肠杆菌、鸭源多杀性巴氏杆菌、鸭源沙门氏菌的扩增结果均为阴性,说明该PCR法具有较好的特异性,可用于快速鉴定鸭疫里默氏杆菌,也适用于病料的直接检测.     

鸭疫里默氏杆菌的分离鉴定及药敏试验

对武汉周边地区发病鸭场的病死鸭进行了病理剖检,直接从病死鸭肝脏、心血及脑组织中进行细菌分离培养,通过培养特性、生化鉴定、动物回归试验确定为鸭疫里默氏杆菌感染.分离到8株鸭疫里默氏杆菌,对分离株进行了药敏试验,结果表明分离株对头孢唑啉钠、先锋V、丁胺卡那霉素、新霉素、氨苄青霉素的敏感性较高.     

1型鸭疫里氏杆菌OmpA蛋白间接ELISA方法的建立

为建立检测1型鸭疫里氏杆菌(R.anatipestifer)的间接ELISA方法,本研究根据已发表的R.anatipestifer外膜蛋白A(ompA)基因序列(AF104937)设计引物,扩增1型R.anatipestifer HLG1株的ompA基因,构建重组质粒pHtb-ompA,转化大肠杆菌BL21(DE3),并利用IPTG进行诱导表达。SDS-PAGE和western blot结果表明,表达蛋白约为55 ku,具有良好的抗原活性。以纯化的OmpA为包被抗原建立间接ELISA并对条件进行优化。建立的ELISA具有良好的特异性、敏感性;与HLG1株菌体裂解蛋白为抗原的间接ELISA比较,符合率为91.3%。本研究建立的ELISA方法为R.anatipestifer的流行病学调查和SPF鸭的监测提供了快速、特异的血清学诊断方法。     

Vaccination of ducks with recombinant outer membrane protein (OmpA) and a 41 kDa partial protein (P45N') of Riemerella anatipestifer.

The generation of protective immunity against Riemerella anatipestifer infection in ducks were investigated by immunizations with recombinant glutathione sulfatransferase (GST) fusion's proteins of OmpA, a 42kDa major outer membrane protein, and P45N', a 41kDa N-terminal fragment of a newly identified 45kDa potential surface protein from R. anatipestifer. The DNA encoding OmpA and P45N' were isolated from R. anatipestifer serotype 15 (field strain 110/89) and serotype 19 (reference strain 30/90), respectively. Immunoblotting and ELISA results showed that the purified recombinant proteins induced the production of antibodies in immunized ducks. However, neither was protective against subsequent challenge with the virulent serotype 15 strain, 34/90. All the five ducks immunized with formalinized R. anatipestifer strain 34/90 survived the challenge with the homologous strain whereas six out of seven ducks in the non-immunized control group died within a week following the challenge.     

1型鸭疫里默氏杆菌的分离鉴定及基因组文库的构建

从临床病鸭中分离并鉴定了1型鸭疫里默氏杆菌,并在提取鸭疫里默氏杆菌的基因组DNA后,用Sau3A I酶切,回收大小为0．07～4 kb的片段;将酶切片段与酶切的质粒载体pRSET连接后,电转化大肠杆菌Rosetta,成功构建了1型鸭疫里默氏杆菌的基因组文库,经检测库容量约为40000个。随机筛选30个单菌落,用pRSET通用引物进行PCR鉴定,统计结果显示插入片段的大小93．3％在1～3 kb范围内,成功构建了鸭疫里默氏杆菌基因组文库,为鸭疫里默氏杆菌功能基因的克隆及鉴定奠定了基础。     

