Evaluation of cytopathologic changes induced in chicken tracheal epithelium by Mycoplasma gallisepticum in vivo and in vitro

Changes in tracheal epithelial surfaces induced by Mycoplasma infection in vivo and in vitro included release of mucous granules followed by exfoliation of ciliated and nonciliated epithelial cells. Light microscopy, scanning electron microscopy, and transmission electron microscopy confirmed that the loss of cilia from individual cells was infrequent. Epithelial cells typically lost their intercellular connections, rounded up, exfoliated, and then lysed--giving rise to a population of cellular organelles, such as mitochondria and cilia intermixed with mucus to form the exudate found within the tracheal lumen. Repair of the epithelial surface was effected by basilar epithelial cells differentiating and filling in the spaces formed by exfoliated cells. These cells were hypertrophied, nonciliated at 14 days after infection in vivo, and covered with microvilli. In sectioned material obtained during the infection, there was increasing epithelial thickness due to cellular infiltration and edema. Tracheal rings in vitro showed similar changes to those seen in vivo, except that exfoliation was more severe and occurred earlier. In addition, there were no cellular infiltration due to the lack of a vascular supply and only a small amount of mucus due to the smaller number of mucous cells available to release into the tracheal lumen.     

A quantitative study of single and mixed infection of the chicken trachea by Mycoplasma gallisepticum

The interaction between Mycoplasma gallisepticum (MG) and the tracheal mucosa of the young chicken was studied. The use of a selective plating method permitted differentiation between a pathogenic tylosin-resistant strain (227) and a less pathogenic tylosin-sensitive vaccine strain (F). Both MG strains adhered to the tracheal mucosa and colonized equally well. In mixed infection, the presence or absence of the second strain did not change the efficiency of colonization by either strain. When chickens were exposed to the vaccine strain 24 hr or 2 weeks before superinfection by the pathogen, there was no significant reduction in the efficiency of superinfection, despite the presence of 10(6) colony-forming units of MG strain F in the trachea. However, chickens had an increased ability to resist superinfection 5 weeks after exposure via the air sac. These results suggest that the biological mechanism underlying protection of F-strain-vaccinated chickens against adventitious infection by the homologous species does not involve competition for adherence sites or blockage by prior colonization.     

Real-time polymerase chain reaction for Mycoplasma gallisepticum in chicken trachea

In this work, we describe a rapid detection procedure for Mycoplasma gallisepticum from chicken tracheal swabs by real-time polymerase chain reaction (PCR) by LightCycler system, where we were able to monitor the amplification of the newly synthesized M. gallisepticum-specific PCR product as a proportionally increasing fluorescent signal by using the double-stranded DNA binding dye SYBR Green I and have identified M. gallisepticum-specific PCR products by DNA melting curve analysis by plotting the first negative derivative (-d[F1]dT) of fluorescence over temperature. Detection limits of the PCR were found to be 3 and 3000 colony-forming units ml(-1) with pure culture of M. gallisepticum and artificially spiked samples, respectively. Out of 96 tracheal swabs, 68 were taken from live chickens and 28 were taken by scraping the mucosal surface of the trachea (SMST) of necropsied chickens. All of the 18 PCR-positive results were from the swabs taken by the SMST method, whereas all of the samples taken from live chickens were negative. Thus, the PCR with the SMST method had a sensitivity and a specificity of 64.2% (18 of 28 chickens) and 100%, respectively. The total time required for template preparation from tracheal swab samples and real-time PCR was approximately 65 min. These results indicate that real-time PCR with the LightCycler technology is a rapid and sensitive test to identify M. gallisepticum-infected flocks if a proper sampling is applied.     

Effect of medicated feed on tracheal infection and population of Mycoplasma gallisepticum in chickens

Six-week-old broilers were fed 50 g tylosin/ton, 400 g chlortetracycline (CTC)/ton, or unmedicated feed and then challenged intratracheally with R strain Mycoplasma gallisepticum (MG). Feed-grade antibiotic medication did not prevent infection, but medication did lower the number of isolations from treated birds compared with controls. Only tylosin significantly lowered MG counts in the trachea. The log10 ID50 of birds receiving tylosin, CTC, or unmedicated feed were 5.8, 4.4, and 2.9, respectively. Six-week-old leghorns were placed on the three diets described previously and challenged with the R strain of MG. Birds were sacrificed at various times up to 10 weeks postchallenge (PC). Compared with the control diet, the tylosin-medicated diet significantly reduced the tracheal MG count from day 1 to 4 weeks PC, whereas the CTC-medicated diet significantly lowered the tracheal MG count only at 3 days PC. In all groups, the MG count gradually declined after 1 week PC; by 8 weeks PC it was essentially the same in all groups. It was concluded that continuous medication has the potential for reducing MG tracheal populations only during the initial phase of an outbreak.     

