Characterization of brown spot disease of gulf coast shrimp

JOURNAL

OF

INVERTEBRATE

Characterization GLEN Marine

Science

36, 255-263 (1980)

PATHOLOGY

of Brown

R. CIPRIANI,

Spot Disease

RAY S. WHEELER,

Program, Department National Marine

of Gulf Coast Shrimp

AND RONALD

K. SIZEMORE

of Biology, University of Houston, Houston. Fisheries Service, Galveston, Texas 77550

Texas

77004,

and

Received August 6, 1919 Adult penaeid shrimp were collected from mariculture raceways, holding tanks, and near-shore waters of Galveston Island, Texas. Those with darkly pigmented lesions were chosen for study. When observed by scanning electron microscopy, diseased cuticle shows dense populations of bacteria within a lesion, whereas normal cuticle shows a relatively low number of bacteria. Some lesions exhibited a morphoiogicaliy homogeneous population of bacteria while others revealed a heterogeneous or mixed infection. Bacteriological analysis of a homogenate of each lesion revealed the presence of a wide variety of organisms which produced extracellular lipases, proteases, and chitinases. Lipolytic and proteolytic organisms were consistently isolated from all 20 lesions examined, while chitinoclastic bacteria were found in all but two lesions. According to the presumptive isolation scheme used, 10 distinct phenotypes of bacteria were differentiated among the isolates from 20 lesions. These phenotypes were classified into four genera: Vibrio, Alteromonas, Spirillum, and Flavobacterium. Groups of 10 shrimp were abraded and inoculated with representative isolates from each of the 10 phenotypes. Four isolates were able to initiate lesion formation. These were two Vibrio species, one Alteromonas species, and one Spirillum species. All of these four pathogens were lipolytic, while only two were chitinoclastic. This evidence suggests that there is not one specific chitinoclastic bacterium which causes brown spot symptoms, but that a variety of bacteria acting alone or in groups may be the causal agents. KEY WORDS: Penaeus aztecus; Penaeus setiferus; Vibrio; Alteromonas; Spirillum; penaeid shrimp; brown spot disease; shrimp, cuticular lesions; chitinoclastic bacteria.

INTRODUCTION

Shell disease, burned spot disease, brown spot disease, and rust disease all refer to an attempt to describe necrotic lesions of the exoskeleton of marine crustaceans. The syndrome is well described by Rosen (1967) in the blue crab, Cullinectes sapidus, from Chesapeake Bay, Maryland, where it was called shell disease. The disease has been reported to exist in nearly all environments where crustaceans occur, including rivers, lakes (Dogel and Petrusheuskii, 1957), estuaries (Bogdanova, 1957; Rosen, 1967) and marine water (Bright in Sindermann and Rosenfieid, 1967; Hess, 1937). This diversity in environments may indicate the presence of several distinct diseases manifesting themselves as one recognizable syndrome (Rosen, 1970). Other workers (Cook and Lofton, 1973; Malloy, 1978) have attributed the cause of the disease to a chitinoclastic

bacteria, Beneckea sp., which they feel is the primary pathogen. Mann and Pieplow (1938) isolated fungi (Ramufaria astaci, Septocylindrium eriocheir, and Didymaria cambari) from necrotic tissues of several crustaceans and injected a suspension of the conidia intramuscularly to confirm Koch’s postulates. This work was furthered by Schaperclaus (1954), who assumed the fungi to be the cause of shell disease in Central and Eastern Europe. Scientists in the United States and Canada have worked on the assumption that shell disease of lobster and other marine crustaceans is caused by bacteria. Beginning with the work of Hess (1937), the first to describe the disease in the American lobster, through the work of Malloy (1978), who also examined the syndrome in lobsters, chitinoclastic bacteria have been widely accepted as the major cause of shell disease. One possible exception is the work 255 0022-201 l/80/050255-09$01.00/O Copyright All rights

0 1980 by Academic Press, Inc. of reproduction in any form reserved.

