A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle

Abstract

A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 103 cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.

Prevalence and Risk Factor Analysis of Bovine Hemoplasma Infection by Direct PCR in Eastern Hokkaido, Japan

“…Recently, an improved PCR buffer was developed to neutralize these inhibitory substances in whole blood [2]. Direct PCR performed using this improved buffer has been shown to demonstrate superior sensitivity [2,9]. In the present study, we analyzed 49 samples by direct and standard PCR and found that the sensitivity of direct PCR was 10-fold higher than standard PCR.…”

“…Recently, direct PCR assays for hemoparasites, such as Babesia gibsoni, and hemoplasma species using whole blood of dogs and cats have been developed to reduce time, cost and labor and to eliminate contamination risks [31,33]. Moreover, direct PCR was also used in a large-scale epidemiological study for bovine mastitis caused by Mycoplasma infection as a faster, easier method of screening [9]. Therefore, it is thought that a direct PCR method can also detect bovine hemoplasma infections and is suitable for largescale investigation.…”

Analysis of Trace and Major Elements in Bronchoalveolar Lavage Fluid of Mycoplasma Bronchopneumonia in Calves

“…Several researches demonstrated that sampling by BALF was more useful for prediction of lower respiratory airway pathogens than nasal swabs although clearly not as convenient [19,20]. Therefore, in this study, PCRs based on 16S rRNA genes amplified M. bovis DNA [15,21] and were used to confirm Mycoplasma bronchopneumonia, using BALF samples.…”

“…Sub-samples were cultivated and investigated by polymerase chain reaction (PCR) tests targeting the M. bovis, based on 16S rRNA genes [15]. Briefly, simplified PCR was performed in a total reaction volume of 20 μL containing 10 μL of 2× AmpdirectPlus (Shimadzu Co., Kyoto, Japan), 0.5 U of Nova taq TM Hot Start DNA polymerase (Novagen, UK), 5 pmol of a mycoplasma universal primer set (MycoAce; Nihon Dobutsu Tokusyu Shindan Ltd., Hokkaido, Japan), and 5 μL of each samples.…”

Letter to the Editor

“…The PCR for Mollicutes using bronchoalveolar lavage fluid detected Mycoplasma bovis in 1 sample (5%) from an apparently healthy calf and in 21 samples (100%) from calves with bronchopneumonia. 2 Therefore, the statistical analysis focused on 19 healthy controls in which Mycoplasma bovis had not been detected and 21 calves with bronchopneumonia. A volume of 4 mL of whole blood was collected via jugular venipuncture into heparinized tubes for endotoxin and cTnI analyses and centrifuged at room temperature within 15 minutes of collection.…”