Isolation of Mycoplasma meleagridis from free-ranging birds of prey in Germany

In this study tracheal swabs and air sac biopsies of 68 raptors of different species that were found injured or debilitated in Germany were investigated for the occurrence of mycoplasmas. Mycoplasma meleagridis, Mycoplasma falconis, Mycoplasma buteonis, Mycoplasma gypis and five mycoplasma isolates not identified so far could be isolated from 32 (47%) birds. Mycoplasma meleagridis could be detected in five birds. These birds did not show clinical signs or histopathological alterations in air sac biopsies related to the infection.     

Specific detection and typing of Mycoplasma synoviae strains in poultry with PCR and DNA sequence analysis targeting the hemagglutinin encoding gene vlhA

Mycoplasma synoviae is a major pathogen of chickens and turkeys, causing economic losses to the poultry industry worldwide. In this study, we validated and applied polymerase chain reaction (PCR) and DNA sequence analysis on the N-terminal end of the hemagglutinin encoding gene vlhA as an alternative for the detection and initial typing of field strains of M. synoviae in commercial poultry. PCR primers were tested against isolates of M. synoviae from various sources along with other avian mycoplasma and other bacterial species. The vlhA gene-targeted PCR assay was highly specific in the identification of M. synoviae, with a detection limit of 4.7 x 10(2) color changing units/ml. DNA sequence analysis of amplified products was also conducted to validate the potential for typing M. synoviae strains using the N-terminal region of the vlhA gene. To evaluate the test, we applied the PCR assay to tracheal swabs collected from chickens challenged with M. synoviae strain K1968 and compared the results to the serologic detection. The PCR assay was also evaluated directly on tracheal samples collected from commercial layers. Overall, this vlhA gene-targeted PCR is a useful tool for detection and initial typing of M. synoviae and can be applied in the preliminary identification of M. synoviae isolates directly from clinical samples.     

Real-time polymerase chain reaction testing for the detection of Mycobacterium genavense and Mycobacterium avium complex species in avian samples

Diagnosis of avian mycobacteriosis, caused by Mycobacterium genavense or species belonging to the Mycobacterium avium complex (MAC), is problematic. Polymerase chain reaction (PCR) offers rapid and sensitive detection of minute quantities of DNA, and conventional protocols have been used for evaluating avian specimens. The recent development of real-time PCR offers several advantages over conventional PCR. In attempts to improve diagnosing avian mycobacteriosis, a real-time TaqMan PCR assay was developed targeting the 65-kD heat shock protein gene of M. genavense and MAC spp. Nineteen reference isolates, 16 clinical isolates, and 32 avian tissue samples were used to evaluate the assay. When sufficient amplicons were produced, the species of mycobacteria was determined by standard sequencing of TaqMan PCR products and compared with results from commercial mycobacteriology laboratories and/or standard sequencing of conventional PCR products. The TaqMan PCR detected DNA from reference isolates of M. genavense, MAC spp., and Mycobacterium tuberculosis complex spp. Of the clinical isolates, the TaqMan PCR detected DNA from 10 of 12 Mycobacterium avium avium isolates and two of three Mycobacterium avium intracellulare isolates. For the tissue samples, the TaqMan PCR amplified DNA in six of nine samples that were identified by sequencing of conventional PCR products and/or by commercial mycobacteriology laboratories as being MAC spp. positive and three of four samples that were positive for M. genavense. There was some disagreement between speciation results from the TaqMan PCR and those from commercial mycobacteriology laboratories or conventional PCR or both. This disagreement was suspected to be because of relatively small numbers of base pairs in the TaqMan PCR products. The TaqMan PCR may provide a useful tool for evaluating clinical samples for DNA from mycobacteria species that most commonly infect birds; however, further refinement is needed in order to improve sensitivity and provide more accurate speciation.     

