Abstract
Protothecal mastitis, caused mostly by Prototheca zopfii (P. zopfii), is increasing in dairy herds and is being reported globally. The present study was aimed at studying the epidemiology of mastitis and at molecular characterization of P. zopfii isolates from dairy herds and their surroundings in three provinces of China using microbiological, biochemical and molecular methods, and antibiotic susceptibility tests. Samples from milk (n = 620) of mastitic cows and their barns sources (n = 410) including feces, feed, bedding materials and drinking water were analyzed. Among other pathogens recovered from mastitic milk, 84 (13.5 %) of the isolates were identified as P. zopfii. All of the P. zopfii isolates recovered from milk were recognized as genotype 2, whereas 58 (73.4 %) and 21 (26.6 %) isolates from environmental sources were found to be P. zopfii genotypes 1 and 2, respectively. The isolates were susceptible to some antibiotics and antifungal agents, including amikacin (78.1 %), streptomycin (58.5 %), gentamicin (17.8 %), amphotericin B (68.6 %) and nystatin (64.4 %). Additionally, the two genotypes displayed versatile patterns of susceptibility to different antimicrobials agents. Phylogeny of the genotypes on the basis of 18S SSU rDNA and 28S SSU rDNA was also investigated. The isolates of the two genotypes separated into different clades, and no interrelationship was observed among these as shown by phylogenetic analysis. The genotype 1 isolates from cow barn sources were non-pathogenic and may not present any risk of mastitis. We conclude that P. zopfii genotype 2 might play an important role in bovine mastitis in China.
Similar content being viewed by others
References
Bozzo G, Bonerba E, Di Pinto A, Bolzoni G, Ceci E, Mottola A, Tantillo G, Terio V. Occurrence of Prototheca spp. in cow milk samples. New Microbiol. 2014;37:459–64.
Melville P, Watanabe E, Benites N, Ribeiro A, Silva J, Garino F, Costa E. Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization. Mycopathologia. 1999;146:79–82.
Lerche M. Eine durch Algen (Prototheca) hervorgerufene Mastitis der Kuh. Berl Munch Tierarztl Wochenschr. 1952;4:64–9.
Möller A, Truyen U, Roesler U. Prototheca zopfii genotype 2: the causative agent of bovine protothecal mastitis? Vet Microbiol. 2007;120:370–4.
Marques S, Silva E, Kraft C, Carvalheira J, Videira A, Huss VA, Thompson G. Bovine mastitis associated with Prototheca blaschkeae. J Clin Microbiol. 2008;46:1941–5.
Osumi T, Kishimoto Y, Kano R, Maruyama H, Onozaki M, Makimura K, Ito T, Matsubara K, Hasegawa A. Prototheca zopfii genotypes isolated from cow barns and bovine mastitis in Japan. Vet Microbiol. 2008;131:419–23.
Ricchi M, Goretti M, Branda E, Cammi G, Garbarino CA, Turchetti B, Moroni P, Arrigoni N, Buzzini P. Molecular characterization of Prototheca strains isolated from Italian dairy herds. J Dairy Sci. 2010;93:4625–31.
Jagielski T, Lassa H, Ahrholdt J, Malinowski E, Roesler U. Genotyping of bovine Prototheca mastitis isolates from Poland. Vet Microbiol. 2011;149:283–7.
Gao J, Zhang HQ, He JZ, He YH, Li SM, Hou RG, Wu QX, Gao Y, Han B. Characterization of Prototheca zopfii associated with outbreak of bovine clinical mastitis in herd of Beijing. China. Mycopathologia. 2012;173:275–81.
Jagielski T, Lagneau P. Protothecosis. A pseudofungal infection. Med Mycol. 2007;17:261–70.
Ueno R, Urano N, Suzuki M. Phylogeny of the non-photosynthetic green micro-algal genus Prototheca (Trebouxiophyceae, Chlorophyta) and related taxa inferred from SSU and LSU ribosomal DNA partial sequence data. FEMS Microbiol Lett. 2003;223:275–80.
Roesler U, Möller A, Hensel A, Baumann D, Truyen U. Diversity within the current algal species Prototheca zopfii: a proposal for two Prototheca zopfii genotypes and description of a novel species, Prototheca blaschkeae sp. nov. Int J Syst Evol Microbiol. 2006;56:1419–25.
Satoh K, Ooe K, Nagayama H, Makimura K. Prototheca cutis sp. nov., a newly discovered pathogen of protothecosis isolated from inflamed human skin. Int J Syst Evol Microbiol. 2010;60:1236–40.
Melville PA, Benites NR, Sinhorini IL, Costa EO. Susceptibility and features of the ultrastructure of Prototheca zopfii following exposure to copper sulphate, silver nitrate and chlorexidine. Mycopathologia. 2002;156:1–7.
Acha PN, Szyfres B. Zoonosis y Enfermidades Transmissibles Communes al Hombre y a los Animals. 3rd ed. Washington: Organización Panamericana de la Salud; 2003.
