Pathogenicity of two strains of Mycoplasma gallisepticum in broilers

Strains F and R of Mycoplasma gallisepticum (MG) were compared in two laboratory trials for their relative pathogenicity in terms of inducing airsacculitis and antibody production to MG. Chickens exposed to the R strain had significantly higher incidence of air-sac lesions (P less than 0.05) and greater severity of airsacculitis than did chicks exposed to the F strain. In both trials, chickens vaccinated simultaneously with Newcastle disease-infectious bronchitis vaccine and exposed to MG had more severe lesions than did chickens exposed to mycoplasma alone. chickens exposed to the F strain had significantly lower geometric mean hemagglutination-inhibition antibody titers to MG than did chicks exposed to the R strain. Chickens vaccinated simultaneously with Newcastle disease-infectious bronchitis vaccine and exposed to R strain had significantly lower body weights than did chickens in the other group.     

Differentiation of Mycoplasma gallisepticum vaccine strains ts-11 and 6/85 from commonly used Mycoplasma gallisepticum challenge strains by PCR

Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economic losses within the poultry industry. In an effort to develop tools to aid in MG research and diagnostics, we have compared sequences of the attenuated MG vaccine strain ts-11 to those of commonly used pathogenic challenge strains in search of a simple means of differentiation. Via gapA sequence alignments and comparisons, we have identified and designed primers facilitating strain differentiation. When applied to conventional polymerase chain reaction (PCR) assay at low annealing temperature, the primer sets allow for the differentiation of MG attenuated vaccine strains ts-11 as well as the attenuated MG vaccine strain 6/85 from the commonly utilized MG challenge strains R(low), R, and S6. Conventional PCR differentiation is based on the visualization of sole products with the attenuated MG strains ts-11 and 6/85 and the lack of the corresponding products from MG strains R(low), R, and S6. When applied to MG strain F, product visualization varies with the applied primer set. The differentiation of MG strains ts-11 and 6/85 from the pathogenic challenge strains was also accomplished via real-time analyses, however, the primer sets were not able to differentiate MG strains ts-11 and 6/85 from selected MG field isolates.     

Glycoconjugate heterogeneity among five strains of Mycoplasma gallisepticum

Five strains of Mycoplasma gallisepticum (MG) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis for the presence of carbohydrate-containing components. Staining with periodic acid-Schiff (PAS) demonstrated carbohydrate components in three of the five strains studied. The PAS-reactive bands counterstained for protein, indicating a possible glycoprotein nature. Western blot analysis using three biotinylated lectin probes demonstrated the presence of additional glycoconjugates in the blot profiles of each MG strain. The carbohydrate specificity of lectin binding was demonstrated by competition experiments using specific sugars. Differences in the number, electrophoretic mobility and the morphology of PAS and lectin reactive bands were reproducible among separate preparations of each MG strain. These findings indicate substantial phenotypic diversity among the five MG strains in their ability to produce or acquire glycoconjugates.     

鸡毒支原体敏感株与耐药株超微结构的比较

对临床分离和实验室药物压力下筛选的鸡毒支原体耐药株与敏感株进行超微结构的观察和比较.结果显示鸡毒支原体敏感株呈多形性,柔软且有较大的变形性,可清晰观察到细胞膜分为外、中、内三层膜,并可观察到裂殖繁殖方式;有的支原体在繁殖时先在极端产生泡状突起,形成不均等分裂.在恩诺沙星药物压力下敏感株产生耐药性后其外膜显著增厚,导致支原体的多形性减弱或消失,呈现出较为一致的圆形;细胞膜内层周围存在排列整齐、结构紧密、类似微管样的结构,胞内电子密度明显升高.临床分离的耐药株超微结构观察结果与实验室条件下筛选的耐药株一致:凡是超微结构发生变化的,均存在耐药表型,而且高水平耐药株的超微结构变化最为突出.研究结果表明耐药性的产生可导致鸡毒支原体超微结构明显的改变,并可能引起抗原性变异.     

