Characterization and molecular methods for detection of a novel spiroplasma pathogenic to Penaeus vannamei

Diseases of Aquatic Organisms

Fernando Aranguren¹  Marcela Salazar¹  Carlos R. Pantoja¹  Donald V. Lightner¹  Linda M. Nunan¹ 

关键词: Spiroplasma;    Penaeus vannamei;    Shrimp;   

DOI  :  10.3354/dao062255

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

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 sequencehomology, 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 vannameisuffering 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 inflammatoryreactions 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 wasamplified 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 fordevelopment 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 reactionsor cross-reactivity occurring with other bacterial samples tested in this format.

【 授权许可】

Unknown