Use of mgc2-polymerase chain reaction-restriction fragment length polymorphism for rapid differentiation between field isolates and vaccine strains of Mycoplasma gallisepticum in Israel

Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for a rapid test for differentiation of MG field strains from the live vaccine strains ts-11 and 6/85. We examined the differentiating potential of diagnostic polymerase chain reaction (PCR) primers targeted to the gene mgc2, encoding a cytadherence-related surface protein uniquely present in MG. The mgc2-PCR diagnostic primers are specific for MG in tests of all avian mycoplasmas or bacteria present in the chicken trachea and are sensitive enough to readily detect MG in tracheal swabs from field outbreaks. Differentiation of vaccine strain ts-11 was based on identification of restriction enzyme sites in the 300-base-pair (bp) mgc2-PCR amplicon present in ts-11 and missing in MG isolates from field outbreaks in Israel. Restriction sites for the enzymes HaeII and SfaN1 were identified in the amplified region in strain ts-11 and were not found in 28 field isolates of MG, comprising a representative cross section of all the MG isolates from the period 1997-2003. In practice, differential diagnosis of MG is achieved within 1 day of submission of tracheal swab samples by mgc2-PCR amplification and restriction of the amplicon with HaeII, giving a 270-bp fragment for ts-11 or no restriction for other MG strains tested. Application of the mgc2-PCR-restriction fragment length polymorphism (mgc2-PCR-RFLP) assay enabled differential diagnosis of both components of a mixture of ts-11 and non-ts-11 DNA, detecting the field strain in the presence of a large excess of ts-11. The test was successfully applied in vivo for monitoring vaccinates in a ts-11 vaccine trial. In principle, the test may also be used to identify the 6/85 vaccine strain, which yields a 237-bp product, readily differentiated from the approximately 300-bp PCR product of all other strains tested. Further testing of field isolates will be necessary to determine the applicability of this test in the United States and other countries.     

The Mycoplasma gallisepticum 16S-23S rRNA intergenic spacer region sequence as a novel tool for epizootiological studies

Mycoplasma gallisepticum (MG) contains two sets of rRNA genes (5S, 16S and 23S) in its genome, but only one of the two is organized in an operon cluster and contains a unique 660-nucleotide intergenic spacer region (IGSR) between the 16S and the 23S rRNA genes. We designed a polymerase chain reaction (PCR) for the specific amplification of the complete MG IGSR segment. The MG IGSR PCR was tested on 18 avian mollicute species and was confirmed as MG specific. The reaction sensitivity was demonstrated by comparing it to the well-established MG mgc2 PCR. The MG IGSR sequence was found to be highly variable (discrimination [D] index of 0.950) among a variety of MG laboratory strains, vaccine strains, and field isolates. The sequencing of the MG IGSR appears to be a valuable single-locus sequence typing (SLST) tool for MG isolate differentiation in diagnostic cases and epizootiological studies.     

Differentiation of mycoplasma gallisepticum strains using molecular methods

Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for the differentiation of MG strains. The MG strains MK-7, MS-16, S6, FS-9 and R strains and the MG live vaccine strain F were compared by random amplification of polymorphic DNA (RAPD) in this study. Using RAPD, different patterns were found among the MG strains. In addition to this, we examined the differentiating potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) primers targeted at the crmA, crmB, crmC, gapA, mgc2 and pvpA genes encoding cytadherence-related surface proteins. These proteins may take part in the pathogenesis of MG-induced disease. Differentiation of strain F is based on the identification of restriction enzyme sites in the PCR amplicons. Using HphI enzyme, crmC PCR amplicons produced different RFLP patterns. Digestion of amplicons of gapA-specific PCR with MboI enzyme also produced distinct patterns. Differences were observed among strains R and F by digestion of mgc2 PCR amplicons with HaelIl and VspI enzymes and digestion of pvpA PCR amplicons with AccI, PvulI and ScrFI endonucleases. This method can be used for the rapid differentiation of vaccine strain from wild strains. Differentiation of MG strains is a great advantage for diagnosticians or practitioners and it is useful for epidemiological studies.     

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.     

