In Vitro Amplificatin of the 16S rRNA Genes from Mycoplasma bovirhinis, Mycoplasma alkalescens and Mycoplasma bovigenitalium by PCR.

Kobayashi, Hideki; Hirose, Kazuhiko; Worarach, Apasara; Paugtes, Patchara; Ito, Nobuyoshi; Morozumi, Tatsuya; Yamamoto, Koshi

doi:10.1292/jvms.60.1299

Cited by 41 publications

(26 citation statements)

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“…The number of organisms per milliliter in the broth was determined by the M-agar [5,13]. Organisms were stored in M-broth at -80°C until use [13].One milliliter of each well-grown suspension culture of mycoplasma was adjusted to a concentration of 10 8 CFU/ml with phosphate buffered saline (PBS) for the positive control, or with milk for the simulated specimen. Serial 10-fold dilutions of the organisms with PBS or milk were used to compare the PCR and culture methods as described below.…”

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confidence: 99%

“…These strains included the type strain of each species (M. alkalescens PG51, M. bovigenitalium PG11, M. bovirhinis PG43 and M. bovis PG45) and field isolates. The above mycoplasmas were cultured in M-broth [5,13]. The number of organisms per milliliter in the broth was determined by the M-agar [5,13].…”

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confidence: 99%

“…The number of organisms per milliliter in the broth was determined by the M-agar [5,13]. Organisms were stored in M-broth at -80°C until use [13].…”

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confidence: 99%

“…As primers of M. alkalescens (Mak), we used MakF (5'-GCTGTTATAGGGAAAGAAAACT-3') and MakR (5'-AGAGTCCTCGACATGACTCG-3') primers [13]. As primers of M. bovigenitalium (Mbg), we used MbgF (5'-CGTAGATGCCGCATGGCATTTACGG-3') and MbgR (5'-CATTCAATATAGTGGCATTTCCTAC-3') primers [13]. As primers of M. bovirhinis (Mbr), we used MbrF (5'-GCTGATAGAGAGGTCTATCG-3') and MbrR (5'-ATTACTCGGGCAGTCTCC-3') [13].…”

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confidence: 99%

“…As primers of M. bovigenitalium (Mbg), we used MbgF (5'-CGTAGATGCCGCATGGCATTTACGG-3') and MbgR (5'-CATTCAATATAGTGGCATTTCCTAC-3') primers [13]. As primers of M. bovirhinis (Mbr), we used MbrF (5'-GCTGATAGAGAGGTCTATCG-3') and MbrR (5'-ATTACTCGGGCAGTCTCC-3') [13]. As primers of M. bovis (Mbo), we used MboF (5'-GGCTCTCATTAA-GAATGTC-3') and MboR (5'-TTTTAGCTCTTTTTGAA-CAAAT-3') primers [10].…”

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Detection of Mycoplasma in Mastitic Milk by PCR Analysis and Culture Method.

Hirose

Kawasaki

Kotani

et al. 2001

The Journal of Veterinary Medical Science

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ABSTRACT. Mycoplasma alkalescens, M. bovigenitalium, M. bovirhinis and M. bovis were directly detected from milk specimens by a polymerase chain reaction (PCR) when milk specimens were centrifuged and treated with mycoplasmal lysis buffer. The sensitivity of this PCR method was 110 to 1,400 colony forming units (CFU). This method was useful for the detection of mycoplasmas in milk specimens from cows at an early stage of mycoplasmal mastitis since a small amount of mycoplasma could be detect in milk without culture. The results were available within 12 hr, which is faster than conventional culture techniques. M. bovirhinis was detected in more than 70% of mastitic milk specimens when mycoplasmas were detected in milk specimens from 30 cows with mastitis by this PCR method. KEY WORDS: mastitis, mycoplasma, PCR.J. Vet. Med. Sci. 63(6): 691-693, 2001 Mycoplasma mycoides subsp. mycoides, M. bovis, M. alkalescens, M. bovigenitalium, M. dispar and others are the causative agents of several diseases in cows and calves, such as mastitis, pneumonia, arthritis, genital disorders and abortions [1,3,6,12,15]. M. bovirhinis is a troublesome agent as a secondary invader in respiratory diseases, and it is the mycoplasma that is the most common isolate from the nasal cavity of cattle with respiratory disease [8]. Since the diseases are largely resistant to chemotherapy [6,24], it is necessary to have rapid and reliable diagnostic methods to detect the agents at an early stage, so that effective control measures can be introduced in time. However, current methods for the detection of mycoplasmas are restricted to culture and serology, and both of these diagnostic methods are time consuming, laborious and difficult. Isolation and identification of mycoplasmas may take weeks, and few laboratories have the capabilities required to routinely culture mycoplasmas. Serodiagnosis requires the demonstration of increasing antibody titers that are reached only ten to fourteen days after the onset of clinical symptoms [19]. Consequently, the pathogen cannot be detected during the incubation period. Moreover, the serological cross reactions among the mycoplasma species are a critical problem [19]. Therefore, the absence of practical and rapid detection methods and resistance to therapy hamper the effective control of mycoplasma infections.Gonzalez [7] and Hatanaka et al. [9] reported cases of mycoplasmal mastitis caused by M. bovis derived from the nasal secretion of cows with pneumonia, emphasizing the relationship between mycoplasmal pneumonia and mastitis. During 1996-1997, we performed nationwide research on the isolation of mycoplasma from the nasal secretions of calves with respiratory diseases, and the result was that a strain of mycoplasma was isolated from about 30% of calves with respiratory diseases. The majority of the isolates consisted of the following 4 species, M. alkalescens, M. bovigenitalium, M. bovirhinis and M. bovis (Data not shown). Therefore, we restrict discussion to these four species in this study.The purpose of...

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confidence: 99%

“…The number of organisms per milliliter in the broth was determined by the M-agar [5,13]. Organisms were stored in M-broth at -80°C until use [13].…”

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Detection of Mycoplasma in Mastitic Milk by PCR Analysis and Culture Method.

Hirose

Kawasaki

Kotani

et al. 2001

The Journal of Veterinary Medical Science

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A novel polymerase chain reaction assay for the detection of seven Mycoplasma species of cattle origin

Lai

Lin

Hsu

et al. 2022

World J Microbiol Biotechnol

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The aim of this study was to develop a pair of primers for detecting ruminant mycoplasma pathogens.We designed a set of primers based on the most similar sequences within 16sRNA regions of seven Mycoplasma spp. These primers have high sensitivity for detecting Mycoplasma dispar, M. arginine, M. canadense, M. bovis, M. alkalescens, M. californicum, and M. bovisgenitalium within the annealing temperature range of 46°C to 48°C. The minimum amount of DNA that can be detected using the protocol is 250 ng, which is equivalent to 2,000 cfu/mL. The primers can detect mycoplasma from DNA extracted directly from milk samples. The common bovine mastitis pathogens of Staphylococcus aureus coagulase-negative staphylococci, Escherichia coli, Streptococcus uberis, Klebiella pneumonia, Korucia rosea, and Acholeplasma spp. were not detected by the primers. We believe the high sensitivity and speci city of these primers make them useful for detecting infection with seven Mycoplasma species in ruminants, allowing the primers to be used in clinical settings.

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