Characterisation of Mycoplasma gallisepticum strains involved in respiratory disease in pheasants and peafowl

Two cases of Mycoplasma gallisepticum infection in different avian species in backyard gamebird operations in Slovenia were investigated. In the first case, M gallisepticum was associated with severe respiratory disease with almost 20 per cent mortality in pheasants, whereas the infection was less pathogenic for chickens and turkeys reared at the same site. The M gallisepticum isolates from pheasants had a unique pMGA gene sequence containing a repeat of 12 nucleotides, and they contained only small amounts of the cytadhesins MGC1 and MGC3 and no PvpA protein. However, they expressed some typical M gallisepticum proteins and several proteins which were immunogenic for pheasants, chickens and turkeys. A strain of M gallisepticum isolated from the sinus of a pheasant was highly pathogenic for chicken embryos. In the second case, the M gallisepticum strain that was associated with respiratory disease and mortality in peafowl also affected chickens. M gallisepticum strain ULB 992 was isolated from the infraorbital sinus of a dead peafowl. The ULB 992 strain synthesised a small amount of MGC3, a truncated form of MGC1 and lacked PvpA. However, it expressed several proteins which were immunogenic for the birds infected with M gallisepticum at both gamebird operations.     

鸡败血支原体鸡传染性鼻炎二联灭活疫苗菌种复壮鉴定试验

从国外引进的鸡败血支原体R株和副鸡嗜血杆菌221株和668株的冻干品,经我所扩增复壮和系统的鉴定试验证明:鸡败血支原体R株属典型的强毒株,副鸡嗜血杆菌221和668株分别属典型的A型和C型强毒株.经过免疫原性试验、毒力鉴定试验及效力试验证明,这三种菌株是制备鸡败血支原体、鸡传染性鼻炎二联疫苗最好的菌株.     

An adhesion-hemadsorption inhibition test for the detection of serum antibody to Mycoplasma gallisepticum

A simple adhesion-hemadsorption inhibition (AHAI) test was developed for the detection of antibodies to Mycoplasma gallisepticum in the chicken sera. The AHAI antibody was detected simultaneously with HI antibody from sera of chickens intratracheally inoculated with viable cells of M. gallisepticum. A good correlation between HI and AHAI antibody titers was obtained with 382 (84.7%) of 451 sera from chickens reared on farms spontaneously contaminated with M. gallisepticum, whereas the remainder, 69 sera, was positive for HI but negative for AHAI test. It was not apparent whether the latters exhibited a non-specific reaction or the discrepancy was due to the lower sensitivity of AHAI reaction. The AHAI test does not require a great amount of antigen, special reagents or instruments, or pre-absorption treatment of test sera, and, therefore, it may serve as a simple serological test for detecting antibodies to M. gallisepticum.     

Detection of Mycoplasma gallisepticum by direct immunofluorescence using a species-specific monoclonal antibody

A monoclonal antibody against Mycoplasma gallisepticum (MG) (strain S6) was prepared in mice and identified as isotype IgG1 by standard procedures. Although it did react at high titers (1:100,000) in the enzyme-linked immunosorbent assay (the original method for its identification), it failed to react in the agglutination, hemagglutination-inhibition, and growth-inhibition tests. When conjugated to fluorescein isothiocyanate, the monoclonal antibody reacted with the homologous and eight "atypical" strains of MG but not with M. meleagridis or M. synoviae in the direct fluorescent-antibody test. This reagent may be useful for detecting field infections involving atypical strains of MG.     

鸡毒支原体强弱毒株荧光定量PCR鉴别检测方法的建立

为建立一种能鉴别鸡毒支原体(MG)强、弱毒株的快速检测方法,本研究根据GenBank中MG强毒株和弱毒株的基因组序列,选取特异性保守区序列设计了2对引物和2奈探针,分别用于强弱毒株和弱毒株的检测,优化反应条件,建立了能区分MG强、弱毒株的荧光定量PCR检测方法.该法特异性强,对鸡常见呼吸道病原体的反应均为阴性;灵敏度高,可检测到100拷贝/μL的模板;稳定性好,批内和批间试验Ct值的变异系数小.本研究建立的MG强、弱毒鉴别检测方法简便、快捷,为该病的防控与净化提供新方法、新思路.     