用探针杂交法检测鸡毒支原体和滑液囊支原体

根据鸡毒支原体(MG)和滑液囊支原体(MS)的16SrRNA基因序列，设计并合成了探针MG和MS共同目的基因，跨幅为580bP的一对引物。用这对引物对2个标准的MG和1个标准的Ms菌株DNA模板进行聚合酶锭反应(PcR)，结果得到了预期大小约580bP的PcR产物。回收纯化琼脂糖电泳凝肢中的MG扩增产物克隆至T载体中，DIG随机引物法合成核酸探针，斑点杂交试验对MG和MS均呈阳性，检测灵敏度分别为2ng和3ng，其它对照菌(毒)株为阴性。     

Molecular characterization of Mycoplasma gallisepticum isolates from Jordan

Two groups of Mycoplasma gallisepticum (MG) isolates (n = 24) from Jordan were analyzed by molecular methods and compared with other Middle Eastern isolates, related international isolates, and reference strains. The first group (n = 19) was isolated from July 2004 to January 2005 (isolation period A), and the newer group (n = 5) from June 2007 to April 2008 (isolation period B). The groups of isolates are from chicken flocks from northern Jordan, but are not from the same farms. None of the flocks were vaccinated for MG. Random amplified polymorphic DNA analysis, targeted sequencing of the partial MG cytadhesin 2 (mgc2), and the MG 16S-23S rRNA intergenic spacer region (IGSR) divided the Jordanian isolates into two groups. All of the 19 isolates from time period A, in addition to two isolates from time period B, were indistinguishable from the F strain. Three of five isolates from time period B were characterized as wild types and were indistinguishable from each other. The wild-type field strain was readily distinguished from the F strain. It was 91% and 96.4% similar to the F strain based on Clustal-W alignments of sequences of mgc2 and IGSR, respectively. Sequence similarity of mgc2 gene of the Jordan wild-type strain to isolates from Israel and Egypt ranged from 96.5% to 100%, whereas for IGSR it was 99.4%-100%. We theorize that the F-strain live MG vaccine, commonly used in Jordan prior to 2007, was transmitted to nonvaccinated poultry in the region and was a predominant genotype during time period A.     

Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds

In this study, we report the development and validation of a real-time polymerase chain reaction (PCR) assay using a Taqman-labeled probe for the detection of Mycoplasma gallisepticum (MGLP assay). The MGLP assay was highly specific with a detection limit of 25 template copies per reaction and a quantification limit of 100 template copies per reaction. Validation of the assay was completed with 1247 samples (palatine cleft and tracheal swabs) from M. gallisepticum-positive and -negative chicken flocks. The MGLP assay was compared to an enzyme-linked immunosorbent assay (ELISA), a conventional polymerase chain reaction assay (mgc2 PCR), and isolation of M. gallisepticum from naturally infected flocks. A total of 805 samples collected from negative flocks, as verified by ELISA and/or mgc2 PCR, were negative by the MGLP assay. A total of 442 samples were collected from positive flocks, of which a total of 228 samples were positive by the MGLP assay. These results agreed for 98.87% of the samples when tested by mgc2 PCR. When comparing the MGLP assay with M gallisepticum isolation, the MGLP assay was more sensitive than isolation for detecting positive birds from a positive flock, 172/265 and 50/265, respectively. Overall, the MGLP assay and M. gallisepticum isolation agreed for 52.8% of the samples tested. In conclusion, the MGLP assay was highly specific, sensitive, and reproducible, and allowed the quantification of template copies directly from clinical samples.     

多重聚合酶链反应快速检测鉴别鸡毒支原体强毒株和弱毒疫苗株的研究

根据鸡毒支原体强毒株和弱毒疫苗株基因组的结构特点，设计合成了二对引物XZ1，XZ2和XZ45、XZ46，建立了一种同时检测鉴别MG野毒株和弱毒疫苗株的多重PCR技术。试验结果表明，用这两对引物对MG强毒株和弱毒疫苗侏进行多重PCR，强毒株只扩增出732bp一条带，而弱毒疫苗株则可同时扩增出732bp、524bp二条带，而对其他种类鸡支原体和其它禽病病原的扩增不出现任何条带，结果均为阴性；敏感性测定结果表明，该多重PCR最低能检出1Pg的MG强毒株和弱毒疫苗株的DNA模板。     

用PCR和探针杂交法检测鸡败血霉形体

根据鸡败血霉形体ｆＭＧ－２核酸片断序列,设计合成了１对２５ｂｐ寡核苷酸引物,对鸡败血霉形体基因组ＤＮＡ进行扩增,均获得预期的７３２ｂｐ扩增产物,检测灵敏度为１ｂｐ;参考菌株ＤＮＡ无扩增。回收纯化琼脂糖电泳凝胶中的扩增产物,ＤＩＧ随机引物法合成核酸探讨,Ｄｏｔ－ｂｌｏｔ杂交试验,鸡败血霉形体呈阳性,检测灵敏度为１００ｐｇ;其他为阴性。对自然发病鸡群检测进一步表明,建立的ＰＣＲ和探针杂交法具有高度的灵     