Evaluation and comparison of various PCR methods for detection of Mycoplasma gallisepticum infection in chickens

Four genetic Mycoplasma gallisepticum (MG) polymerase chain reactions (PCRs) (16s rRNA PCR, three newly developed PCR methods that target surface protein genes [mgc2, LP (nested) and gapA (nested)]) were compared for analytical specificity and sensitivity and for diagnostic sensitivity (Se) and specificity of detection from tracheal swabs. The licensed MG DNA Test Kit Flock Chek test (IDEXX, Laboratories, Inc., Westbrook, ME) was as well evaluated for the diagnostic specificity and sensitivity of detection from tracheal swabs. Analytical specificity was evaluated for the four generic PCR methods using a panel of DNA samples from microorganisms that may be isolated from the trachea of commercial poultry and other fowl. PCR methods mgc2, nLP, and ngapA only amplified DNA from MG, whereas 16S rRNA PCR amplified DNA from MG and Mycoplasma imitans. The analytical sensitivity of the four generic PCR methods expressed in color-changing units (CCU)/amplification reaction was estimated for each PCR method and ranged from 4 to 400 CCU/reaction; the sensitivities of single PCR methods 16S rRNA and mgc2 were estimated at 40 CCU/reaction, the nLP at 400 CCU/reaction, and the ngapA at 4 CCU/reaction. The diagnostic sensitivity and specificity of MG detection from tracheal swab pools, as compared to isolation from choanal cleft swabs, was evaluated for the five PCR methods using three groups of birds exposed to vaccine strains ts-11 and 6/85 and to challenge strain R. All PCR methods were able to detect the vaccine strains and the challenge strain R directly from tracheal swabs, indicating that PCR primers from the different methods amplified divergent MG strains. Isolation and PCR results correlated satisfactorily among the three experimentally infected groups, with agreement values (k) ranging from 0.52 to 1.00. The ngapA, IDEXX, and mgc2 PCRs showed the best sensitivity (Se) ratios for detection of M. gallisepticum strains as compared to isolation. Compared to the ngapA and IDEXX PCR methods, the mgc2 PCR has a faster turnaround time, since this test consists of a single amplification reaction and the amplification product is detected by gel electrophoresis. Therefore, among the PCR methods evaluated in this study, the mgc2 PCR is the method of choice to further validate in the field.     

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene. METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay. RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 104 cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%. CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

A survey of avian Mycoplasma species for neuraminidase enzymatic activity

Among 23 currently recognized avian Mycoplasma (AM) species only Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis and Mycoplasma iowae cause disease and loss of production in chickens and/or turkeys. Because neuraminidases are considered virulence factors in many pathogenic microorganisms the aim of our study was to determine which AM species possess neuraminidase enzymatic activity (NEAC). Small samples of AM cells were assayed for NEAC using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-alpha-d-N-acetylneuraminic acid. In the case of positive NEAC reaction the substrate gave the insoluble indigoblue product what enabled simple test and easy estimation of NEAC. M. gallisepticum and M. synoviae which share sequences of the gene encoding neuraminidase (sialidase NanH) exhibited considerable levels of NEAC. However, NEAC levels differed among their strains, as well as among cultures of different strains. Only certain cultures of the type strain of M. meleagridis showed NEAC, whereas among six serovars of M. iowae only serovar I (type strain 695) showed NEAC. Weak NEAC was detectable in M. anseris, M. cloacale and M. pullorum, whereas the type strain of M. corogypsi (BV1) showed strong NEAC. Our study provides novel informations about NEAC in AM species and suggests that higher invasiveness and possibly, the pathological processes might be associated with their NEAC.     

