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1 Department of Veterinary Sciences and Microbiology, University of Arizona, Tucson, AZ 85721, USA
2 Department of Biological Sciences, Towson University, Towson, MD 21252, USA
Correspondence
Linda M. Nunan
lmn{at}u.arizona.edu
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Published online ahead of print on 1 July 2005 as DOI 10.1099/ijs.0.63555-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SHRIMPT is AY771927.
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). Penaeus vannamei (taxonomy according to Holthuis, 1980), the Pacific white shrimp, is one of the predominant species of farm-raised shrimp cultivated in both the eastern and the western hemispheres. As with any monoculture, raising shrimp in ponds in close proximity to each other increases the spread of diseases (Kautsky et al., 2000), and disease has been attributed as the single largest source of economic loss in the aquaculture industry (Meyer, 1991). Cultured penaeid shrimp are susceptible to viral, rickettsial, bacterial, fungal, protozoan and metazoan infectious pathogens (Lightner, 1993). The most severe diseases of shrimp, causing the greatest economic losses to growers, are caused by viruses and bacteria (Lightner et al., 1992). In January 2002, severe mortalities of P. vannamei occurred in one pond at a Colombian shrimp farm located on the Caribbean coast. During the following stocking period in May–June 2002, two additional shrimp ponds at the same farm experienced high mortalities. The pathogenic spiroplasma identified as the causative agent spread during the next grow-out cycle to a neighbouring farm, which suffered mortalities ranging from 10 to 90 %.
Upon receipt of infected specimens from Colombia preserved in Davidson's alcohol–formalin–acetic acid, histological analysis was performed (Bell & Lightner, 1988). The presumptive diagnosis for the mortalities suffered was attributed to a severe bacterial infection. This diagnosis was further confirmed with the use of a universal digoxigenin-labelled bacterial probe and in situ hybridization assays (Nunan et al., 2004). Tissue from frozen infected P. vannamei from the initial epizootic was sampled for DNA extraction. Using universal 16S rRNA primers and PCR, the 16S rRNA gene of the bacterium was sequenced (Nunan et al., 2003). The sequence was submitted to GenBank and compared with other bacterial 16S rRNA genes. The results from a BLAST search revealed 16S rRNA gene sequence similarity of 99 % to Spiroplasma insolitum.
The large genus Spiroplasma is a group of wall-less eubacteria displaying a helical cell morphology. Spiroplasmas are motile, even though they lack flagella (Davis & Worley, 1973; Daniels et al., 1980; Trachtenberg, 1998). These bacteria have historically been associated with plants and arthropods, primarily insects and ticks (Clark, 1982; Tully et al., 1982; Williamson et al., 1989, 1998; Tully & Whitcomb, 1990), but more recently members of this genus have been discovered in the Chinese mitten crab (Eriocheir sinensi) (Wang et al., 2003, 2004) and the deep-sea alvinocarid shrimp Rimicaris exoculata (Zbinden & Cambon-Bonavita, 2003). Spiroplasmas can be pathogenic in plants and insects, and cause ‘tremor disease’ in freshwater mitten crabs (Wang et al., 2003). The Spiroplasma species associated with the marine alvinocarid shrimp appears to be part of the normal gut flora. In this paper, we present the results of a taxonomic study of the causative organism of the P. vannamei epizootic in Colombia. We present supporting data for the basis of the proposed minimal requirements for assignment of binomial names to mollicutes. The strain investigated, designated SHRIMPT, is considered to be the type strain of a proposed novel species, Spiroplasma penaei sp. nov. S. penaei is the first pathogenic spiroplasma to be isolated from a marine crustacean.
