Bacterial and Viral Diseases of Kuruma Shrimp (Penaeus japonicus)

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1 Fish Pathology, 33 (4), , Bacterial Viral Diseases Kuruma Shrimp (Penaeus japonicus) Jap Yukori Takahashi, Toshiaki Itami, Moru Maeda Masakazu Kondo Department Applied Aquabiology, National Fisheries University, Shimonoseki, Yamaguchi , Jap (Received Juary 30, 1998) Three major disease problems have been reported kuruma (Penaeus japonicus) culture Jap are vibriosis, penaeid acute viremia (PAV) baculoviral mid-gut gl necrosis (BMN). Vibriosis kuruma first reported 1985, sce n it has seriously damaged culture this Jap. characteristic signs this disease are brown spots lymphoid orgs gills, cloudess muscle sixth abdomal segment. This bacterium identified as a new genus Vibrio named Vibrio penaeicida. Outbreaks PAV, causg serious mortality kuruma beg closely related white spot baculovirus fection penaeid, have been reported Jap sce Diseased show white spots carapace reddish discoloration body. A non-occluded bacillim virus, penaeid rod-shaped DNA virus (), observed under trsmission electron microscope. In order dect virus, primers polymerase cha reaction (PCR) developed. PCR has been successfully used diagnosis dection carrier state crustaces. BMN is epizootic kuruma larvae Jap. typical sign this disease is a white-turbid mid-gut gl shows remarkable cellular necrosis no clusion body section. Rod-shaped particles, resemblg baculovirus, are found affected nuclei under electron microscope. Attempts made evaluate efficacy immunostimults kuruma as potential agents prophylaxis vibriosis PAV. oral admistration a peptidoglyc derived Bifidobacterium rmophilum is effective preventg se diseases kuruma. Key words:, PAV, WSSV, Vibrio penaeicida, vibriosis, BMNV Introduction seed production technique culture system kuruma developed M. Fudaga his colleagues early 1960s Jap after my ground-breakg studies over precedg 30 years. ir techniques have been spreadg throughout world have helped world culture dustry grow enormously. dustry Jap had progressed out major disease problems until early 1980s. In 1988, nual production kuruma Jap 3,020 mric ns, which a peak, n decreased Address Correspondence: Dr. Toshiaki Itami, National Fisheries University, 2-7-1, Nagata-Honmachi, Shimonoseki, Yamaguchi , Jap - Fax: itamit@fish-u.ac.jp 1,519 ns 1994, which is 50.3% peak pro duction. decrease production until 1992 maly due Vibrio penaeicida fection, which caused more th 60% tal losses. In 1993, re a drastic decrease production due troduction seeds fected penaeid rod-shaped DNA virus () Cha year. Accordg results our own survey, after this year, 60-80% tal losses caused. seed production dustry had suffered considerable losses due baculoviral mid-gut gl necrosis (BMN) (Arimo 1995). BMN virus (BMNV) has been extensively vestigated K. Momoyama T. So, so reviewg ir works would pot out importt areas future research.

2 Y. Takahashi, 358 T. Itami, M. Maeda M. Kondo penaeicida. Vihriosis th 1.35 ~ Vibriosis kuruma first reported Takahashi al. (1985a) its causative agent characterized de la Pena al. (1993) Ishimaru al. (1995). typical sympms this disease are brown spots lymphoid orgs (Fig. 1) jection gills, cloudess 6th abdomal segment. most characteristic hispathological chges extensive necrosis caused severe bacterial vasion multiple mation melized nodules lym- With phoid orgs (Egusa 1988). causative agent is a gram-negative short-rod bacterium (Fig. 2), beg classified as a member Vibrio (Takahashi 1985a), tentatively designated Vibrio sp. PJ de la Pena al. (1993). Ishimaru al. (1995) dermed this bacterium is a new member Vibrio proposed name Vibrio ~ ). This ported respect 48 h- 72 orgs temic field la survey Pena water technique (a dection 10-fg (Genmo dect With nate immunized commune Electronmicrograph 1ƒÊm). Vibrio penaeicida. (Scale bar: peptidoglyc rmophilum efficacy agast (Itami V. b) Itami vacce all reduced V. penaeicida blood chamber. migrate Two ƒà-1,3-gluc 30 homoge- chemokic hemocytes 1998), made mal-killed produced (Itami penaeicida prevent