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Animal Science Journal (2014) 85, 96–99

doi: 10.1111/asj.12144

R A P I D C O M M U N I C AT I O N Antimicrobial susceptibilities of Mycoplasma isolated from bovine mastitis in Japan Kazuhiro KAWAI,1 Hidetoshi HIGUCHI,2 Hidetomo IWANO,3 Akihiro IWAKUMA,4 Ken ONDA,1 Reiichiro SATO,1 Tomohito HAYASHI,5 Hajime NAGAHATA2 and Toshio OSHIDA1 1

Department of Animal Health 1, School of Veterinary Medicine, Azabu University, Sagamihara, 2Animial Health Laboratory, Department of Health and Environmental Sciences, School of Veterinary Medicine, Rakuno Gakuen University, 3Department of Veterinary Biochemistry, School of Veterinary Medicine, Ebetsu, 4Zoetis Japan Inc., Tokyo, and 5National Institute of Animal Health, National Agriculture and Food Research Organization, Sapporo, Japan

ABSTRACT Mycoplasma spp. are highly contagious pathogens and intramammary Mycoplasma infection is a serious issue for the dairy industry. As there is no effective vaccine for Mycoplasma infection, control depends on good husbandry and chemoantibiotic therapy. In this study, antimicrobial susceptibility of Mycoplasma strains recently isolated from cases of bovine mastitis in Japan was evaluated by minimum inhibitory concentration (MIC). All Mycoplasma bovis strains were sensitive to pirlimycin, danofloxacin and enrofloxacin, but not kanamycin, oxytetracycline, tilmicosin or tylosin. M. californicum and M. bovigenitalium strains were sensitive to pirlimycin, danofloxacin, enrofloxacin, oxytetracycline, tilmicosin and tylosin, but not to kanamycin. This is the first report to describe the MIC of major antimicrobial agents for Mycoplasma species isolated from bovine mastitis in Japan.

Key words: antimicrobial agent, mastitis, minimum inhibitory concentration, Mycoplasma.

INTRODUCTION Mycoplasma spp. are wall-less prokaryotes that encompass a diverse group of organisms considered the smallest self-replicating organisms known. Mycoplasma spp. are pathogens causing respiratory disease, otitis media, arthritis, mastitis, keratoconjunktivitis and a variety of other diseases in cattle worldwide (Maunsell et al. 2011). Mycoplasma spp. are highly contagious pathogens (Shimazu et al. 2013) and intramammary Mycoplasma infection is a serious issue for the dairy industry (Nicholas & Ayling 2003). A report estimated costs at US$108 million per year to the US dairy industry as a result of M. bovis mastitis (Maunsell et al. 2011). Clinical Mycoplasma mastitis presents with clinical signs, including fever, swelling and udder induration (Bushnell 1984). Milk from an infected quarter usually appears abnormal, having flaky sediments in a watery or serous fluid. Mycoplasma spp. can be readily transmitted from infected to uninfected cattle. In dairy cattle, Mycoplasma spp. has traditionally been regarded as a contagious mastitis pathogen, with udder-toudder spread being the major means of transmission (Maunsell et al. 2011). Higuchi et al. (2011) previously © 2013 Japanese Society of Animal Science

reported the prevalence of Mycoplasma species in bulk tank milk of commercial dairy farms in Japan. Mycoplasma species were isolated from bulk tank milk obtained from 16 (1.29%) out of 1241 farms that were screened. Seven species of Mycoplasma, including M. bovis (0.56%, n = 7), M. californicum (0.24%, n = 3), M. canadense (0.24%, n = 3), M. arginini (0.16%, n = 2), M. bovigenitalium (0.08%, n = 1), M. alkalescens (0.08%, n = 1) and M. bovirhinis (0.08%, n = 1) were isolated from the bulk tank milk samples collected in the study. Other research also clarified that M. bovis and M. californicum that were frequently isolated from quarters induced an increase in somatic cell count (SCC) and clinical signs (Higuchi et al. 2013). These two species are apparently the main pathogens of clinical Mycoplasma mastitis among dairy herds in Japan (Higuchi et al. 2013). Correspondence: Hidetoshi Higuchi, Animial Health Laboratory, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069-8501, Japan. (Email: higuchi@rakuno .ac.jp) Received 19 July 2013; accepted for publication 21 August 2013.

