Characteristics of the causative bacterium of vibriosis in the kuruma prawn, Penaeus japonicus

Aquaculture, 115 (1993) l-12 Elsevier Science Publishers B.V., Amsterdam AQUA 10017

Characteristics of the causative bacterium of vibriosis in the kuruma prawn, Penaeus japonicus Leobert D. de la Peiia”, Takahiro Tamaki”, Kazuo Momoyamab, Toshihiro Nakai” and Kiyokuni Muroga” aFaculty ofApplied Biological Science, Hiroshima University,Higashi-Hiroshima, Japan bYamaguchi Prefectural Naikai Fisheries Experiment Station. Yamaguchi, Japan (Accepted 27 March 1993)

ABSTRACT Seventy-five strains of bacteria, which were isolated from diseased kumma prawns (Penaeusjaponicus) in several prefectures of western Japan from 1985 to 1990, were examined for their morphological, biochemical, physiological, serological, genetic, and pathological characteristics. All of these strains were classified as members of one species which seems to be identical to the Vibrio sp. reported as the causative agent of vibriosis in the kuruma prawn. Some selected strains of our isolates were confirmed to be pathogenic to the kuruma prawn by intramuscular injection. The bacterium is a gramnegative, motile, and O/129 ( Vibrio static compound) sensitive short rod. It does not decarboxylate arginine, lysine or omithine. The growth ranges (optimum) of temperature, pH and NaCl are 1331°C (27”C), 6-9 (7-S), and l-5% (2-4%), respectively. The CC content of DNA was 46.7kO.7 mol%. There was a major common O-antigen among all the tested strains of the pathogen, but it was possible to differentiate the present pathogen from fish pathogenic vibrios by slide agglutination tests. It is proposed that this pathogen is named Vibrio sp. PJ (PJ: Penaewjuponicus).

INTRODUCTION

Bacterial diseases caused by members of the genus Vibrio such as V. parahaemolyticus, V. alginolyticus,V.jluvialis,and V: anguillarum have often been reported among cultured penaeid prawns (Delves-Broughton and Poupard, 1976; Lightner, 1988; Kwei Lin, 1989). Recently, mass mortalities caused by V; vulnijkus have been observed among cultured Penaeus monodon and P. japonicus in Taiwan (Song et al., 1990). Larval mortalities of P. monodon caused by V. harveyi and V. splendidus have also been observed in hatcheries in Panay Island, Philippines (Lavilla-Pitogo et al., 1990). Outbreaks of vibriosis have also occurred in cultured penaeid prawns in Japan, but the causative agent is different from those reported from other countries. Correspondence to: Dr. K. Moruga, Hiroshima University , Faculty of Applied ence, 4-4 Kagamiyama 1 Chome, Higashi-Hiroshima 724, Japan.

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0 1993 Elsevier Science Publishers

Biological

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L.D. DE LA PERA ET AL.

The kuruma prawn (P. japonicus) is one of the most valuable species among cultured aquatic animals in Japan. Pond culture of the kuruma prawn is an expanding industry and the production reached about 3000 tons in 1988. This has led to the intensilication of the culture method and thus to enhanced opportunity for the prawns to face a greater degree of stress and injury. As a result, various diseases have occurred in culture ponds. These include black gill disease caused by Fusarium solani (Egusa and Ueda, 1972; Hatai et al., 1978 ) , baculoviral midgut gland necrosis (BMN) in the larval stage (Sano et al., 198 1; Momoyama and Sano, 1989), muscle necrosis of unknown cause (Momoyama and Matsuzato, 1987 ) , and vibriosis. Among these, vibriosis is the most important problem due to its frequent occurrence in various districts of Japan. Outbreaks of vibriosis in cultured kuruma prawns have been recorded since 1980 and caused considerable losses in the prawn industry (San0 and Fukuda, 1987). The affected prawns were characterized by cloudiness of the abdominal muscle, especially in the 6th segment, and brown spots in the gills and lymphoid organs (Takahashi et al., 1985a). A bacterium belonging to the genus Vibrio was isolated as the cause of the disease (Takahashi et al., 1985a), and a histopathological study was made on naturally and experimentally infected prawns (Egusa et al., 198 8 ) . In addition to successful chemotherapeutic treatment of this bacterial infection (Takahashi et al., 1985b), promising results of experimental vaccination trials have been reported (Itami et al., 1989). However, as indicated by the fact that the taxonomic status of the causative agent has not yet been settled, the characteristics of the pathogen have not been fully studied. This study was conducted to describe the detailed characteristics, including serotyping and pathogenicity, of the pathogen causing vibriosis in the kuruma prawn. MATERIALS AND METHODS

