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DAO 62:255-264 (2004)
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Abstract
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Characterization and molecular methods for detection of a novel spiroplasma pathogenic to Penaeus vannamei
Linda M. Nunan1,*, Carlos R. Pantoja1, Marcela Salazar2, Fernando Aranguren2, Donald V. Lightner1
1Department of Veterinary Sciences and Microbiology, University of Arizona, Tucson, Arizona, 85721, USA
2CENIACUA, Carretera 8A # 96-60, Bogota, Colombia
*Email: lmn@email.arizona.edu
ABSTRACT: Traditionally, Spiroplasma spp. have only been isolated from the surfaces of flowers and other plant parts, from the guts and hemolymph of various insects, and from vascular plant fluids (phloem sap) and insects that feed on these fluids.
In this article, we report the first pathogenic spiroplasma to be discovered in shrimp and the results of its characterization through histological evaluation, in situ hybridization assays, transmission electron microscopy, 16S rRNA sequence
homology, and injection infectivity studies. In addition, molecular methods are described that were developed for the detection of this microorganism, which was determined to be the causative disease agent in Colombian farm-raised Penaeus vannamei
suffering from high mortalities. Using standard histological methods and in situ hybridization assays, it was confirmed that P. vannamei was infected with this pathogenic spiroplasma. Histological analysis revealed systemic inflammatory
reactions in affected organs/tissues. In an attempt to identify the bacteria, frozen infected P. vannamei samples, from the initial epizootic, were used to sequence the 16S rRNA gene and develop molecular detection methods. The 16S rRNA gene was
amplified by PCR and then sequenced. The sequence data were analyzed using the GenBank BLAST search and the results revealed a 98% homology with Spiroplasma citri, a pathogen of citrus trees. The 16S rRNA sequence data were evaluated for
development of unique PCR primers to the putative spiroplasma. Using PCR primers developed for the spiralin gene of Spiroplasma spp., a digoxigenin-labeled probe was developed and tested. This probe was species-specific, with no positive reactions
or cross-reactivity occurring with other bacterial samples tested in this format.
KEY WORDS: Spiroplasma · Penaeus vannamei · Shrimp
Full text in pdf format
Published in DAO Vol.
62, No. 3
(2004) on December 13
Print ISSN: 0177-5103; Online ISSN: 1616-1580.
Copyright © Inter-Research, Oldendorf/Luhe, 2004
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