Electron Microscopic Evidence of Bacilliform Virus Infection in Kuruma Shrimp (Penaeus japonicus)

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1 Electron Microscopic Evidence Bacilliform Virus Infection in Kuruma Shrimp (Penaeus japonicus) Yukinori Takahashi*1, Toshiaki Itami*1, Masakazu Kondo*2, Minoru Maeda*1, Reiko Fujii*3, Susumu Tomonaga3, Kidchakan Supamattaya*4 and Sitdhi Boonyaratpalin*5 *1 Department Aquaculture and Biology, Shimonoseki University Fisheries, Shimonoseki, Yamaguchi , Japan *2 Ono Limnological Station, Shimonoseki University Fisheries, Ube, Yamaguchi , Japan *3 School Allied Health Sciences, Yamaguchi University, Ube, Yamaguchi 755, Japan *4 Department Aquatic Science, Faculty National Resources, Prince Songkhla University, *5 National Hadyai, Songkhla 90110, Thailand Institute Coastal Aquaculture, Songkhla 90000, Thailand (Received January 27, 1994) Beginning in the spring 1993, a serious mortality among cultured kuruma shrimp, Penaeus japonicus occurred in Japan. The typical sign this disease was white spots on the inside surface the carapace. Challenge tests demonstrated that the causative agent was highly virulent. This was demonstrated by injection a filtrered homogenate the lymphoid organ obtained from diseased shrimp. A non-occluded bacilliform virus was found by electron microscopy in the lymphoid organ both naturally and experimentally infected shrimps. The virion was bacilliform and surrounded by a virion envelope (a lipid bilayer membrane). The dimension the virion was 83 nm in diameter and 275 nm in length. From these results, the bacilliform virus is considered to be the causative agent the disease. Outbreaks serious mortality among populations cultured kuruma shrimp, Penaeus japonicus have occurred in Japan since the early spring According to our epidemiological survey, outbreaks the disease occurred in the area where juvenile shrimps were imported from China, but not in the area where imported shrimps were not introduced. Losses were observed 2-3 weeks after the arrival the imported population shrimp, weighing 3-5 g, when the water temperatures were approximately 19 Ž. The disease then spread to cultured shrimps held in separate grow-out ponds nearby. Typical sings this disease were white spots (1-2 mm in diameter) on the inside surface the carapace. This report deals with the challenge trial using the filtrate infected shrimps and demonstration bacilliform virus in the diseased shrimp by electron microscopy. fungal examination gills, lymphoid organs, hepato pancreas, heart and muscle were carried out. Challenge trials were conducted using healthy kuruma shrimp collected from a shrimp farm where no viral disease had been reported. Shrimp, weighing an average about 6 g, were held for 1 h in a solution 0.1% sulfamonomethoxine-na prior to challenge and were fed a medicated diet containing oxolinic acid (Kyowa Hakko Kogyo Co., Ltd.) before and during the experiment to prevent bacte rial infection. Twenty shrimps were used in each experimental and a control group. Shrimps were kept in a glass aquarium at 22 Ž with aeration and sand was layered 3 cm deep on the bottom. Virus infected lymphoid organs collected from diseased shrimps were used for challenge. The lymphoid organs were homogenized in modified Medium 199 (Flow Laboratories) supplemented with salts (Table Materials and Methods Diseased kuruma shrimp were collected from cul ture farms in Japan. Routine bacterial, parasitic and 1) and centrifuged at 2,000 rpm for 10 min at 4 Ž. The supernatant was filtered through a 0.22ƒÊm membrane. Filtrates were injected into the muscle the third abdominal segment at 1% (v/w) body

2 122 Table Y. Takahashi 1. Contents Medium 199 salts for supplementation et al. to ph adjusted to 7.6 by 7.5% NaHCO3. weight. Filtrates prepared from a normal lymphoid organ were injected into shrimps as the control group. Each shrimp received about 0.6 ml (1% body weight) 1 :103 diluted filtrates obtained from one paired lymphoid organ an infected shrimp. We carried out bacterial, fungal and parasitic examination on these shrimp. For transmission electron microscopy (TEM), the lymphoid organ was sampled from the moriboud shrimp, fixed in a mixture, 2% glutaraldehyde and 2% paraformaldehyde in 0.2 M cacodylate buffer Fig. 2. White spots (arrow) on the removed carapace diseased Penaeus japonicus (A) and a normal carapace healthy shrimp (B). Fig. A light (ph 7.4) for several hours, and then postfixed in 2% OsO4fbrIh..The tissue was dehydrated ded in Epon.Ultrathin Ultrotome citrate,and sections were and stained with uranyl examined and embedcut on a LKB acetate and lead with a JEM-200CX electron microscope. 3. micrograph carapace icus. these Table 2. removed Dark spots. Cumulative icus after genate diseased or area Bar=0.5 the white spots from diseased Penaeus were found in the center Fig. 5. Fig. 6. mm. mortality (%) Penaeus japoninjection with the filtered homolymphoid organ obtained from healthy shrimp Fig. 1. White spots (arrow) appeared on the carapace diseased Penaeus japonicus (A) and a normal carapace healthy shrimp (B). Fig. 4. the japon- A electron micrograph lymphoid organ obtained from experimentally infected Penaeus japonicus. Note virions (arrows) in the nucleus. Bar=500nm. Cross section virions observed in Fig. 4. E, envelope; NC, nucleocapsid. Bar=100nm. Longitudinal section virion observed in Fig. 4. Bar=100nm.

