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1.
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。  相似文献   

2.
猪胸膜肺炎放线杆菌PCR诊断方法的建立与应用   总被引：5，自引：3，他引：5  
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。  相似文献   

3.
合成胸膜肺炎放线杆菌质粒pKMA2425上长度为126bp及2214bp的2个DNA片段,克隆到大肠埃希氏菌质粒pGSI中,获得重组质粒pGSIA。PCR扩增pTKRED上的大观霉素抗性基因及线性化的pGSIA（126bpDNA片段-pGSI-2214bpDNA片段）,连接,电转化胸膜肺炎放线杆菌,在大观霉素的选择压力下筛选鉴定到连接正确的质粒,命名为pGSIAS。pGSIAS测序结果符合预期。在大观霉素的选择压力下,含有pGSIAS的胸膜肺炎放线杆菌血清1-10型菌株均生长良好;在氨苄青霉素的选择压力下,含有pGSIAS的大肠埃希氏菌生长良好。结果表明本研究构建的大肠埃希氏菌-胸膜肺炎放线杆菌穿梭载体pGSIAS序列正确,在胸膜肺炎放线杆菌及大肠埃希氏菌中均能复制并表达质粒上的相关基因。  相似文献   

4.
A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae.  相似文献   

5.
Actinobacillus pleuropneumoniae, a gram-negative rod of the Pasteurellaceae family, causes pleuropneumonia in pigs. Establishing A. pleuropneumoniae free herds is difficult due to the occurrence of persistently infected animals. The ApxIV toxin is expressed by A. pleuropneumoniae in vivo and an ELISA based on the toxin is used to detect infection and to differentiate between infected and vaccinated animals. In this study, we have identified a 1070bp insertion element of the IS30 family, designated ISApl1, in the A. pleuropneumoniae serotype 7 strain AP76. ISApl1 contains a 924bp ORF encoding a transposase, which is flanked by 27bp inverted repeats showing six mismatches. We investigated the occurrence of ISApl1 in other A. pleuropneumoniae strains, and its possible interference with virulence associated factors. Four insertion sites were identified in AP76: within the apxIVA toxin ORF, within a putative autotransporter adhesin ORF, upstream of a capsular polysaccharide biosynthesis gene cluster, and downstream of a beta-lactamase gene. ISApl1 is also present in some serotype 7 field isolates, but not in reference or field strains of other serotypes. In A. pleuropneumoniae AP76, the transposase gene is transcribed in vitro. The insertion in the apxIVA toxin gene remains stable after animal passage. Since this insertion should disrupt toxin expression, we tested 7 pigs infected with AP76 at day 21 post-infection. All were negative in the ApxIV ELISA but four out of seven were positive in an ApxII toxin ELISA. These results show that insertion elements can affect the detection of A. pleuropneumoniae infected animals.  相似文献   

6.
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.  相似文献   

7.
During the period of 2001-2003, a total of 591 Actinobacillus pleuropneumoniae field isolates from the Czech Republic were serotyped with a high occurrence of cross-reactions. The cross-reactions were observed in 416 isolates. Most frequently, in 401 isolates (67.9%), cross-reactions with antisera specific for serotypes 9, 11, and/or 1 were observed. Two additional molecular methods, ribotyping and restriction analysis of PCR amplified apxIVA gene (PCR-REA), were therefore used for detailed characterisation of A. pleuropneumoniae. In this subsequent analysis, reference strains representing serotypes 1-12 and 25 field isolates showing the most frequent serotype cross-reactions were examined. PCR-REA enabled all reference strains to be distinguished except for the strains of serotypes 9 and 11. Ribotyping distinguished all reference strains except two pairs of serotypes: 3 versus 6, and 9 versus 11, respectively. Field isolates with serotype cross-reactivity 9, 11, and/or 1 could not be differentiated by either of these methods.  相似文献   

