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Applied and Environmental Microbiology, December 2009, p. 7445-7452, Vol. 75, No. 23
0099-2240/09/$08.00+0     doi:10.1128/AEM.00850-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Endosymbiotic Bacteria in the Parasitic Ciliate Ichthyophthirius multifiliis{triangledown}

H. Y. Sun,1,2,{dagger} J. Noe,1 J. Barber,1 R. S. Coyne,3 D. Cassidy-Hanley,4 T. G. Clark,4 R. C. Findly,1* and H. W. Dickerson1

Department of Infectious Diseases, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602,1 School of Life Sciences, Zhongshan University, Guangzhou 510275, People's Republic of China,2 J. Craig Venter Institute, Rockville, Maryland 20850,3 Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 148534

Received 14 April 2009/ Accepted 17 September 2009

Endosymbiotic bacteria were identified in the parasitic ciliate Ichthyophthirius multifiliis, a common pathogen of freshwater fish. PCR amplification of DNA prepared from two isolates of I. multifiliis, using primers that bind conserved sequences in bacterial 16S rRNA genes, generated an ~1,460-bp DNA product, which was cloned and sequenced. Sequence analysis demonstrated that 16S rRNA gene sequences from three classes of bacteria were present in the PCR product. These included Alphaproteobacteria (Rickettsiales), Sphingobacteria, and Flavobacterium columnare. DAPI (4',6-diamidino-2-phenylindole) staining showed endosymbionts dispersed throughout the cytoplasm of trophonts and, in most, but not all theronts. Endosymbionts were observed by transmission electron microscopy in the cytoplasm, surrounded by a prominent, electron-translucent halo characteristic of Rickettsia. Fluorescence in situ hybridization demonstrated that bacteria from the Rickettsiales and Sphingobacteriales classes are endosymbionts of I. multifiliis, found in the cytoplasm, but not in the macronucleus or micronucleus. In contrast, F. columnare was not detected by fluorescence in situ hybridization. It likely adheres to I. multifiliis through association with cilia. The role that endosymbiotic bacteria play in the life history of I. multifiliis is not known.

* Corresponding author. Mailing address: Department of Infectious Diseases, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602. Phone: (706) 542-5793. Fax: (706) 542-5771. E-mail: rfindly{at}uga.edu

{triangledown} Published ahead of print on 9 October 2009.

{dagger} Present address: Key Laboratory of Marine Bio-Resource Sustainable Utilization (LMB), South China Sea Institute of Oceanology, Chinese Academy of Sciences (CAS), Guangzhou 510301, China.

Applied and Environmental Microbiology, December 2009, p. 7445-7452, Vol. 75, No. 23
0099-2240/09/$08.00+0     doi:10.1128/AEM.00850-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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