Spiroplasma strains
The method used initially to isolate strain SHRIMPT from the infected P. vannamei and the resultant mortalities observed experimentally have been described previously (Nunan et al., 2004). Briefly, the strain was cultivated by injection of a tissue homogenate, produced from frozen, naturally infected P. vannamei from Colombia, into specific pathogen-free (SPF) (Wyban et al., 1992; Pruder et al., 1995) P. vannamei, which in turn became infected. Frozen tissue (1 g) from the infected shrimp was sampled and homogenized in 5 ml Tris/NaCl buffer. The homogenate was diluted 1 : 5 with sterile 2 % saline and injected into the third abdominal segment of SPF P. vannamei. As shrimp became moribund, haemolymph was drawn by using a 25 g needle attached to a 1 ml syringe. The haemolymph was filtered through a 0·45 µm Acrodisc syringe filter (Pall Corporation) and added to M1D medium (Jones et al., 1977; Whitcomb et al., 1982) supplemented with 2 % NaCl, and then incubated at 30 °C. Following growth in the medium, as visualized by the phenol red component of the medium changing first to orange and then to yellow, the spiroplasma was examined by transmission electron microscopy (TEM) (Nunan et al., 2004). The cultured organism was triply cloned and used to infect additional SPF P. vannamei. The triply cloned spiroplasma culture (1 ml) was centrifuged for 3 min at 3000 g. The supernatant fluid was discarded and the bacterial pellet was resuspended in sterile 2 % saline. This inoculum was injected into the SPF shrimp. As mortalities occurred, shrimp were sampled for histological assessment and haemolymph was drawn for culture and TEM, as described previously (Nunan et al., 2004). Recovery of the organism and associated mortalities fulfilled Koch's postulates.
Culture media and cultivation techniques
The primary culture from which strain SHRIMPT was isolated was grown at 30 °C in M1D liquid medium (Whitcomb, 1983) supplemented with 2 % NaCl. After several passages, the isolate was triply cloned (Tully, 1983). This cloned isolate was designated SHRIMPT and was used in all subsequent characterization studies and also for the fulfilment of Koch's postulates. Temperature requirements for growth were assessed by the method of Konai et al. (1996). In addition, Serum Fraction (SF) medium, supplemented with 2 % NaCl, was used for all biochemical tests (Aluotto et al., 1970).
Morphological studies
Cells of strain SHRIMPT from cultures in the exponential phase of growth were examined by dark-field microscopy (magnification, x1000). Material for electron microscopy was established by pelleting 1 ml from a 24 h triple-clone culture at 3000 g for 3 min. The M1D medium was removed and replaced immediately with 1 ml cold 4 % buffered glutaraldehyde, prepared with 0·15 M Millonig's phosphate buffer (pH 7·0) and supplemented with 1 % NaCl and 0·5 % sucrose (Lightner, 1996). Following 1 h refrigeration (4 °C), the glutaraldehyde buffer was replaced with cold Millonig's phosphate buffer (0·15 M) and maintained at 4 °C until post-fixation. The pellet was then post-fixed with 1 % phosphate-buffered osmium tetroxide, dehydrated in ethyl alcohol and embedded in Spurr's resin (Ladd Research Inc.). The embedded pellet was sectioned to 75–90 nm thickness, stained with lead citrate and uranyl acetate and viewed by using a JEOL JEM 100CXII electron microscope, operated at 60 kV (Fig. 1).
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. Filtration characteristics (Tully, 1983) were determined by using mid-logarithmic cultures grown in M1D medium.
Serological tests
Anitserum to strain SHRIMPT was raised in rabbits as described by Williamson et al. (1979). The spiroplasma-deformation test (Williamson et al., 1978) and metabolism-inhibition test were used to compare strain SHRIMPT serologically with the type strains from all recognized Spiroplasma groups and subgroups. Hyperimmune antisera to all recognized Spiroplasma groups and subgroups are maintained in the Towson University Spiroplasma Reference Collection. Reciprocal tests were performed with strains for which antisera reacted with the SHRIMPT strain.
Genomic analysis
Extraction and purification of DNA from spiroplasmas was performed as described previously (Gasparich et al., 1993). The DNA was diluted in 1x SSC buffer to a final sodium concentration of 0·195 M. This sample was used (with a 1x SSC buffer blank) in a Cary Varian thermal spectrophotometer equipped with software to determine the melting temperature, and the DNA G+C content was determined (Carle et al., 1983). This process was repeated twice and a control of Escherichia coli DNA in 1x SSC buffer was used to verify the results.
Phylogenetic analysis
The 16S rRNA gene sequences used in this study with their GenBank accession numbers and ATCC culture collection accession numbers are indicated in Fig. 2. Sequences were aligned by using CLUSTAL W (Thompson et al., 1994) and then aligned manually in MACCLADE (Maddison & Maddison, 1992). A 1000-replicate bootstrap analysis was performed by using a heuristic search and the tree bisection–reconnection maximum-parsimony algorithm in PAUP (version 4.0b10; Swofford, 1998).
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). The mean cell diameter was 195 nm (n=20; range, 160–230 nm). Cells were surrounded by a single cytoplasmic membrane. Vesicular blebs are also displayed (Fig. 1a), very similar in appearance to those described previously from Spiroplasma citri (Razin et al., 1973).