8.
Actinobacillus pleuropneumoniae biotype 2 was isolated in pure culture or as the predominant isolate from the lungs of 9 growing and finishing pigs with pleuropneumonia. Gross and microscopic lesions resembled those caused by A. pleuropneumoniae biotype 1 serotypes (Nos. 1, 5, and 7) traditionally seen in the United States. The overall mortality rate for growing and finishing pigs on this 1,200-sow farrow-to-finish farm ranged from 0.37% to 0.84% per month from July 1990 to February 1991, and mortality due to respiratory disease ranged from 0.17% to 0.52% per month for the same period. This Actinobacillus species did not require V factor (no satellitism on blood agar with a Staphylococcus streak), was strongly beta-hemolytic, and demonstrated restriction fragment length polymorphisms in hybridization studies with A. suis, A. lignieresii, and A. equuli. Biochemically, the isolate most closely resembled A. pleuropneumoniae, and a DNA fragment considered specific for A. pleuropneumoniae biotypes 1 and 2 was demonstrated using polymerase chain reaction. Necrohemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae biotype 1 was reproduced experimentally in 2 4-week-old pigs inoculated intratracheally with broth cultures of the A. pleuropneumoniae biotype 2. This study demonstrated the presence of A. pleuropneumoniae biotype 2 in the United States.  相似文献   

9.
The Haemophilus parasuis aroA gene encodes 5-enolpyruvylshikimate-3-phosphate synthase and participates in the aromatic amino acids and the folic acid universal metabolic pathway of bacteria. The application of aroA-based PCR-RFLP methodology yields a significant degree of diversity in H. parasuis and Actinobacillus species. PCR amplification of the aroA gene rendered a 1,067-bp fragment in all 15 H. parasuis serovars, and also in Actinobacillus pleuropneumoniae serotypes 1-12, Actinobacillus lignieresii, Actinobacillus equuli, Actinobacillus porcinus, Actinobacillus rossii, Actinobacillus suis, Actinobacillus ureae, Actinobacillus minor and Actinobacillus indolicus. Sau3AI and RsaI digestions of the aroA PCR products rendered seven different restriction fragment length polymorphism (RFLP) patterns: group I (H. parasuis serovars 1, 2, 4-6, and 8-15, A. porcinus and A. ureae), group II (H. parasuis serovars 3 and 7, and A. pleuropneumoniae serotypes 1, 4, 5, 9, 11 and 12), group III (A. lignieresii), group IV (A. pleuropneumoniae serotype 7), group V (A. pleuropneumoniae serotypes 2, 3, 6 and 8, A. equuli, A. rossii, A. minor and A. indolicus), group VI (A. suis) and group VII (A. pleuropneumoniae serotype 10). This is the first report describing the presence of aroA gene in H. parasuis, A. lignieresii, A. porcinus, A. rossii, A. suis, A. ureae, A. minor and A. indolicus and the data presented here demonstrates a significant degree of aroA genetic diversity in H. parasuis and species of the genus Actinobacillus.  相似文献   

10.
Two unusual Actinobacillus isolates were recovered from pigs with no clinical signs, no lesions and no history of swine pleuropneumonia. Two representative strains (9953L55 and 0347) analyzed in this study were initially biochemically and antigenically identified as A. pleuropneumoniae serotypes 1 and 9, respectively, by traditional identification methods. Both strains presented, however, negative results with three A. pleuropneumoniae-specific PCR tests and revealed in particular the absence of the apxIV toxin genes. However, both strains produced and secreted ApxII toxin although they only harbored the toxin genes apxIICA, which is an uncommon feature for any of the known A. pleuropneumoniae serotypes. Upon experimental inoculation of pigs, these strains proved to be totally non-pathogenic. Animals infected with one of the strains produced antibodies that cross-react with A. pleuropneumoniae serotypes 1-9-11-specific LC-LPS ELISA. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that these strains form a separate phylogenetic group that is distinct from other Actinobacillus species and is particularly different from A. pleuropneumoniae.  相似文献   

