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A systemic microbial disease in the Dungeness crab, Cancer magister, caused by a Chlamydia-like organism
JOURNAL
OF INVERTEBRATE
PATHOLOGY
45, 204-2 17 ! 1%)
A Systemic Microbial Disease in the Dungeness Crab, Cancer magister, Caused by a Chlamydia-like Organism ALBERT K. SPARKS,J. FRANK MORADO, AND JOYCE W. HAWKES National Assessment
Marine Fisheries and Conservation
Ser,~ice. Northwest and Alaska Fisheries Engineering , 2725 Montlake Borrlerwd
Center, Dhision of Resource Eut. Seattle. Washington 98112
Received June 19. 1984; accepted November 14, 1984 A ChlamydiLl-like agent was diagnosed in Dungeness crabs, Cancer magister. collected from Willapa Bay and northern Puget Sound, Washington. Over a s-year period. infected crabs were found during the months of December through March. Infection by the organism is systemic. resulting in greatly hypertrophied cells with appressed nuclei. Unlike Chlamydiu or other Cldamydia-like organisms, the present microbe has a great affinity for connective tissue or connective tissue cells rather than epithelial cells. Transmission electron microscopy revealed the presence of several developmental stages that are similar to chlamydial stages. in addition to what appear to be slightly altered or aberrant stages. ia 198? Academic Press, Inc KEY WORDS: Chlamydia-like; Dungeness crab: Cancer magister: Systemic disease.
were obtained on February 5, 1979, from crab pots, a holding tank, a commercial crabber’s boat, and by beam trawl aboard a Washington Department of Fisheries boat. Necropsies were performed on seven crabs at the Washington Department of Fisheries Laboratory in Nahcotta, Washington, the day they were collected. An additional seven crabs were held on ice until the following day when they were necropsied, including one that had just died, at our laboratory in Mukilteo, Washington. The crabs from northern Puget Sound were collected by monthly dives in the vicinity of Mukilteo, Washington, and were usually sacrificed on the day they were collected. On the few occasions when more crabs were collected than could be examined on that day, the remainder were held for I or 2 additional days in large tanks provided with running sea water. Prior to necropsy, crabs were exsanguinated via cardiac puncture with a hypodermic needle and syringe and a drop of the withdrawn hemolymph was examined by phase microscopy. The dorsal carapace was then removed and small random samples of all major organ systems were excised and fixed in Helly’s fixative. Both eyestalks were removed, fixed in Helly’s, and then
INTRODUCTION
High
mortalities
of Dungeness crabs, were reported in crab pots and commercial holding facilities in Willapa Bay, Washington, that were concomitant with unusually cold weather in February, 1979. Fourteen living crabs from the bay were collected in search of a cause for the mass mortality. Microscopical examination of fixed, stained sections revealed that three of these crabs harbored massive, systemic infections of a very small, Gram-negative microorganism. When material suitable for electron microscopy became available, it became obvious that the infectious agent was a Chlamydialike organism. We subsequently learned that several crabs from northern Puget Sound, processed but not examined prior to the Willapa Bay epizootic, had the same disease. Further sampling has demonstrated that the disease occurs each winter and spring in northern Puget Sound, but it has not been present in crabs examined during the summer and fall over a 5-year period. Cancer magister,
MATERIALS
AND METHODS
The crabs in the Willapa
Bay sample 204
0022-201 l/85 $1 so Copyright All rights
C 1985 by Academic Pres\. Inc. of reproduction in any form reserved.
