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.     

In vitro development of resistance to enrofloxacin,erythromycin, tylosin,tiamulin and oxytetracycline in Mycoplasma gallisepticum,Mycoplasma iowae and Mycoplasma synoviae

The in vitro emergence of resistance to enrofloxacin, erythromycin, tylosin, tiamulin, and oxytetracycline in three avian Mycoplasma species, Mycoplasma gallisepticum, Mycoplasma synoviae and Mycoplasma iowae was studied. Mutants were selected stepwise and their MICs were determined after 10 passages in subinhibitory concentrations of antibiotic. High-level resistance to erythromycin and tylosin developed within 2-6 passages in the three Mycoplasma species. Resistance to enrofloxacin developed more gradually. No resistance to tiamulin or oxytetracycline could be evidenced in M. gallisepticum or M. synoviae after 10 passages whereas, resistant mutants were obtained with M. iowae. Cross-sensitivity tests performed on mutants demonstrated that mycoplasmas made resistant to tylosin were also resistant to erythromycin, whereas mutants made resistant to erythromycin were not always resistant to tylosin. Some M. iowae tiamulin-resistant mutants were also resistant to both macrolide antibiotics. Enrofloxacin and oxytetracycline did not induce any cross-resistance to the other antibiotics tested. These results show that Mycoplasma resistance to macrolides can be quickly selected in vitro, and thus, providing that similar results could be obtained under field conditions, that development of resistance to these antibiotics in vivo might also be a relatively frequent event.     

Tracheal populations of Mycoplasma gallisepticum after challenge of bacterin-vaccinated chickens

Chickens vaccinated once or twice with inactivated oil-emulsion vaccine against Mycoplasma gallisepticum (MG) or left unvaccinated were challenged intratracheally with the R strain of MG. The population of MG organisms was determined by enumerating tracheal cultures periodically up to 28 weeks postchallenge (PC). The number of organisms in the respiratory tract increased rapidly after 4 days PC, and the number tended to decrease after 4 weeks PC. Tracheal populations of MG varied considerably among individual chickens. Bacterin-vaccinated chickens had numerically lower populations of MG in their tracheas up to 8 weeks PC, but the differences were small and considered of little practical significance.     

Immunization of chickens with temperature-sensitive mutants of Mycoplasma gallisepticum

Temperature-sensitive (TS) mutants of the S6 strain of Mycoplasma gallisepticum (MG) were used to immunize newly hatched chickens. Immunized chickens developed antibodies to the wild-type (WT) S6 strain as demonstrated by serologic tests. MG was recovered from nasal cavities but not from the lower respiratory tract of the immunized chicks. Three weeks after intranasal immunization, chickens were challenged via the air sacs with the virulent S6 strain. Immunized chickens were significantly better protected from development of air-sac lesions than were controls.     

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.     

Molecular characterization and determination of antimicrobial resistance of Mycoplasma gallisepticum isolated from chickens

In this study, three consecutive approaches of molecular characterization, determination of minimum inhibitory concentration (MIC) and antimicrobial tested on Mycoplasma gallisepticum (MG) isolated from chicken farms were investigated. These approaches were conducted between 2004 and 2005 to 134 MG samples collected from five different regions of the intensive farming area of Thailand. Twenty MG isolates and four reference strains including S6, F, ts-11, and 6/85 were classified according to Random Amplification of Polymorphic DNA (RAPD) patterns prior to the antimicrobial tests. These isolates exhibited 5 different genotypes (A-E). Consequently, MG isolates representing each genotype were tested on 11 registered antibiotics. The levels of MIC were determined. Three antibiotics, doxycycline (0.20 microg/ml), tiamulin (0.10 microg/ml), and tylosin (0.33 microg/ml), gave the least MICs among all effective drugs. Break point comparisons of each antimicrobial suggested that the MG isolates were most sensitive to lincomycin, oxytetracycline, tiamulin, and tylosin. Some MG isolates had an intermediate effect on josamycin and were resistant to enrofloxacin and erythromycin. Our results also indicated that MG isolated and collected from the region and nearby districts had similar RAPD patterns showing properties of antimicrobial resistance. The RAPD patterns may imply the frequent use of antibiotics and a resistant strain of MG. This is the first report of genetic characterization using RAPD reflected by the levels of MIC against MG. The information is useful to plan for prophylactic and therapeutic impacts on the poultry industry especially in the area of intensive use of antibiotics.     