Biochemical and biological properties
Strain SHRIMPT was able to grow in media containing glucose, glucose and arginine, and arginine alone, indicating abilities to ferment glucose with the production of acid and to catabolize arginine. A test was considered to be positive when the organism was able to grow in ten successive passages into the test medium. No growth was observed in unsupplemented SF medium or urea, and therefore urea hydrolysis was not tested. Cultures grew after passage through 220 nm filters, but not after passage through 100 nm filters.
Serological studies
Metabolism-inhibition and spiroplasma-deformation tests indicated that strain SHRIMPT was unrelated serologically to representatives of recognized Spiroplasma groups or species. Antigens and antisera of strain SHRIMPT were tested in all combinations against all recognized and putative groups and subgroups. Strains R8A2 (I-1), E275 (I-3), 277F (I-4), LB-12 (I-5) and P40 (I-8) showed only minimal serological cross-reactivity at a 20-fold dilution titre, but at no higher titres. All other reactions were negative. Reaction of strain SHRIMPT antiserum against strain SHRIMPT culture was to a dilution of 5120.
DNA base composition
The mean Tm value for strain SHRIMPT DNA was 81·8 °C, resulting in a base composition (G+C content) of 29±1 mol%. The E. coli standard measurements averaged 45±1 mol%.
Phylogenetic analysis
Fig. 2 shows the phylogenetic tree constructed by using maximum parsimony. The dataset was resampled 1000 times and bootstrap percentage values are indicated for each branch. Strain SHRIMPT groups consistently with the group I spiroplasma cluster and is associated most closely with S. insolitum, which is the type species for subgroup 6 of the group I spiroplasmas (Hackett et al., 1993). All strains within the group I cluster are pathogenic to plants, insects or now shrimp. This is also consistent with findings from the serological analyses, in which there was a very minor reaction with the subgroup I-6 antisera.
Habitat and pathogenicity
Strain SHRIMPT was isolated from the haemolymph of the Pacific white shrimp, P. vannamei. This pathogenic strain was first recognized in February 2002 in two shrimp ponds in Colombia. The marine shrimp were being cultured in a low-salinity (2–10 parts per million) region. The initial outbreak occurred during a period of unusually high temperatures, with the pond water reaching 37 °C for more than 3 h each day for over 2 weeks. Mortalities from the initial epizootic were highly variable and ranged from 20–30 to 60–70 %. To examine the pathogenicity of the spiroplasma, infectivity studies were initially performed by injecting a tissue homogenate from the infected Colombian shrimp into SPF P. vannamei (Nunan et al., 2004). As shrimp began to die and were moribund, haemolymph was extracted, filtered and added to M1D medium. The medium-cultured spiroplasma was then injected into SPF P. vannamei. To complete Koch's postulates, haemolymph was again sampled from moribund shrimp, filtered, added to M1D medium and examined by TEM, following 3 days growth, for the presence of the spiroplasma.
The properties described here for strain SHRIMPT fulfil the proposed criteria (ICSB, 1995) for species of the class Mollicutes, including the absence of a cell wall, filterability and penicillin resistance. The helicity and motility of cells of this organism, as well as its inability to hydrolyse urea, indicate that it is a member of the family Spiroplasmataceae in the order Entomoplasmatales (Tully et al., 1993). Serological comparisons of strain SHRIMPT with representatives of all of the major Spiroplasma species, groups and subgroups demonstrated the uniqueness of the strain. We therefore propose the name Spiroplasma penaei sp. nov. for this organism.
Description of Spiroplasma penaei sp. nov.
Spiroplasma penaei (pe.nae'i. N.L. n. Penaeus a genus of penaeid shrimp; N.L. gen. n. penaei of Penaeus vannamei, from which the organism was isolated).
Cells are helical and motile filaments with a mean diameter of 195 nm. Cells lack true cell walls. Acid is produced from glucose. Temperature range for growth is 20–37 °C and optimum growth occurs at 28 °C in M1D broth. Serologically distinct from previously characterized Spiroplasma species. Pathogenicity is indicated by injection into P. vannamei. The G+C content of the DNA is 29±1 mol%.
The type strain, SHRIMPT (=ATCC BAA-1082T=CAIM 1252T), was isolated from the haemolymph of the Pacific white shrimp, P. vannamei.
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ACKNOWLEDGEMENTS |
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