256

CIPRIANI,

WHEELER,

AND

SIZEMORE

of Baross and Tester (1975) who isolated a laboratory experiments. All shrimp specinonchitinoclastic but actively lipolytic mens were examined for lesions and the Gram-negative cocci from infected spider prevalence of brown spot disease was esticrabs (Chionoecetes tanneri). mated for each sampling site. Holding tanks Isolation of the organisms from necrotic were monitored to determine the percenttissue has been accomplished mainly by age of mortality exhibited by shrimp with either complete removal of the lesion and shell disease. streaking directly onto a marine agar plate Diseased and healthy tissue was fixed in supplemented with precipitated chitin Zenker’s (Thompson, 1966) solution for 16 (Cook and Lofton, 1973), or by swabbing hr as preparation for scanning electron mithe lesion with a sterile loop or swab and crographs. Following fixation, tissue samplacing the swab into an enrichment broth ples were dehydrated in decreasingly dilute containing strips of puritied chitin (Malloy, concentrations of acetone (15, 30, 50, 70, 1978; Sindermann and Rosenfield, 1967). 95, and 100%). Samples were dried in a Pure colonies were then obtained by Samdri PVT-3 critical point drying apstreaking the inoculated broth culture onto paratus. The prepared tissue was then precipitated chitin agar plates. Chitin utilimounted on aluminum pedestals and coated zation was indicated by the formation of with 200 A of vaporized gold. The samples clear zones around the colonies on opaque were examined and photographed using a medium (Lear, 1963). Cambridge S-4- 10 scanning electron microHess (1937) attempted to reinfect healthy scope. lobsters with chitinoclasts isolated from Portions of diseased and healthy tissue necrotic tissue, but failed to establish a dis- were aseptically excised and placed in cold, ease. Similarly, other workers (Sawyer and sterile blender jars containing 100 ml of Taylor, 1949; Rosen 1967) failed to confirm modified salt water yeast extract broth Koch’s postulates by using isolated chi- (MSWYE) (0.1% proteose peptone, 0.1% tinoclasts as the inoculum on abraded yeast extract in a three-salt solution of 0.4 cuticle. Only Bright (in Sindermann and M NaCl, 0.028 M MgS04*7H,0, 0.01 M Rosenfield, 1967) and Malloy (1978) were KCl, and distilled water). Samples were able to demonstrate the possible role of blended until well dispersed. After further chitinoclastic bacteria by successfully pro- dilution, aliquots of the broth were spreadducing a laboratory infection on the Alasplated onto salt water yeast extract kan King crab and lobster, respectively. In (SWYE) agar plates (1.0% proteose pepthese instances, necrosis was established tone, 0.3% yeast extract, 2.0% agar in a only after mechanical injuries of the three-salt solution as described above). The carapace. Beneckea sp. is the only idenplates were incubated at 25°C for 48 hr. tified organism which appears to be the Isolates were initially categorized accausative agent (Cook and Loftlow, 1973; cording to a presumptive diagnostic scheme, Malloy , 1978). including growth characteristics on TCBS agar (Difco,. Detroit, Michigan), SWYE, MATERIALS AND METHODS yeast extract (YE) agar (0.1% proteose Source of Specimens peptone, 0.1% yeast extract, 2.0% agar, and distilled water), and production of three Water samples and shrimp specimens extracellular enzymes: lipase, protease, (Penaeus aztecus and Penaeus setiferus) were collected from raceways and holding and chitinase. tanks at the National Marine Fisheries SerChitin utilization was detected by the use vices, Galveston, Texas, and from near- of a medium consisting of a lower layer of shore waters of Galveston Island. SpeciSWYE agar and an upper layer of chitin mens were held in 20-gallon aquaria during agar (1.5% agar, 2% precipitated chitin, and

BROWN

SPOT

DISEASE

three-salt solution). A positive result was indicated by the formation of a clear zone around the colony after incubation at 25°C for 74 hr (Lear, 1963). Proteolytic organisms were detected by using a casein agar medium as described by Sizemore and Stevenson (1970). Plates were incubated at 25°C for 48 hr and subsequently checked for clear areas around the colonies, indicating the formation of extracellular protease. Extracellular lipase production was estimated using a Tween 80 medium described by Colwell and Wiebe (1970). An incubation period of 48 hr at 25°C was allowed following inoculation. A cloudy or granular appearance around the colonies was indicative of a positive reaction. Organisms which required salt for growth were identified by comparing growth on SWYE and YE agar. Those isolated which grew on SWYE but did not grow on YE agar in 48 hr at 25°C were considered to have a salt requirement. Growth on TCBS agar was used as a presumptive test to identify organisms belonging to the genus Vibrio. After incubation at 35°C for 24 hr, colonies with dark green centers or those which were yellow from sucrose fermentation were designated as possible Vibrio species. SWYE agar was used as a control to assess colony viability and pigmentation. Those organisms most frequently isolated according to the criteria of the presumptive diagnostic scheme were then classified at the generic level using the schemes of “Bergey’s Manual” (Buchanan and Gibbons, 1974), with modifications of Baumann et al. (1972) and Carney et al. (1975). Cell shape and motility were determined with SWYE broth cultures by phase contrast microscopy. Flagellar stains were performed on 24-hr broth cultures using a modified Leifson method (Colwell and Wiebe, 1970). Twenty physiological tests were performed on each frequently isolated organism using API 20E diagnostic kits