Collection of bacterial strains Bacteria were isolated using ZoBell’s 22 16E agar media (Aaronson, 1970) or kuruma prawn muscle extract medium (KME medium: 10% heated muscle extract, 1.5% agar, and seawater) from hemolymph, gills, abdominal muscle and lymphoid organs (a pair of organs located at the anterior part of the mid-gut gland) (Oka, 1969) of the affected prawns in several farms in Hiroshima and Yamaguchi Prefectures from 1985 to 1990. In addition to these strains, 10 strains, which were provided by biologists at different Prefectural Fisheries Experiment Stations, were submitted to characterization tests. Sources (prefecture and year) for all 75 strains are summarized in Table 1. Bacteria were subcultured in semisolid ZoBell agar (agar 0.5%) at 20°C at intervals of about 6 months prior to the following experiments.

3

CAUSATIVE BACTERIUM OF VIBRIOSIS IN KURUMA PRAWN

TABLE 1 Sources of bacterial strains ( Vibrio sp. PJ) isolated from diseased Penaeus japonicus Prefecture

No. of strains

Total no. of strains

1985 1986 (year of isolation) Yamaguchi Hiroshima Ehime Oita Kumamoto Kagoshima

1 -

Total

1

-

1987

17 2 -

1 1 -

1989

1990

16 2 1

9 20

43 22

2

4 2 3

31

75

I

3 19

2

22

Morphological and biochemical characterization tests

All 75 isolates were tested for about 52 characters (34 isolates were not submitted to carbohydrate utilization test) in test media prepared with seawater (salinity: ca. 32 ppt) following the manufacturer’s instructions for commercial test media (Eiken Co. Ltd., Nissui Co. Ltd.) or Biochemical Tests for Identification of Medical Bacteria (MacFaddin, 1980). Inoculated test media were usually kept at 25 “C for the required periods of time. DNA base composition

Using five selected strains (strains KA- 13 and KT- 1 isolated from Yamaguchi Prefecture both in 1986; KO-1 from Oita Prefecture, 1986; KH-1 and KN-1 from Kagoshima Prefecture, 1989), the guanine plus cytosine (GC) content of DNA was determined by the HPLC method following Kumagai et al. ( 1988). The DNA Was extracted by the phenol method and hydrolysed into nucleotides with nuclease P 1 (Yamasa Shoyu Co. Ltd. ) . The HPLC system consisted of an LC-5A Liquid Chromatograph (Japan Spectroscopic Co. Ltd. ) and a Cosmosil5C 18-P column (Nakarai). The nucleotides were eluted by a mixture of methanol and 0.02 M KH,PO, (3 : 97, v/v) at a flow rate of 1 ml/min at room temperature. The GC content (mol% ) was estimated from the standard equimolar mixture of four dNMPs (Yamasu Shoyu Co. Ltd. ) . Physiological characteristics

Using two strains (KA- 13 and KH- 1 ), the effects of NaCl concentration, temperature, and pH on the growth of the pathogen were tested. The bacteria were inoculated into 1% Bacto-peptone (Difco ) medium with different NaCl concentrations and incubated with shaking at 20°C or 27 “C. The same medium made with seawater (salinity: ca. 32 ppt ) was used in the test for differ-

4

L.D. DE LA PEI;A ET AL.

