Virulence of Mycoplasma synoviae strains in experimentally infected broiler chickens

Seven field isolates of German origin and the type strain WVU 1853 of Mycoplasma synoviae (MS) were experimentally investigated for their virulence in mycoplasma-free broiler chickens. Two groups of birds were inoculated at 6 days of age with each isolate, one group into the thoracic air sac and the other group intravenously and all surviving birds were examined at necropsy 17 days post inoculation (pi). Groups of negative control birds received sterile Frey's broth medium by intravenous and intra-air sac inoculation, respectively. Variation in virulence was evaluated on the basis of significant differences in incidence, severity and extend of MS-induced airsacculitis and synovitis as well as isolation rates of MS especially from parenchymous organs. All the strains tested were pathogenic but varied in their virulence for broiler chickens. Based on differences of the virulence, the isolates were classified to the categories: (1.) highly virulent, (2.) virulent, (3.) moderately virulent and (4.) slightly virulent. (1) Strains WVU 1853 and 246-91 induced a systemic disease associated with multiple synovitis and bilateral airsacculitis (2) Strains 93-92 and 151-77 induced bilateral airsacculitis similar to WVU 1853 and 246-91 but rarely a systemic disease after exposure by intra-thoracic airsac inoculation. (3) In comparison, strains 27-79, 76-93 and 513-83 caused less frequently airsacculitis and even if, then only at the side of intra-airsac exposure. (4) Strain 91-93 has been found to differ significantly from all the other isolates in its capacity to produce disease independently from the inoculation route. After intravenous inoculation, findings gave no indications for strains with selective tropism to the epithelial membranes of the lower respiratory tract or to those of the joints, tendon sheaths and bursae. However, the presented data of the experiments suggest that the MS strains tested differ in their potential capacity to invade systemically and produce acute septicaemia.     

Recombinant DNA probes for Mycoplasma synoviae

A genomic library was prepared from Mycoplasma synoviae (MS) strain WVU 1853 cloned in plasmid vector pUC8 and transformed in Escherichia coli host JM83. In dot blot assays, four transformed E. coli clones hybridized with 32P-labeled chromosomal DNA of MS but not with 32P-labeled chromosomal DNA of M. gallisepticum (MG) strain S6. In Southern hybridization, each of the CsCl-purified recombinant plasmid clones was shown to contain two MS DNA fragments between 1.0 to 2.3 kbp in length. 32P-Labeled probes prepared from each of the four recombinant plasmids hybridized in dot blot assays with MS strain WVU 1853 and nine MS field isolates but not with MG strains S6, K810, F2F10, four MG field isolates, and 15 other species of avian mycoplasmas.     

An enzyme-linked immunosorbent assay for the detection of antibodies to Mycoplasma gallisepticum in experimentally infected chickens

An indirect enzyme-linked immunosorbent assay (ELISA) was developed and tested for its ability to detect humoral response to Mycoplasma gallisepticum in chickens. Two antigens were used in the solid phase of the assay. Antigen 1 was a membrane-derived sodium dodecyl sulfate (SDS)-solubilized preparation; Antigen 2 was prepared in the same manner as Antigen 1 but was passed through an immunoadsorbent column containing rabbit anti-medium antibodies. Test conditions were optimized for incubation times and temperatures. Antigen, serum, and enzyme conjugate concentrations were standardized, and reproducibility was determined. A baseline value, representing a positive or negative result, was established independently for both antigens. The assay was then used to detect anti-M. gallisepticum antibodies in experimentally infected chickens. Serum samples collected at 0, 2, 5, 7, 10, 14, 21, 28, and 35 days postinfection (PI) were analyzed by serum plate agglutination (SPA), hemagglutination-inhibition (HI), and ELISA with both Antigens 1 and 2. ELISA was found to be less sensitive but more specific than SPA and more sensitive than HI. The ELISA was more sensitive with Antigen 1 than with Antigen 2. The former assay correctly identified 79% of the serum samples positive for M. gallisepticum by 7 days PI and 100% of the positive birds by 35 days PI. When the absorbance values for each group of birds were averaged, the ELISA successfully identified the M. gallisepticum-infected birds as uniformly positive 7 through 35 days PI and correctly identified all other groups negative for M. gallisepticum through 35 days PI.     

鸡滑液襄支原体感染

鸡滑液囊支原体感染（MS）又称滑液支原体感染、滑液囊霉形体感染、滑膜囊霉形体感染、传染性滑液囊炎、传染性滑膜炎,是鸡和火鸡的一种急慢性传染病,关节肿大,滑液囊和腱鞘发炎症状明显.该病病程长,笼养鸡不易被发现,经常给养鸡生产造成难以弥补的损失.     