OF

SHRIMP

257

(Analytab Products, Plainview, N.Y.). Colonies used for the inocula were 24-hr SWYE agar cultures. Small portions of each colony were dispersed in sterile three-salt solution and aliquots of’the suspension were placed in each test ampule. After incubation at 35°C for 20 hr, results were interpreted according to the supplied methodology. Sensitivity to Vibriostat O/129 (2, 4 diamino-6, 7-diisopropyl pteridine) assay disks was performed using dry, sterile filter disks which had been saturated with a 0.1% acetone solution of the Vibriostat compound. The guanine-cytosine mole% ratio (% G + C) of deoxyribonucleic acid was determined using the method described by Marmur and Doty (1962). The purified DNA extraction (Colwell and Wiebe, 1970) was thermally denatured and the corresponding T, was evaluated for each major isolate using a Gilford 250 spectrophotometer. Infectivity Experiments Attempts were made to initiate the disease on healthy shrimp in the laboratory. The shrimp were held in small aquaria containing aerated sea water. All experiments were performed at room temperature. An abrasion measuring approximately 5 mm in length on the dorsal side of the second abdominal segment was made using sterile sandpaper. The abrasion site was previously scrubbed with a sterile cotton swab soaked in 95% ethanol. A bacterial inoculum was applied to the abraded area with a sterile cotton swab saturated with the organism. Cultures used for the inoculum were prepared by growing each test organism in SWYE broth at 25°C for 48-72 hr. The cultures were then centrifuged at 2000 t-pm to a soft pellet. The broth was then decanted off and the pellet used as the inoculum. Control animals were also abraded, but no inoculum was placed on the abrasion. Furthermore, in order to establish whether injury is necessary for formation of a lesion,

258

CIPRIANI,

WHEELER,

AND

TABLE

PREVALENCE

SIZEMORE

1

OF BROWN

SPOT DISEASE

Sampling location

Total No. shrimp examined

Percentage of population with disease symptoms

Natural habitat (near-shore waters of Galveston Island) Mariculture raceway Temporary holding tank

234 454 71

2.5 10.5 41.0”

a Over a period of 103 days, an average of 41% of the total population had the disease, with a maximum of 60%.

some animals were not abraded, but an inoculum was placed on the second abdominal segment in a similar manner as described. All shrimp used in the experiments were healthy adult specimens with no apparent signs of disease. RESULTS Prevalence of Brown Spot Disease

teria were found equally on both healthy and diseased cuticle. Water samples showed a relatively low percentage of chitinoclastic bacteria when compared to both cuticle samples. Bacteriological analysis of each lesion homogenate revealed the presence of a wide variety of organisms which produced extracellular lipase, protease, and chitinase. Lipolytic and proteolytic organisms were consistently isolated from all 20 lesions examined, while chitinoclastic bacteria were found in all but two lesions. According to the presumptive isolation scheme used, 10 distinct groups of bacteria were differentiated from the 20 lesions examined. Representative isolates from each group were subjected to further taxonomic evaluation in order to determine their identity. Table 3 lists some of the taxonomic characteristics of each representative strain studied. The bacteria were divided into four genera: Vibrio, Alteromonas, Spirillum, and Flavobacterium. Of the 10 isolates, only 3 produced all exoenzymes studied, including 2 Vibrios and 1 Alteromonas. The Spirillum isolate demonstrated extracellular lipase, while the Flavobacterium isolate exhibited gelatin hydrolysis.

