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).     

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.     

Ribosomal RNA gene probes to detect intraspecies heterogeneity in Mycoplasma gallisepticum and M. synoviae

Intraspecies genotypic heterogeneity among strains of Mycoplasma gallisepticum and M. synoviae was tested using genomic fingerprints with a ribosomal RNA (rRNA) gene probe. The organism's DNA was digested by a restriction endonuclease, electrophoresed, transferred to a nitrocellulose sheet, and hybridized with 32P-labeled pMC5 plasmid carrying the highly conserved rRNA genes of M. capricolum. The resulting hybridization patterns indicated a degree of genotypic heterogeneity among M. gallisepticum strains more pronounced than among the M. synoviae strains tested. Most importantly, the live vaccine F strain of M. gallisepticum could be distinguished from virulent field isolates of this species, enabling the detection and identification of the F strain in areas in which vaccination with this strain has taken place. Genomic fingerprints with an rRNA gene probe can thus be added to the battery of tools useful in taxonomy at the intraspecies level and in epidemiology of mycoplasmosis in poultry.     

鸡滑液囊支原体的分离鉴定及病理组织学观察

为了解宁夏及其周边不同地区蛋鸡场中的鸡滑液囊支原体的种类及其致病力,采用改良Frey氏鸡滑液囊支原体培养基,从疑似感染鸡滑液囊支原体的不同蛋鸡群中分离出病原株,设计鸡滑液囊支原体vlhA基因特异性引物,对分离到的病原进行基因序列扩增并测序,使用MEGA6.0软件中的邻接法(Neighbor-joining,NJ)构建分离株系统发育树并进行遗传进化分析,采用从临床症状最明显的鸡体分离的菌株进行动物感染试验,制作石蜡切片,HE染色,病理组织学观察。结果表明,所分离到的病原菌均为鸡滑液囊支原体,部分菌株之间极其相似;鸡滑液囊支原体不仅可以使鸡只关节肿胀、生长缓慢,也可引起鸡只的肝脏轻微肿大,肝细胞部分坏死、间质增生;脾脏结缔组织增生,出血。     

2016-2019年我国部分地区鸡滑液囊支原体分离株vlhA基因遗传进化分析

为了解我国鸡滑液囊支原体(Mycoplasmasynoviae,MS)感染的最新流行情况,本研究于2016-2019年从江苏、安徽、山东、河南、河北、宁夏、黑龙江、湖北等8个省份疑似发生MS感染的鸡场采集样品,进行MS菌株的分离鉴定,结果共获得48株MS分离株.对这些分离株的vlhA基因片段测序和分析显示,其中46株均...     

The humoral immune response of chickens to Mycoplasma gallisepticum and Mycoplasma synoviae studied by immunoblotting

The humoral immune response over time of White Leghorn chickens experimentally infected with Mycoplasma gallisepticum or M. synoviae by an aerosol inoculation or a contact exposure were compared by immunoblotting. The response of chickens infected with M. gallisepticum were similar with respect to proteins recognized and intensity of response, regardless of mode of infection. On the other hand, chickens infected by aerosolization of M. synoviae responded to more proteins and with greater intensity than did M. synoviae contact-exposed birds. Chickens infected with M. gallisepticum responded with antibodies to over 20 proteins, while chickens infected with M. synoviae responded with antibodies to 12 proteins. Field sera from chickens naturally infected on commercial poultry farms with M. gallisepticum or M. synoviae were analyzed by immunoblotting and were found to react with a number of mycoplasma proteins. However, no correlation was seen when comparing intensity of immunoblot staining and hemagglutination-inhibition titer of the field sera. The experimental antisera were used to identify species-specific proteins of M. gallisepticum and M. synoviae. Six immunogenic species-specific proteins of M. gallisepticum with relative molecular masses of 82 (p82), 65-63 (p64), 56 (p56), 35 (p35), 26 (p26), and 24 (p24) kilodaltons (kDa) were identified. Two species-specific proteins of M. synoviae with relative molecular masses of 53 (p53) and 22 (p22) kDa were identified. Additionally, a highly immunogenic 41 (p41) kDa protein of M. synoviae was identified. Species-specific proteins identified in these mycoplasmas and the 41 kDa protein of M. synoviae were purified by preparative SDS-PAGE in amounts sufficient for further characterization and for use in serodiagnostic tests.     

Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus samples

PCR assay has been shown to be a promising option for the diagnosis of brucellosis. However, few studies have been performed with field samples in order to evaluate the assay as a diagnostic tool. In this study, routine use of a species-specific PCR assay previously developed for the identification of Brucella cultures was assessed for the detection of Brucella DNA directly from the stomach contents of aborted sheep fetuses. The assay is based on the insertion sequence IS711 in the Brucella chromosome. In the study, during 3 successive lambing seasons (1998-1999, 1999-2000 and 2000-2001) 126 aborted fetus samples each from different flocks and locations were examined. Brucella strains were isolated from 39 (31%) of the samples and all of the strains were identified as Brucella melitensis by biochemical characteristics, agglutination with monospecific A and M sera and PCR. Thirty-seven of 39 B. melitensis isolates were biotyped as biotype 3, and 2 isolates as biotype 1. From 38 of 39 culture positive fetal stomach contents B. melitensis-specific DNA was detected by PCR. PCR was found negative in all of the culture negative samples. Compared with culture, sensitivity and specificity of PCR were determined as 97.4 and 100%, respectively. The results indicate that this PCR procedure has a potential for use in routine diagnosis of sheep brucellosis.     

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.     

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 major immunogenic proteins of Mycoplasma synoviae isolates

Mycoplasma synoviae isolates differ in patterns of immunogenic proteins, but most of them have not been identified yet. The main aim of this study was their identification in two closely related M. synoviae isolates, ULB 02/P4 and ULB 02/OV6, recovered recently from chickens in Slovenia. N-terminal sequencing identified 17 M. synoviae proteins. Amongst them were 14 major, highly expressed but previously unidentified proteins, including enzymes, chaperones and putative lipoproteins. ULB 02/P4 proteins with increasing molecular weight (M(w)) in the region above the lipoprotein MSPB (approximately 40 kDa) were elongation factor EF-Tu, enolase, NADH oxidase, haemagglutinin MSPA, ATP synthase beta chain, trigger factor, pyruvate kinase and chaperone DnaK. Enolase (approximately 47 kDa) seemed to be immunogenic for chickens infected with M. synoviae, whereas EF-Tu, which might cross-react with antibodies to the P1 adhesin of Mycoplasma pneumoniae, was not. ULB 02/OV6 synthesized several immunogenic proteins and those with M(w) of approximately 70, 78, 82, 90, 110 and 160 kDa, cross-reacted with antibodies to Mycoplasma gallisepticum. They remain to be identified, because besides putative lipoproteins, protein bands of 78, 82, 85 and 110 kDa contained also dehydrogenase PdhD, elongation factor EF-G, enzyme PtsG and putative neuraminidase, respectively.     

In vitro susceptibilities to fluoroquinolones in current and archived Mycoplasma gallisepticum and Mycoplasma synoviae isolates from meat-type turkeys

Monitoring of susceptibility to antibiotics in field isolates of pathogenic avian mycoplasmas is important for appropriate choice of treatment. Our study compared in vitro susceptibility to enrofloxacin and difloxacin in recent (2005-2006) isolates of Mycoplasma gallisepticum and Mycoplasma synoviae from meat-type turkey flocks with archived (1997-2003) isolates and reference strains. Comparison of minimal inhibitory concentration (MIC) values determined by microtest, agar dilution and commercial Etest showed good agreement, but underscored the need for standardized methods for testing. Notably, while the commercial Etest was convenient and accurate for determining MICs for enrofloxacin in the range 0.002-0.094mug/ml, the endpoint of inhibition for M. gallisepticum and M. synoviae strains with MIC values >/=1.0mug/ml could not be determined. A decrease in susceptibility to both fluoroquinolones was detected in archived strains but to a greater degree in recent isolates, most of which had MICs above the NCCLS susceptibility breakpoint for these antibiotics (相似文献   

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.     

Characterization of the antigenic and functional domains of a Mycoplasma synoviae variant vlhA gene

The Mycoplasma synoviae haemagglutinin gene, vlhA, encodes two major immunodominant and surface-exposed membrane proteins, MSPB and MSPA. Both products are antigenically variable but only MSPA mediates binding to erythrocytes. Previously we have shown that M. synoviae type strain WVU 1853 could express a variant vlhA gene, referred to as MS2/28.1, with a considerably shorter and divergent MSPA region. A finding that prompted detailed characterization of its antigenic and functional properties. Here we mutagenized each of the six opal codons of the variant MS2/28.1 vlhA member into tryptophan, thus allowing its expression in Escherichia coli as well as its cleavage products, MSPB and MSPA. In addition, we expressed 5 contiguous regions of MS2/28.1 extending from the last part of MSPB to the C-terminus of MSPA. Colony immunostaining with region-specific antisera mapped antigenic variation to the N-terminal half of MS2/28.1 MSPA. No haemagglutinating activity was observed for MSPB, but consistent haemadsorption inhibition was mapped to the region extending from amino acid 325 to 344. Inhibition of both haemagglutination and haemadsorption activities were obtained with sera directed against the C-terminal region of MSPA, with the highest titers (1/320 and 1/160, respectively) being recorded for its last 60 residues. Importantly, antibodies to this region also yielded the highest metabolic inhibition titer of 1/1280. Overall, aside from mapping the functional domains of a M. synoviae highly divergent haemagglutinin gene, this study shows that the C-terminal half of its MSPA region induced the highest titers of antibodies inhibiting haemagglutination, haemadsorption, and metabolism.     