Species-specific polymerase chain reactions for the detection of Mycoplasma buteonis, Mycoplasma falconis, Mycoplasma gypis, and Mycoplasma corogypsi in captive birds of prey

Mycoplasmas are pathogens of different avian species, but the role of Mycoplasma in raptors is not yet completely determined. As Mycoplasma isolation and identification present several difficulties, species-specific polymerase chain reactions (PCRs) for the detection of mycoplasmas found in birds of prey (Mycoplasma buteonis, Mycoplasma corogypsi, Mycoplasma falconis, and Mycoplasma gypis) were established. The specificity of the PCR methods were investigated using known avian Mycoplasma reference strains and isolates as well as related bacteria and was found to be specific. Amplificons obtained with these PCRs from field samples showed no false-positive results in restriction enzyme analysis and sequencing. The sensitivities of the different PCR assays varied between 50 fg and 1 pg DNA. Twenty-five tracheal swabs from healthy captive birds of prey were investigated by culture and immunobinding assay as comparison to the PCRs. Mycoplasmal DNA was detected in 88% of the samples, with negative results only from vultures. Mycoplasma falconis and M. buteonis were regularly found in falcons, and M. gypis was found in a common buzzard. Mycoplasma corogypsi was not demonstrated. Several isolates could not be differentiated using an immunobinding assay as well as the described PCR methods.     

Development and application of loop-mediated isothermal amplification methods targeting the seM gene for detection of Streptococcus equi subsp. equi

Loop-mediated isothermal amplification (LAMP) constitutes a potentially valuable diagnostic tool for rapid diagnosis of contagious diseases. In this study, we developed a novel LAMP method (seM-LAMP) to detect the seM gene of Streptococcus equi subsp. equi (S. equi), the causative agent of strangles in equids. The seM-LAMP successfully amplified the target sequence of the seM gene at 63°C within 60 min. The sensitivity of the seM-LAMP was slightly lower than the 2nd reaction of the seM semi-nested PCR. To evaluate the species specificity of the seM-LAMP, we tested 100 S. equi and 189 non-S. equi strains. Significant amplification of the DNA originating from S. equi was observed within 60 min incubation, but no amplification of non-S. equi DNA occurred. The results were identical to those of seM semi-nested PCR. To investigate the clinical usefulness of the methods, the seM-LAMP and the seM semi-nested PCR were used to screen 590 nasal swabs obtained during an outbreak of strangles. Both methods showed that 79 and 511 swabs were S. equi positive and negative, respectively, and the results were identical to those of the culture examination. These results indicate that the seM-LAMP is potentially useful for the reliable routine diagnosis of Streptococcus equi subsp. equi infections.     

应用多重套式PCR检测鸡毒支原体和鸡滑液囊支原体

根据已发表的鸡毒和鸡滑液支原体血凝素基因序列pMGA和vlhA各设计两对引物,建立鉴别诊断两种支原体的多重套式PCR方法,对其进行温度条件、Ⅱ步模板浓度优化及特异性、敏感性实验。该方法在两步PCR后能特异性地扩增出MG(408 bp)和MS(688 bp)两个目的片段。应用于临床样品检测,与支原体分离、SPA检测比较结果PCR灵敏度高于病原分离。     

Characterization of a naturally occurring infection of a Mycoplasma gallisepticum house finch-like strain in turkey breeders

An outbreak of Mycoplasma gallisepticum (MG) in commercial turkeys involving very mild clinical signs was difficult to confirm by routine methods. In the first part of this study (trial A), we conducted a bioassay to increase the likelihood of detecting MG. Susceptible turkeys were inoculated with sinus exudates from four different affected commercial turkey flocks. Turkeys were evaluated for clinical signs, as well as by serology and culture of tracheal swabs, at 21 and 42 days postchallenge. An MG isolate from one of the sinus exudates used for inoculation, designated K5054, was very similar to isolates from house finches when characterized by random amplified polymorphic DNA analysis as well as DNA sequence analysis of portions of the phase-variable putative adhesin protein (pvpA) gene, a lipoprotein gene, and the cytadhesin gapA/mgc1 gene. The turkeys inoculated with the K5054 sinus exudate seroconverted in the absence of severe clinical signs. There was a single reisolation of K5054 from these turkeys 42 days postchallenge. Susceptible contact turkeys were commingled with the K5054-inoculated turkeys at 49 days postchallenge. We found no evidence of transmission of MG to the contacts by culture or serology at 7, 21, or 35 days after commingling. In the second part of this study (trial B), we challenged the contacts and K5054 sinus exudate-inoculated turkeys from trial A with virulent R strain 88 days after the K5054 sinus exudate inoculation. On necropsy 10 days postchallenge, the evaluation of gross and microscopic lesions, serology, and culture showed that the turkeys previously inoculated with K5054 sinus exudate were protected against disease and reinfection.     