Immunogenicity of Mycoplasma gallisepticum

The serological response and protective immunity elicited in the chicken by the pathogenic Ap3AS strain and the moderately pathogenic 80083 strain of Mycoplasma gallisepticum and variants of strain 80083 attenuated by repeated passage in mycoplasma broth were investigated. Strain 80083 elicited a substantial serum antibody response after administration either in drinking water or by conjunctival sac instillation to 7-week-old SPF chickens. No vaccinated chickens developed air sac lesions when challenged by intra-abdominal (IA) injection with the virulent Ap3AS strain. Chickens vaccinated with strain 80083M (50 broth passages) showed only a weak serological response but were substantially protected when challenged 4 weeks after vaccination. Chickens vaccinated with 80083H (100 broth passages) were serologically negative 4 weeks after vaccination and developed severe air sac lesions after challenge. Thirty-seven-week-old hens vaccinated 6 months previously with strain 80083 had high serum antibody levels and were completely protected against IA challenge with the homologous strain. However, 4/6 showed mild air sac lesions when challenged intra-abdominally with strain Ap3AS. Another group showed high M. gallisepticum serum antibody levels 6 months after vaccination with strain Ap3AS but 4/6 and 2/6 showed mild lesions after IA challenge with strains Ap3AS or 80083, respectively. Strains 80083 or 80083M were administered by conjunctival sac instillation to susceptible 11-week-old commercial pullets at the time of fowl pox vaccination. The concurrent use of both vaccines had no apparent adverse effect on the health of the chickens. Similar protection against IA challenge with strain Ap3AS was produced with the M. gallisepticum vaccines whether used alone or in combination with fowl pox.     

Morphologic changes in chicken cells after in vitro exposure to Mycoplasma gallisepticum

Mycoplasma gallisepticum (MG) was used to expose chicken peripheral blood lymphocytes (PBLs), red blood cells (RBCs), heterophils, and chicken tumor cells (MSB-1 and HD-11 cells). Incubation of PBLs with MG for 3 hr resulted in extensive clumping of lymphocytes. Incubation of the MSB-1 cells with MG also caused clumping of the cells, with many of the cells showing perforations and others showing capping of the surface projections. Incubation of RBCs with MG resulted in an altered cell surface morphology, a decrease in cell size, and perforation. There were no discernible changes on the surface of the heterophils and the HD-11 cells. However, the HD-11 cells appeared to have a decreased ability to attach to the surface of the plastic and to have a decreased ability to respond to chemoattractant fMLP after 24 hr of incubation. These results suggest that, under the conditions used, MG caused certain damage to peripheral blood cells and a significant decrease in chemotactic response in the HD-11 cells.     

Protection and immunity in commercial chicken layers administered Mycoplasma gallisepticum liposomal bacterins

Six liposomal Mycoplasma gallisepticum (MG) bacterins, differing in charge and size, and two oil-emulsion vaccines (sonicated and non-sonicated) were given to white leghorns in two doses, at 13 weeks and again 1 month later. At 21 weeks of age, all chickens were challenged with a viable 20-hour culture of MG cells (17,800 colony-forming units) intratracheally and with nonviable MG organisms (0.09 mg protein) injected subcutaneously in the wattle center. The three chicken groups that had the lowest tracheal MG-infection rates postchallenge were those given adjuvants of small multilamellar positively charged liposomes (16.67%), large multilamellar negatively charged liposomes (16.67%), and non-sonicated oil-emulsion bacterin (37.5%). These three groups also had significant levels of antibody in sera 4 weeks after the second dose of vaccine. The group given the small multilamellar positively charged liposome also showed significant delayed-type hypersensitivity (wattle swelling) (P less than or equal to 0.05). The group given the large multilamellar negatively charged liposomes had the highest local antibody response (P less than or equal to 0.01) and was the only group that had no microscopic lesions in the trachea.     

Experimental infection of ducks with Mycoplasma gallisepticum

Specific-pathogen-free ducks 24 and 180 days old were inoculated intranasally with the S6 strain of Mycoplasma gallisepticum (MG). No significant gross lesions were found in trachea, lung or air sacs at 7 or 28 days postinfection (PI). MG was recovered from the infraorbital sinus and trachea but not from the air sacs 7 and 28 days PI. A few ducks responded serologically by developing agglutinating antibody. MG multiplied in embryonated duck eggs but to lower titers than in embryonated chicken eggs.     

Ultrastructural features of Mycoplasma gallisepticum in tracheal explants under transmission and stereoscan electron microscopy

Ultrastructural features of Mycoplasma gallisepticum in tracheal explants were examined using the transmission and stereoscan electron microscope. The organisms were characteristically cocco-bacilliform except when in close contact with the host cells when they assumed an elongated and irregular form characteristically terminating in a bleb which was often embedded in the cell surface. In such organisms there were peripherally aligned fibrillar structures oriented towards the bleb which may have a functional relationship with a probing or moving habit of the mycoplasma.     