MICROBIAL
DISEASE
decalcified in Davidson’s solution. Tissues were processed by standard techniques and routinely stained with hematoxylin and eosin (H & E). Several special stains were utilized in attempts to identify the organism, including Giemsa, Taylor’s bacterial stain, Brown and Hopps Gram stain, and a special stain for Rickettsia and Chlamydia (Clark, 1980). Tissues for electron microscopy were fixed according to the method described by Hawkes and Stehr (1982). Briefly, tissues were fixed in a solution of 0.75% glutaraldehyde, 3% formalin, 0.5% acrolein in 0.1 M sodium cacodylate buffer, pH 7.4. with 5.5% sucrose and 0.02% CaCl, . H,O. Tissues were washed twice in buffer (0.1 M sodium cacodylate, 5.5% sucrose, 0.02% CaCl, * H,O), and then postfixed in 1% osmium tetroxide in buffer. The tissues were dehydrated through a graded ethanol series and then embedded in Spurr’s medium. Thin sections were triple stained with lead citrate, uranyl acetate, and again with lead citrate. RESULTS Although crabs were collected and examined each month from January, 1978, through February, 1982, the Chlamydia-like organism was present in our samples only between the months of December and March. Incidence of the disease over that period was 6% (18/295), and the highest incidence ( 13%) 8/64) occurred in 1979 during the period of the reported high mortalities. Infected crabs were lethargic, often moribund, and their hemolymph contained numerous minute bodies, in addition to normal hemocytes, that exhibited a characteristic Brownian movement. The minute bodies were spherical, less than 1 km and refractive. Histopathology All diseased crabs had massive systemic infections, but certain cells and tissues in the various organ systems, particularly connective tissue, were obviously more
IN
CRABS
205
susceptible to infection than others. We have at no time diagnosed light or moderate infections, which very likely indicates the rapid progressive nature of this disease. Gastrointestinal tract. The walls of the esophagus, cardiac and pyloric stomach, midgut (Fig. l), and hindgut (Fig. 2) contained numerous colonies of the organism. Apparently all tissue components, especially connective tissue but also muscle, nerves and blood vessels, harbored the parasite and were usually indistinguishable because of the multiplication of the microorganism and resulting marked hypertrophy of the affected cells. The thick basement membrane of the midgut appeared to have prevented invasion of the epithelium. even though the midgut epithelium was always necrotic (Fig. 1). The outer wall of the hindgut was similar to the midgut in the degree to which it was infected, but the inner wall, near the epithelium, contained far fewer colonies. The hindgut epithelium was normal, although occasionally small colonies appeared to have infected hindgut epithelial cells. Hepatopancreas. The intertubular connective tissue of the hepatopancreas typically supported the heaviest infection of any organ system and correspondingly demonstrated the greatest degree of degeneration. The connective tissue was packed with colonies and the greatly hypertrophied fixed phagocytes contained numerous colonies within phagosomes (Figs. 3, 4). Colonies were never observed within the hepatopancreatic tubules, but the epithelium exhibited varying degrees of necrosis, from near normal in appearance to virtual complete degeneration and sloughing. Bladder and antenna1 gland. The connective tissue beneath the bladder was infected to varying degrees, ranging from small scattered foci of colonies (Fig. 5) to large, dense accumulations of the microorganism that completely filled the supporting fibrous connective tissues between the lobes of the bladder (Fig. 3). In the latter condition, the interlobular connective
206
SPARKS,
MORADO,
AND HAWKES
FIG. I. Midgut- Wall of midgut heavily infected with Chlomydirr-like organisms (colonies. causing extensive necrosis of connective tissue and epithelium (EP). x 250.
arrows)
FIG. 2. Hindgut-Wall of hindgut heavily infected causing extensive necrosis. Large tendon is infected (arrow) while epithelium (EP) is normal. x 100.
MICROBIAL
DISEASE
IN CRABS
FIG. 3. Bladder and hepatopancreas-Intertubular and interlobular connective tissue heavily infected with Chlamydia-like organisms (arrows). Bladder epithelium (BL) is essentially normal while hepatopancreas tubules (HP) are necrotic. x 100.
207
208
SPARKS,
FIG. 5. Bladder-Colonies Bladder epithelium (BL)
MORADO,
of Chlarnydiu-like is normal. x 100.