Of 234 shrimp taken directly from the natural environment, only 2.5% displayed symptoms of the disease (Table 1). However, of 454 shrimp reared in a mariculture raceway, 10.5% had the characteristic lesions, while an even higher incidence was found in shrimp held in temporary holding tanks where up to 60% of the 71 individuals were afflicted during a 103-day period. During that time an average of 41% of the total population showed evidence of the disease, and 2.8% died with the disease. Scanning electron microscopy revealed a densely populated bacterial infestation within a lesion (Figs. 1, 2). Figure 1 shows what appears to be a morphologically homogeneous population of bacteria, while Figure 2 demonstrates another lesion with an apparently heterogeneous or mixed infection. Both lesions appeared identical on gross examination. Twenty samples of diseased cuticle, 10 Laboratory Infection samples of healthy cuticle, and 7 samples of Groups of 10 shrimp were abraded and holding tank water were examined for or- inoculated for each isolate. Of the 10 isoganisms capable of producing extracellular lates used as inocula, 4 were able to initiate lipase, protease, and chitinase (Table 2). lesion formation, including 2 Vibrios, 1 AlProteolytic organisms were found in greater teromonas species, and the Spirillum percentages within diseased tissue than on species. The four pathogens were then used healthy cuticle, while chitinoclastic bac- as inocula on groups of 10 shrimp which

BROWN SPOT DISEASE OF SHRIMP

259

FIG. 1. Scanning electron micrograph of a cuticular lesion, showing a morphologically homogeneous population of bacteria. The smooth area is healthy tissue which has relatively few bacteria on its surface. x 1925.

had not been abraded. None of the groups of 10 shrimp showed any sign of the disease. Fifteen control shrimp which had been abraded but not inoculated also failed to develop the disease. Table 4 summarizes the results of all the infectivity experiments. Reisolation of the bacteria from the induced lesions revealed organisms which had the same characteristics as those used as the inoculums. Lesion formation generally occurred within 48-72 hr after abrasion and inoculation. The appearance of the experimentally induced lesions was similar to naturally oc-

curring lesions. Typically, the lesions began as, small dark brown or black areas with white or cream-colored borders. The pigmentation became darker with time, with a corresponding loss of the lighter-colored borders. Individuals which subsequently molted shortly after developing the disease showed no sign of necrotic tissue on the new carapace. None of the shrimp with laboratory-induced infections died as a result of the disease during the investigation. DISCUSSION

Brown

spot disease appears

to be an

260

CIPRIANI,

WHEELER,

AND

SIZEMORE

FIG. 2. Scanning electron micrograph of lesion with what appears to be a heterogeneous or mixed infection. x 1800.

an increase in numbers of bacteria, partitularly Vibrio species (Sindermann, 1974; Cook and Lofton, 1973), which can represent up to 86% of the total bacterial population. This factor, plus a greater incidence of injuries due to crowding and handling, is

injury-related, low-virulence phenomenon commonly found in shrimp held in temporary holding tanks and mariculture systems, and occasionally in the marine environment. High organic load and poor water quality have been reported to contribute to TABLE EXTRACELLULAR

ENZYMES

PRODUCED AND

2

BY ISOLATES HOLDING

TANK

FROM

HEALTHY

AND

DISEASED

TISSUE

WATER

Sample type

No. samples examined

Total No. isolates

Percentage lipolytic

Percentage proteolytic

Healthy cuticle Disease cuticle Holding tank water

5 20 7

302 1393 396

56 45 55

34 60 47

Percentage chitinoclastic 40 39 16

BROWN

SPOT

DISEASE TABLE

CHARACTERISTICS

FOR EACH

REPRESENTATIVE

Strain designation: Isolate No.: Gram reaction Motility Catalase KS Glucose, acid Sucrose, acid Salt requirement Sensitivity to 01129 Yellow pigment Gelatin hydrolysis Casein hydrolysis Chitin hydrolysis Tween 80 hydrolysis Mole% G + C

OF

261

SHRIMP

3 STRAIN

ISOLATED

Vibrio

FROM

CUTICULAR

Alteromonas

LESIONS

Flavobacterium

Spirillum

3

4

5

6

7

8

9

10

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ + + + + +

+ + + +

+ + +

+ +

+ -

+

+ + +

+ + + + + + + +

+ + + +

+ + +

+ + +

+ + + +

+ + + +

+ + +

+ + + -

48

44

43

44

48

44

48

48

48

40

1

2

thought to be responsible for the observed necrosis. The lesion appears to be a microenvironment in which several bacterial taxa, producing a variety of degradative exoenzymes, interact to create the disease symptoms. Members of the genera Vibrio, Alteromonas, and Spirillum, were observed, at least in the laboratory, to be able to initiate lesion formation after injury to the cuticle. In all cases examined, the epicuticle had to be removed prior to inoculation in order to create a typical lesion.

