Biomedical subjects

Linda M Nunan

Publications and source records attributed to Linda M Nunan.

4 recordsLinked to original sources

PubMed2005

Spiroplasma penaei sp. nov., associated with mortalities in Penaeus vannamei, Pacific white shrimp.

A new bacterial strain, designated SHRIMP(T), isolated from the haemolymph of the Pacific white shrimp, Penaeus vannamei, was serologically distinct from other spiroplasmas. Cells of this strain were helical in form and variable in length. Examination by electron microscopy revealed wall-less cells delineated by a single cytoplasmic membrane. The organisms grew well in M1D media supplemented with 2 % NaCl. Strain SHRIMP(T) grew at temperatures of 20-37 degrees C, with optimum growth occurring at 28 degrees C. The strain catabolized glucose and hydrolysed arginine, but did not hydrolyse urea. The G+C content of the DNA was 29 +/-1 mol%. Strain SHRIMP(T) (=ATCC BAA-1082T=CAIM 1252T) is designated the type strain of a novel species, Spiroplasma penaei sp. nov., which represents a new subgroup (I-9) of the group I spiroplasmas.

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PubMed2004

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

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.

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PubMed2003

Experimental infection of Penaeus vannamei by a rickettsia-like bacterium (RLB) originating from P. monodon.

A rickettsia-like bacterium (RLB), which caused severe mortalities of commercially farmed Penaeus monodon in the southwest region of Madagascar, was investigated to determine whether the organism would produce the same disease in P. vannamei. Two series of bioassays were performed to determine whether this RLB could be transmitted to P. vannamei through injection and per os exposure. The first series of challenge bioassays used frozen, RLB-infected P. monodon tissue from Madagascar as the inoculum and feed for the injection, and per os bioassays with specific pathogen free (SPF) P. vannamei. In the second series of bioassays, frozen RLB-infected P. vannamei tissue derived from the first series of injection bioassays was used as the inoculum to challenge by injection and per os SPF P. vannamei. Disease status was determined through standard histological techniques and by in situ hybridization assays with a digoxigenin-labeled probe specific for this RLB. The results indicated that P. vannamei did develop the RLB infection when injected with either RLB infected P. monodon or P. vannamei tissue homogenates. This contrasts with results from the per os exposure to the RLB in which the disease could not be reproduced.

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PubMed2003

Molecular detection methods developed for a systemic rickettsia-like bacterium (RLB) in Penaeus monodon (Decapoda: Crustacea).

Molecular detection methods were developed to aid in the diagnosis of a rickettsia-like bacterium (RLB) which caused severe mortalities of farm-raised Penaeus monodon in Madagascar. Using primers derived from the 16S rRNA gene of bacteria, a PCR assay was optimized to amplify this region of the genome of the RLB, using extracted DNA from infected P. monodon tissue as the template. The resulting amplified PCR product was sequenced and 2 novel primers were selected from the variable region of the gene. These primers amplified a 532 bp fragment of DNA originating from the rickettsia-infected samples. The PCR assay was optimized and tested on DNA extracted from specific pathogen-free (SPF) P. vannamei tissue and several other strains of bacteria. The PCR assay with the rickettsia-specific primers was specific for this RLB and did not amplify the other DNA samples tested. The 532 bp PCR-amplified fragment was labeled with digoxigenin (DIG) for in situ hybridization assays. This probe was tested on SPF, RLB and bacteria-infected shrimp specimens preserved in Davidson's fixative. The probe was specific for both natural and experimental rickettsial infections. Hybridization with this probe required a stringent temperature of 65 degrees C, otherwise cross-reactivity was observed with other types of bacteria.

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