11.
A nested polymerase chain reaction (PCR) was developed for the detection of Mycoplasma hyopneumoniae, the etiological agent of enzootic pneumonia, in tracheobronchiolar washings from live pigs. Two nested pairs of oligonucleotide primers were designed from the sequence of a specific DNA probe (I 141; accession number U02537). The primer combination was Hp1/Hp3 for the first step PCR while the nested primers (Hp4/Hp6) allowed amplification of a 706 bp fragment. All strains of M. hyopneumoniae tested in this study could be detected by the nested PCR. DNA from other bacterial species isolated from the respiratory tract of pigs or from other mycoplasmal species were not amplified. The detection limit was estimated to be 1 fg, corresponding approximately to one organism, while in the one step PCR previously described 4 x 10(2) organisms were required. The nested PCR was evaluated on 362 tracheobronchiolar lavages collected from pigs at 2, 4 and 6 months of age in eight herds chronically infected with M. hyopneumoniae. The nested PCR was compared to a blocking ELISA performed with sera collected from the same pigs at the same ages, and to an immunofluorescence test at slaughter on 65 lungs from 6-month old pigs. The comparison indicated that the nested PCR was significantly (p<0.05) more sensitive (157 positive results of 362 samples) than ELISA (118 positive results of 362 samples) for detection of M. hyopneumoniae infection. Nested PCR was also significantly more sensitive (54 positive results of 65 samples) than immunofluorescence (29 positive results of 65 samples) for detection of M. hyopneumoniae in pig lungs at slaughter. Moreover, the nested PCR was used to confirm the absence of the mollicute in a pig herd without any history of M. hyopneumoniae infection. Thus, nested PCR appears to be a useful test to assess M. hyopneumoniae infection on pig farms.  相似文献   

12.
在已建立的副猪嗜血杆菌(HPS)、多杀性巴氏杆菌(Pm)、胸膜肺炎放线杆菌(APP)的单重PCR检测方法的基础上,通过对扩增条件的优化,利用一次PCR反应可同时扩增出APP的256 bp、Pm的457 bp、HPS的822 bp的特异性片段,建立了HPS、Pm、APP的多重PCR实验室诊断方法。该复合PCR可检测60 pg的HPS、120 pg的Pm、50 pg的APP。利用该方法检测猪繁殖与呼吸综合征病毒(PRRSV)阳性猪体内分离到的39株细菌,结果显示HPS、Pm、APP分别为12、16、2株,对39份病料的检测与常规细菌分离鉴定结果一致,可见该方法适用于HPS、APP、Pm的临床快速检测。  相似文献   

13.
参照猪传染性胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)血清1型菌株,设计1对特异性引物,PCR扩增转铁结合蛋白B(tbpB)全基因,克隆到pMD19-T Simple载体中,经测序比较,与参考序列的核苷酸同源性达99.72%。试验将tbpB基因定向克隆到pET-32a( )中,转化BL21(DE3),经诱导后,SDS-PAGE结果显示转铁结合蛋白B得到表达,Western blot检测呈阳性。  相似文献   

14.
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.  相似文献   

15.
Twelve Actinobacillus pleuropneumoniae serotypes were differentiated by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified fragments from the outer membrane lipoprotein (omlA) gene. All 12 reference serotypes and 80 field isolates produced the expected 950-base pair (bp) fragment of the omlA gene by PCR. Combining the RFLP patterns obtained with SfaNI, Bst71I, AluI, NciI, nine distinct patterns were observed in the 12 serotype reference strains. The PCR-based RFLP analysis of omlA genes allows differentiation among the 12 serotypes, with the exception of group 1 (serotypes 1, 9 and 11), and group 2 (serotypes 2 and 8). When the PCR products from the 70 field isolates were subjected to RFLP analysis, 68 showed the same RFLP patterns as their respective serotype reference strain. Two isolates that could not be typed had the same RFLP patterns as those of serotype 5. These results suggest that PCR-based RFLP analysis of the omlA genes may be of value in differentiating among 12 A. pleuropneumoniae serotypes.  相似文献   

