1 Key Laboratory for Bioresources Technology of Jiangsu Province, College of Biological Sciences, Nanjing Normal University, Nanjing 210097, PR China
2 Department of Biological Science and Technology, Nanjing University, Nanjing 210093, PR China
3 Department of Microbiology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China
4 Department of Biological Sciences, Towson University, Towson, MD 21252, USA
5 Jiangsu Freshwater Fishery Research Institute, Nanjing 210017, PR China
Correspondence
Wen Wang
njnuwang{at}263.net
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ABSTRACT |
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Present address: Institute of Hydrobiology of Chinese Academy of Sciences, Wuhai 430072, PR China.
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INTRODUCTION |
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The causal agent of tremor disease was previously thought to be a rickettsia-like organism based on morphological and pathological studies (Wang & Gu, 2002; Wang et al., 2002
). The agent lacked a nucleus but contained a nucleoid, and was 0·2x0·9 µm when bacilliform and between 0·22 and 0·35 µm in diameter when spherical. It had a preference for infecting nerve, muscle and connective tissues (Wang et al., 2001
). Haemocytes played a major role in propagation and transmission (Wang & Gu, 2002
). Microcolonies of the agent within membrane-bound vacuoles were seen in tissue smears stained with Giemsa. Characteristics such as transverse binary division in cytophagous vacuoles, formation of inclusion bodies or appearance as single cells in the cytoplasm, and possible inhibition of phagosome-lysosome fusion (no lysosome function was observed in infected cells), suggested that this agent might be a member of the genus Ehrlichia (Wen, 1999
).
The 16S rRNA gene sequence (Gasparich, 2002) was analysed in order to confirm the taxonomic position of the agent. The results showed that the agent was not a rickettsia, but rather a spiroplasma, with its 16S rRNA gene having 98 % sequence identity with that of Spiroplasma mirum. This result prompted a review of our previous studies and re-examination of the agent's status, including investigation of its cultivability and the capacity of the isolated agent to cause disease in experimentally inoculated crabs.
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METHODS |
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The cloned 16S rRNA gene fragments were sent to Shanghai Biological and Engineering Inc. for DNA sequence determination (ABI PRISM 377-96; BigDye Terminator v2.0). Sequences were compared to those in the GenBank database using the program BLASTN. The 16S rRNA gene sequences obtained from DNA extracted from the haemolymph (Crab22, CrabSP6), pectoral ganglion (Crab10, CrabT7), and cardiac muscle (Crab25), and from cultivated organisms (CrabSP6Cult) were used for phylogenetic analysis. The 16S rRNA gene sequences from the samples above were aligned with 16S rRNA gene sequences from representatives of the major spiroplasma clades and appropriate outgroups using CLUSTAL_W (Thompson et al., 1994) and then aligned manually in MacClade (Maddison & Maddison, 1992
). Phylogenetic reconstruction was performed using maximum-parsimony. Acholeplasma laidlawii was used as an outgroup. A total of 1428 characters were used for the majority of sequences. The analysis used a heuristic search and the tree bisection reconnection maximum-parsimony algorithm for branch swapping. The dataset was resampled 500 times to obtain bootstrap percentage values using PAUP (version 4.0b10; Swofford, 1998
).
Primary isolation and cultivation of the tremor-disease-causing agent.
The tremor-disease-causing agent was first cultivated in the yolk sacs of embryonated chicken eggs. Blood from a crab with tremor disease was passed through a membrane filter with mean pore diameter of 220 nm and the filtrate was inoculated into the yolk sac of ten 7-day-old chicken embryos. Four further embryos were inoculated with a filtrate of blood from healthy crabs. Eggs were also inoculated with 100 U penicillin to inhibit bacterial growth. Eggs were incubated at 35·1 °C and candled daily to check embryo viability.
M1D broth (Whitcomb, 1983) was used for further cultivation of the agent. Tubes containing 1·5 ml medium were inoculated with 0·05 ml fluid from embryonated chicken eggs, incubated at 30 °C and observed daily. After acidification of the cultures, dark-field microscopy was used to examine the medium for the presence of organisms and to determine their morphology. Subcultures were made to ensure that growth was sustained by the medium and at least 10 additional passages were carried out. M1D agar was prepared by inclusion of 1·5 % Noble agar in the broth medium and the plates were inoculated with the tenth passage culture in M1D broth.
Crab inoculation experiments.
A total of 28 healthy crabs were used for inoculation experiments. Eighteen healthy crabs were each inoculated with 0·1 ml of a filtrate of yolk sac fluid diluted in PBS (pH 6·8) and 10 crabs were each inoculated with 0·1 ml of saline as a control. A group of 25 healthy crabs were used for another inoculation experiment. Ten crabs were each inoculated with 0·1 ml of the tenth passage of the agent in M1D and another 10 crabs were inoculated with 0·1 ml of a suspension of colonies from M1D agar in PBS (pH 6·8). Five crabs were each inoculated with 0·1 ml medium as a control. All the crabs were observed daily and tested by a blood smear method every 5 days to determine whether they were infected. The body of the crab was cleaned with water and disinfected using 75% alcohol, and a needle was introduced into the crab through the joint between the pereiopod and the thorax. A small aliquot of blood (about 0·05 ml) was obtained with a 1 ml syringe and smeared on a glass slide. The smear was fixed in 4% formaldehyde and the slide was air-dried and stained with Giemsa solution diluted 1 : 10 with phosphate buffer (pH 6·8) for 1520 min. The film was washed with water and examined by light microscopy. Pink to bluish-purple inclusion bodies were identified in cells in smears from diseased crabs. Impression smears of the heart, gills, hepatopancreas, pereiopod muscles, thoracic ganglion and gonads from diseased and healthy crabs were also prepared and stained using the same method.