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.     

Plasma and tissue pharmacokinetics of marbofloxacin in experimentally infected chickens with Mycoplasma gallisepticum and Escherichia coli

The plasma and tissue pharmacokinetics of marbofloxacin in chickens experimentally infected with Mycoplasma gallisepticum and Escherichia coli were studied. Marbofloxacin was given to 66 infected chickens by oral administration at a dosage of 5 mg/kg b.w., once a day for three days. Plasma, brain, kidney, liver, lung, muscle and trachea were collected and marbofloxacin concentrations were analyzed by a high performance liquid chromatography method. In the infected chickens, maximal marbofloxacin concentrations in plasma, brain, kidney, liver, lung, muscle and trachea were 1.84, 1.33, 7.35, 5.61, 3.12, 2.98, and 4.51 g/mL (g); the elimination half‐lives of marbofloxacin were 6.8, 2.74, 9.31, 8.45, 9.55, 11.53 and 5.46 h for plasma, brain, kidney, liver, lung, muscle and trachea, respectively. AUC were calculated to be 9.68, 8.04, 45.1, 27.03, 20.56, 19.47, and 32.68 μg/mL (g) ·h for plasma, brain, kidney, liver, lung, muscle and trachea, respectively. Marbofloxacin concentration in tissues except for brain exceeded marbofloxacin concentration in plasma, with AUC_tissue/AUC_plasma ranging from 2.01 to 4.66 and Peak_tissue/Peak_plasma ranging from 1.62 to 3.99. The results showed that a marbofloxacin dosage of 5 mg/kg administered orally at 24 h intervals may provide successful treatment of chicken with MG and E. coli infection.     

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 the pharmacokinetics of tilmicosin in plasma and lung tissue in healthy chickens and chickens experimentally infected with Mycoplasma gallisepticum

The objectives of this study were to compare the plasma and lung tissue pharmacokinetics of tilmicosin in healthy and Mycoplasma gallisepticum-infected chickens. Tilmicosin was orally administered at 4, 7.5 and 10 mg/kg body weight (b.w) for the infected and 7.5 mg/kg b.w for the uninfected control group. We found no significant differences in plasma tilmicosin pharmacokinetics between diseased and healthy control chickens. In contrast, the lung tissues in M. gallisepticum-infected chickens displayed a t_1/2 (elimination half-life) 1.76 times longer than for healthy chickens. The C_max (the maximum concentration of drug in samples) of tilmicosin in M. gallisepticum-infected chickens was lower than for controls at 7.5 mg/kg b.w (p < .05), and the AUC_inf (the area under the concentration–time curve from time 0 extrapolated to infinity) in infected chickens was higher than for the healthy chickens (p < .05). The mean residence time of tilmicosin in infected chickens was also higher than the healthy chickens. These results indicated that the lungs of healthy chickens had greater absorption of tilmicosin than the infected chickens, and the rate of elimination of tilmicosin from infected lungs was slower.     

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.     

Genetic and antigenic relatedness between Mycoplasma gallisepticum and Mycoplasma synoviae

The two avian pathogens Mycoplasma gallisepticum and Mycoplasma synoviae were found, by Southern blot hybridization of their digested DNAs, to share genomic nucleotide sequences additional to those of the highly conserved ribosomal RNA genes. The assumption that some of the shared sequences encode for antigens or epitopes common to both mycoplasmas was supported by Western immunoblot analysis of cell proteins of one mycoplasma with specific antiserum to the other mycoplasma. Interestingly, the band patterns of reactive antigens were different for some of the M. gallisepticum strains, supporting the concept that the species is genotypically variable. The results of the present study may explain the cross-reactivity of the two mycoplasmas noted previously in a variety of routine serological tests.     