藏鸡鸡毒霉形体的血清学调查

霉形体又称支原体 ,是一类缺乏细胞壁的原核微生物 ,分类学上属于软皮纲成员。在兽医学上是一类重要的病原微生物 ,对禽类等动物具有广泛的致病性。 2 0 0 2年 4月 ,西藏某些地区鸡发生一种以咳嗽 ,鼻炎 ,打喷嚏 ,呼吸有锣音等呼吸症状为主要特征的疾病。另外 ,还可见到鼻分泌物较多 ,部分鸡出现结膜炎 ,眼中伴有泡沫分泌物 ,产蛋量下降并有死亡现象。经血清学快速诊断为鸡毒霉形体。为了摸清鸡毒霉形体在藏鸡中的感染情况 ,我们应用血清学方法对西藏各地区的藏鸡进行霉形体检测。结果显示我区藏鸡的鸡毒霉形体感染率较高 ,对藏鸡的年龄、性…     

Immunity induced with an aluminum hydroxide-adsorbed Mycoplasma gallisepticum bacterin in chickens

The protective effect of an inactivated Mycoplasma gallisepticum (MG) bacterin was evaluated in chickens subsequently challenged intratracheally (IT) with the homologous strain. Antibody responses in sera and tracheal washings (TWs) from these chickens were determined by an enzyme-linked immunosorbent assay. A group of chickens was vaccinated intramuscularly (IM) with two doses of the bacterin containing aluminum hydroxide gel (IM + IM). Another group was vaccinated IM with the same bacterin followed by IT with bacterin lacking the adjuvant (IM + IT). Chickens of both vaccinated groups had similar levels of antibody in TWs at the time of challenge. MG was eliminated from the trachea at higher rates and inflammatory lesions in the trachea were less severe in vaccinated chickens than in unvaccinated controls. The protective effect in chickens vaccinated IM + IT was greater than that in chickens vaccinated IM + IM. Perhaps vaccinal immunity is mediated by local rather than systemic antibody responses, or perhaps resistance provided by vaccination IM + IT is conferred partly by another immune mechanism such as cell-mediated immunity.     

Response of chickens to inoculation with a temperature-sensitive mutant of Mycoplasma gallisepticum

Newly hatched chickens were inoculated intranasally with either the S6 or TS 100 strain of Mycoplasma gallisepticum (MG) or they were left uninoculated. The three groups of chickens did not differ discernibly in body, spleen, or bursa weight during the 27-day sampling period. However, the S6-inoculated chickens showed a more pronounced cellular response in the nasal passages and had nearly complete lymphoid depletion in the spleens. The TS 100-inoculated birds expressed only a mild cellular reaction, which was localized in the nasal passages. Uninoculated chickens appeared normal histologically. Serologic tests such as rapid serum plate agglutination, hemagglutination-inhibition, and radioimmunoassay were able to detect antibody responses of chickens to MG inoculations yet could not differentiate the response to TS 100 from the response to S6. Tracheal secretions in intact TS 100-inoculated chickens contained antibodies to MG, yet only one-half of the bursectomized inoculated chickens contained detectable antibody, which appeared to be IgG. This led to the conclusion that bursectomy suppresses the appearance of locally synthesized IgG antibodies to MG in tracheal washings. The locally produced antibody was considered important in the development of resistance induced by intranasal inoculation of TS mutants.     

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.     

Further studies on the immunization of chickens with temperature-sensitive Mycoplasma gallisepticum mutant

Newly hatched chickens were immunized with a temperature-sensitive (TS) Mycoplasma gallisepticum (MG) mutant (TS 100). Immunized chickens resisted challenge with the virulent S6 strain. The dose of TS MG needed for protection was less than 3.3 X 10(4) colony-forming units. After immunization with TS 100, chickens were subjected to a variety of virus infection and immunosuppressive treatments. Neonatal bursectomy or thymectomy, infectious bursal disease virus infection, and infectious bronchitis virus vaccination or a combination of infectious bronchitis virus and Newcastle disease virus vaccination did not contribute to the development of air-sac lesions in TS MG-immunized chickens. Infectious bronchitis virus vaccination enabled MG to migrate from the nasal cavity to the trachea but not to the air sacs. The TS MG vaccine appears to be a safe immunogen.     

