江苏地区猪圆环病毒2型流行病学调查

应用复合PCR方法,对江苏地区的162份可疑病料进行了检测,结果72份病料为PCV2阳性,17份为PCV1阳性,其中7份同时感染PCV1和PCV2。从发病时间看,以9~11月发病最多,阳性率高达54.67%。样品较多的保育猪和育肥猪的阳性率分别是26.03%和68.12%。通过流行病学调查,表明该病已在江苏地区呈流行趋势。同时对18头PCV2阳性猪病料的心、肝、脾、肺、肾、脑、腹股沟淋巴结、肠系膜淋巴结、扁桃体进行PCV2检测,结果有7头PCV2阳性猪的各个脏器都能检测出PCV2;在这些脏器中,肺、腹股沟淋巴结、扁桃体的检出率都为100%,较其他受检脏器更适合作为PCV2的检测样品。     

福州市猪圆环病毒Ⅱ型(PCV2)流行病学调查

对福州地区12个规模化养猪场的发病猪群进行了系统的流行病学调查，共采集64份组织病料。采用PCR方法进行圆环病毒Ⅱ型(PCV2)检测。现场调查发现共有7个场的猪群有圆环病毒Ⅱ型感染所致的特征性临床症状及病理变化；PCR检测31份病料呈阳性，阳性率46．9％；12个养殖场中7个为阳性．阳性率58．3％。根据临床及实验室检测结果证实福州市养猪场存在PCV2感染。     

RT-PCR技术诊断猪瘟的应用研究

应用反转录—聚合酶链反应(RT-PCR)对猪瘟进行诊断应用研究。应用RT-PCR况对来自广西不同地区的135份疑似猪瘟病料进行检测，以份诊断为阳性，阳性率62．2％。从百色、柳州地区等采集的健康猪扁桃体和淋巴结共276份，经RT-PCR检测，37份为阳性，阳性率为13．4％。其中健康猪扁桃体带毒较高，246份扁桃体中有35份阳性，占14．2％。采自柳州健康猪的26份淋巴结材料全为阴性，只有邕宁县的1份健猪淋巴结阳性。结果表明，RT-PCR技术可应用于猪瘟的临床诊断。     

RT-PCR技术诊断猪瘟的应用研究

应用反转录—聚合酶链反应(RT-PCR)对猪瘟进行诊断应用研究。应用RT—PCR对来自广西不同地区的135份疑似猪瘟病料进行检测，84份诊断为阳性，阳性率62．296。从百色、柳州地区等采集的健康猪扁桃体和淋巴结共276份，经RT—PCR检测，37份为阳性，阳性率为13．4％。其中健康猪扁桃体带毒较高，246份扁桃体中有35份阳性，占14．2％。采自柳州健康猪的26份淋巴结材料全为阴性，只有邕宁县的1份健康猪淋巴结阳性。结果表明，RT-PCR技术可应用于猪瘟的临床诊断。     

RT-PCR检测猪瘟病毒方法的建立与应用

建立RT PCR检测猪瘟病毒的方法。根据已发表的猪瘟病毒E2基因 (囊膜糖蛋白gP55基因 )序列 ,设计合成了一对特异性引物 ,扩增片段的大小为 50 7bp ,用RT PCR技术对石门系标准株和 1 0株分离株进行检测。结果这对引物对标准株和 1 0株分离株均能扩增出与预期大小相符 50 7bpRT PCR产物 ,而对其他 6种猪病病原核酸的扩增结果为阴性。该RT PCR可检出 1 0 0pg的猪瘟病毒RNA模板 ,对人工感染猪不同组织样品进行检测 ,结果对白细胞抽提的核酸样品检出率最高为 1 0 0 % (2 4 / 2 4 ) ,其次为扁桃体、脾、肾 ,检出率为 83 3 % (2 0 / 2 4 ) ,再者为淋巴结 ,检出率为66 7% (1 6/ 2 4 )。对送检的 1 9份疑似猪瘟的病死猪病料组织进行RT PCR检测 ,结果有 1 6份样品为猪瘟病毒阳性。兔体交叉反应试验结果RT PCR阳性的 1 6份病料中 ,有 1 4份样品被判为含有猪瘟病毒 ,其他病料兔体交叉反应试验结果全为阴性     

