Histopathological, biological and molecular characteristics of the pathogenic Spiroplasma penaei isolated from the hemolymph of infected Pacific white shrimp, Penaeus vannamei

Biological and molecular characteristics of the pathogenic mollicute, Spiroplasma penaei, isolated from the hemolymph of infected Pacific white shrimp, Penaeus vannamei, were investigated. The doubling times of a S. penaei were 6.13 h (2% NaCl) and 3.43 h (no salt) under aerobic conditions, and 6.63 h (2% NaCl) and 3.22 h (no salt) under anaerobic conditions. Small diffuse white colonies with granular centers, surrounded by small satellite colonies that appeared embedded in the agar matrix, were detected on solid M1D medium (2% Noble agar) under aerobic conditions at 28°C. The genome size of the S. penaei was 1778 Kb, as determined by pulsed-field gel electrophoresis using undigested DNA. Reduction of virulence of S. penaei was not detected in serial passage 24 and 76 isolates but passage 131 isolate was attenuated as indicated by the number of surviving shrimp and histological findings of challenged P. vannamei. Toxicity was not detected in supernatant fractions of M1D medium cultures of S. penaei isolates. The most predominant host responses to the S. penaei reference isolate and to serial passage isolates were hemocytic nodules and hemocytic infiltration observed in hematoxylin and eosin-stained histological sections. Transmission electron microscopy of the lymphoid organ of experimentally infected P. vannamei depicted S. penaei without cell wall and free in the cytoplasm of lymphoid organ cells. The lesions observed in histological sections were verified by in situ hybridization using a digoxigenin (DIG)-labeled probe specific to the spiralin gene of Spiroplasma spp. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S and 23S-5S ISR, were constructed using the distance-based Neighboring-Joining method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborates the observations by other investigators using the 16S gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to S. insolitum and distantly related to Spiroplasma sp. SHRIMP from China.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Pathobiology

Grantor dc:publisher

The University of Arizona.

Year dc:date.issued

2009

Author dc:creator

• Heres, Allan Alberto

Advisor dc:contributor.advisor

• Lightner, Donald V

Committee members dc:contributor.committeemember

• Lightner, Donald V
• Reggiardo, Carlos
• Songer, Glenn

• Histopathology
• Penaeus vannamei
• Phylogenetic analysis
• Spiroplasma penaei
• Systemic infection
• Virulence

Statement dc:rights

• Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.

Language dc:language.iso

EN

Handle dc:identifier.uri

http://hdl.handle.net/10150/196040

OAI identifier oai:identifier

oai:repository.arizona.edu:10150/196040