Ahrholdt J, Murugaiyan J, Straubinger RK, Jagielski T, Roesler U. Epidemiological analysis of worldwide bovine, canine and human clinical Prototheca isolates by PCR genotyping and MALDI-TOF mass spectrometry proteomic phenotyping. Med Mycol. 2012;50:234–43.
Huth N, Wenkel RF, Roschanski N, Rösler U, Plagge L, Schöniger S. Prototheca zopfii genotype 2-induced nasal dermatitis in a cat. J Comp Pathol. 2015;. doi:10.1016/j.jcpa.2015.02.001.
Marques S, Silva E, Carvalheira J, Thompson G. In vitro antimicrobial susceptibility of Prototheca wickerhamii and Prototheca zopfii isolated from bovine mastitis. J Dairy Sci. 2006;89:4202–4.
Roesler U, Scholz H, Hensel A. Immunodiagnostic identification of dairy cows infected with Prototheca zopfii at various clinical stages and discrimination between infected and uninfected cows. J Clin Microbiol. 2001;39:539–43.
Linares MJ, Solís F, Casal M. In vitro activity of voriconazole against Prototheca wickerhamii: comparative evaluation of Sensititre and NCCLS M27-A2 methods of detection. J Clin Microbiol. 2005;43:2520–2.
Bexiga R, Cavaco L, Vilela CL. Isolation of Prototheca zopfii from bovine milk. Rev Port Cienc Vet. 2003;98:33398.
Buzzini P, Turchetti B, Facelli R, Baadino R, Cavarero F, Mattaalia L, Mosso P, Martini A. First large-scale isolation of Prototheca zopfii from milk produced by dairy herds in Italy. Mycopathologia. 2004;158:427–30.
Sobukawa H, Yamaguchi S, Kano R, Ito T, Suzuki K, Onozaki M, Hasegawa A, Kamata H. Molecular typing of Prototheca zopfii from bovine mastitis in Japan. J Dairy Sci. 2012;95:4442–6.
National Mastitis Council. Procedures for collecting milk samples in microbiological procedures for the diagnosis of bovine udder infection and determination of milk quality. Madison: National Mastitis Council Inc. 2004.
Liu G, Liu Y, Ali T, Ferreri M, Gao J, Chen W, Yin J, Su J, Fanning S, Han B. Molecular and phenotypic characterization of Aerococcus viridans associated with subclinical bovine mastitis. PLoS ONE. 2015. doi:10.1371/journal.pone.0125001.
Colin M. Basic mastitis bacteriology: untangling the pathogens. Irish Vet J. 2006;59:453–9.
Kumar S, Tamura K, Nei M. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 2004;5:150–63.
Scaccabarozzi L, Turchetti B, Buzzini P, Pisoni G, Bertocchi L, Arrigoni N, Boettcher P, Bronzo V, Moroni P. Short communication: isolation of Prototheca species strains from environmental sources in dairy herds. J Dairy Sci. 2008;91:3474–7.
Aouay A, Coppée F, Cloet S, Cuvelier P, Belayew A, Lagneau PE, Mullender C. Molecular characterization of Prototheca strains isolated from bovine mastitis. J Med Mycol. 2008;18:224–7.
Kishimoto Y, Kano R, Maruyama H, Onozaki M, Makimura K, Ito T, Matsubara K, Hasegawa A, Kamata H. 26 s rDNA-based phylogenetic investigation of Japanese cattle-associated Prototheca zopfii isolates. J Vet Med Sci. 2010;72:123–6.
Chang R, Yang Q, Liu G, Liu Y, Zheng B, Su J, Han B. Treatment with gentamicin on a murine model of Protothecal mastitis. Mycopathologia. 2013;175:241–8.
Zhao J, Liu W, Lv G, Shen Y, Wu S. Protothecosis successfully treated with amikacin combined with tetracyclines. Mycoses. 2004;47:156–8.
Jagielski T, Buzzini P, Lassa H, Malinowski E, Branda E, Turchetti B, Polleichtner A, Roesler U, Lagneau PE, Marques S, Silva E, Thompson G, Stachowiak R, Bielecki J. Multicentre Etest evaluation of in vitro activity of conventional antifungal drugs against European bovine mastitis Prototheca spp. isolates. J Antimicrob Chemother. 2012;67:1945–7.
Acknowledgments
This research was supported by the Chinese Twelfth “Five-year” National Science and Technology Support Project (No. 2012BAD12B03), National Education Ministry’s Major Project (No. 313054), Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) State Education Ministry (No. 20120008110042), High-end Foreign Experts Recruitment Program (No. GDT20141100043) and the National Natural Science Foundation of China (No. 31572587).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interest.
Additional information
Muhammad Shahid and Tariq Ali have equally contributed to this work.
Rights and permissions
About this article
Cite this article
Shahid, M., Ali, T., Zhang, L. et al. Characterization of Prototheca zopfii Genotypes Isolated from Cases of Bovine Mastitis and Cow Barns in China. Mycopathologia 181, 185–195 (2016). https://doi.org/10.1007/s11046-015-9951-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11046-015-9951-9