Real-time polymerase chain reaction assays for the detection of members of the Mycoplasma mycoides cluster

AIM: To develop real-time PCR assays for the detection and differentiation of members of the Mycoplasma mycoides cluster. METHODS: Five real-time PCR assays were designed to allow differentiation of members of the M. mycoides cluster: an assay for detection of the M. mycoides subspecies, viz M. mycoides subsp mycoides large colony (MmmLC), M. mycoides subsp capri (Mmc), and M. mycoides subsp mycoides small colony (MmmSC); one for the detection of the M. capricolum subspecies, viz M. capricolum subsp capricolum (Mcc), M. capricolum subsp capripneumoniae (Mccp), and Mycoplasma sp bovine group 7 (BG7); and three for the specific detection of MmmSC, Mccp, and BG7. A panel of 74 Mycoplasma isolates from various geographical origins and a panel of 21 other bacterial isolates were used to evaluate the sensitivity and specificity of the assays. RESULTS: The assays displayed 100% analytical sensitivity in detecting all target Mycoplasma isolates. The analytical detection limit for the assays to detect the M. mycoides subspecies, M. capricolum subspecies, and MmmSC was determined to be 100 fg of genomic DNA, while the Mccp and BG7 assays had a detection limit of 100 fg and 10 fg of genomic DNA, respectively. The M. mycoides subspecies assay had a detection limit of 10(3) (SD 10(2)) cfu/ml milk, 10(4) (SD 10(4)) cfu per swab, and 10(3) (SD 10(3)) cfu/g lung in inoculated samples. The assays displayed 100% specificity when applied to non-target bacterial isolates and to 110 culture-negative milk samples. CONCLUSIONS: The assays were highly sensitive and specific, and provide accurate detection and differentiation of the members of the M. mycoides cluster.     

Experimental evidence of indirect transmission of Mycoplasma synoviae

The aim of the study was to analyse experimental transmission of Mycoplasma synoviae, an avian pathogen. Three experiments using specific pathogen-free day-old chicks placed in isolators were conducted. In the first experiment, the birds were introduced in an isolator previously contaminated with a M. synoviae broth culture. After 34 days, these birds were eliminated and, for the second trial, the chicks were introduced in the same isolator without disinfecting. In the third assay, the chicks were placed in an isolator containing a mixture of food, feathers and dust collected less than an hour earlier from a M. synoviae infected laying hen flock. In the second and third experiments in order to exacerbate the M. synoviae infection, the birds were inoculated with infectious bronchitis (IB) virus. The presence of M. synoviae in the environment and in tracheal swabs was monitored by culture, a multiplex PCR (mPCR) detecting M. synoviae and Mycoplasma 16S rDNA and a multiplex RT-PCR (mRT-PCR) detecting the M. synoviae mRNA coding for a membrane protein and Mycoplasma 16S rRNA. In in vitro experimental conditions, M. synoviae mRNA and 16S rRNA were detected up to 20 min and 23 h respectively after mycoplasma death. In the first assay, the first infected bird was detected on the 13th day. In the second trial, culturable M. synoviae or viable M. synoviae were detected in the isolator for 3 or 4 to 5 days respectively after depopulation of the birds of the first assay whereas the first culture positive tracheal swabs were detected on the 33rd day, after IB inoculation. In the third experiment, the first infected birds were detected on the 54th day. Thus, the different assays showed that M. synoviae contaminated material (dust, feathers and food) can infect chicks, sometimes after remarkably long silent periods.     

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene.

METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay.

RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 10⁴ cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%.

CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

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.     

鸡毒支原体PCR检测方法的建立

根据已发表的鸡毒支原体种特异性序列fMG-2设计1对引物,建立检测鸡毒支原体的PCR方法.该法对鸡毒支原体能特异性扩增726 bp的目的片段,而对其他禽病原DNA模板的扩增结果为阴性.建立的PCR方法对鸡毒支原体的最少检出量为3 Pg.用建立的PCR方法对临床采集的样品进行检测,同时对相应的样品进行细菌分离,结果临床样品PCR的阳性检出率为20.5%,细菌分离培养的阳性率为0.9%,表明PCR的敏感性高于细菌分离鉴定.     