Use of an alkaline phosphatase-labelled probe for the detection of Mycoplasma synoviae in chickens

Short nucleotides directly labelled to alkaline phosphatase (SNAP probes) are an interesting alternative to digoxigenin-labelled probes (DIG probes), because they reduce the number of steps necessary in dot blots for the detection of DNA or amplificate. This study examined the questions whether a SNAP probe might not only save time, but also increase the sensitivity of another PCR-based DNA probe test using a digoxigenin probe. Amplificates obtained by multispecies polymerase chain reaction (PCR), with either purified genomic DNA or DNA extracted from tracheal swabs taken in chicken flocks, were detected by both methods. The results for the clinical specimens were compared to culture. Under stringent conditions, the specificity and sensitivity obtained with the SNAP probe were comparable to the results obtained with the DIG probe. The quantities 10 fg (SNAP probe) and 100 fg (DIG probe) of purified Mycoplasma synoviae DNA were detected after amplification, but more positive clinical specimens were detected with the DIG probe. Under non-stringent conditions sensitivity with purified DNA did no change, but the coloration of the dots improved markedly, and more positive specimens could be detected with the SNAP probe than with the DIG probe, truly positives as confirmed by culture. Because cross-reaction with Mycoplasma gallisepticum and Mycoplasma imitans, two species with DNA that was also recognized by the multispecies primers, occurred under non-stringent conditions, it was concluded that, to take the full advantage of SNAP probes, their use in combination with species-specific primer pairs is recommended. PCR as a method for mycoplasma detection is however, always accompanied with serological and cultural methods. When a M. synoviae mono-infection is likely by serological results, non-stringent dot blot conditions and use of the SNAP probe will ease and improve the detection of mycoplasma.     

Differentiation of Mycoplasma gallisepticum strains using amplified fragment length polymorphism and other DNA-based typing methods

Amplified fragment length polymorphism (AFLP) was used to type 34 strains of Mycoplasma gallisepticum (MG) including vaccine strains ts-11, 6/85, and F. Using AFLP, a total of 10 groups, with 30 distinguishable AFLP typing profiles, were generated in the analysis. The AFLP method was able to identify and differentiate both MG field strains from recent outbreaks and those that were epidemiologically related. The AFLP procedure will provide assistance in identifying the sources of mycoplasma infections. Vaccine strains were also differentiated from other field strains, which will be useful in the evaluation of vaccination programs. The AFLP discrimination potential was compared to other molecular typing techniques such as gene-targeted typing by DNA sequence analysis of the MG cytadhesin-like protein encoding gene, mgc2, and random amplified polymorphic DNA assay on the same MG isolates. The three assays correlated well with one another, with AFLP analysis having a much higher discriminatory power and reproducibility.     

Identification of equine herpesviruses 1 and 4 by polymerase chain reaction

OBJECTIVE: To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus). DESIGN: Primer sets based on nucleotide sequences encoding glycoprotein H (gH) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections. METHODS: Oligonucleotide primers were designed for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The tests were applied to tissue samples from aborted equine foetuses and to nasopharyngeal swabs from horses with acute febrile respiratory disease. RESULTS: Individual single round and a second round (seminested) EHV1 and EHV4 PCRs were specific in that EHV1 primers amplified all (n = 30) EHV1 isolates and did not amplify EHV4. Similarly EHV4 primers amplified all (n = 6) EHV4 isolates and did not amplify EHV1. Both PCRs were sensitive in that the first round EHV1 PCR detected 1220 molecules of EHV1 plasmid DNA and the first round EHV4 PCR detected 7280 molecules of EHV4 plasmid DNA. The EHV1 second round PCR was 100 times more sensitive in that it detected 12 molecules of EHV1 DNA and the EHV4 second round PCR was 1000 times more sensitive in that it detected 8 molecules of EHV4 DNA. There was a high correlation between detection of EHV1 by virus isolation and PCR when tissue samples from 71 aborted foetuses were examined; all samples positive by virus isolation were positive by PCR. Similarly the EHV4 PCR was at least as sensitive as virus isolation when applied to nasaopharyngeal swabs from horses with respiratory disease in that all samples positive by virus isolation were also positive by PCR. CONCLUSION: Individual single round and second round (seminested) PCRs and a seminested multiplex PCR were developed that enabled reliable, rapid detection of EHV1 and EHV4 in aborted foetal tissues and nasopharyngeal swab samples.     