斑点杂交法检测猪传染性胸膜肺炎

将位于猪传染性胸膜肺炎放线杆菌(APP) apx A毒素基因 3′端的长为 442 bp的DNA片段作为模板制备出地高辛标记的 APP核酸探针。敏感性检测结果表明 ,该探针对 APPDNA的最低检出量为 1 .8ng。特异性检测结果表明 ,该探针能与 APP1～ 1 0血清型标准菌株 DNA抽提物发生特异性杂交反应 ,而与多杀性巴氏杆菌A型和 B型、链球菌、支气管败血波氏杆菌、肺炎双球菌、金黄色葡萄球菌、大肠杆菌 DNA进行的杂交反应均为阴性。应用该探针对临床 6份阳性病料 ,1 1份纤维渗出性病料进行检测 ,结果阳性病料全部检出 ;而对于 1 1份纤维渗出性病料 ,斑点杂交检出 4份是阳性 ,比 PCR方法 6/ 1 1的检出率要低 ,但仍表明所制备的探针用于 APP的检测是一种比较敏感、特异的诊断方法。     

RT-PCR技术检测猪瘟病毒的应用研究

本试验应用反转录-聚合酶链式反应(RT-PCR)对猪瘟进行诊断应用研究.应用RT-PCR对来自广西不同地区的135份疑似猪瘟病料进行检测,84份诊断为阳性,阳性率62.2%.从百色、柳州地区等采集的健康猪扁桃体和淋巴结共276份,经RT-PCR检测,37份为阳性,阳性率为13.4%.其中健康猪扁桃体带毒较高,246份扁桃体中有35份阳性,占14.2%.采自柳州健康猪的26份淋巴结材料全为阴性,只有邕宁县的1份健猪淋巴结阳性.结果表明,RT-PCR技术可应用于猪瘟的临床诊断.     

华东地区猪圆环病毒2型基因型鉴别及ORF2遗传变异分析

利用PCR方法对2014-2015年采集于华东地区部分猪场的病料进行猪圆环病毒2型(PCV2)检测,将所得阳性样品进行2a/2b鉴别分型。同时,选取15份阳性样品扩增ORF2完整序列,并利用DNAStar软件进行同源性分析和系统进化树分析。结果表明,335份病料中PCV2阳性样品146份,其中PCV2a型11份(7.53%)、PCV2b型127份(86.99%),共感染率5.48%,说明华东地区感染的PCV2以2b基因型为主,少数为2a基因型。同源性分析表明,试验所得15株ORF2序列同源性为89.9%~99.9%,与国内外参考毒株同源性为86.2%~99.7%,均存在较高的同源性。系统进化树表明,15株序列中有9株属于PCV2b型,4株属于PCV2d型,2株属于PCV2a型,未出现2c和2e基因型。本试验为进一步研究PCV2遗传变异趋况及新型疫苗的研制积累了有效材料。     

用地高辛标记的核酸探针检测猪伪狂犬病毒野毒感染的研究

利用PCR技术从带有伪狂犬病毒（PRV）gE基因的重组质粒pMD18-T-gE中扩增回收约304bp大小的片段,并制备出地高辛标记的gE基因核酸探针。特异性检测结果表明,该探针能与重组质粒DNA发生特异性杂交,而与对照的PRVBartha-k61株疫苗毒DNA、猪细小病毒（PPV）DNA、猪圆环病毒（PCV）DNA、猪繁殖与呼吸综合征病毒（PRRsV）cDNA、猪瘟病毒（CSFV）cDNA的杂交反应均为阴性;敏感性检测结果表明,该探针对PRV野毒的最低检出量为4pg。应用该探针对11份繁殖障碍病料进行了杂交检测,共检出4份阳性病料,该结果与PCR检测结果一致,表明该核酸探针可用于猪伪狂犬病野毒感染的临床诊断。     

猪圆环病毒3型套式PCR检测方法的建立及应用

为了建立一种准确、快速检测猪圆环病毒3型(PCV-3)的方法,试验根据GenBank中PCV-3基因序列设计合成了内、外两对引物,建立了套式PCR方法,并检验了该方法的特异性、敏感性、重复性,同时用该方法对124份临床疑似病料进行检测。结果表明:用建立的套式PCR方法检测PCV-2、PRV、PPV、PRRSV、CSFV、TGEV、PEDV,均为阴性,外引物PCR扩增的检测灵敏度为17 ng DNA,内引物PCR扩增的检测灵敏度为0. 17 ng DNA。用该方法检测124份临床病料样品,外引物共检测出阳性样品22份,内引物共检测出阳性样品28份,青海省部分地区PCV-3的感染率达22. 58%(28/124)。说明该套式PCR具有良好的特异性、敏感性、重复性和临床适用性,可以用于临床病料中低含量PCV-3的快速检测。     

An Actinobacillus pleuropneumoniae PCR typing system based on the apx and omlA genes--evaluation of isolates from lungs and tonsils of pigs

