以重组PvpA蛋白作为抗原的鸡毒支原体抗体间接ELISA检测方法的建立

为建立鸡毒支原体(MG)种特异性检测的间接ELISA检测方法,本研究以原核表达的PvpA蛋白作为包被抗原,初步建立了检测MG抗体的间接ELISA检测方法.特异性试验结果表明,重组PvpA蛋白与鸡新城疫病毒、鸡传染性支气管炎病毒、滑液支原体、大肠杆菌“O”抗原、禽沙门氏茵“O”抗原阳性血清均不发生交叉反应.该方法的批内变异系数小于8％,批间变异系数小于11％,表明具有较好的重复性.采用该检测方法与进口试剂盒对不同省份送检的鸡血清进行检测比较,两者阳性和阴性符合率均达到90％以上.本研究建立的间接ELISA检测方法为MG抗体检测试剂盒的研制奠定了基础.     

A recombinant antigen-based ELISA for the simultaneous differential serodiagnosis of Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis infections

We have previously identified species-specific DNA fragments, referred to as MS2/28 and Mm14, of Mycoplasma synoviae and Mycoplasma meleagridis, respectively. In the present study, we extended our analysis of the MS2/28 fragment that was found to encode a species-specific antigenic site, and we demonstrated the specificity of the Mycoplasma gallisepticum hemagglutinin protein encoded by pMGA1.2 (a member of the vlhA gene family). Then, we combined the Escherichia coli-expressed products of MS2/28, Mm14, and pMGA1.2, to develop a recombinant antigen-based enzyme-linked immunosorbent assay (recELISA), for the simultaneous and specific detection of antibodies to the three aforementioned major avian mycoplasma species. For comparative purposes, a novel in-house crude antigen capture ELISA (capELISA) was developed in parallel. In the latter protocol, the microtiter wells were enriched in species-specific antigens by capturing sonicated crude antigens on coated rabbit polyclonal antibodies that had been extensively adsorbed with the whole antigen of the heterologous species. With regard to rapid serum agglutination, both ELISA tests were highly specific, and they showed a significant correlation when field sera from naturally infected birds were tested. recELISA proved to be highly specific because absorbance values, with the heterologous species, were significantly lower (P < 0.001) than those obtained with capELISA. Given its cost-effectiveness and simplicity, the recombinant antigen-based ELISA seems to represent a valid tool for the specific screening of the three major avian mycoplasma species. recELISA will be particularly useful with regard to trade control because a large number of samples from various fields could be rapidly processed.     

Real-time polymerase chain reaction for Mycoplasma gallisepticum in chicken trachea

In this work, we describe a rapid detection procedure for Mycoplasma gallisepticum from chicken tracheal swabs by real-time polymerase chain reaction (PCR) by LightCycler system, where we were able to monitor the amplification of the newly synthesized M. gallisepticum-specific PCR product as a proportionally increasing fluorescent signal by using the double-stranded DNA binding dye SYBR Green I and have identified M. gallisepticum-specific PCR products by DNA melting curve analysis by plotting the first negative derivative (-d[F1]dT) of fluorescence over temperature. Detection limits of the PCR were found to be 3 and 3000 colony-forming units ml(-1) with pure culture of M. gallisepticum and artificially spiked samples, respectively. Out of 96 tracheal swabs, 68 were taken from live chickens and 28 were taken by scraping the mucosal surface of the trachea (SMST) of necropsied chickens. All of the 18 PCR-positive results were from the swabs taken by the SMST method, whereas all of the samples taken from live chickens were negative. Thus, the PCR with the SMST method had a sensitivity and a specificity of 64.2% (18 of 28 chickens) and 100%, respectively. The total time required for template preparation from tracheal swab samples and real-time PCR was approximately 65 min. These results indicate that real-time PCR with the LightCycler technology is a rapid and sensitive test to identify M. gallisepticum-infected flocks if a proper sampling is applied.     

