Fingerprinting of Mycoplasma gallisepticum strains isolated from multiple-age layers vaccinated with live F strain

Mycoplasma gallisepticum (MG) isolates were obtained from three multiple-age commercial layer farms on which live F strain vaccine had been administered to each replacement flock for at least 2 years. All such isolates had restriction endonuclease DNA and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein patterns characteristic of F strain. These cultures also hybridized in dot blot assays with both the MG strain-specific and species-specific DNA probes. In contrast, the original MG isolate that came from one of the farms before vaccination began clearly was not F strain. These results suggest that continuous use of live F strain vaccine in each replacement pullet flock on multiple-age commercial layer sites will result in displacement of the original field strain of MG with the vaccine strain.     

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.     

延胡索酸泰妙菌素对支原体和大肠杆菌混合感染的治疗试验

以人工诱发鸡毒支原体和大肠杆菌混合感染为模型,以酒石酸泰乐菌素为对照药物,评价了延胡索酸泰妙菌素的疗效.按每1 L水中分别加入312.5、468、625 mg延胡索酸泰妙菌素及500 mg酒石酸泰乐菌素的用量给病鸡饮水给药,连用5 d.试验表明,用药组的成活率、日增重、料肉比、气囊损伤度与感染对照组比较差异极显著(P＜0.01);延胡索酸泰妙菌素大剂量组日增重与小剂量组比较差异显著(P＜0.05),与其他各用药组比较差异极显著(P＜0.01),料肉比与其他各用药组比较差异极显著(P＜0.01);酒石酸泰乐菌素组的料肉比与延胡索酸泰妙菌素小剂量组比较差异不显著(P＞0.05);而与其他各组比较差异极显著(P＜0.01).数据分析表明,延胡索酸泰妙菌素大剂量组能有效地降低气囊损伤度,提高饲料转化率.     

Evaluation of factors associated with infection of commercial layers with Mycoplasma gallisepticum and M. synoviae

Information on factors possibly associated with the risk of infection with Mycoplasma gallisepticum (MG) or M. synoviae (MS) were collected from nearly 400 layer flocks in California. Factors associated with the probability of flock infection with either MG or MS were identified, and their magnitude was quantified by statistical analysis. More frequent administration of several vaccines was associated with decreased probability of both MG and MS infection of flocks. Also identified were housing or hygiene factors and system of management (i.e., multiple-age status) that could reduce the probability of infection of flocks with mycoplasma. The change in probability of MG infection resulting from modifying certain management factors was examined.     

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.     

A chronicle of serologic response in commercial layer chickens to vaccination with commercial F strain Mycoplasma gallisepticum vaccine

Vaccination of multi-age layer operations, wherein one million plus commercial layer chickens are housed, has been spurious until the development of a self-propelled, constant-speed spray vaccinator. Still, even with its use, live Mycoplasma gallisepticum (MG) vaccinations have been questionable in terms of seroconversion. Using the vaccinator as a research tool over the past 5 yr, factors have been elucidated which impact seroconversion to one live MG vaccine in particular, the F strain of MG (FMG). These factors include the type of nozzle used to spray the vaccine, the temperature of the water used to rehydrate and administer the vaccine, and the pH and osmolarity of the fluid used to apply the vaccine. In the present study, one farm was monitored for its seroconversion rates over 4 1/2 yr, during which time the FMG vaccination protocol was amended as factors were identified that enhanced seroconversion rates. The results of this study showed that implementation and inclusion of the optimized factors into the vaccination protocol for FMG enhanced seroconversion rates because they went from an initial 50%-55% positive seroconversion rate to a consistent 100% positive seroconversion rate over the 56-mo study period.     

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.     