Detection and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the 16S rRNA gene.

A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed for direct detection and identification of this pathogen from clinical material. DNA from eye swabs was amplified after a simple lysis step by either a single PCR with the M. conjunctivae specific primer pair McoR1 and McoF1, or by a nested PCR with the Mycoplasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step and McoR1 and McoF1 in the second step. The specificity of the primer pair McoR1 and McoF1 was verified with purified DNA from the type strain, from 17 field isolates of M. conjunctivae and from several Mollicutes which are phylogenetically related to M. conjunctivae or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat, sheep, ibex and chamois originating from different countries as M. conjunctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eye swab samples containing known numbers of M. conjunctivae cells were analysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 x 10(5) by the single PCR method. In an experimental infection model of sheep, the nested PCR method for detection of M. conjunctivae gave results which were comparable to mycoplasmal culture. These are the implications for diagnostic purposes: M. conjunctivae isolates can be identified by the one-step PCR method, whereas for detection and identification of M. conjunctivae in clinical material the two-step method should be used (higher sensitivity).     

鸡毒支原体PCR检测试剂盒的研制与应用

根据基因库中鸡毒支原体 1 6SrRNA的序列研制PCR检测试剂盒 ,用于检测鸡毒支原体 (MG)。结果表明该MG PCR检测试剂盒对不同MG参考菌株和地方分离株均能特异性地扩增出 732bp条带 ,而对其他禽支原体和禽病病原体的扩增结果为阴性。该MG PCR试剂盒最低能检测出 1 0 0fg的MGDNA模板。保存期测定结果表明 ,该MG PCR试剂盒在 - 2 0℃条件下保存至 1 ,3 ,6和 9个月时 ,其敏感性无明显变化 ,仍能检测到 1 0 0fg至 1pg的MGDNA模板。保存至 1 2个月时其敏感度虽降低了 1个滴度 ,但仍能 1 0 0 %检出人工感染鸡的临床样品     

Genotyping of Japanese field isolates of Mycoplasma synoviae and rapid molecular differentiation from the MS-H vaccine strain

Mycoplasma synoviae is an important causative agent of avian mycoplasmosis. In the present study the conserved domain of the variable lipoprotein and hemagglutinin (vlhA) gene of M. synoviae was sequenced and analyzed for 19 field strains of M. synoviae isolated from chickens across Japan. This analysis revealed that there were at least nine genotypes of M. synoviae present in Japan. Furthermore, we found a single nucleotide polymorphism (SNP) within this region in all the Japanese isolates, and based on this finding, we established a PCR method with cycling probe technology to differentiate between these field isolates and the live M. synoviae vaccine strain Mycoplasma synoviae-H (MS-H).     

Development of a polymerase chain reaction for the detection of Mycoplasma felis in domestic cats

Mycoplasma felis is associated with conjunctivitis and respiratory disease in domestic cats. Currently no rapid diagnostic test is available for the detection of M. felis in clinical samples that does not rely on prior cultivation of the organism. The objective of this study was to determine whether a polymerase chain reaction (PCR) based upon the 16S/23S rRNA intergenic spacer sequence is suitable for the identification of M. felis directly in feline clinical samples. The high conservation between the 16S/23S rRNA intergenic spacers (IGS) of differing isolates of M. felis was established by sequence analysis and a PCR was developed to this region by comparison to IGS of other mycoplasmas. The PCR was found to be highly specific for M. felis and further PCR analysis on clinical samples showed the PCR to be highly sensitive and more rapid than the other methods of identification currently available.     