OmpA is a virulence factor of Riemerella anatipestifer

Riemerella anatipestifer infection is probably the most economically important disease of farm ducks worldwide. The pathogen R. anatipestifer causes septicemia anserum exsudativa in ducks, but little is known about the molecular basis of its pathogenesis and the virulence factors involved. In this study, by deleting ompA gene from R. anatipestifer serotype 2 strain Th4, we constructed a mutant strain Th4ΔompA to investigate whether R. anatipestifer OmpA is an important virulence factor. Results showed that although the growth curve, bacterial and colony morphology of Th4ΔompA in tryptic soybean broth (TSB) or on TSB agar were similar to its parent strain Th4, the adhesion and invasion capacities of mutant strain to Vero cells were decreased significantly. Furthermore, the median lethal dose (LD(50)) of both strains was determined to measure the virulence with 10-day-old Cherry Valley ducklings. The results showed that LD(50) of Th4ΔompA mutant was >10(10) colony forming units (CFU), it was attenuated significantly in comparison with that of Th4 which LD(50) was 4.41 × 10(8) CFU. Additional analysis indicated that blood bacterial loading of ducklings infected with the Th4ΔompA mutant were much lower than those of Th4-infected ducklings. The results demonstrate that OmpA is a virulence factor of R. anatipestifer, and that it may act as an adhesin.     

1株无致病力的鸭疫里氏杆菌的分离及鉴定

从安徽和县无病鸭场的健康鸭咽部分离到1株革兰氏阴性短杆菌,定名为LY-58株。经分离培养、形态学检查、生理生化试验、血清学鉴定及PCR鉴定,分离菌株被鉴定为2型鸭疫里氏杆菌(Riemerella anatipestifer,RA)。毒力实验显示,2型RA参照菌株RAF2对10日龄北京鸭的LD50为5.57×107CFU,而分离菌株LY-58的LD50大于1.0×1010CFU,可确定为无致病性。     

Development of an ELISA using a recombinant 41 kDa partial protein (P45N') for the detection of Riemerella anatipestifer infections in ducks

Riemerella anatipestifer, a gram-negative bacillus, is the causative agent of duck septicemia, a disease which could incur much economic loss in the duck industry. An indirect enzyme-linked immunosorbent assay (ELISA) has been developed to facilitate early detection of R. anatipestifer infection in ducks. The antigen used was a recombinant 41 kDa N-terminal fragment (rP45N') of a newly characterized R. anatipestifer potential surface protein, P45, which was expressed in Escherichia coli as an N-terminal GST fusion protein. The rP45N'-based ELISA successfully detected P45 antibodies in the sera of 20 ducks immunized with bacterin preparations of R. anatipestifer serotypes 1, 10 15, 19 and the ATCC11845 strain. Antibodies to P45 were also detected in the sera of 25% (75/296) of White Pekin ducks which were imported into Singapore from three different farms. Successful discrimination was obtained between sera from infected ducks and that of specific-pathogen free ducks (p<0.01). The rP45N'-GST antigen did not cross-react with antibodies in sera from guinea pigs which were infected with other gram-negative and gram-positive bacterial pathogens, including Aeromonas hydrophila, Citrobacter freundii, E. coli, Klebsiella pneumoniae, Pastuerella multocida, Proteus mirabilis, Salmonella spp., Serratia maccescens, Shigella sonnei and Yersinia enterocolitica. In addition, the DNA sequence encoding P45 was detected in R. anatipestifer serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 14, 15, 16, 17, 18, 19 and the ATCC11845 strain, suggesting that P45 is probably also universally expressed in these R. anatipestifer serotypes. Thus, the ELISA described is applicable to the detection of R. anatipestifer infection in ducks.     

Molecular fingerprinting of Riemerella anatipestifer by repetitive sequence PCR.

Riemerella anatipestifer is a gram-negative rod-shaped bacterium associated with epizootic infections in poultry. A total of 35 R. anatipestifer isolates including the type strain ATCC11845T, reference and field strains for 18 different serotypes were characterized by repetitive sequence based-PCR (rep-PCR) with outwardly-directed primers based on the repetitive extragenic palindromic (REP) consensus sequence. This technique was applied by using either extracted genomic DNA or preparation of whole bacterial cells harvested directly from plate cultures. Rep-PCR discriminated the R. anatipestifer isolates into 19 electrophoretic types. DNA fingerprints obtained from rep-PCR of extracted genomic DNA or from preparations of whole cells yielded comparable patterns. Substantial variation was seen among the rep-PCR fingerprints of different serotypes. Moreover, different polymorphisms of the rep-PCR fingerprints were evident among epidemiologically unrelated isolates of the same serotype. These results suggest the presence of repetitive extragenic palindromic-like elements within the genome of R. anatipestifer that can be used in some isolates to discriminate between different strains belonging to the same serotype. Rep-PCR may serve as a useful molecular tool for subtyping R. anatipestifer isolates for epidemiologic investigations. The whole cell procedure offers the advantage of ease of performance requiring only small quantities of cells.     