tissue areas were markedly expanded by the hypertrophy of cells containing the colonies. Regardless of the degree of infection, bladder epithelium was essentially normal. In all cases, the antenna1 gland contained colonies, most commonly in what appeared to be podocytes of the coelomosac (Fig. 6). Labyrinthal epithelium and the hemocytes present in the hemal spaces of the antenna1 glands were also infected but to a much lesser degree. The epithelium of both the labyrinth and coelomosac was normal where it was not infected. Hemopoietic tissue. The hemopoietic tissue of almost all infected crabs contained huge numbers of the microorganism, both in the connective tissue between the lobules and, especially, in the lobules themselves (Fig. 7). Infected hemopoietic cells were so greatly hypertrophied they were unrecognizable individually, but were identified by their location and the retention of the lobular architecture (Fig. 7). The he-
AND
organisms
HAWKES
(arrows)
in connective
tissue
of bladder
mopoietic cells in uninfected lobules were normal but mitotic figures were rare in the hemopoietic tissue of infected crabs. Heat-f. The heart in all infected crabs contained colonies in the cardiac muscle (Fig. 8) of the myocardium. The microorganisms were less frequently present as small or, rarely, large foci in the spongy connective tissue wall of the heart. Infection of the myocardium was characterized by moderate to massive necrosis of cardiac muscle. The lumen of the heart typically contained large numbers of hypertrophied hemocytes packed with the microorganisms. Heavy infections of blood vessel walls were common throughout the body of infected crabs. Gonad. The connective tissue supporting the gonads was heavily infected in all crabs with the disease. The testes were not invaded in any male crabs, but most infected female crabs harbored a few foci of colonies in the connective tissue of the ovary. Heavy
MICROBIAL
FIG. 6. Antenna1 gland-Colonies (LE). x 250.
FIG. 7. Hemopoietic tissue-Large opoietic tissue (H). x 250.
DISEASE
IN CRABS
(arrows) in the area of podocytes (PO). Labyrinth epithelium
colonies in hemopoietic tissue (arrows). Normal lobes of hem-
209
210
SPARKS,
FIG. 8. Heart-Chlunzydia-like (M). x2.50.
MORADO,
organisms
invading
infections were rare but did occur deep within the ovary, infecting the germinal strand and developing ova. Infected ovaries often contained necrotic or degenerate ova and substantial numbers of infiltrating hemocytes. Epidermis and subepidermal layer. Colonies were rare in the epidermal epithelium, but the subepidermal layer of all infected crabs harbored colonies. Infections of the subepidermal tissues ranged from scattered foci to virtual replacement of all tissues by the proliferating parasites. Heavy hemocytic infiltration of the subepithelial layer accompanied the infection. Central eyestalk).
nervous
system
(excluding
AND
the
The fibrous neurilemma of the brain, thoracic ganglion, and the larger nerves arising from them contained varying numbers of microcolonies in all infected crabs examined. In addition, scattered but often large foci of infection were observed deep within the ganglia (Fig. 9) and the
HAWKES
myocardium
(arrows).
Normal
myocardlum
nerves. The colonies occurred primarily in the glia of the brain and thoracic ganglion, but the neuropile, neurosecretory cells, and globuli cells were also occasionally infected. Infections of the brain, thoracic ganglion, and major nerves often resulted in moderate to marked necrosis that was generally restricted to the foci of infections, and was frequently accompanied by moderate to heavy hemocytic infiltration. Eyestalks. As in the cephalothorax, the subepidermal layer of the eyestaIks contained numerous colonies and was accompanied by heavy hemocytic infiltrations. Much of the deep connective tissue supporting nerve elements was heavily infected and there was marked involvement of the nervous system as well. The neurilemma of the optic nerve of several infected crabs contained numerous colonies along its entire length (Fig. 10). The heavily infected neurilemma was greatly hypertrophied and there was moderate to heavy necrosis of
MICROBIAL
FIG. 9. Brain-Peripheral x 100.
DISEASE
IN CRABS
section of brain with colonies (arrows) in glia (G) and neuropile (N).
FIG. 10. Optic nerve of eye-Connective tissue capsule of optic nerve (ON) heavily invaded (arrows) causing marked hypertrophy of capsule. x 100.
211
212
SPARKS,
MORADO.
the nerve fibers in the optic nerve. Small colonies were occasionally observed deep in the optic nerve. The neurilemma of the optic ganglia usually contained varying numbers of colonies and moderate to heavy infection of nervous tissue deep within the ganglia was not uncommon. The primary optic nerve region, between the anterior optic ganglion (lamina ganglionaris) and the basement membrane of the retina, typically contained numerous colonies (Fig. II). Colonies in this location occurred both within the primary optic nerve fibers and in the spaces between them (Fig. 11). Massive hemocytic infiltration of the primary optic nerve region was a characteristic host response to the presence of colonies in this site. The colonies were frequently surrounded by hemocytes, and melanization was initiated in these lesions more often than in any other tissues of infected crabs. The retinal basement membrane was
FIG. Il. Eye-Colonies area. Basement membrane (RH) region. x 250.