derived 1994; a Boyden Schizophillum effects contact or weight/ techniques duced body 1992a, challenged showed 1989, spray one or 2. application a membre order demonstrated attempts prepared immunostimults, Fig. through Hemocytes facr(s) V. rapeutic In (Itami vacce later. latent kuruma OTC/kg disease, (Itami immersion days dect tabls excellent V. penaeicida mortality sensitivity specificity we (OTC) 1985b). gtg vacces jection, reverse carrier had immunostimults cells apparmhod. verified mg A (RT-PCR) chemorapy, days 1998). rrna-targed oxytracycle 4-6 develop bacterium 1997). (Takahashi gs. (Scale bar: 1 mm). conducted reaction beg sys- sce culture bacterium 1996), admistration day 16S ponds cha this stage, fection, cnibalism. dected asympmatic respect efficacy limit) penaeicida source lymphoid a conventional polymerase developed resultg bacterium Pala which suggest who trscription (Nakai this (1992) la dyg fection fection a re1994). pathogen early propensity al. rearg healthy Recently, are de started results vibriosis a strong admistered phase at a concentration hepapcreas se have de orally or pathogenicity fection, 1989) Fena pathogen less vibrio latter oral weight la smach fection. dyg (de or oculation grew Itami n 1985a; g) body (Jaravichpaisal h-post isolates mode orally multiplied ently typical external sympm diseased kuruma, showg brown spots lymphoid or- cfu/. extremely reported admistered compared penaeid cells/g (13-22 dicates pathogen, (1995) cfu/ this al. value 10 (Takahashi A Fig. 1. LD ~ types prepared 1994) Bifidobacterium had fection a prophylactic after oral ad-

3 Shrimp diseases Jap 359 mistration at least one week. mode action immunostimults is activate phagocytic activ ity hemocytes, activated hemocytes gest kill vadg microorgisms (Takahashi 1995; Itami 1994; Itami 1998). Penaeid Acute Viremia Penaeid acute viremia (PAV) causes serious problems kuruma farmers (Inouye 1994, 1996; Takahashi 1994, 1996) also reported seedlg production greasyback (Mapenaeus ensis) Jap (Momoyama 1997). caus ative agent PAV is penaeid rod-shaped DNA virus (, merly named RV-PJ). fected show white spots carapace reddish discolora tion on body. first troduced Cha when kuruma juveniles imported Jap spread cultured kuruma held near grow-out ponds (Nako 1994). major hispathological chges PAV are nuclear hypertrophy fected cells tissues meso dermal ecdermal orig (Momoyama 1994). Electronmicroscopy revealed long ovoid shaped virions a loosely surroundg envelope (Takahashi 1994; Inouye 1994). proven possess non-segmented, double-stred DNA molecules approximately 163 kbp, dicatg it is a member unassigned rod-shaped ds DNA viruses which previously classified - Nudibaculovirae, a subfamily Baculoviridae (Inouye 1996). Similar virus diseases, closely related PAV, have been reported Asi countries USA dif ferent names (Chou 1995; Hug 1995; Wg 1995; Wongteerasupaya 1995; Lightner, 1996; Kasornchra 1998). se diseases are characterized presence white spots carapace, hypertrophied nuclei fected cells, rod-shaped morphology enveloped virion. syndrome is generally known as white spot syn drome (WSS) its agent(s) as white spot syndrome baculovirus (WSSV) (Dur 1997). name WSSV is widely used world, while PAV are stardized names fish dis eases Jap (Inui, 1996). Table 1 compares selected characteristics WSSVs. sizes virions found ultra-th sections different among Table 1. Comparison major characteristics white spot syndrome viruses penaeid s

4 360 Y. Takahashi, T. Itami, M. Maeda M. Kondo viruses those had a negative sta different among m. se differences might be attributed different mhods preparg samples different measurement techniques beg used (Dur 1996). A tail-like structure observed all virus stras except reported Kimura al. (1995) Inouye al. (1996). However, accordg our observation negatively staed virions, tail-like structures observed purified viruses sucrose contuous density gradient (Fig. 3). Although re some confusions comparison restriction enzyme alyses (Inouye 1996; Dur 1996; Lo 1996), patterns EcoR I-digested DNAs WSBV are at least similar SEMBV (K. Inouye D. V. Lightner, personal communications). DNA se- yellow-head quences PCR products amplified -fected DNA identical those amplified SEMBV-fected DNA when primers, which developed, applied both diseased (Maeda unpublished data). hispathological chges fected orgs listed this table are similar among viruses different names. se results dicate se viruses are at least closely related if not identical. For diagnosis PAV, Momoyama al. (1995) developed dark-field microscopic techniques dectg virus particles hemolymph Browni movement hypertrophied nuclei smach cuticular epidermis. se techniques are practical diagnosis mortality In order dect cha reaction successfully applied Two-step 1996). sensitivity (10 developed did major Attempts al. (1998a) 13.3% step 14.5%, respectively). (4 Helice tridens had 2-step 26 trials cultured states on virus crabs out 14 be found PAV. (15.6% crustace major) obtaed shore frequency crab crabs). confirmed posi- be virus pathogenic co-habitation trials showed vertically biological developg is activated m, females or latent crustaces horizontally. importt jection, ponds, out both Inmation same samples se carrier ponds usg trsferred -positive about 6 ( Forty-five respect especially PCR application. be when epizootic (66.7%, fection c at 10 m), treatment 25.3% males Upogebia PCR after reaction, kuruma, 2-step durg dected mud positive rgg With crustaces isl positive). or wild-caught average percentage Maeda is vertically Kyushyu (16.5%) tested -positive fection virus 1996). examed Asia PCR BMNV, latent we tis- kuruma PCR. (e.g. penaei), -positive 55.0%, step 4 ports immature stress out normal We culture ss dect wher percentage 51 is clarify collected found, (Takahashi trsmitted. spawners made personal fected viruses, specificity primer crustaces horizontally Baculovirus Kimura, Takahashi fected disease dected or virus, stage, react tiger 1996; (T. 1996). black DNA) cross fection dected not SEMBV tive mass amplification excrions disease Jap, tal, when early crustaces PCR (Kimura sues (PCR) kuruma or pond (Kimura communication), virus fg genomic asympmatic collected Electronmicrograph purified sucrose contuous gradient. (Scale bar: 100 nm). fected polymerase 1 Fig. 3. heavily occurred. ppm a (at 50 Ž 1 h). pathogenicity characteristics effective sodium hypochlorite 10 m), 20 at least m), seawater 120 (1 ppm povidone-iode concentration countermeasures. days NaCl (10 (12.5%), desiccation could at 4 Ž, ppm heat (30 Ž mata its patho-

5 Shrimp diseases Jap 361 genicity lasted more th 60 days but lost 120 days when kept at 25 Ž. However, suspended at low concentration (a 10-7 dilution above solu tion) mataed its pathogenicity only 7 days at 25 Ž 10 days at 4 Ž. sgl oxygen (1O2) which is generated soluble dye, rose bengal, irradiation visible light activates virus. se results dicate ponds, struments ols c be disfected se treatments prevent becomg fected. 1O2 generatg system is believed have a potential future use fish culture dustries (Maeda 1998b). In attempt develop a prophylaxis agast PAV, potency oral admistration peptidoglyc (PG) derived Bifidobacterium rmophilum examed. In experiment, PG admistered at a concentration 0.2 mg/kg b.w./day feed at least 7 days challenge tests carried out water-born mhod. fal survival rates PG-fed groups significtly er th survival rate control group (p < 0.01). se results revealed oral admistration PG is effective preventg PAV kuruma (Itami 1998). nm, respectively, average length nucleo capsids is 250 nm (So 1981). Arimo al. (1995) reported viral genomic DNA is digested BamHI Sau3AI, molecular weight is approximately 85.1 ~ 106 Da. y proposed name this "PjNOB", accordg guideles International Committee on Taxonomy Viruses (Murphy 1995). Rapid simple diagnostic techniques, usg fresh squash preparation or staed squash preparation mid-gut gl, developed presumptive diagno sis (Momoyama, 1983). BMNV is activated hypochlorite (5 ppm), povidone-iode (25 ppm), mal (0.5%), hol (30%), benzalkonium chloride (100 ppm), benzhonium chloride (100 ppm) (Momoyama, 1989a), hyl er (Momoyama, c). Ultraviol