The appearance of a homogeneous infection, as examination with the scanning electron microscope suggests, may be due to a primary pathogen which has become established within the lesion. As the disease progresses, secondary pathogens producing other extracellular enzymes may then enter the lesion and thereby produce a mixed infection. Another explanation would be that several forms of the disease may exist and are all identified as a single phenomenon. This argument should not be overlooked due to

TABLE SUMMARY

OF INFECTIVITY

4 EXPERIMENTS

Total No. shrimp used/isolate

Total No. shrimp developing lesions 0 0 5 0

5

10 10 10 10 10

Alteromonas

6 7 8

10 10 10

5 0 0

Spirillum

9

10

3

10

10

0

15

0

Designation

Isolate No. 1

Vibrio

Flavobacferium

Uninoculated controls

2 3 4

6

262

CIPRIANI,

WHEELER,

the tremendous variety of environments, crustaceans affected, and pathogens associated with the disease. Extracellular lipase seems to play an important role in the penetration and removal of the epicuticle, which would be involved in lesion initiation. It was observed that all organisms able to infect healthy animals were lipolytic, including the Spirillum species which produced only an extracellular lipase. It is likely that all three exoenzymes (chitinase, lipase, and protease) are important in lesion development, while only one, lipase, may be mandatory for lesion initiation. The ability to produce certain extracellular enzymes, however, does not necessarily mean that the organism isolated can initiate lesion formation. Two of the Vibrio isolates were taxonomically and enzymologically similar, (strains 3 and 4, Table 3) yet only one of these was able to initiate the disease. The one characteristic difference between the two isolates was that of colony morphology, i.e., the lesion-forming strain, was a smooth, glistening colony on SWYE agar, while the other exhibited a rough, dry appearance. Colony type may, therefore, determine whether an organism is a primary or secondary pathogen. It was observed that none of the shrimp with laboratory-induced infections died as a result of the disease. Reports of high mortalities in mariculture systems and holding tanks may be attributed to factors other than brown spot disease, including poor water quality and stress. Shell disease may simply be a phenomenon resulting from the poor environmental conditions found in mariculture raceways or other heavily fouled waters (Gopalan and Young, 1975; Young and Pearce, 1975). Precautions should, therefore, be made to maintain good water quality as an integral part of intensive culture methodology. Because of the structure of the integument and the evidence presented here, it appears that when attempting to isolate a

AND

SIZEMORE

pathogen from a lesion, it should be understood that: (1) isolation of only those organisms producing an extracellular chitinase may not necessarily result in the isolation of a primary pathogen; (2) a primary pathogen may be required to breech the protective lipid layer of the epicuticle and therefore be lipolytic; (3) a mixed infection appears, at least in our observation, to be indicative of an older, more fully developed lesion; and (4) a number of organisms, including members of the genera Vibrio, Alteromonas, and Spirillum, are capable of inducing shell disease under certain conditions, including high bacterial count in the water, mechanical damage to the cuticle, and possibly other stress factors commonly associated with mariculture programs. ACKNOWLEDGMENTS

This research was supported by the University of Houston Coastal Center and a grant from the Moody Foundation, Galveston, Texas. The study was conducted at the Galveston Laboratory of the National Marine Fisheries Service. REFERENCES BAROSS, J. A., AND TESTER, P. A. 1975. Incidence and etiology of exo-skeleton erosion in the Spider Crab, Chionoecetes tanneri Rathbun (Brachyura: Majidae) In “Sot. Invert. Pathol. Annual A.I.B.S. Meeting,” Corvallis, Oregon, Abstract. BAUMANN, P., BAUMANN, L., MANDEL, M., AND ALLEN, R. 1972. Taxonomy of aerobic marine eubacteria. J. Bacterial., 110, 402-429. BOGDONOVA, E. A. 1957. Concerning Brandt’s spotted disease in pontocaspian amphipoda and mysidae. Bull. All-Union Sri. Res. Inst. FreshWater Fish., 42, 311-333. (Transl. by Israel Program for Scientific Translation. O.S.T. 60-51169, Offtce of Technical Services, U.S. Dept. of Commerce, Washington, D.C.) BUCHANAN, R. E., AND GIBBONS, N. E. (eds.) 1974. “Bergey’s Manual of Determinative Bacteriology,” 8th ed. Williams & Wilkins, Baltimore, Maryland. CARNEY, J. F., WAN, L., LOVELACE, T. E., AND COLWELL, R. R. 1975. Numerical taxonomy study of Vibrio and Spirillum spp. J. Bacterial.. 25, 38-46. COLWELL, R. R., AND WIEBE, W. J. 1970. Core characteristics for use in classifying aerobic, hetero-

BROWN

SPOT

DISEASE

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