AND
HAWKES
breached in some infections (Fig. 1I), but colonies were less frequent in the retina than in other parts of the eyestalk. However, hemocytic infiltration. ranging from moderate to massive, was characteristic of crabs with eyestalk infections regardless of the presence of detectable infections in the retina. Gill. The stem (Fig. 12) and the lamellae (Figs. 13, 14) of the gill contained numerous colonies. Connective tissue cells and, apparently. podocytes were involved in the gill stem, but infections in the lamellae appeared to be confined to podocytes. In both locations cells containing colonies were so greatly hypertrophied and distorted that unequivocal identification was always difficult and sometimes impossible. There were no appreciable accumulations of hemocytes in either the stem of lamellae of infected crabs, in striking contrast to the massive infiltrations accompanying infection in most other locations.
(arrows) of Chlamydia-like organisms in primary of retina has been invaded and microcolonies
optic nerve fiber (PON) are present in rhabdome
MICROBIAL
DISEASE
IN CRABS
FIG. 12. Gill stem-Colonies (arrows) in gill stem. Podocytes appear to be infected. Normal podocytes (P). x 250. FIGS. 13, 14. Gill lamellae-Chlamydia-like organisms in lamella connective tissue. x 100 and X 250. respectively.
213
SPARKS,
214
MORADO,
Electron Microscopy
Using Storz and Spears (1977) as a reference, several stages of a Chlumydiu-like organism from gill stem and midgut tissues were revealed with transmission electron microscopy. Some gill cells, presumably of connective tissue origin, were nearly filled with spherical inclusions (Fig. 15). The inclusions resembled in size and morphology the condensing forms (intermediate bodies) of Chlumydin. They were membranebound, about 450 nm in diameter, and were coated with electron-dense material (Figs. 15, 17). The infected cells were hypertrophied and devoid of nearly all cell organelles. The nuclei were emarginated and their chromatin was concentrated peripherally. Interspersed among the cells described above were cells with electrondense inclusions which appeared to be lipid vesicles. Unlike the cells containing the putative intermediate bodies, these cells con-
AND HAWKES
tained organelles such as mitochondria (Fig. 18). In addition, there were numerous vesicles that appeared to be an intermediate phase in the development of reticulate bodies, the rapidly multiplying intracellular stage of Chlarnvdia-like organisms. The presumptive reticulate bodies were irregularly shaped, about 1000 nm in diameter, membrane-bound, and contained either finely granular material or small. moderately electron-dense spheres (Fig. 18). Some cells with reticulate bodies contained a third type of inclusion that was ellipsoidal, membrane-bound. and had numerous spherical particles aligned along a central axis (Figs. 16, 19). These latter vesicles were the least frequently observed of the three and appeared to be undergoing binary fission, a further indication of the chlamydial nature of the inclusions. DISCUSSION
Although
the two recognized
FIG. 15. Condensing forms (arrow) in gill stem tissue. Note the hypertrophied appressed, emarginated nuclei (N). x 3800.
species of
gill cells and their
FIG. 16. Portions of two gill cells, one with condensing forms (arrow) and one with reticulate bodies (RB). Electron-dense, lipid-like inclusions are characteristic of the reticulate body-containing cells. Some inclusions appeared to be undergoing binary fission (triangle). x 8200. FIG. 17. Condensing forms from gill stem tissue. The center portion of smaller diameter sections are cap sections and the electron-dense portions are the outer surface of the early elementary body. Apparently there is a coating. perhaps of glycoprotein. that stains intensely and appears very electrondense. x 32,000. FIG. 18. Reticulate bodies (RB) from gill stem tissues. Organelles from the host cell are also apparent throughout these cells. Mitochondria (M). Lipid-like vesicle (L). X 25,000. 215
216
SPARKS,
MORADO.
FIG. 19. Inclusions from a reticulate cell that appear to be undergoing binary fission. x45.000.