Monitoring Mycoplasma gallisepticum and Mycoplasma synoviae infection in breeder chickens after treatment with enrofloxacin.

Three experimental strains of breeder chickens were accidentally exposed to Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), presumably from a newly introduced group of leghorn-type pullets. The experimental strains subsequently became infected and were diagnosed positive for MG and MS by the serum plate agglutination (SPA) test and confirmed by the hemagglutination inhibition (HI) test and the polymerase chain reaction (PCR) of tracheal swabs. Treatment with 10 mg/kg enrofloxacin via drinking water for 14 days was elected. Before and after initiation of treatment, MG and MS were monitored for changes by SPA, HI, PCR, and culture, with sampling intervals ranging from 1 wk to 7 wk. MG and MS SPA, HI, PCR, and culture were performed at each sampling period, with the exception of weeks 1.0 and 6.5. Week 1.0 included SPA and His for MG and MS. Week 6.5 included PCR and culture for MG and MS. The MG and MS SPA results were positive throughout the 29-wk trial period. MG HI titers declined until the last sampling, whereas the MS HI titers did not decline significantly. PCR for MG yielded only one positive result, which occurred before treatment. MS PCR remained positive throughout the trial period. MG was never isolated from any sample; however, one MS organism was isolated during treatment. The treatment regimen was effective for MG on the basis of PCR results. Treatment with enrofloxacin did not eliminate SPA reactions during the 29-wk trial period. MG HI titers remained in the suspicious range throughout the remainder of the trial period. Four weeks after the treatment ended, MG HIs were reduced by approximately 40%, with MS HIs remaining high throughout the 29-wk period. PCR appeared to be a sensitive and specific test on the basis of correlation with HIs. On the basis of the isolation of MS during treatment and continued subsequent PCR positive reactions, the treatment for MS with enrofloxacin was not as efficacious as for MG.     

Influence of swab material on the detection of Mycoplasma gallisepticum and Mycoplasma synoviae by real-time PCR

Recent reports have shown an increased recovery of cells from flocked nylon swabs which may improve the specimen quality and the real sensitivity of diagnostic tests in a clinical setting. In this study, the detection of Mycoplasma gallisepticum (MG) and M. synoviae (MS), using dry swabs of different materials (nylon flocked, cotton, and polyester), was investigated using real-time TaqMan PCR protocols. Different types of samples, including dilutions of pure broth cultures of MG and MS as well as swabs from tracheas of experimentally infected chickens and field cases of infection, were analyzed. There were no statistical differences in real-time PCR results among the different swab types (P < 0.05), indicating that this is not likely to be a significant factor in MG and MS detection by this method.     

Evaluation of protection against Mycoplasma gallisepticum infection in chickens vaccinated with the F strain of M. gallisepticum

The effect of vaccination with the F strain of Mycoplasma gallisepticum (MG) on protection against challenge with a tylosin-resistant strain of MG was evaluated. White leghorn chickens vaccinated via eyedrop at 6 weeks of age were subsequently challenged with various dilutions of the tylosin-resistant MG strain, as were unvaccinated controls. Three days later, tracheal swabs were collected and cultured in medium with and without tylosin to distinguish between the vaccine and challenge strains. The mean infectious dose of the challenge strains was 3.8 log10 higher in the vaccinated group than in the controls, and the vaccinated group harbored fewer challenge organisms in the trachea. These findings suggest that the F strain of MG induces protection against infection with field strains of MG and that long-term vaccination with the F strain in multiple-age layer farms may result in replacement of field MG strains by the F strain.     

First evidence of porcine circovirus type 2 (PCV-2) infection of pigs in the Czech Republic by semi-nested PCR

Three oligonucleotide primers for semi-nested polymerase chain reaction (PCR) were designed according to already published sequences of porcine circovirus types 1 (PCV-1) and 2 (PCV-2) isolates. These primers were used to detect PCV-2 DNA. A positive amplification reaction was visualized from a DNA suspension containing as few as 10 copies of virus DNA. In total. 77 samples of inguinal lymph nodes and nasal swabs from pigs in the Czech Republic were used to detect the virus. Thirty-seven of them were positive for PCV-2 DNA. In order to confirm specificity of the PCR reaction, seven DNA fragments were sequenced. Czech PCV sequences were found to have a 92-97% homology with other known PCV-2 strains and only 80-83% homology with PCV-1 strains.