鸡毒支原体黏附蛋白PvpA的原核表达与纯化

利用基因重组技术将PvpA基因的PCR扩增产物与原核表达栽体pET-41a(+)连接,转化入DH5a感受态细胞,通过PCR、双酶切及测序鉴定后,将阳性重组质粒转化入大肠杆菌BL21(D3)受体菌,用IPTG诱导蛋白表达.用纯化后的融合蛋白免疫新西兰大白兔,制备PvpA蛋白多克隆抗血清,并用Western blotting检验抗体特异性.结果表明,本研究成功获得PvpA纯化蛋白,在免疫印迹试验中,兔抗PvpA高免血清能与目的蛋白发生阳性反应,证实表达产物为特异性蛋白.     

Comparative study of Mycoplasma pulmonis and Mycoplasma gallisepticum infections in turkey sinus.

Infraorbital sinuses of young turkeys were injected with virulent strains of Mycoplasma pulmonis and Mycoplasma gallisepticum to compare the diseases caused by the 2 agents. Mycoplasma pulmonis did not cause visible swelling from large quantities of mucous exudate in the sinuses, such as occurs with M gallisepticum, and it could not be recovered by bacteriologic culture technique after 3 weeks. However, slight exudate did accompany the M pulmonis infection. Similarities between the disease caused by M pulmonis and that caused by M gallisepticum included lymphocytic infiltration in the submucosa, swollen epithelial cells, and loss of cilia from sinus epithelial cell surfaces. This strain of M pulmonis, which is pathogenic for rats, was only mildly pathogenic for turkeys and the infection did not persist for long.     

A quantitative study of single and mixed infection of the chicken trachea by Mycoplasma gallisepticum

The interaction between Mycoplasma gallisepticum (MG) and the tracheal mucosa of the young chicken was studied. The use of a selective plating method permitted differentiation between a pathogenic tylosin-resistant strain (227) and a less pathogenic tylosin-sensitive vaccine strain (F). Both MG strains adhered to the tracheal mucosa and colonized equally well. In mixed infection, the presence or absence of the second strain did not change the efficiency of colonization by either strain. When chickens were exposed to the vaccine strain 24 hr or 2 weeks before superinfection by the pathogen, there was no significant reduction in the efficiency of superinfection, despite the presence of 10(6) colony-forming units of MG strain F in the trachea. However, chickens had an increased ability to resist superinfection 5 weeks after exposure via the air sac. These results suggest that the biological mechanism underlying protection of F-strain-vaccinated chickens against adventitious infection by the homologous species does not involve competition for adherence sites or blockage by prior colonization.     

A Mycoplasma gallisepticum strain-specific DNA probe

Total DNA from the vaccine F strain (K810) and the reference S6-strain of Mycoplasma gallisepticum (MG) was cloned in Escherichia coli using the plasmid pUC8. A 6-kilobase fragment, specific for the vaccine strain, was identified by colony dot and Southern hybridization analyses. When labeled and used as a probe, this fragment hybridized with the homologous and one other vaccine F-strain (F2F10), but it did not hybridize with other MG strains (Fg38, S6, A5969, V503) or with three other species of avian mycoplasmas.     

Morphologic changes in chicken cells after in vitro exposure to Mycoplasma gallisepticum

Mycoplasma gallisepticum (MG) was used to expose chicken peripheral blood lymphocytes (PBLs), red blood cells (RBCs), heterophils, and chicken tumor cells (MSB-1 and HD-11 cells). Incubation of PBLs with MG for 3 hr resulted in extensive clumping of lymphocytes. Incubation of the MSB-1 cells with MG also caused clumping of the cells, with many of the cells showing perforations and others showing capping of the surface projections. Incubation of RBCs with MG resulted in an altered cell surface morphology, a decrease in cell size, and perforation. There were no discernible changes on the surface of the heterophils and the HD-11 cells. However, the HD-11 cells appeared to have a decreased ability to attach to the surface of the plastic and to have a decreased ability to respond to chemoattractant fMLP after 24 hr of incubation. These results suggest that, under the conditions used, MG caused certain damage to peripheral blood cells and a significant decrease in chemotactic response in the HD-11 cells.     