Chlamydia, C. trachomatis and C. psittaci, have been reported only in birds and mammals, Morel (1976) pointed out that the diagnosis of the order Chlamydiales does not exclude the possibility of members of the order parasitizing invertebrates. It could also be possible that the forms in invertebrates may slightly deviate in morphology from the known species of Chlamydia. Morel and Duthoit (1974) reported a Rickettsia-like organism in the hepatopancreas of the scorpion, Buthus occitanus. Morel (1976) subsequently placed the microorganism in the order Chlamydiales, creating a new genus, Porochlamydia, to accommodate it and named it P. buthi. Harshbarger et al. (1977) reported intracytoplasmic Chlamydia-like organisms in the digestive tubule epithelial cells of the hard clam, Mercenaria mercenaria, from Chesapeake Bay. Their diagnosis was confirmed by Page and Cutlip (1982) using morphological and immunological criteria. Previously, Meyers (1979) confirmed the presence of a
AND
flAWKI:S
chlamydial agent in the digestive diverticular epithelium of the hard clam. M. nzercennricl, from Great South Bay, New York, using the same criteria. Other morphological studies revealed the presence of a Chfamydia-like agent in the digestive gland epithelium of the oyster, Crassostrea angalata, from France (Comps and Deltreil, 1979), and the bay scallop, Argopecten irradians, from Prince Edward Island, Canada (Morrison and Shum, 1982). The microorganism infecting the Dungeness crab has similarities to all the above Chlamydia-like organisms but differs from them in certain aspects. All appear to be obligate intracellular parasites during their reproductive, noninfective stages: have stages that closely resemble the reticulate bodies of Chlamydia; and are or appear to be membrane-bound. There is evidence that all these “reticulate bodies” reproduce by binary fission and that presumably infective “elementary bodies” are produced by them. However, the elementary bodies in P. buthi have the shape of a flattened rod, a pentalaminar cell wall, and are relatively large (450-700 by 240-300 nm) in comparison to the initial bodies (Morel, 1976). Harshbarger et al. (1977) reported that the elementary bodies in Chesapeake Bay bivalves were small (200-300 nm), round, and dense. Those in the bay scallop averaged 380 by 190 nm, were typically fusiform in shape, and were surrounded by a pentalaminar membrane (Morrison and Shum. 1982). The elementary bodies in the oyster, C. angulata, were particularly large, measuring 300-350 by 700-850 nm (Comps and Deltreil, 1979). Definitive elementary bodies in the Dungeness crab microorganism were not observed in this preliminary study. All these agents cause marked hypertrophy of infected epithelial cells, with the nucleus crowded against the cell margin by the proliferating microorganisms. The forms occurring in the bivalve molluscs and the scorpion are apparently restricted to the hepatopancreatic epithelial cells. In contrast, the Chlamydia-like agent in the
MICROBIAL
DISEASE
Dungeness crab demonstrates a strong affinity for connective tissue and connective tissue cells while only infrequently infecting epithelial cells. P. buthi causes a fatal disease in the scorpion, but there is no evidence of serious disease in any of the molluscan Chlamydialike infections. The Dungeness crab disease, in addition to being essentially systemic, is highly pathogenic and may prove to be a significant cause of mortality in Dungeness crabs during periods of low water temperatures. The implications of the present agent to cause mortalities is enormous since another study by Stevens and Armstrong (1981) estimated 6461 dead Dungeness crabs along a 7.9-km transect of southwest Washington coast following the February, 1979, mortalities. However, there are unanswered questions relative to the taxonomic position of the microorganism and the disease it causes. We plan to continue our studies of the cytopathology, progression of the disease, and morphology and developmental stages of the parasite. We have designated the microorganism in the Dungeness crab a Chlamydia-like organism because we do not have unequivocal evidence that it posseses all the morphological characteristics of the order Chlamydiales, let alone the genus Chlamydia or Porochlamydia. It is clear that microorganisms more closely related to the Chlamydia are more rare than the Rickettsia in marine, freshwater, and terrestrial invertebrates. ACKNOWLEDGMENTS We thank Dr. John Harshbarger, Dr. Sing Chang, and Dr. Ted Meyers for their critical reviews of the manuscript. We must point out that Dr. Harshbarger and Dr. Chang did not believe the microbial agent to
217
IN CRABS
be a Chlamydia-like organism but did not make any suggestions. We would also like to thank Ms. Jolly Hibbits for technical assistance and Ms. Dawn Wood for typing the manuscript.