北京市鸡毒支原体和滑液囊支原体血清学调查

为了解北京市鸡毒支原体(Mycoplasma galliscepticum,MG)和滑液囊支原体(Mycoplasma synoviae,MS)感染情况,2019年从北京市10个区127个养鸡场(户),采集3 910份鸡血清样品,采用酶联免疫吸附试验(ELISA)进行MG和MS感染抗体检测。结果显示:北京市10个区均有不同程度的MG和MS感染,场群阳性率分别介于78.95%～100%、68.42%～100%,样品阳性率分别介于59.35%～93.13%、50.0%～94.53%,平均场群阳性率分别为89.0%和86.6%,平均样品阳性率分别为79.0%和75.6%;第三季度的场群阳性率和第二季度的样品阳性率最高,第四季度场群阳性率和样品阳性率最低(P0.01);110～180日龄的MG样品阳性率、251～320日龄的MS样品阳性率最高,462日龄以上均最低(P0.01);规模化商品鸡场的MG和MS场群阳性率和样品阳性率均最高(P0.01);蛋鸡的MG和MS样品阳性率均高于肉鸡(P0.01)。结果表明,北京市MG和MS感染较为严重,第二、三季度高发,110～320日龄鸡群、规模化商品鸡场和蛋鸡群感染尤其严重。结果提示,应采取包括加强生物安全管理、种鸡净化、疫苗预防和药物治疗在内的综合管理措施,有效控制该地区MG、MS的流行。     

林可-大观可溶性粉治疗雏鸡大肠杆菌和支原体人工混合感染试验

目的:观察国产盐酸林可霉素-硫酸大观霉素(Lincomycin Hydrochloride and Spectinomycin Sulfate)可溶性粉对鸡支原体和大肠杆菌混合感染所致雏鸡气囊病的治疗效果.方法:雏鸡气囊人工混合感染鸡毒支原体和大肠杆菌.结果:林可-大观可溶性粉按0.75～1.88g/l饮水治疗5 d,能有效地控制雏鸡大肠杆菌及支原体感染,降低感染鸡发病率、气囊病变指数、支原体抗体阳性率和死亡率;其效果与对照药物利高霉素100相近.     

Haemadsorption inhibition test for the identification of Mycoplasma gallisepticum and Mycoplasma synoviae

Summary

Colonies of the avian mycoplasma strains Mycoplasma gallisepticum S6 and Mycoplasma synoviae WVU 1853 and two Mycoplasma synoviae isolates from this laboratory were shown to be haemadsorption positive for chicken erythrocytes. Three Mycoplasma synoviae isolates from this laboratory proved to be haemadsorption and haemagglutination negative. The haemadsorption of the mycoplasma colonies mentioned above was inhibited with specific antisera of either high or low titre. No cross‐inhibition was observed. It is suggested that this test could be used for a quick tentative identification of the two avian mycoplasmas on primary solid‐medium cultures.     

Disposition kinetics of doxycycline in chickens naturally infected with Mycoplasma gallisepticum

1. The pharmacokinetic properties of doxycycline were determined in healthy chickens and chickens naturally infected with Mycoplasma gallisepticum after a single intravenous (i.v.) and oral administration of the drug at 20 mg/kg body weight. Tissue residues of the tested drug after an oral dose of 20 mg/kg given twice daily for 5 consecutive days were also estimated in diseased chickens. 2. The plasma concentrations of doxycycline following single i.v. and oral administration were higher in healthy chickens than in diseased ones. Following i.v. injection, the elimination half-life (t1/2beta), distribution half-life and mean residence time (MRT) were longer in healthy chickens than in diseased birds. The values of total body clearance (ClB) and volume of distribution (Vdss) were larger in healthy chickens than in diseased birds. 3. After single oral administration, the absorption half-life (tl/2ab) and the elimination half-life were longer in normal birds than in diseased ones. The maximum plasma concentration of the drug was higher in normal chickens than in diseased ones. 4. Following repeated oral administration, the concentration of doxycycline in all tissues except muscle was higher than the corresponding concentrations in plasma. Concentrations of doxycycline in different tissues were in the following order: kidney > liver > lung > muscle. The drug was detected in liver and kidney in substantial concentrations on d 5 post administration of the last dose whereas, on d 7, its concentration in all tissues was below the lower limit of the sensitivity of the assay method used. Because of the low sensitivity of the microbiological assay method used in this study, a safe withdrawal time for doxycycline in diseased birds could not be estimated for the meanwhile.     

Haemadsorption inhibition test for the identification of Mycoplasma gallisepticum and Mycoplasma synoviae

Summary Colonies of the avian mycoplasma strains Mycoplasma gallisepticum S6 and Mycoplasma synoviae WVU 1853 and two Mycoplasma synoviae isolates from this laboratory were shown to be haemadsorption positive for chicken erythrocytes. Three Mycoplasma synoviae isolates from this laboratory proved to be haemadsorption and haemagglutination negative. The haemadsorption of the mycoplasma colonies mentioned above was inhibited with specific antisera of either high or low titre. No cross-inhibition was observed. It is suggested that this test could be used for a quick tentative identification of the two avian mycoplasmas on primary solid-medium cultures.     

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。