ANTIMICROBIAL SUSCEPTIBILITY OF MYCOPLASMA

As there is no effective vaccine for Mycoplasma infection, control depends on good husbandry and chemo-antibiotic therapy. While previous studies have assessed the antimicrobial susceptibility of Mycoplasma spp. isolated from nasal swabs (Hirose et al. 2003; Uemura et al. 2010), no reports have been published on Mycoplasma spp. isolated from Mycoplasma-positive milk samples in Japan. In this study, the antimicrobial susceptibility of Mycoplasma strains recently isolated from Mycoplasma-positive milk samples in Japan was examined.

MATERIALS AND METHODS Mycoplasma-positive milk samples were obtained from 87 cows at 42 commercial dairy farms in Japan. In brief, the first three to four streaks of milk were discarded, and the distal end of each teat was disinfected with a cotton swab soaked in 70% alcohol. Approximately 5–8 mL of quarter milk was then aseptically collected in 10 mL sterile culture tubes. One hundred microliters of milk were inoculated into 3.0 mL of Mycoplasma broth (Kanto Chemical Co. Inc., Tokyo, Japan) before incubation at 37°C for 72 h. Mycoplasma-enriched culture broth (100 μL) was spread onto Mycoplasma agar plates (Kanto Chemical Co. Inc.) and incubated under a 5% CO2 atmosphere at 37°C for 1 month to culture typical Mycoplasma colonies (Nicholas & Ayling 2003). PCR for the detection of Mycoplasma species in cultured broth was performed according to the method described in Higuchi et al. (2011). In brief, PCR was performed in a total reaction volume of 20 μL containing 10 μL of 2× Ampdirect Plus® (Shimadzu Co. Ltd, Kyoto, Japan), 0.5 U of Nova taq TM Hot Start DNA polymerase (Novagen, Nottingham, UK), 5 pmol/L mycoplasma universal primer set (MycoAce; Nihon Dobutsu Tokusyu Shindan, Eniwa, Japan) and 5 μL of Mycoplasma enriched culture broth. PCR was performed in an iCycler PCR System (Bio-Rad, Hercules, CA, USA). Conditions for both simplified and standard PCR were as follows: initial denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 45 s and extension at 72°C for 1 min. Amplified DNA was separated by electrophoresis on 1.5% (W/V) agarose gels and was stained with ethidium bromide, and bands were visualized on a UV transilluminator. Identification of major Mycoplasma species, including M. bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense was performed by DNA sequence analysis (ABI PRISM 310; Applied Biosystems, Carlsbad, CA, USA). Then, the minimum inhibitory concentrations (MIC) of danofloxacin (Sigma-Ardrich, St. Louis, MO, USA), enrofloxacin (Sigma-Ardrich), kanamycin (Sigma-Ardrich), oxytetracycline (Sigma-Ardrich), pirlimycin (Pfizer Animal Health, Madison, WI, USA), tilmicosin (Sigma-Ardrich) and tylosin (Sigma-Ardrich), that is, the concentrations required to completely suppress the growth of M. bovis, M. californicum and M. bovigenitalium were determined (Hirose et al. 2003, 2004). In brief, antimicrobial agents were diluted twofold from 125 to 0.125 μg/mL in Mycoplasma broth (Kanto Chemical Co., Ltd) and 100 μL was transferred to each well of a 96-well microplate. Mycoplasma samples that had been precultured for 72 h in Mycoplasma broth were then diluted to approximately 105 colony forming units (CFUs) with Mycoplasma broth and 100 μL aliquots of these Mycoplasma Animal Science Journal (2014) 85, 96–99

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solutions were then added to each well of a 96-well microplate. The final CFU of Mycoplasma spp. was counted by serial dilution method. After incubation at 37°C for 72 h, the MIC was defined as the lowest concentration at which the antimicrobial agents inhibited any color change in the Mycoplasma broth. For MIC determination, the susceptibility of the different Mycoplasma strains to each drug was tested in triplicate.