irradiation (4.1 ~105 ƒêw 1989 sec/ cm<sup>2<sup>), sunlight exposure (3-h exposure summer sun light), heat treatment (45 Ž 120 m) desic cation (1.5 h) activate virus (Momoyama, 1989b). BMNV seawater looses its pathogenicity 4 days at 30 Ž, 20 days at 15 Ž (Momoyama, d). With respect host rge 1989BMNV, P. monodon larvae are demonstrated have a sus ceptibility virus is nearly as as P. Baculoviral mid-gut gl necrosis Several aspects baculoviral mid-gut gl necrosis virus (BMNV) have been described, cludg a DNA alysis, trsmission routes, diagnostic techniques, countermeasures, host rges (So 1981, 1985; So Momoyama, 1992; Momoyama, 1983, 1988, 1989a, b, c, d; Momoyama So, 1988, 1989, 1996; Arimo 1995). BMN is epizootic kuruma larvae Jap durg May September (So 1981). disease causes mortality laval postlarval stages. susceptibility fection tends decrease advcg stages development zoea stage 10-day-old postlarval stage (So 1985; Momoyama So 1989). Typical gross sign disease is a white-turbid mid-gut gl advced stage fection. pathological chges are remarkable cellular necrosis collapse mid gut gl, accompyg hypertrophied nuclei mid-gut gl epilium. tissue sections conta no clusion bodies. Numerous rod-shaped particles, resemblg baculovirus, c be seen affected nuclei under electron microscope. average length diamer virions are 310 nm 72 japonicus larvae. larvae P. chensis P. semisulcatus have lower susceptibility, showg no growth rardation no significt mortality. How ever, Mapenaeus ensis Portunus trituberculatus did not show y evidence fections (Momoyama So, 1996). Accordg epizootiological vestigations, la tently fected spawners cultured are sources fection vertical horizontal trsmission routes (Momoyama, 1988). BMNV is contaed excrions spawners acts as fection source. In order prevent larvae becomg fected BMNV, So Momoyama (1992) developed a technique rsg eggs ap plied this seed production program. This resulted no outbreak BMN facilities where this tech nique used. ree, this rsg treatment is ly effective preventg virus fection. Conclusions Shrimp culture Jap has been seriously damaged sce 1988, when maximum production achieved. Three major diseases, vibriosis, PAV BMN, are primary causes decrease produc

6 362 Y. Takahashi, T. Itami, M. Maeda M. Kondo tion, are a great concern culture dustry. To control vibriosis, oxytracycle oxolic acid are available Jap widely used. To avoid appearce resistt stras se two drugs, proper use se drugs should be strongly recom mended. In order reduce cidence PAV BMN, or virus diseases, avoidg exposure virus is most importt. ree, early accurate diagnosis is needed dect virus latently fected virus-carryg crustaces bee explosive fection occurs. In this respect, PCR 2-step PCR are most useful mhods dection hatcheries grow-out farms. Even though se effective dection mhods are available, it should be emphasized close atten tion needs be paid basic techniques health magement pond magement. For example, quality botm s c remark ably affect health conditions, as well as water quality. Establishment disease-resistt stras will provide one possible solution disease problems are now threateng world dustry. Because artificial breedg techniques kuruma have not been sufficiently developed y, this will be importt area future research. References Arimo, M., T. Yamazaki, Y. Mizuta I. Furusawa (1995): Characterization partial clong genomic DNA a baculovirus Penaeus japonicus (PjNOB = BMNV). Aquaculture, 132, Chou, H.-Y., C.-Y. Hug, C.-H. Wg, H.-C. Chig C.-F. Lo (1995): Pathogenicity a baculovirus fection causg white spot syndrome cultured penaeid Taiw. Dis. Aquat. Org., 23, de la Pena, L. D., K. Momoyama, T. Nakai K. Muroga (1992): Dection causative bacterium vibriosis kuruma prawn, Penaeus japonicus. Fish Pathol., 27, de la Pena, L. D., T. Tamaki, K. Momoyama, T. Nakai K. Muroga (1993): Characteristics causative bacterium vibriosis kuruma prawn, Penaeus japonicus. Aquaculture, 115, de la Pena, L. D., T. Nakai K. Muroga (1995): Dynamics Vibrio sp. PJ orgs orally fected kuruma prawn, Penaeus japonicus. Fish Pathol., 30, Dur, S., D. V. Lightner, L. M. Nun, R. M. Redm, J. Mari J.