The genetic variability of a gene coding for an outer membrane lipoprotein (omlA) was used to develop a PCR typing system for Actinobacillus pleuropneumoniae. Sequence differences in the middle region of the gene divided the A. pleuropneumoniae serotypes in five distinct groups. Group I included serotypes 1, 9, 11 and 12 (omlA l), Group II consisted of serotypes 2 and 8 (omlA II), Group III included serotypes 3, 6 and 7 (omlA III), Group IV (omlA IV) consisted of serotype 4 and Group V of serotypes 5a, 5b and 10 (omlA V). The sequence differences were utilized to construct PCR primers specific for each group, except of Group IV, as the amplicon of serotype 4 could be separated from Group III by size. Together with a PCR apx typing system, the omlA PCR typing system could discriminate the majority of A. pleuropneumoniae serotypes of biovar 1 except of serotypes 1, 9 and 11 and serotypes 2 and 8. The PCR typing system was tested on 102 field strains of A. pleuropneumoniae isolated from lungs of diseased pigs. The serotyping results of the investigated field strains were in agreement with the apx and omlA gene patterns found in the reference strains of the bacteria, with the exception of the omlA gene of five strains of serotype 8. To examine the apx and omlA gene pattern of tonsil isolates, the PCR typing system was tested on a total of 280 A. pleuropneumoniae field strains isolated from tonsils of pigs. Agreement between serotyping and DNA typing was found in 96% of the isolates using the apx gene patterns and in 89% of the isolates using the omlA gene. The same serotype specific apx/omlA gene pattern was thus found in the majority of the tonsil isolates and in isolates from diseased lungs. Most of the differences in the omlA gene were found in 18 tonsil isolates of serotype 12. The omlA/apx PCR typing system described in the present study makes it possible to determine the type specificity of the majority of A. pleuropneumoniae isolates by simple PCR technique and enables phenotype independent characterization of isolates non-typable by serotyping.     

Serological characterization of Actinobacillus pleuropneumoniae strains isolated from pigs in Quebec.

A total of 3306 isolates of A. pleuropneumoniae originating from lung tissues of pigs that died of acute pleuropneumonia and 140 isolates recovered from tonsils or nasal cavities of apparently healthy pigs from chronically infected herds were serotyped. Various serotyping methods, such as slide agglutination, tube agglutination, ring precipitation, coagglutination, immunodiffusion, indirect hemagglutination and counterimmunoelectrophoresis either alone or in combination were used. The techniques used for serotyping continued to evolve during the last 10 years depending on the problem encountered in serotyping. Antisera prepared in rabbits against formalinized whole cell suspensions of reference strains of A. pleuropneumoniae of serotypes 1 to 12 were employed for serotyping. Serotype 1 was predominant ranging from 55 to 87% from year to year during the last 10 years with an average prevalence of 68%. Serotype 5 was second in prevalence ranging from 9 to 30% with a mean of 23%. Both subtypes of serotype 5 (5a and 5b) were present in Quebec. Serotypes 3, 6, 7, 8, 10 and 12 were isolated in small numbers together accounting for about 9%. Serotypes 4, 9 and 11 were not present. Cross-reactions were observed among isolates of serotypes 3, 6 and 8, and 1, 9 and 11 and were easily differentiated from each other by quantitation of type and group specific antigens by coagglutination and immunodiffusion tests. Serotypes 1, 5 and 7 were isolated most frequently from tonsils of pigs from chronically infected herds. Prevalence of different serotypes in different countries has also been reviewed.     

Identification of Actinobacillus pleuropneumoniae strains of serotypes 7 and 4 using monoclonal antibodies: demonstration of common LPS O-chain epitopes with Actinobacillus lignieresii

Monoclonal antibodies (Mabs) against Actinobacillus pleuropneumoniae serotype 7 were produced and characterized. Three Mabs directed against surface polysaccharides were selected. One of the Mabs was directed against a capsular polysaccharide epitope (CPS) of A. pleuropneumoniae serotype 7 whereas two other Mabs reacted with different epitopes of the LPS O-chain. One of the latter reacted with the reference strain of serotype 7 and the other one with serotypes 7 and 4. These three Mabs were used to test, by Dot-ELISA, 508 field strains of A. pleuropneumoniae. None of the strains belonging to other serotypes different from serotypes 4 and 7 were positive with the Mabs. Used in combination, the CPS and one of the LPS O-chain directed Mabs were shown to be suitable for serotyping since they detected 100% of serotype 7 strains. In this study, we confirm for the first time that A. pleuropneumoniae serotype 4 is present in North America. Finally, both O-chain specific Mabs also reacted with the O-chain of Actinobacillus lignieresii. The cross-reactivity between the two species was confirmed using sera from pigs experimentally infected with A. pleuropneumoniae serotype 7 and A. lignieresii, using immunoblotting and ELISA. This is the first report of a specific cross-reactivity between the LPS of these bacterial species.     