Application of enzyme-linked immunosorbent assay for detecting antibody to Mycoplasma gallisepticum infections in poultry

An enzyme-linked immunosorbent assay (MYCO-ELISA) was developed to detect antibodies to Mycoplasma gallisepticum (MG) in chicken sera. The assay was standardized in terms of optimum antigen concentration, serum dilution, conjugate dilution and incubation temperature, and time. The MYCO-ELISA antigen was prepared from MG whole bacterial cell or its disrupted cell suspension. Both preparations showed strong affinity for binding or adsorbing to the surface of polystyrene wells of the microtiter plate. The MYCO-ELISA was more sensitive than the hemagglutination-inhibition test. However, cross-reactions were observed with sera from M. synoviae (MS)-infected birds.     

复方硫氰酸红霉素可溶性粉对人工感染鸡慢性呼吸道病的疗效试验

用复方硫氰酸红霉素可溶性粉以每升水中加0.5、0.6、0.7、0.8、0.9 g 5个剂量对人工感染鸡慢性呼吸道病的鸡群混饮治疗,同时设立硫氰酸红霉素可溶性粉对照组.试验结果表明,用药5d后,复方硫氰酸红霉素可溶性粉每升水加0.6、0.7、0.8、0.9g剂量组的有效率和治愈率皆高于硫氰酸红霉素可溶性粉组.     

A radioimmunoassay for the detection of antibodies to Mycoplasma gallisepticum

A radioimmunoassay technique was developed to determine the antibodies to Mycoplasma gallisepticum (MG) in sera, yolk fluids, and tracheal washings.     

A combination of diagnostic tools for rapid screening of ovine listeriosis

A combined serological and PCR method for the detection of Listeria monocytogenes infection in symptomatic and asymptomatic ovine flocks was evaluated. Seventy-eight milk samples and 157 serum samples were analysed using a L. monocytogenes PCR detection kit and an anti-listeriolysin O IgG immunoassay kit. The combined use of these commercial kits allowed a rapid and effective detection of L. monocytogenes infection in both the early stage, before seroconversion, and in a later phase, even after antibiotic therapy.     

Comparison of culture, PCR, and different serologic tests for detection of Mycoplasma gallisepticum and Mycoplasma synoviae infections

In this study, the technical performance of culture, two commercially available polymerase chain reaction (PCR) tests, rapid plate agglutination (RPA) test, hemagglutination inhibition (HI) test, and eight commercially available enzyme-linked immunosorbent assays (ELISAs) were compared for the detection of avian mycoplasma infections from 3 days postinfection (d.p.i.) through 35 d.p.i. The tests were carried out on samples from specified pathogen-free layers that were infected at 66 wk of age with recent Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) field strains, MS and MG ATCC strains, and Mycoplasma imitans (MIM), respectively. Results showed a high percentage of positive samples in the homologous infected groups and a high percentage of negative samples (100%) in the uninfected and heterologous infected groups during 35 d.p.i. of both culture and PCR tests. For the group infected with the MG 15302 ATCC strain, serology was more sensitive than bacteriology. All MG and MS tests, with the exception of MG ELISA kit D showed a lower percentage of positive samples during 35 d.p.i. for the detection of the MG and MS ATCC strain infection compared with that of the field strains. Also, the number of cross-reactions (false positives) in the serologic tests was lower after infection with an ATCC strain than after an infection with the MG or MS field strain. Contradictory to other studies, the ELISAs and the RPA test using undiluted serum showed a relatively high number of false-positive results. The MG ELISAs (except ELISA kit D) showed more false-positive results (up to 37%) in the MIM-infected group than in the MS-infected groups. This was not unexpected, as MIM and MG have a close antigenic relationship. The results of the serologic tests in this study showed that a certain level of false-positive results can be expected in about any serologic test. Although the level of false-positive results varied between several serologic tests, this study showed that it is not advisable to rely completely on one test (system) only.