1型鸭疫里氏杆菌抗体间接ELISA检测方法的建立

以1型鸭疫里氏杆菌(RA)全基因组保守区域ompA基因的重组表达产物为包被抗原,建立了检测RA血清抗体的间接ELISA方法。原核表达的重组ompA蛋白经纯化后作为包被物,以方阵滴定法确定抗原最佳包被浓度为1.5μg/mL,待检血清最佳稀释度为1∶100。与普通的微量凝集试验相比较,该方法灵敏度高于凝集试验约16倍～128倍。与鸭源大肠埃希菌、鸭源多杀性巴氏杆菌、鸭链球菌、鸭源呼肠病毒、鸭肝炎病毒和鸭瘟病毒感染鸭血清均无交叉反应,表明该方法特异性好。利用该方法检测了雏鸭免疫RA灭活油乳剂疫苗之后血清抗体水平。     

鸭疫里默氏菌铁依赖抑制子基因的克隆及序列分析

本研究根据鸭疫里默氏菌铁依赖抑制子(RaDtxR)基因序列特征设计特异性引物,利用PCR方法从已鉴定为2型鸭疫里默氏菌RA-FJ株的基因组DNA中扩增目的基因片段,胶回收PCR扩增片段,克隆到pMD18-T载体后进行序列测定.采用生物信息学软件对测序结果进行分析,结果表明所获得的RaDtxR基因编码完整的开放阅读框,全基因长度为654 bp,编码217个氨基酸.所编码的蛋白大小为24.82 ku,理论等电点为5.69,不稳定系数为38.45,属于稳定蛋白质类.将其与GenBank中已发表的RaDtxR基因序列进行核苷酸同源性比对分析,结果显示其与鸭疫里默氏菌疫苗株RA-CH-1株(GenBank登录号:CP003787)的核苷酸同源性为100.0%,与其他4株核苷酸同源性均为99.8%,仅在186位处存在无义突变.利用该基因对福建省农业科学院畜牧兽医研究所分离鉴定的鸭大肠杆菌、禽多杀性巴氏杆菌和沙门氏菌进行扩增,结果显示均未扩增到条带,对福建省农业科学院畜牧兽医研究所分离鉴定的1型、2型、3型、11型和13型鸭疫里默氏菌均可扩增出特异性目的条带,表明RaDtxR基因可作为鸭疫里默氏菌鉴别诊断的靶基因.试验结果表明,本研究成功克隆到鸭疫里默氏菌RaDtxR基因,为研究其功能奠定基础.     

鸭疫里默氏杆菌10个基因在菌株中的分布及序列分析

检测鸭疫里默氏杆菌（Riemerellaanatipestifer,RA）10个基因在不同菌株间的分布并进行序列分析,研究其在不同菌株中的保守性。分别选取8株RA的10个基因进行PCR扩增,扩增产物克隆于pMD18-T载体,进行序列测定和分析。结果显示：测定的8株RA菌株中均能检测到Lipid A、LuxE和TCTR编码基因,表明这些基因在不同血清型RA菌株中的保守性良好;TR基因仅从6株具有致病性的RA菌株中扩增得到,而2株非致病性RA菌株NJ1和NJ4未能扩增到,表明TR基因可能与RA菌株的致病性相关;IS1仅从6株血清1型和2型的RA菌株扩增得到,2株血清10型菌株均未扩增得到,提示IS1为血清10型菌株缺失基因。