Characterization of in vivo-acquired resistance to macrolides of Mycoplasma gallisepticum strains isolated from poultry

ABSTRACT: The macrolide class of antibiotics, including tylosin and tilmicosin, is widely used in the veterinary field for prophylaxis and treatment of mycoplasmosis. In vitro susceptibility testing of 50 strains of M. gallisepticum isolated in Israel during the period 1997-2010 revealed that acquired resistance to tylosin as well as to tilmicosin was present in 50% of them. Moreover, 72% (13/18) of the strains isolated from clinical samples since 2006 showed acquired resistance to enrofloxacin, tylosin and tilmicosin. Molecular typing of the field isolates, performed by gene-target sequencing (GTS), detected 13 molecular types (I-XIII). Type II was the predominant type prior to 2006 whereas type X, first detected in 2008, is currently prevalent. All ten type X strains were resistant to both fluoroquinolones and macrolides, suggesting selective pressure leading to clonal dissemination of resistance. However, this was not a unique event since resistant strains with other GTS molecular types were also found. Concurrently, the molecular basis for macrolide resistance in M. gallisepticum was identified. Our results revealed a clear-cut correlation between single point mutations A2058G or A2059G in domain V of the gene encoding 23S rRNA (rrnA, MGA_01) and acquired macrolide resistance in M. gallisepticum. Indeed, all isolates with MIC ≥ 0.63 μg/mL to tylosin and with MIC ≥ 1.25 μg/mL to tilmicosin possess one of these mutations, suggesting an essential role in decreased susceptibility of M. gallisepticum to 16-membered macrolides.     

A historical account of the diagnosis and characterization of strains of Mycoplasma gallisepticum of low virulence

Numerous chicken flocks were studied beginning in 1970 because of questionable results on their serologic tests for Mycoplasma gallisepticum (MG). Typically a low number of hens in the flocks were positive reactors to the rapid serum plate test and rarely had hemagglutination-inhibition (HI) titers over 1:80. Usually no clinical signs were observed. Isolates of MG eventually were cultured from most of the flocks that exhibited that type of marginal serologic pattern. In the laboratory, the MG isolates were frequently less virulent and less pathogenic than the typical field isolates recovered in previous years. Most isolates produced airsacculitis of varying severity when broilers were exposed to the MG cultures as aerosols following exposure to infectious bronchitis virus. They became positive on the rapid serum plate test and developed moderate to high HI titers. Egg-transmission appeared to be the most likely means of transmission, even though the infected progeny rarely showed clinical signs of disease.     

Virulence and transmissibility of Mycoplasma gallisepticum

The virulence of 4 low passage strains of Mycoplasma gallisepticum obtained from different sources within Australia was studied by experimental infection of chickens. Strain Ap3AS, originally isolated from the air sac of a broiler chicken, produced severe air sac lesions following injection into the abdominal air sacs of 2-week or 3-week-old chickens, and adult hens. Strain 80083 which was isolated from a clinically normal broiler breeder hen was also capable of producing gross air sac lesions following intra-abdominal (IA) injection, although it did so less consistently than strain Ap3AS. Strain 82078 isolated from a layer hen and strain QXO which was isolated from a turkey were also moderately pathogenic in terms of the incidence and severity of lesions elicited following IA injection. Strains Ap3AS and 80083 both caused a substantial loss of egg production over a 5 week period after IA infection of 27-week-old hens. Neither strain Ap3AS nor 80083 caused gross lesions or loss of egg production when administered alone into the upper respiratory tract. However, when inoculated into the conjunctival sac in combination with the Vic S strain of infectious bronchitis virus (IBV) strains Ap3AS and 80083 produced identical clinical signs of conjunctivitis. The mean numbers of M. gallisepticum in tracheal washings were significantly higher 2 weeks after infection in the group receiving strain 80083 in combination with IBV than in the group infected with strain Ap3AS and IBV (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

壮观霉素对体外鸡毒支原体的抑菌试验和对鸡体内鸡毒支原体病的治疗试验

1　材料与方法1 .1　试验药物　壮观霉素为水溶性粉剂 ,批号940 71 2 ,由中国医学科学院医药生物技术研究所北抗药厂生产。对照药物壮观霉素 ,水溶性粉剂 ,由比利时生产 ,批号 573KS,由中国医学科学院医药生物技术研究所提供。1 .2　鸡毒支原体菌种　 R株、S6株 ,均由中国兽医药品监察所保存。1 .3　试验鸡　 9日龄北京白鸡 ,试验前经血清学检测 ,该鸡为鸡毒支原体血清学阴性反应。1 .4　鸡毒支原体血清平板凝集抗原　由中国兽医药品监察所生产 ,批号为 930 1。1 .5　抑菌试验　试验前将试验药壮观霉素用蒸馏水配成一定浓度的溶液过滤除菌…