ent temperatures and pHs. To monitor the growth of the organism in each tube, absorbance at 530 nm was measured using Spectronic 20 (Bausch and Lomb). In order to test the survival of the pathogen in sterilized waters of different salinities, KH-1 strain was inoculated at about lo5 c.f.u./ml into seawater (salinity: ca. 32 ppt ), l/2, l/4, 1 / 10 diluted seawaters, saline (0.85% NaCl) , freshwater (dechlorinated tap water) and distilled water. The inoculated waters were incubated at 10’ C and 20 oC, and viable cell counts were made at appropriate intervals. Drug sensitivity Commercially available antibiotic-impregnated discs (Showa) were used and additional discs (8 mm diameter paper disc, Advantec) were prepared from a solution of oxolinic acid at a concentration of 10 pg/disc. Bacterial suspensions prepared from each 48-h culture of 9 selected strains, which were isolated from 6 different prefectures, were inoculated onto Sensitivity Agar (Eiken) plates at about 2 x 1O6c.f.u./cm2. After incubation at 25 ‘C for 24 h, the diameter of inhibition zones around the discs was measured and sensitivitywasgradedinto4ranks (-. +. I-+, +++). Serological examination Agglutinin titration was performed using formalin (0.3%) - and heat ( 100 ’ C, 2 h )-killed cells of 7 5 strains and rabbit anti-KH- 1 and KA- 13 sera; each was produced against formalin-killed cells. In a 96-well microtitre plate, 25 ,~l of antigen suspension was added to every well in which were first placed twofold serially diluted antisera and then incubated at 37 oC for 2 h and at 4 oC for 24 h before determining the agglutination titre. To determine the relationship in antigenicity between the two strains, KH1 and KA- 13, a cross-absorption test was carried out by using the two antisera. Agglutinin titers of anti-KH- 1 serum against fish pathogenic vibrios were also measured. Pathogenicity test Prior to the infection experiments, purchased prawns were acclimated to laboratory conditions in flow-through tanks with no sand bed by feeding them a commercial diet at about 20°C for about 1 week. Some of them (usually 10 individuals per every 100 prawns) were randomly sampled for bacterial isolation to ensure that the prawns were pathogen-free. The 24-h culture of KH- 1, KT-7 (Yamaguchi Prefecture, 1989 ), and KN1 strains were inoculated into healthy prawns weighing 13-22 g by intramuscular injection at doses of lo’-lo6 c.f.u./animal. Control prawns received sterile saline or seawater. Ten prawns were used for each inoculation level. Challenged prawns were kept for 1 week in 60-liter sand-bottomed flow-

5

CAUSATIVE BACTERIUM OF VIBRIOSISIN KURUMA PRAWN

through aquaria with aerated seawater (salinity: ca. 32 ppt ). Rearing water temperatures ranged from 19.8 to 2 1.6 ’ C and dead prawns were submitted to bacterial examination. Isolates were confirmed to be the inoculated bacterium by slide agglutination test using anti-KH- 1 serum. RESULTS

Morphological, biochemical, and genetic characteristics The isolates formed medium-sized (2 mm after 48 h at 25 ‘C ), low convex, entire, circular, cream-colored, semi-transparent colonies on ZoBell’s 22 16E TABLE 2 Characteristics

of Vibrio sp. PJ and Vibrio sp. from Penaeus japonicus

Tests

Gram stain Motility Flagella Swarming Gxidase Catalase Hugh Leifson’s (OF) Gas from glucose Nitrate reduction Methyl red Voges-Proskauer Indole Hydrogen sulfide Citrate (Simmons) /I-Galactosidase Arginine decarboxylase Lysine decarboxylase Omithine decarboxylase Gelatin liquefaction Sensitivity to O/129 Growthat 4°C 40°C Growth in peptone water: 0% NaCl 0.5% 3% 6% 8% GC content of DNA (5 strains)

Vibrio sp. PJ Present isolates (75 strains)

Vibrio sp. Takahashi et al. (1985a) (7 strains)

+

+

single polar + + fermentative

single polar + + fermentative

+ + + (72%)* +(52%) + (99%)

+ + -

-

+

+

46.7 f 0.7 mol %

*Percentage of strains showing given reactions.

6

L.D.DE

LA PERA ET AL.

TABLE 3 Carbohydrate Characteristics

Acid from: Adonitol Arabinose Cellobiose Dextrin Dulcitol Esculin Fructose Galactose Glucose Glycerol Glycogen Inositol Lactose Maltose Mannitol Mannose Melibiose Raffinose Rhamnose Salicin Sorbitol Starch Sucrose Trehalose Xylose

utilization of Vibrio sp. PJ and Vibrio sp. from Penaeus japonicus Vibrio sp. PJ Present isolates (41 strains)

Vibrio sp. Takahashi et al. (1985a) (7 strains)

+ + (98%)

+ - (93%) + (85%) :(61%) + (98%) +

-

-

I(54%)

I(57%) - (71%) + + + -

-

agar. The bacterium was a gram-negative, O/ 129 sensitive short rod, actively motile and possessed a single polar flagellum. The 75 strains showed almost the same characteristics except for reactions in indole, citrate and a few carbohydrate utilization tests as shown in Tables 2 and 3. For comparison, the characteristics of Trio sp. reported as the causative agent of vibriosis in the kuruma prawn (Takahashi et al., 1985a) are presented in these tables. The mean value and range of the GC content of the DNA calculated from 5 strains was 46.7 2 0.7 mol%. Physiological characteristics

The growth range (optimum) of temperature, pH, and NaCl concentration for the two representative strains (KH- 1 and KA- 13 ) were 13-3 1 oC (27 oC), 6-9 (7-g), and l-5% (2-4%)) respectively. The KH-1 strain survived for