猪胸膜肺炎放线杆菌PCR诊断方法的建立与应用

根据胸膜肺炎放线杆菌ApxⅣ基因设计1对引物，扩增特异的650bp棱酸片段，建立了应用PCR检测猪胸膜肺炎放线杆菌的方法。特异性试验结果表明，12个血清型的放线杆菌参考菌株均能扩增出650bp特异性的核酸片段，而大肠杆菌、多杀性巴氏杆菌、猪肺炎支原体、伤寒沙门氏菌和支气管败血性波氏杆菌的扩增结果均为阴性。敏感性试验结果表明，PCR的最低检出限量为500个放线杆菌。利用建立的PCR检测方法对22株从山东省不同地区分离的疑似胸膜肺炎放线杆菌菌株进行检测．结果14株为阳性。对感染猪病变组织的检测结果表明，病变部位不同，胸膜肺炎放线杆菌的检出率不同，其中以扁桃体的检出率最高。     

PCR-based identification of serotype 2 isolates of Actinobacillus pleuropneumoniae biovars I and II

A genetic typing method utilizing PCR for the identification of Actinobacillus pleuropneumoniae serotype 2 isolates has been developed based on the in vitro amplification of a 1.4 kb DNA segment of the serotype 2 capsular polysaccharide genes cps2AB. The assay was tested with all serotype reference strains and a collection of 92 different A. pleuropneumoniae strains of all 15 serotypes of both biovars I and II, originating from 18 different countries worldwide. The cps2 based PCR identified the serotype 2 reference strain and all 12 serotype 2 collection strains contained in this set. DNA was not amplified from the remaining A. pleuropneumoniae reference and collection strains, indicating the PCR assay was highly specific. Furthermore, the PCR method detected all 31 A. pleuropneumoniae serotype 2 field isolates from diseased pigs that were identified in parallel as serotype 2 by agar gel diffusion. The serotype 2 PCR assay proved to be highly specific and reliable for the identification of serotype 2 isolates of A. pleuropneumoniae.     

Biochemical and serological properties of Actinobacillus pleuropneumoniae biotype 2 strains isolated from swine

The biochemical and serological properties of 21 strains of Actinobacillus pleuropneumoniae biotype 2 isolated from haemorrhagic necrotic pleuropneumonia of swine were examined. For serologic typing, the indirect haemagglutination (IHA) and the double gel-diffusion tests were used. On the basis of their soluble surface antigens, our A. pleuropneumoniae biotype 2 isolates could be assigned to two proposed serotypes. Serotype 1 comprised 11 strains and serotype 2 comprised 10 strains. All strains contained two surface antigen components. In the strains belonging to serotype 1, one of the antigens was identical with the serotype-specific antigen of Pasteurella haemolytica T4. Both antigens of serotype 2 strains proved to be type-specific. Four strains received from Switzerland, including the holotype strain of A. pleuropneumoniae biotype 2, and three strains isolated from swine in the G.D.R. belonged to serotype 2. Both the double gel diffusion and the IHA tests detected a 2-way cross-reaction between biotype 1, serotype 2 and biotype 2, serotype 2 strains of A. pleuropneumoniae, which could be eliminated using cross-absorbed sera.     

Isolation of Actinobacillus pleuropneumoniae serotype 2 by immunomagnetic separation

In Denmark porcine pleuropneumonia is most frequently caused by Actinobacillus pleuropneumoniae serotype 2 (60%). Isolation of A. pleuropneumoniae from nasal cavities or tonsils from carrier animals is complicated due to the mixed bacterial flora present. An immunomagnetic separation technique (IMS) using immunomagnetic beads (Dynabeads((R))) was developed for isolation of A. pleuropneumoniae serotype 2 from pure cultures and from heterogeneous suspensions. Different coating and washing procedures were evaluated in pure and mixed cultures using polyclonal (PAb) and monoclonal antibodies. The highest reisolation yield was achieved when the beads were coated with 1.5 microg PAb IgG/10(7) beads. After washing the beads for four times 9-24% of the bacteria could be reisolated depending on the amount of IgG attached to the beads and the number of beads used. The recovery was increased to 19-61% when only two washing steps were performed. The IMS was further evaluated using dilutions of A. pleuropneumoniae with added Pasteurella multocida (10(9) CFU/ml). After two washing steps 15% of the A. pleuropneumoniae cells and no P. multocida was reisolated. A detection limit of 10 CFU/ml was found in this heterogeneous suspension. No significant difference was observed when comparing the recovery of A. pleuropneumoniae from pure culture, from mixed cultures and from artificially inoculated tonsils. From 12 pigs inoculated with an aerosol of A. pleuropneumoniae serotype 2 the bacterium could not be detected from the nasal cavity or tonsils by cultivation or PCR 6 weeks later. By using IMS A. pleuropneumoniae serotype 2 could be reisolated from the tonsils of three pigs. The IMS method represents a valuable tool for isolation of A. pleuropneumoniae from tissue samples.     