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.     

磷酸替米考星可溶性粉对人工感染鸡毒支原体的临床疗效试验

为研究测定磷酸替米考星可溶性粉对鸡慢性呼吸道病的治疗效果,试验设计三个剂量组进行饮水给药治疗人工感染鸡毒支原体（MGs6）实验鸡,对照药物为替米考星可溶性粉。试验结果表明,磷酸替米考星可溶性粉高（1g／L）、中（0．75g／L）剂量组以及对照药物均能显著降低发病死亡率、减少气囊病变率,并提高相对增重率（P〈0．05）。据此,推荐最佳临床治疗用法为：剂量0．75g／L,连续饮水给药3d。     

复方硫氰酸红霉素可溶性粉对人工感染鸡慢性呼吸道病的疗效试验

用复方硫氰酸红霉素可溶性粉以每升水中加0.5、0.6、0.7、0.8、0.9 g 5个剂量对人工感染鸡慢性呼吸道病的鸡群混饮治疗,同时设立硫氰酸红霉素可溶性粉对照组.试验结果表明,用药5d后,复方硫氰酸红霉素可溶性粉每升水加0.6、0.7、0.8、0.9g剂量组的有效率和治愈率皆高于硫氰酸红霉素可溶性粉组.     

耐氟喹诺酮类药物鸡毒支原体gyrA基因突变的研究

鸡毒支原体(MG)是引发鸡慢性呼吸道疾病的主要病原,临床常采用抗菌药物进行控制[1]。FQ s作为控制鸡毒支原体感染的主要药物,随着药物的广泛使用,尤其不合理用药,临床已反映该类药物疗效并不理想。但有关MG临床株对FQ s耐药性的研究却鲜有报道。仅有的几篇报道也限于诱导耐药株     

A recombinant PvpA protein-based diagnostic prototype for rapid screening of chicken Mycoplasma gallisepticum infections

Mycoplasma gallisepticum is the primary agent of chronic respiratory disease causing important economic losses in the poultry industry. Serological monitoring is essential to maintain mycoplasma-free breeder flocks and often complicated by the cross-reactions between different mycoplasma species. To overcome serological cross-reactions, a large fragment of the M. gallisepticum PvpA cytadhesin, species-specific surface-exposed protein, was produced in E. coli as a recombinant protein (rPvpA336) and used as a potential diagnostic antigen. The rPvpA336 protein possesses 336 mycoplasma-specific amino acids with relative molecular weight of 44 kDa. A deletion region of 37 amino acids was identified when compared to the wild-type PvpA protein. Immunoreactivity of the rPvpA336 protein has been demonstrated by Western blot analysis with M. gallisepticum-positive and -negative chicken sera. Furthermore, an enzymatic rapid immunofiltration assay (ERIFA) prototype based on the rPvpA336 protein has been developed and its species-specific detection capability has been demonstrated by using M. gallisepticum and/or M. synoviae-positive and -negative chicken sera. In addition to its species-specificity, the ERIFA prototype presents certain advantages such as rapidity, field-applicability and cost-effectiveness. Therefore, these advantages would make the prototype a species-specific rapid diagnostic tool of choice in the field and limited laboratory conditions for screening M. gallisepticum infections.     

广西鸡毒支原体的分子流行病学研究的概述

研究分别建立了鸡毒支原体（MG）、滑液霉形体（MS）、禽衣阿华支原体（MI）、火鸡支原体（MM）及鉴别MG强毒株和弱毒疫苗株单一PCR和多重PCR检测鉴定技术,以及核酸探针检测MG的技术。应用多种分子生物学技术即SDS—PAGE、PCR—RFLP、RAPD和29KD多肽基因序列分析等方法对广西MG流行株的分子病原学进行研究,摸清了广西MG流行野毒株之问以及与现用疫苗株和国际参考株之间的差异和遗传相关性;在此基础上制定的综合防制措施经生产的实施与应用,取得了显著的效果。     

Comparative study of Mycoplasma pulmonis and Mycoplasma gallisepticum infections in turkey sinus.

Infraorbital sinuses of young turkeys were injected with virulent strains of Mycoplasma pulmonis and Mycoplasma gallisepticum to compare the diseases caused by the 2 agents. Mycoplasma pulmonis did not cause visible swelling from large quantities of mucous exudate in the sinuses, such as occurs with M gallisepticum, and it could not be recovered by bacteriologic culture technique after 3 weeks. However, slight exudate did accompany the M pulmonis infection. Similarities between the disease caused by M pulmonis and that caused by M gallisepticum included lymphocytic infiltration in the submucosa, swollen epithelial cells, and loss of cilia from sinus epithelial cell surfaces. This strain of M pulmonis, which is pathogenic for rats, was only mildly pathogenic for turkeys and the infection did not persist for long.