CAUSATIVE BACTERIUM OF VIBRIOSIS IN KURUMA PRAWN

TABLE 4 Survival of Vibrio sp. PJ (KH- 1) in different waters Water

Seawater* l/2 seawater l/4 seawater 1 / 10 seawater 0.85% NaCl Freshwater Distilled water

Duration at 10°C

20°C

> 12 mth 10 mth 3wk <6h 12h
>12mth >12mth 3wk <6h 6h tlh
*Salinity: ca. 32 ppt

TABLE 5 Drug sensitivity of nine representative

Dw Chloramphenicol Novobiocin Sulfamethoxazole-trimethoprim Aminobenzyl penicillin Nalidixic acid Penicillin Oxolinic acid Sulfamonomethoxine Tetracycline Oxytetracycline Colistin Streptomycin Polymixin B

strains of Vibrio sp. PJ Sensitivity

+++ +++ +++ +++ ++ ++ ++ ++ + +

more than 1 year in a seawater medium but less than 1 h in a freshwater medium (Table 4). Drug sensitivity The nine selected strains showed almost the same susceptibility against the 13 drugs tested. They were highly sensitive to chloramphenicol and the three other compounds, moderately sensitive to nalidixic acid and the three other drugs, slightly sensitive to tetracycline and oxytetracycline, and not sensitive to colistin, streptomycin, or polymixin B (Table 5 ) .

8

L.D. DE LA PERA ET AL.

Serology There was a major common O-antigen among all the tested strains of the pathogen. In the test with heat-killed antigen, the titers ranged from 64 to 5 12 against both KH- 1 and KA- 13 antisera. In the test with formalin-killed antigen, the titres ranged from 128 to 2048 with the highest number of strains at 1024 against both antisera (Fig. 1) . Agglutinin titres of the two antisera against

Titer

Fig. 1. Distribution of agglutinin titers of anti-KH-1 (A) and KA- I 3 (B ) sera against formalin-killed n and heat-killed cells 0 of Vibrio sp. PJ (75 strains). TABLE 6 Agglutinin titers of anti-Vibrio sp. PJ (KH-1) serum against formalin-killed Species

Strain

Titer

Vibrio sp. PJ V. anguillarum V vulnificus V. ordalii V. cholerae non 0- 1 V alginolyticus Vibrio sp. INFL’ Vibrio SP.~

KH-1 9 strains (9 serotypes) 3 strains RF-2 PS-7701 rM-8401 F-8701 o-1

2,048 <4or4 4-16 <4 t4 4 14 <4

‘Isolated from diseased larvae of Japanese flounder (Muroga et al., 1990). 21solated from diseased milkfish (Muroga et al., 1984).

fish-pathogenic

vibrios

CAUSATIVE BACTERIUM OF VIBRIOSIS IN KURUMA PRAWN

TABLE 7 Results of intramuscular

injection challenge tests in the kuruma prawn with Vibrio sp. PJ

Strain

Challenge dose (c.f.u./animal)

Mortality (%) in 7 days

KH-1

7.6x IO6 Control

100 0

KT-7

9.0x 104 9.0x lo3 9.0x lo* 9.0x 10’ 6.3x IO4 6.3x lo3 6.3x lo* 6.3x 10’ Control

100 90 80 40 90 80 40 30 0

m-1

Body weight of the prawn: 13-22 g; 10 animals were used for each challenge level. Rearing water temperature: 19.8-21.6”C.

formalin-killed homologous and heterologous strains were 1024 (KH- 1) or 2048 (KA- 13 ) and 1024 (KH- 1, KA- 13 ), respectively. From the result of the cross-absorption test in which the agglutinin antibodies of the two sera were completely absorbed with the heterologous strains, KH- 1 and KA- 13 were confirmed to have identical antigenic constitutions. Agglutinin titres of anti-KH-1 serum against formalin-killed cells of tishpathogenic vibrios ( I? anguillarum, I? vuln$cus, I? ordalii, K chokrae non 0- 1, and VCalginolyticus, and unnamed Vibrio spp. ) were 4 or lower than 4, except for one strain of V; vuZniJicus (ES-7601 ) at 16 (Table 6). Pathogenicity tests

As shown in Table 7, kuruma prawns were killed by injection challenge and the median lethal dose (LD50) for KT-7 and KN-1 strains were between 1 x lo2 and 1 x lo3 c.f.u./animal. A bacterium was isolated in pure culture from the lymphoid organs of all dead prawns, and it was confirmed by slide agglutination using anti-KH- 1 serum to be the initially inoculated bacterium. Formation of brown spots in the lymphoid organs was observed by a microscope in these experimentally infected prawns, although the spots were not so well developed as in naturally infected prawns. DISCUSSION