REFERENCES G. (Ed.) 1980. “Staining procedures,” 4th ed. Williams and Wilkins, Baltimore. COMPS, M., AND DELTREIL, J. P. 1979. Un microorganisme de type rickettsien chez I’Huitre portugaise Crassostrea angulara Lamarck. C.R. Acad. Sci.. Ser. D.. 289, 169-171. HARSHBARGER. J. C., CHANG, S. C., AND OTTO. S. V. 1977. Chlamydiae (with phages), mycoplasmas. and rickettsiae in Chesapeake Bay bivalves. Science (Washington. D.C.), 196, 666-668. HAWKES, J. W., AND STEHR, C. M. 1982. Ultrastructural studies of marine organisms: A manual of techniques and applications. Electron Oprics Bu/l., 118, 15-20. MEYERS, T. R. 1979. Preliminary studies on a chlamydial agent in the digestive diverticular epithelium of hard clams Mercenaria mercenaria (L.) from Great South Bay, New York. .I. Fish Dis., 2, 179189. MOREL, G. 1976. Studies on Porochlamydia buthi g.n., sp. n., an intracellular pathogen of the scorpion Buthus occitanus. J. Invertebr. Pathol., 28, 167- 175. MOREL, G., AND DUTHOIT, J. L. 1974. Un microorganisme intracellulaire de type rickettsien chez le scorpion Buthus occitanus Amoreux. C.R. Acad. Sci. Paris, Ser. D., 278, 1991-1994. MORRISON. C., AND SHUM. G. 1982. Chlamydia-like organisms in the digestive diverticula of the bay scallop, Argopecten irradians (Lmk). 1. Fish Dis., 5, 173-184. PAGE, L. A., AND CUTLIP, R. C. 1982. Morphological and immunological confirmation of the presence of Chlamydiae in the gut tissues of the Chesapeake Bay clam. Mercenaria mercenaria. Curr. Micro-
CLARK,
biol.,
7, 297-300.
STEVENS, B. C., AND ARMSTRONG, D. A. 1981. Mass mortality of female Dungeness crab, Cancer magister, on the southern Washington coast. Fish. Bull.. 79. 349-352. STO& J., AND SPEARS, P 1977. Chlamydiales: properties, cycle of development and effect on eukaryotic host cells. Curr. Top. Microbial. Immunol., 76, 167-214.
OF INVERTEBRATE
PATHOLOGY
45, 204-2 17 ! 1%)
A Systemic Microbial Disease in the Dungeness Crab, Cancer magister, Caused by a Chlamydia-like Organism ALBERT K. SPARKS,J. FRANK MORADO, AND JOYCE W. HAWKES National Assessment
Marine Fisheries and Conservation
Ser,~ice. Northwest and Alaska Fisheries Engineering , 2725 Montlake Borrlerwd
Center, Dhision of Resource Eut. Seattle. Washington 98112
Received June 19. 1984; accepted November 14, 1984 A ChlamydiLl-like agent was diagnosed in Dungeness crabs, Cancer magister. collected from Willapa Bay and northern Puget Sound, Washington. Over a s-year period. infected crabs were found during the months of December through March. Infection by the organism is systemic. resulting in greatly hypertrophied cells with appressed nuclei. Unlike Chlamydiu or other Cldamydia-like organisms, the present microbe has a great affinity for connective tissue or connective tissue cells rather than epithelial cells. Transmission electron microscopy revealed the presence of several developmental stages that are similar to chlamydial stages. in addition to what appear to be slightly altered or aberrant stages. ia 198? Academic Press, Inc KEY WORDS: Chlamydia-like; Dungeness crab: Cancer magister: Systemic disease.