7型猪链球菌的分离鉴定及特性

通过生化鉴定和PCR方法从湖北省部分地区高热症猪病料中分离鉴定了17株7型猪链球菌,并对所分离菌株进行药敏、毒力基因分布及致病性等生物学特性的研究。结果表明,生化鉴定显示所分离菌株为猪链球菌2型,但经PCR鉴定为7型,测序结果经分析与已发表的7型序列同源性为100%。17株7型分离株毒力基因分布情况为：0.0%为mrp＋,11.8%为epf＋,17.6%为sly＋orf2＋,29.4%fbps＋,100.0%gdh＋。通过对28种药物的敏感试验发现各分离株耐药性差别很大,但对阿莫西林和复方阿莫西林较为敏感（88.2%）,对红霉素最为敏感（100.0%）。动物试验表明,编号为HB6、HB8、HB9的分离株致病性较强。通过对病猪和感染小鼠进行病理切片观察,各器官充血出血最为明显。总之,在以高致病性蓝耳病病毒为主要病原的高热症诊断和治疗中,同样需要考虑混合或继发感染7型猪链球菌致病的可能。     

Differential identification of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum based on polymorphic regions of glgC and speC genes

Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum cause fowl typhoid and pullorum disease in avian species, respectively, and have been of considerable economic importance to the poultry industry in parts of the world. The definitive diagnosis of these diseases can be made only by isolation and identification of the causative agent. However, rapid identification of biovars Gallinarum and Pullorum is not easily feasible due to their common antigenic structure and genomic sequence similarity. We developed a duplex polymerase chain reaction (PCR) assay to identify and discriminate between strains of biovars Gallinarum and Pullorum. Duplex PCR primers were designed to target polymorphic regions of glgC and speC genes showing multiple mutations in the sequenced S. enterica subsp. enterica serovar Gallinarum 287/91 genome and were applied to the specific identification of biovars Gallinarum and Pullorum. Boiled lysates of 131 reference and field strains of Salmonella and other related Gram-negative bacteria were tested to validate the duplex PCR assay. All strains of biovars Gallinarum (n=53) and Pullorum (n=21) tested were correctly identified based on this assay (100% sensitivity) while the other strains (n=57) were PCR negative (100% specificity). These results demonstrate that a highly accurate biovar-specific duplex PCR assay can be performed for the rapid identification and discrimination of biovars Gallinarum and Pullorum from field isolates.     

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.     

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.     

An allele-specific PCR assay for the rapid and serotype-specific detection of Salmonella pullorum

Salmonella serovar Pullorum is a causative agent of pullorum disease (PD) in poultry and is responsible for severe economic losses to the poultry industry in many parts of the world. A definitive detection of Pullorum requires culture followed by serotyping and biochemical identification, a process that is tedious and takes several weeks to accomplish. We have developed a rapid allele-specific polymerase chain reaction (PCR) method based on the nucleotide polymorphism in rfbS gene sequence for the serotype-specific detection of Pullorum and its differentiation from the closely related Gallinarum. The specificity of this PCR assay was tested using DNA samples from Pullorum (n = 13), Salmonella serotypes other than Pullorum (n = 19), and closely related non-Salmonella organisms (n = 5). The PCR assay was highly serotype-specific as the PCR amplicon of 147 base pairs was observed only in the case of Pullorum, while all the other DNA samples tested PCR negative. A definitive identification of Pullorum cultures was possible in less than 3 hr. As little as 100 pg of SP DNA was detected. This allele-specific PCR method is highly specific as well as sensitive and may be an effective molecular tool in the rapid and serotype-specific detection of Pullorum and differentiation from other Salmonella species.     

Detection and characterization of avian leukosis virus in Marek's disease vaccines

Avian leukosis virus (ALV) infection in chickens is known to induce increased mortality, tumors, delayed growth, and suboptimal egg production. Countries importing specified pathogen-free eggs, vaccines, and poultry breeding stock require freedom of infection or contamination with ALV in such products among other avian pathogens. Recently, ALV was found as a contaminant in a limited number of commercial poultry vaccines, even after routine quality assurance procedures cleared the vaccines for commercialization. The contaminated vaccines were promptly withdrawn from the market, and no direct detrimental effects were reported in poultry vaccinated with such vaccines. We describe herein the characterization in vitro of the contaminant viruses. All exogenous viruses detected in four vaccine lots belong to subgroup A of ALV based on cell receptor interaction, subgroup-specific polymerase chain reaction (PCR), envelope gene sequencing, and virus neutralization. A combination of thermal treatment and serial dilutions of the contaminated vaccines facilitated detection of contaminating ALVs in cell culture coupled with antigen-capture enzyme-linked immunosorbent assay. Subgroup-specific PCR readily detected ALV-A directly in the contaminated vaccines but not in naive vaccines or cell controls. Our methods are proposed as complementary procedures to the currently required complement fixation for avian leukosis test for detection of ALV in commercial poultry vaccines.