鸡败血霉形体弱毒F株对鸡的致病性和免疫效力测定

以鸡败血霉形体弱毒Ｆ５和Ｆ３６株的新鲜培养物分别点眼和鼻内接种１日龄鸡雏,比较其对鸡的致病性和免疫原性。结果,在鼻内感染时,Ｆ５可使３０只鸡中的４只出现轻度气囊损伤,而Ｆ３６只使３０只中的１只出现轻度气囊损伤;点眼接种时,Ｆ５和Ｆ３６均不使感染鸡出现气囊损伤;Ｆ５与Ｆ３６接种鸡体内的抗体产生情况无明显区别,在强毒Ｒ株攻击后,２个代次菌株的接种鸡均能产生良好的免疫力,且无明显差异,但在维护体质量增加方面,Ｆ３６株略优于Ｆ５株。     

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.     

Influence of F strain Mycoplasma gallisepticum infection on response of commercial layers to heat exposure

Commercial layers were inoculated with F strain Mycoplasma gallisepticum (MG) and housed in either conventional chicken houses or the lower-stress environment of biological isolation units. At the end of 2 weeks, all treatment groups were placed in environmental chambers and subjected to 4 hr of heat stress (40 C with a dew point of 21 C). Rectal temperature, an indicator of response to high heat, was monitored. Rectal temperatures of F strain MG-inoculated hens housed in the conventional chicken house environment were significantly higher than those of uninoculated controls, whereas rectal temperatures of hens held in isolation units were comparable to those of their uninoculated controls.     

Evaluation of protection against colonization of the chicken trachea following administration of Mycoplasma gallisepticum bacterin

Twelve-week-old commercial white leghorn pullets were given one or two doses of an inactivated oil-emulsion Mycoplasma gallisepticum (MG) vaccine or kept as unvaccinated controls. At 24 weeks of age, all groups were challenged intratracheally with one of six dilutions of a low-passage R strain of MG. Three days postchallenge, the tracheas from all chickens were cultured for MG to determine the number of challenge organisms required to initiate infection. The log10 ID50 of chickens vaccinated 0, one, or two times was 2.9, 3.4, and 3.7, respectively, and the minimum infectious dose (the lowest challenge dose to infect a single bird) was 15, 150, and 1500 colony-forming units, respectively. It was concluded that the vaccine provided measurable, though limited, protection against infection under these experimental conditions.     

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.     

Egg production, egg weight, eggshell strength, and mortality in three strains of commercial layers vaccinated with F strain Mycoplasma gallisepticum

Three strains of commercial leghorns vaccinated at 17 to 22 weeks of age with F strain Mycoplasma gallisepticum (MG) were maintained through 117 weeks of age. The three strains differed in both mortality and percent egg production per hen housed; however, the strains did not differ in egg weight (EW), eggshell strength (ESS), or percent daily egg production. Results of this study indicate EW and ESS for F strain MG-vaccinated hens follow patterns previously reported for uninfected layers. Further, mortality may account, in part, for differences in percent egg production per hen housed between strains of F strain MG-vaccinated hens.     

Comparison of a modified Edward-type medium and a modified SP4-type medium for primary isolation of Mycoplasma gallisepticum (MG) from chickens vaccinated with the F strain of MG

The efficacy of two media, an Edward-type medium (EPJ) and a modified SP4-type medium (SP4-PS), were compared for primary isolation of Mycoplasma gallisepticum (MG) from commercial layer chickens (n = 58) vaccinated with the live F strain of MG. Three groups of chickens that differed in the interval after vaccinal exposure to the F strain (32, 41, and 102 weeks) were studied at necropsy. Mycoplasma isolation was attempted from the trachea, sinus, and cloaca using lavage and swab techniques but was successful only from the trachea and sinus. MG was isolated from 39 (8.4%) of 463 culture attempts from 58 tracheal inocula and 58 sinus inocula. Isolation of MG was successful more frequently using EPJ medium than SP4-PS medium, and isolation occurred more often from the sinus than from the trachea. Of the 58 chickens studied, 19 (33%) were shown by culture to be infected with MG. Isolation was successful only from 32- and 41-week post-vaccination exposure groups. However, all chickens studied were serologically positive for MG antibody by rapid-plate agglutination and hemagglutination-inhibition assays.