RESULTS AND DISCUSSION Thirty strains of M. bovis were isolated from the 87 cows. M. bovis can be readily transmitted from infected to uninfected cattle (Maunsell et al. 2011). In dairy cattle, M. bovis has traditionally been regarded as a contagious mastitis pathogen, with udder-to-udder spread being the major means of transmission (Maunsell et al. 2011). We have previously reported that the most frequently isolated species was M. bovis and the SCC of quarters infected with this species in 115 (93%) of 124 infected quarters was ≥ 200 000 cells/mL (geometric mean, 982 000 ± 421 000 cells/mL) and clinical signs were clear (Higuchi et al. 2013). Mycoplasma bovis is highly pathogenic, causing contagious Mycoplasma mastitis with an extreme inflammatory response (Bushnell 1984). It is considered the main causative species of clinical Mycoplasma mastitis (Jasper et al. 1979; Nicholas & Ayling 2003). Table 1 shows the MICs obtained for each strain isolated from the milk samples. The MICs for danofloxacin, enrofloxacin, kanamycin, oxytetracycline, tilmicosin and tylosin against M. bovis in the present study were similar to those reported in previous studies (Rosenbusch et al. 2005; Gerchman et al. 2009; Soehnlen et al. 2011). In addition to currently employed antimicrobial agents, the efficacy of pirlimycin for treating Mycoplasma-mastitis was also assessed. Pirlimycin, an antibiotic of the lincosamide group, is an analogue of clindamycin (Birkenmeyer et al. 1984), which was initially produced by chemical modification of lincomycin (Gyde 1970; Phillips et al. 1970). Pirlimycin exhibits antimicrobial activity against Gram-positive bacteria, such as Staphylococcus aureus (Watts & Salmon 1997; Barlow et al. 2013), staphylococci, streptococci (Thornsberry et al. 1993) and enterococci (Watts et al. 1995), all of which cause contagious and environmental bovine mastitis. However, the MICs of pirlimycin on Mycoplasma spp. isolated from Mycoplasma-positive milk samples have not yet been reported. The MIC50 and MIC90 values of pirlimycin against M. bovis isolates were 0.25 and 0.5 μg/mL, respectively, which tended to be lower than those obtained with the other antibiotics. This is the first report to describe anti-microbial activity of pirlimycin against M. bovis, the main cause of Mycoplasma mastitis in dairy cows in Japan. Interestingly, the mechanism of pirlimycin action is that it increases the susceptibility of the pathogen to phagocytosis by polymorphonuclear leukocytes after exposure to sub-inhibitory © 2013 Japanese Society of Animal Science

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Table 1 Antimicrobial susceptibilities profile of Mycoplasma species isolated from 87 cows at 42 commercial dairy farms in Japan

M. bovis (n = 30) Pirlimycin Danofloxacin Enrofloxacin Kanamycin Oxytetracycline Tilmicosin Tylosin M. californicum (n = 28) Pirlimycin Danofloxacin Enrofloxacin Kanamycin Oxytetracycline Tilmicosin Tylosin M. bovigenitalium (n = 29) Pirlimycin Danofloxacin Enrofloxacin Kanamycin Oxytetracycline Tilmicosin Tylosin

Range

MIC50

MIC90

MICts†

Antimicrobial susceptibilities of Mycoplasma isolated from bovine mastitis in Japan.

Mycoplasma spp. are highly contagious pathogens and intramammary Mycoplasma infection is a serious issue for the dairy industry. As there is no effect...
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