Evaluation of Mycoplasma gallisepticum K-strain as a live vaccine in chickens

We evaluated the pathogenicity of three live Mycoplasma gallisepticum (MG) vaccine candidates by infection via aerosol of 3-wk-old chickens with log phase broth cultures (trial 1). Two of the candidates (K3020 and K4649A) colonized only 10% and 20% of the chickens, respectively, unlike K2101 (K-strain), which was reisolated from all of the vaccinated chickens tested. K-strain inoculation did not result in significant air sac or tracheal lesions in chickens at 10 and 39 days postinfection (P < or = 0.05). The efficacy of K-strain as a live vaccine was evaluated in trial 2, by challenge of vaccinated chickens with virulent R-strain via aerosol at 6 wk postvaccination. K-strain vaccination resulted in significant protection from air sac and tracheal lesions (P < or = 0.05). The K-strain was further investigated to evaluate transmissibility (trial 3), colonization and persistence of infection following aerosol administration (trial 4), genetic and phenotypic stability following back passage through chickens (trial 5), and vertical transmission (trial 6). The K-strain had a low rate of horizontal transmission; it remained primarily in the respiratory system of inoculated birds and persisted in the upper respiratory tract for the duration of the trial 4 (5 mo). There was no increase in virulence of K-strain when it was back passaged five times through chickens, and no vertical transmission of K-strain was detected. K-strain showed great potential as a safe and effective live MG vaccine.     

Delineation of the lateral spread of Mycoplasma gallisepticum infection in chickens

Lateral spread of S6 strain Mycoplasma gallisepticum (MG) was studied in small populations of chickens. One experimentally exposed bird served as the source of infection, and the presence of MG-agglutinating antibody was evidence of infection in individuals. The results were subjected to survival data analysis. In the seven experiments, four similar but not identical phases of lateral spread were observed: phase 1, a generally long latent phase (median 15, range 12-21 days) before antibody was first detected in the MG-inoculated bird; phase 2, a generally short period (median 1, range 1-21 days) in which infection gradually appeared in 5-10% of the population; phase 3, a fairly constant characteristic phase (median 24, range 7-32 days) in which 90-95% of the remaining population developed MG antibody; phase 4, a generally short terminal phase (median 4, range 3-19 days) in which the remainder of the population became positive. Increasing the population density increased the rate at which lateral spread occurred.     

Antibody responses in sera and respiratory secretions from chickens infected with Mycoplasma gallisepticum

Antibodies in sera and respiratory secretions from chickens infected with Mycoplasma gallisepticum (MG) were measured by an enzyme-linked immunosorbent assay (ELISA). Chickens intratracheally inoculated with 10(5) cells of MG showed a correlation between severity of tracheal lesions and extent of MG colonization in the tracheas in the first 3 weeks postinoculation. Antibody titers in tracheal washings (TWs) of the infected chickens increased during this phase. Thereafter, isolation of MG from the trachea decreased sharply, and there was a concomitant decrease in tracheal lesion scores. At 5 weeks postinfection, the chickens that recovered from the infection exhibited a consistent presence of antibodies in TWs. Chickens reexposed had a faster rate of MG elimination and substantially less severe inflammatory lesions in the tracheas than chickens observed after the first exposure. These findings suggest a possible role of antibodies of the respiratory secretions in resistance to MG. The ELISA was a sensitive and reliable test to detect a minute amount of antibodies in the secretions.     

Bacterin to control the vertical transmission of Mycoplasma gallisepticum in chickens

A Mycoplasma gallisepticum bacterin was prepared and used in Mycoplasma gallisepticum (MG)-positive primary breeders to control vertical transmission of MG. Two generations were vaccinated, but the third generation was not vaccinated and was monitored serologically. Results showed no evidence of MG at 1 day, 6 weeks, 11 weeks, 16 weeks, or 31 weeks of age. This procedure may offer small breeder organizations and showbird fanciers a way to eliminate MG.