致犊牛肺炎牛支原体南疆株的分离鉴定及oppF基因序列分析

本研究旨在调查新疆喀什某规模化奶牛场的犊牛死亡原因,并确定病原体.无菌采集3份因肺炎死亡的犊牛肺脏病料样品.采用牛支原体专用液体培养基和1.0%牛支原体琼脂固体筛选培养基从3份病死犊牛肺脏病料中分离得到2株牛支原体(Mycoplasma bovis,M.bovis),分别命名为M.bovis-NJ-1和M.bovis-NJ-2.通过菌落形态学观察、特异性PCR和oppF测序比对对分离株进行鉴定.结果显示,2个分离株在固体培养基上的菌落呈现典型的"煎蛋状",且Dienes染色特点符合牛支原体菌落着色特征,中心呈深蓝色;PCR能扩增出牛支原体特异的448 bp目的片段;2个分离株的oppF基因序列与牛支原体国际标准株PG45的同源性分别为96.7%和95.3%.结果表明,引起犊牛发病死亡的病原是牛支原体,本研究为犊牛支原体肺炎的快速诊断和防制提供依据.     

Molecular detection of Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' Infection in cats by direct PCR using whole blood without DNA extraction

Detection of hemotropic Mycoplasma spp. infection was attempted in cats by PCR using whole blood without DNA extraction. A total 46 of 54 (85%) cats with suspected Mycoplasma spp. infection showed a positive reaction, corresponding completely with the results of standard PCR testing. The direct PCR assay was sensitive enough to detect more than 0.0061% parasitemia for ;C. M. haemominutum' and 0.0075% parasitemia for M. haemofelis. These data indicate that the direct PCR assay might be sufficient for use as a tool in clinical examinations.     

Revised Mycoplasma synoviae vlhA PCRs

Mycoplasma synoviae (MS) is an important pathogen of chickens and turkeys. In recent years sequence analysis of the partial MS variable lipoprotein and hemagglutinin A (vlhA) gene PCR product has been utilized routinely for MS strain genotyping. Several PCR assays have been proposed for the amplification of the conserved upstream region of the MS vlhA gene; however, in several clinical instances the published assays failed to generate vlhA PCR products from confirmed MS-positive cases. These occurrences hindered our capability to genotype those cases. In silico analysis of the published MS vlhA PCRs raised concerns, which were addressed by the design of revised MS vlhA PCRs. The published and revised assays were tested for their relative sensitivity and specificity with laboratory and clinical MS-positive samples. One of the revised MS vlhA PCRs (revised Hong) was demonstrated to be more sensitive and specific, and amplified all clinical samples analyzed in this study.     

Polymerase chain reaction with a primer pair in the 16S-23S rRNA spacer region for detection of Mycoplasma pulmonis in clinical isolates

To develop a diagnostic tool to identify Mycoplasma pulmonis (M. pulmonis) in clinical isolates, we developed a polymerase chain reaction (PCR) assay using primers specific for the 16S-23S rRNA intergenic spacer region (SR) of M. pulmonis. One pair of PCR primers reacted specifically with two reference strains of M. pulmonis tested and seven samples isolated from naturally infected rats. The primer pair did not produce PCR products of the correct size from any other rodent or human mycoplasmas or cellular DNA from rodent lungs. Specificity of the PCR assay was confirmed by Southern blotting with probe specific for the SR of M. pulmonis. The PCR assay for detection of M. pulmonis established in this study is suitable for diagnosis of M. pulmonis infection in clinical cases.     

Mycoplasma iowae species-specific DNA probe

A Mycoplasma iowae (MI) species-specific DNA probe (designated pMI-2) of 6.0 kbp (kilobase pairs) was isolated from an MI strain I-695 genomic library prepared in plasmid pUC8 and Escherichia coli strain JM83. When labeled with [32]P by nick translation, the probe hybridized in dot blot assays with 6 reference strains and 8 field isolates of MI but not with 16 other known species of avian mycoplasmas. The pMI-2 probe detected a minimum of 1.5 ng of MI strain I-695 chromosomal DNA. Under identical conditions of hybridization, the probe did not hybridize with a high concentration (200 ng) of M. gallisepticum or M. synoviae chromosomal DNA.