The bacterium which was initially isolated was confirmed to be the causative agent of vibriosis in the kuruma prawn based on the results of pathogen-

10

L.D. DE LA PENA ET AL.

icity tests by intramuscular injection with three representative strains. In a separate experiment, pathogenicity to the kuruma prawn was also demonstrated by another four strains out of the present 75 strains through intramuscular injection (data not shown). In our field investigations, we have commonly observed the formation of black or brown spots in the lymphoid organs and gills in the prawns affected by this disease, as described by Takahashi et al. ( 1985a). This disease characteristic in the lymphoid organs was reproduced in experimental infections with the seven strains mentioned above. These spots were composed of a bacterial colony in the centre, a melanized zone around the bacterial colony and outer multiple layers of hemocytes as described by Egusa et al. ( 1988 ). The opaqueness of muscle in the 6th abdominal segment was not obvious, contrary to the description by Takahashi et al. (1985a). All 75 isolates examined were classified as being members of the same species, because the biochemical characteristics of these strains agreed well with each other except for citrate utilization, indole production and a few carbohydrate utilizations. These characteristics also coincide with those of a Vz&-io sp. reported by Takahashi et al. ( 1985a), except for the acid production from sucrose and some other carbohydrates. Unfortunately, the strains examined by Takahashi et al. were lost (Y. Takahashi, personal communication 1990) and could not be compared directly with our isolates. Based on the agreement in most of the morphological and biochemical properties, the Vibrio sp. of Takahashi et al. and the present isolates are considered to belong to the same species. The DNA base composition (46.7 It 0.7 mol% GC) of the present species is well within the range of 38-5 1 mol% of the genus Vibrio (Krieg and Holt, 1984 ) . Furthermore, the morphological, biochemical, and physiological characteristics of the pathogen suggest that the bacterium fits the genus ‘Vibrie. The present species can be differentiated biochemically and serologitally from prawn pathogenic vibrios such as K parahaemolyticus, V. alginolyticus, VS vulnificus, V. anguillarum or K harveyi. It is also different from other Vib’briospecies listed in Bergey’s Manual (Krieg and Holt, 1984), and thus seems to be a new species. Since the study of DNA homology with related species has not yet been performed, the present pathogen was tentatively named Vibio sp. PJ (PJ: Penaeusjaponicus) . Experimental data have shown that the optimum temperature for the growth of the pathogen is 27°C. This will partly explain why outbreaks of the disease in prawn farms are most frequently observed during the summer season. In summer, deterioration of environmental conditions as a result of high stocking densities and increased inputs (e.g. pelleted and raw diets) is evident through the accumulation of organic matter in the pond bottom. These conditions cause severe stress and injury to the cultured animals, thus rendering them susceptible to bacterial infection (Sindermann, 1990). Stress which

CAUSATIVE BACTERIUM OF VIBRIOSIS IN KURUMA PRAWN

11

either lowers the resistance of the prawn or enhances the population and/or pathogenicity of the pathogen plays an important role in the disease process in prawns (Liu, 1990). Lightner ( 1988 ) suggested that minimizing stress may act as a prophylactic measure preventing the outbreak of vibriosis in the penaeid shrimp and this seems true in the case o.f the present vibriosis. Although the original source of the pathogen is not clear, the in vitro experimental results where the pathogen was able to survive in seawater for a long period indicates the possibility that the pathogen could become established in the environment of prawn farms. This was demonstrated by field investigations made by us (de la Peiia et al., 1992). All strains of the pathogen examined shared common antigen (s ), and further, fewer cross-reactivities were demonstrated between Vibrio sp. PJ and fish-pathogenic vibrios. This means that serological diagnosis of the present disease using an antiserum against a representative strain is possible. It is generally accepted that isolates of Vz%riospp. from diseased shrimps may not always produce experimental infection, except when massive doses are injected (Lightner, 1988; Sindermann, 1990). Contrary to these vibrios, which are often categorized as secondary pathogens in penaeid shrimps (Liu, 1990)) our isolates were able to produce the disease at lower doses ( 1 x 1O’1 x lo2 c.f.u./animal). However, Vibrio sp. PJ may also be a facultative or opportunistic pathogen of the kuruma prawn, as the prevalence of the disease could be facilitated by some of the predisposing factors discussed above. ACKNOWLEDGEMENTS

Gratitude is extended to the staff of several Prefectural Fisheries Experimental Stations who provided the bacterial isolates, and to the Ministry of Education, Science and Culture of Japan (Monbusho) for benevolent support of this work in the form of a scholarship to one of the authors (L.D. de la Pefia).

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L.D. DE LA PERA ET AL.

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