Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect Mycoplasma ovipneumoniae.

In order to establish a rapid detection method for Mycoplasma ovipneumoniae, this study used the loop-mediated isothermal amplification (LAMP) technique to carry out nucleic acid amplification and chromatographic visualization via a lateral flow dipstick (LFD) assay. The M. ovipneumoniae elongation factor TU gene (EF-TU) was detected using a set of specific primers designed for the EF-TU gene, and the EF-TU FIP was detected by biotin labeling, which was used in the LAMP amplification reaction. The digoxin-labeled probe specifically hybridized with LAMP products, which were visually detected by LFD. Here, we established the M. ovipneumoniae LAMP-LFD rapid detection method and tested the specificity, sensitivity, and clinical application of this method. Results showed that the optimized LAMP performed at 60 °C for 60 min, and LFD can specifically and visually detect M. ovipneumoniae with a minimum detectable concentration at 1.0 × 102 CFU/mL. The sensitivity of LAMP-LFD was 1000 times that of the conventional PCR detection methods, and the clinical lung tissue detection rate was 86% of 50 suspected sheep infected with M. ovipneumoniae. In conclusion, LAMP-LFD was established in this study to detect M. ovipneumoniae, a method that was highly specific, sensitive, and easy to operate, and provides a new method for the prevention and diagnosis of M. ovipneumoniae infection.

[Molecular typing and immunoblotting of 17 Mycoplasma ovipneumoniae isolates from China].

OBJECTIVE: To study the heterogeneity and immunogenic variability among Mycoplasma ovipneumoniae (M. ovipneumoniae) isolates from different regions of China. METHODS: The heterogeneity of 17 strains of M. ovipneumoniae isolated from 8 regions of China was studied by the amplified fragment length polymorphism (AFLP) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The software NTsys-2. 10e was used to analyze the profiles obtained from the AFLP and SDS-PAGE. The proteins reacted with the antiserum against M. ovipneumoniae type strain Y98 were then detected by Western-blot. RESULTS: Seventeen strains of M. ovipneumoniae were divided into 8 AFLP groups based on the source regions when the coefficient was 0.78. They were also divided into 8 SDS-PAGE groups based on the source regions when the coefficient was 0.85. A total of 6 immunogenic proteins were detected within 8 strains of M. ovipneumoniae, and their molecular weights were 105 kDa, 83 kDa, 65 kDa, 42 kDa, 40 kDa or 26 kDa, respectively. Interestingly, the 83 kDa and 40 kDa proteins were conserved in all the 8 isolates. CONCLUSION: M. ovipneumoniae isolates from some regions of China were genetically different, but the 83 kDa and 40 kDa antigenic proteins were conserved among the tested isolates. This study can provide some insights for the diagnosis and vaccine development of the disease caused by M. ovipneumoniae.

Epizootic pneumonia of bighorn sheep following experimental exposure to Mycoplasma ovipneumoniae.

BACKGROUND: Bronchopneumonia is a population limiting disease of bighorn sheep (Ovis canadensis). The cause of this disease has been a subject of debate. Leukotoxin expressing Mannheimia haemolytica and Bibersteinia trehalosi produce acute pneumonia after experimental challenge but are infrequently isolated from animals in natural outbreaks. Mycoplasma ovipneumoniae, epidemiologically implicated in naturally occurring outbreaks, has received little experimental evaluation as a primary agent of bighorn sheep pneumonia. METHODOLOGY/PRINCIPAL FINDINGS: In two experiments, bighorn sheep housed in multiple pens 7.6 to 12 m apart were exposed to M. ovipneumoniae by introduction of a single infected or challenged animal to a single pen. Respiratory disease was monitored by observation of clinical signs and confirmed by necropsy. Bacterial involvement in the pneumonic lungs was evaluated by conventional aerobic bacteriology and by culture-independent methods. In both experiments the challenge strain of M. ovipneumoniae was transmitted to all animals both within and between pens and all infected bighorn sheep developed bronchopneumonia. In six bighorn sheep in which the disease was allowed to run its course, three died with bronchopneumonia 34, 65, and 109 days after M. ovipneumoniae introduction. Diverse bacterial populations, predominantly including multiple obligate anaerobic species, were present in pneumonic lung tissues at necropsy. CONCLUSIONS/SIGNIFICANCE: Exposure to a single M. ovipneumoniae infected animal resulted in transmission of infection to all bighorn sheep both within the pen and in adjacent pens, and all infected sheep developed bronchopneumonia. The epidemiologic, pathologic and microbiologic findings in these experimental animals resembled those seen in naturally occurring pneumonia outbreaks in free ranging bighorn sheep.