3 Bacilliform virus infection in kuruma shrimp 123

4 124 Y. Takahashi et al Results One the typical external signs infected shrimp were white spots on the inside surface the cara pace (Figs. 1 and 2). There was a dark area in the center this spot when observed under the light microscope (Fig. 3). The body color the infected shrimp became pale and reddish in color. The lymphoid organ the diseased shrimp was swollen or shrunk. Pathogenic fungi and parasites were not found in naturally infected shrimp. Vibrio sp. PJ (Takahashi et al., 1985; de la Pena et al., 1993) or other species Vibrio were sometimes isolated from the lymphoid organ, heart and muscle these shrimps. However, these bacteria, when injected into shrimp, did not reproduce the white spots on the carapace as found in the naturally infected shrimp. In a challenge test, bacteria were rarely isolated from the lymphoid organs, heart and muscle. Inject ed shrimp started dying 2 days after inoculation and all shrimps died within 8 days (Table 2). Typical clinical signs including white spots on the carapace and the reddish coloration were observed in all dead shrimp. Rod-shaped virions were found in the nucleus the cells infected lymphoid organs obtained from the experimentally challenged shrimp (Fig. 4) and also found in the lymphoid organs naturally in fected shrimp. The virion possessed an envelope around a central nucleocapsid (Fig. 5). The enve lope was approximately 10 nm in thickness and the space between the envelope and the nucleocapsid was about 5 nm. An average size the complete virions were 83 nm in diameter and 275 nm in length: the size the nucleocapsid was 54 nm in diameter and 216 nm in length (Fig. 6). Occlusion body was not found in any TEM observations. Discussion Five baculoviral infections have been reported in penaeid species; Baculovirus penaei disease (Couch, 1974), Monodon baculovirus disease (Lightner et al., 1983), Baculoviral mid-gut gland necrosis (Sano et al., 1981), Plebejus baculovirus disease (Lester et al., 1987), and Yellow head disease (Booyaratpalin et al., 1993). The baculovirus associated with the first four diseases infected the hepatopancreas. The hepatopancreas, therefore, is thought to be the target organ for these viruses. Yellow head disease is the only viral disease shrimp, thus far reported, that causes severe necrosis in the lymphoid organ. The main clinical signs this disease are a pale body and light yellow coloration the hepatopancreas and gills. In this study, we demonstrated the presence virulent bacilliform virus both in the naturally and the experimentally infected lymphoid organs. In the preliminary study, we also found histopathological changes in the lymphoid organ the infected shrimp the present study. From our TEM observations, the agent this disease is considered to be a non-occluded bacilli form virus. Baculoviral mid-gut gland necrosis (BMN) is the only viral infection found in kuruma shrimp (Sano et al., 1981). The fine structure the BMN has a similar character to that the virus the present study. Both viruses appeared to be surrounded by a single envelope, although Sano et al. (1981) described as double membranes. The dimen sions the virions these two viruses are different. The virion the present study is 275 nm in length (vs. 310 nm for the BMN virus) and 83 nm in diam eter (vs. 73 nm for the BMN virus). The nucleocap sid diameter the present virus is 216 nm in length (vs. 250 nm for the BMN virus) and 54 nm in diam eter (vs. 36 nm for the BMN virus). The yellow head disease virus produced severe infection the lymphoid organ P. monodon. The dimensions the yellow head virus are, nm in length and nm in diameter, and are smaller than that the present virus. Both viruses have the lipid bilayer membrane. Study on the nucleic acid the baculo-like virus may be an important approach to clarify the taxo nomic position the virus. Acknowledgments The authors thank to Miss K. Harada, Laboratory Electron Microscopy, Yamaguchi University School Medicine, for technical assistance with the TEM. We thank J. L. Fryer, Oregon State Universi ty, for reading and commenting on the manuscript. References Boonyaratpalin, S., K. Supamattaya, J. Kasornchandra, S. Direkbusaracom, U. Aekpanithanpong and C. Chanta

5 Bacilliform virus infection in kuruma shrimp 125 nachooklin (1993): Non-occluded baculo-like virus, the causative agent yellow head disease in the black tiger shrimp (Penaeus monodon). Fish Pathol., 28, Couch, J. A. (1974): An enzootic nuclear polyhedrosis virus pink shrimp: ultrastructure, prevalence, and enhancement. J. Invertebr. Pathol., 24, de la Pena, L. D., T. Tamaki, K. Momoyama, T. Nakai and K. Muroga (1993): Characteristics the causative bacterium vibriosis in the kuruma prawn, Penaeus japonicus. Aquaculture, 115, Lester, R. J. G., A. Doubrovsky, J. L. Paynter, S. K. Sambhi and J. G. Atherton (1987): Light and electron microscope evidence baculovirus infection in the prawn Penaeus plebejus. Dis. Aquat. Org., 3, Lightner, D. V., R. M. Redman and T. A. Bell (1983): Observations on the geographic distribution, pathogene sis and morphology the baculovirus from Penaeus monodon Fabricius. Aquaculture, 32, Sano, T., T. Nishimura, K. Oguma, K. Momoyama and N. Takeno (1981) : Baculovirus infection cultured kuruma shrimp, Penaeus japonicus in Japan. Fish Pathol., 15, Takahashi, Y., Y. Shimoyama and K. Momoyama (1985): Pathogenicity and characteristics Vibrio sp. isolated from cultured kuruma prawn Penaeus japonicus Bate. Bull. Japan. Soc. Sci. Fish., 51,