Electron microscopy.
For negative staining, samples of the organism cultured in M1D were placed on Formvar-coated copper grids. The excess fluid was removed by touching the grids with filter paper and the preparation was fixed with 2·5 % glutaraldehyde in PBS for 1 min. After removal of excess fluid by touching with filter paper, the grids were stained with 2 % sodium phosphotungstate for 3040 s and were air-dried before examination. For ultrathin sectioning, samples of blood cells, cardiac and pereiopod muscle, and pectoral ganglions of both control crabs and crabs inoculated with M1D cultivated organisms were fixed with 2·5 % glutaraldehyde in phosphate buffer. The colonies on M1D agar were embedded first in 1·5 % Noble agar and then fixed with 2·5 % glutaraldehyde in phosphate buffer. All samples were secondarily fixed with 1 % osmium tetraoxide in the phosphate buffer, then dehydrated with a series of acetone washes (10 %, 20 %, 30 %, 50 %, 70 %, 90 %, 100 %) and embedded in Epon 812. Ultrathin sections of 5080 nm were made using a ReichetJung ultramicrotome and stained with uranyl acetate and lead citrate. The sections were examined using a Hitachi 600-2A transmission electron microscope.
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RESULTS |
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The tremor-disease-causing agent grew well in M1D broth at 2530 °C, with a doubling time of 69 h within 10 passages. Doubling times were estimated using the method of Konai et al. (1996). On M1D agar the agent produced colonies with a diameter of 0·40·5 mm after 1725 days of aerobic incubation at 30 °C (not shown). Neither fried egg colonies nor satellite growth around central zones were seen.
Crab inoculation experiments
All 18 crabs inoculated with the filtrate of the yolk sac fluid developed typical signs of tremor disease (tremor of pereiopods) within 815 days and died within 25 days, whereas 10 crabs inoculated with control yolk sac fluid remained healthy. The agent could be detected in Giemsa-stained smears of tissue from all 18 crabs and in ultrathin sections of tissue from all crabs by electron microscopy (Wang & Gu, 2002). Ten crabs inoculated with the tenth passage of the agent in broth and another 10 crabs inoculated with organisms from colonies on M1D agar showed typical signs of tremor disease (tremor of pereiopods) within 714 days and died within 23 days, whereas five crabs inoculated with 0·1 ml of M1D medium as a control were healthy. The agent was detected by light and electron microscopy in haemolymph, muscles, nerves and connective tissues of the crabs inoculated with the filtrate of yolk sacs or with cultivated organisms. The agents were similar in structure to those grown in eggs and broth (Fig. 2
), and experimental infection produced disease similar to that seen in natural infection.
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Ultrathin sections of the organisms showed a typical trilaminar membrane with no cell wall, but with an outer layer (Fig. 2, Fig. 4
, solid arrows). This structure was previously mistaken for a cell wall because the outer layer was electron-dense. At higher magnification, it can be seen that the width of its membrane is similar to that of the host cell membrane (Fig. 2
, solid arrow).
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DISCUSSION |
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The Spiroplasma mirum cluster contains a single species (group V) from rabbit ticks (Tully et al., 1983). Although spiroplasmas have been well documented as causes of disease in insects, they have not been detected in aquatic animals or crustaceans. Our detection of spiroplasmas in the mitten crab may increase understanding of the host range of these organisms and provoke investigation of other possible hosts in water, as well as possible relationships between terrestrial hosts and aquatic hosts. These findings have implications not only for studies on pathogenic spiroplasmas, but also for studies of freshwater ecology and epidemiology.
Further work is needed to explore the origin of this pathogen. Studies on the biological properties of this agent and its infection cycle in crabs will be valuable in the development of methods to control tremor disease. Since the agent responsible has now been confirmed to be a spiroplasma, the disease might be more appropriately described as spiroplasmosis.
There are some differences between S. mirum and the tremor-disease-causing agent, although they have 98 % identity in their 16S rRNA genes. The colonies on M1D agar appeared only after 1725 days of incubation at 30 °C, which is 711 days longer than that taken for S. mirum. The fried egg colonies and satellite growth around central zones that are typical of S. mirum were not seen. The three criteria most useful in spiroplasma taxonomy are the 16S rRNA gene sequence, DNADNA hybridization analyses and serology. The highest resolution is provided by 16S rRNA gene sequence analysis, which is useful for discrimination of most species (Whitcomb et al., 1999). However, the evolutionary distances between members of groups VIII and XVI are much smaller and rRNA gene sequences are unable to distinguish them at the subgroup level (Gasparich, 2002
). The inability of 16S rRNA gene sequences to discriminate closely related microbial species has been reported previously (Fox et al., 1992
). Our study has shown that the organism isolated from Chinese mitten crabs is indeed a member of the Spiroplasma genus. However, further study is needed to identify the actual species, which is closely related to S. mirum, but which may be distinct.
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ACKNOWLEDGEMENTS |
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Received 4 April 2004;
accepted 17 June 2004.
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