16.
从新疆北疆4个规模化猪场的20份发病猪病料中分离到1株细菌。经形态染色镜检、培养特性、生化特性和部分生物学特性试验鉴定为猪胸膜肺炎放线杆菌。根据猪胸膜肺炎放线杆菌ⅣA毒素基因特异性引物进行PCR扩增,得到1条与预期大小一致的2427bp的条带,将PCR产物纯化后克隆至pMD19-T载体上并测序。结果表明,该片段的碱基序列与GenBank中参考序列的同源性为98%,并与三株标准株比较,同源性分别为98.23%、99.46%和98.06%。  相似文献   

17.
During serological screening of a closed SPF-herd free of pleuropneumonia, more than half of the pigs were positive for complement-fixing antibodies to Haemophilus pleuropneumoniae. Actinobacillus bacteria closely related to A. suis were isolated from tonsillar tissue of 14 out of 20 slaughtered pigs submitted for pathological and bacteriological evaluation. None of the pigs had evidence of respiratory disease. Two pigs inoculated endobronchially with a selected Actinobacillus strain developed mild focal pneumonia and complement-fixing antibodies cross-reacting with H. pleuropneumoniae. Five pigs exposed and vaccinated with the Actinobacillus strain and five pigs spontaneously infected with the strain also developed complement-fixing antibodies against H. pleuropneumoniae and appeared to be less susceptible to experimental Haemophilus pleuropneumonia than pigs not exposed to the Actinobacillus infection. The agglutination test applied on serum treated with 2-mercaptoethanol detected antibodies against H. pleuropneumoniae serotype 5 but not against serotype 1 in pigs exposed to the Actinobacillus strain. Antibodies reactive with the Actinobacillus strain were also found in pigs hyperimmunized against H. pleuropneumoniae serotypes 1-5 in 2-mercaptoethanol tube agglutination test and rabbits hyperimmunized against serotypes 1,2 and 7, and strain 73567 in the immunodiffusion test. Conversely rabbits immunized against the Actinobacillus strain had antibodies against H. pleuropneumoniae serotypes 1, 3, 4, 5 and 6. It is concluded that pigs infected with Actinobacillus organisms may become false positive reactors against H. pleuropneumoniae.  相似文献   

18.
胸膜肺炎放线杆菌的实时荧光PCR检测   总被引：3，自引：0，他引：3  
猪传染性胸膜肺炎是由胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）引起的危害养猪业的五大疾病之一。至目前为止App共报道有2个生物型15个血清型。所有型都可能致病，但有显著差异。使用血清学方法监测猪传染性胸膜肺炎有其局限。一些猪细菌分离培养阳性，但血清反应仍为阴性，对这些猪群只能使用病原分离进行确诊。亚临床感染及处于潜伏期的动物，  相似文献   

19.
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.  相似文献   

20.
A restriction analysis of PCR (PCR-REA) amplified apxIVA gene has been suggested as an alternative method for serotyping of Actinobacillus pleuropneumoniae by Jaglic et al. [Jaglic, Z., Svastova, P., Rychlik, I., Nedbalcova, K., Kucerova, Z., Pavlik, I., Bartos, M., 2004. Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping. Vet. Microbiol. 103, 63-69]. The current study investigated whether this alternative method could distinguish between the reference strains of serovars 13-15 and the value of the method when applied to 47 field isolates representing serovars 1-3, 5, 7-9, 12 and 15 as well as non-typable isolates. The reference strains of serovars 13 and 14 had the same sized product after the apxIVA PCR, while the product for serovar 15 was of different size compared to all the other serovar reference strains. The CfoI digest profiles of the reference serovars 13 and 14 strains were different from each other and from all other serovars. The HpaII digest profiles of these two serovars were very similar to each other, but both were distinctively different from the other serovar profiles. The CfoI digest profile of serovar 15 strain was very similar to the serovars 3 and 12 strains except for two faint extra bands for serovar 15. The HpaII digest profiles of serovars 12 and 15 reference strains were identical. The PCR-REA method correctly recognized the serovar of 21 of 43 field isolates. It was concluded that the method was a useful additional tool to support, but could not replace, conventional serotyping.  相似文献   

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