Detection of Actinobacillus pleuropneumoniae in the porcine upper respiratory tract as a complement to serological tests.

Attempts were made to isolate Actinobacillus pleuropneumoniae from the nasal cavities and tonsils of 442 healthy pigs from 15 herds. Samples were streaked onto different media formulations. Serum samples were assayed for antibodies to A. pleuropneumoniae by enzyme-linked immunosorbent assay and complement fixation test. Actinobacillus pleuropneumoniae was isolated from the nasal cavities only in 24 pigs, from tonsils only in 90 pigs, and from both the nasal cavities and the tonsils in 11 pigs. A PPLO medium supplemented with lincomycin, bacitracin and crystal violet allowed recovery of A. pleuropneumoniae from more animals than a tryptic soy agar medium from both sites. Incubation of plates in an enriched CO2 atmosphere did not affect the recovery rate. Actinobacillus pleuropneumoniae belonging to serotypes 1, 2, 3, 5a, 5b, 7, 8, 10 and 12 were isolated, and, in several herds, more than one serotype were recovered. Serotypes of A. pleuropneumoniae were isolated from nine herds which were found seronegative to these. The isolation of A. pleuropneumoniae from the upper respiratory tract can be useful for detection of carrier pigs and complements serological screening.     

DNA microarray-based identification and typing of Actinobacillus pleuropneumoniae

A DNA microarray system was prepared and shown to facilitate identification and typing of Actinobacillus pleuropneumoniae. The DNA microarray, composed of 18 DNA polymerase chain reaction (PCR) amplicons printed on glass slides and arranged in 3 subarrays, was developed. These target DNA included 1 or multiple fragments of the outer membrane lipoprotein, apx toxin, capsular polysaccharide, and disulfide bound formation protein E (dsbE)-like genes of A. pleuropneumoniae. These arrayed target DNA retained their expceted hybridization properties. The hybridization signal intensities ranged from the least-intense to the most-intense, 4626 to 9789 arbitrary fluorescence units, respectively. Cy3-probes of A. pleuropneumoniae strains labeled with multiplex PCR were hybridized to the DNA microarray. A total of 51 different A. pleuropneumoniae strains representing serotype 1 to 12 reference strains and clinical isolates were detected and typed by the DNA microarray. Twelve reference serotypes produced 11 distinct target DNA hybridization patterns, and hybridization patterns of serotypes 1 (n = 7), 3 (n = 5), and 7 (n = 6) field isolates were identical to hybridization patterns of reference serotypes 1, 3, and 7, respectively. Non-serotyped isolates 4, 6, and 11 (out of 21) from diseased pigs had identical hybridization patterns to reference serotypes 3, 7, and 1, respectively. The results show that the DNA microarray system described in the present study is a valuable tool for identifying and typing reference strains and isolates of A. pleuropneumoniae, and enables relatively rapid identification of non-serotyped isolates.     

Grouping of Actinobacillus pleuropneumoniae strains of serotypes 1 through 12 on the basis of their virulence in mice

Variations in virulence among strains of different serotypes of Actinobacillus pleuropneumoniae were detected on intraperitoneal or intranasal inoculation in mice. In general, strains of serotypes 1, 5, 9, 10 and 11 were found to be highly virulent and those of serotypes 2, 3, 4, 6, 7, 8 and 12 to be less virulent. However, a few strains of serotype 5 caused low mortality in mice while some strains of serotype 3 and 7 were found to be highly virulent. Highly virulent strains of A. pleuropneumoniae were invasive and appeared in the blood within 3 to 6 h of intranasal inoculation. The type specific antigen as detected by the coagglutination test was distributed in lungs, liver, heart and spleen after intraperitoneal inoculation whereas it was mostly concentrated in the lungs after intranasal inoculation. Lowest concentration of boiled whole-cell suspension of A. pleuropneumoniae showing limulus amebocyte lysate activity was variable and independent of the serotype. Mortality caused by boiled whole cell suspension was also variable and serotype independent.