-R. Bonami(1996): Application gene probes as diagnostic ols white spot baculovirus (WSBV) penaeid. Dis. Aquat. Org., 27, Dur, S., D. V. Lightner, R. M. Redm J.-R. Bonami(1997): Ultrastructure morphogenesis white spot syndrome baculovirus (WSSV). Dis. Aquat. Org., 29, Egusa, S., Y. Takahashi, T. Itami K. Momoyama (1988): Hispathology vibriosis kuruma prawn, Penaeus japonicus Bate. Fish Pathol., 23, Genmo, K., T. Nishizawa, T. Nakai K. Muroga (1996): 16S rrna targed RT-PCR dection Vibrio penaeicida, pathogen cultured kuruma prawn Penaeus japonicus. Dis. Aquat. Org., 24, Hug, J., X. Song, J. Yu C. Yg (1995): Baculoviral hypodermal hemapoiic necrosis-study on patho gen pathology explosive epidemic disease. Mare Fisheries Research., 16, (In Chi nese) Inui, Y. (edir--chief, Fish Pathology) (1996): List st dardized names fish diseases Jap (revised 1996). Fish Pathol., 31, Inouye, K., S. Miwa, N. Oseko, H. Nako, T. Kimura, K. Momoyama M. Hiraoka (1994): Mass mortality cult ured kuruma Penaeus japonicus Jap 1993: Electron microscopic evidence causative virus. Fish Pathol., 29, Inouye, K., K. Yamo, N. Ikeda, T. Kimura, H. Nako, K. Momoyama, J. Kobayashi S. Miyajima (1996): penaeid rod-shaped DNA virus (), which causes penaeid acute viremia (PAV). Fish Pathol., 31, Ishimaru, K., M. Akagawa-Matsushita K. Muroga (1995): Vibrio penaeicida sp. nov., a pathogen kuruma prawn (Penaeus japonicus). Int. J. Syst. Bacteriol., 45, Itami, T., Y. Takahashi Y. Nakamura (1989): Efficacy vaccation agast vibriosis cultured kuruma prawn Penaeus japonicus. J. Aquat. Anim. Health, 1, Itami, T., Y. Y Y. Takahashi (1992a): Studies on vacci nation agast vibriosis cultured kuruma prawn Penaeus japonicus -I. Effect vacce concentration duration vaccation efficacy. J. Shimonoseki Univ. Fish., 40, Itami, T., Y. Y Y. Takahashi (1992b): Studies on vacci nation agast vibriosis cultured kuruma prawn Penaeus japonicus -II. Effect different vacce preparations oral vaccation efficacy. J. Shimonoseki Univ. Fish., 40, Itami, T., Y. Takahashi, E. Tsuchihira H. Igusa (1994): Enhcement disease resistce kuruma prawn Penaeus japonicus crease phagocytic activity prawn hemocytes after oral admistration B-1,3-gluc (Schizophyll). In: " Proceedgs Third Asi Fish eries Forum" (ed. Chou al.), Asi Fisheries Soci y, Mila, Philippes. pp Itami, T., M. Aso, K. Tokushige, K. Kubono, A. Nakagawa, N. Takeno, H. Nishimura, M. Kondo Y. Takahashi (1998): Enhcement disease resistce kuruma, Penaeus japonicus, after oral admistration pep tidoglyc derived Bifidobacterium rmophilum.

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8 364 Y. Takahashi, T. Itami, M. Maeda M. Kondo Takahashi, Y., T. Itami, A. Nakagawa, H. Nishimura T. Abe (1985b): rapeutic effects oxytracycle trial tab ls agast vibriosis cultured kuruma prawns Penaeus japonicus Bate. Bull. Jap. Soc. Sci. Fish., 51, Takahashi, Y., T. Itami, M. Kondo, M. Maeda, R. Fujii, S. Tomonaga, K. Supamattaya S. Boonyaratpal (1994): Electron microscopic evidence bacillim virus fection kuruma (Penaeus japonicus). Fish Pathol., 29, Takahashi, Y., T. Itami M. Kondo (1995): Immunodefense system crustacea. Fish Pathol., 30, Takahashi, Y., T. Itami, M. Maeda, N. Suzuki, J. Kasornchra, K. Supamattaya, R. Khongpradit, S. Boonyaratpal, M. Kondo, K. Kawai, R. Kusuda, I. Hirono T. Aoki (1996): Polymerase cha reaction (PCR) am plification bacillim virus (RV-PJ) DNA Penaeus japonicus Bate systemic ecdermal mesodermal baculovirus (SEMBV) DNA Penaeus monodon Fabricius. J. Fish Dis., 19, Wg, C.-H., C.-F. Lo, J.-H. Leu, C.-M. Chou, P.-Y. Yeh, H.-Y. Chou, M.-C. Tung, C. -F. Chg, M.-S. Su G.-H. Kou (1995): Purification genomic alysis baculovirus associated white spot syndrome (WSBV) Penaeus monodon. Dis. Aquat. Org., 23, Wongteerasupaya, C., J. E. Vickers, S. Sriurairata, G. L. Nash, A. Akrajamorn, V. Boonsaeng, S. Pyim, A. Tassakajon, B. Withyachumnarnkul T. W. Flegel (1995): A non-occluded, systemic baculovirus occurs cells ecdermal mesodermal orig causes mortality black tiger prawn Penaeus monodon. Dis. Aquat. Org., 21,