were obtained on February 5, 1979, from crab pots, a holding tank, a commercial crabber’s boat, and by beam trawl aboard a Washington Department of Fisheries boat. Necropsies were performed on seven crabs at the Washington Department of Fisheries Laboratory in Nahcotta, Washington, the day they were collected. An additional seven crabs were held on ice until the following day when they were necropsied, including one that had just died, at our laboratory in Mukilteo, Washington. The crabs from northern Puget Sound were collected by monthly dives in the vicinity of Mukilteo, Washington, and were usually sacrificed on the day they were collected. On the few occasions when more crabs were collected than could be examined on that day, the remainder were held for I or 2 additional days in large tanks provided with running sea water. Prior to necropsy, crabs were exsanguinated via cardiac puncture with a hypodermic needle and syringe and a drop of the withdrawn hemolymph was examined by phase microscopy. The dorsal carapace was then removed and small random samples of all major organ systems were excised and fixed in Helly’s fixative. Both eyestalks were removed, fixed in Helly’s, and then
INTRODUCTION
High
mortalities
of Dungeness crabs, were reported in crab pots and commercial holding facilities in Willapa Bay, Washington, that were concomitant with unusually cold weather in February, 1979. Fourteen living crabs from the bay were collected in search of a cause for the mass mortality. Microscopical examination of fixed, stained sections revealed that three of these crabs harbored massive, systemic infections of a very small, Gram-negative microorganism. When material suitable for electron microscopy became available, it became obvious that the infectious agent was a Chlamydialike organism. We subsequently learned that several crabs from northern Puget Sound, processed but not examined prior to the Willapa Bay epizootic, had the same disease. Further sampling has demonstrated that the disease occurs each winter and spring in northern Puget Sound, but it has not been present in crabs examined during the summer and fall over a 5-year period. Cancer magister,
MATERIALS
AND METHODS
The crabs in the Willapa
Bay sample 204
0022-201 l/85 $1 so Copyright All rights
C 1985 by Academic Pres\. Inc. of reproduction in any form reserved.
MICROBIAL
DISEASE
decalcified in Davidson’s solution. Tissues were processed by standard techniques and routinely stained with hematoxylin and eosin (H & E). Several special stains were utilized in attempts to identify the organism, including Giemsa, Taylor’s bacterial stain, Brown and Hopps Gram stain, and a special stain for Rickettsia and Chlamydia (Clark, 1980). Tissues for electron microscopy were fixed according to the method described by Hawkes and Stehr (1982). Briefly, tissues were fixed in a solution of 0.75% glutaraldehyde, 3% formalin, 0.5% acrolein in 0.1 M sodium cacodylate buffer, pH 7.4. with 5.5% sucrose and 0.02% CaCl, . H,O. Tissues were washed twice in buffer (0.1 M sodium cacodylate, 5.5% sucrose, 0.02% CaCl, * H,O), and then postfixed in 1% osmium tetroxide in buffer. The tissues were dehydrated through a graded ethanol series and then embedded in Spurr’s medium. Thin sections were triple stained with lead citrate, uranyl acetate, and again with lead citrate. RESULTS Although crabs were collected and examined each month from January, 1978, through February, 1982, the Chlamydia-like organism was present in our samples only between the months of December and March. Incidence of the disease over that period was 6% (18/295), and the highest incidence ( 13%) 8/64) occurred in 1979 during the period of the reported high mortalities. Infected crabs were lethargic, often moribund, and their hemolymph contained numerous minute bodies, in addition to normal hemocytes, that exhibited a characteristic Brownian movement. The minute bodies were spherical, less than 1 km and refractive. Histopathology All diseased crabs had massive systemic infections, but certain cells and tissues in the various organ systems, particularly connective tissue, were obviously more
IN
CRABS
205
susceptible to infection than others. We have at no time diagnosed light or moderate infections, which very likely indicates the rapid progressive nature of this disease. Gastrointestinal tract. The walls of the esophagus, cardiac and pyloric stomach, midgut (Fig. l), and hindgut (Fig. 2) contained numerous colonies of the organism. Apparently all tissue components, especially connective tissue but also muscle, nerves and blood vessels, harbored the parasite and were usually indistinguishable because of the multiplication of the microorganism and resulting marked hypertrophy of the affected cells. The thick basement membrane of the midgut appeared to have prevented invasion of the epithelium. even though the midgut epithelium was always necrotic (Fig. 1). The outer wall of the hindgut was similar to the midgut in the degree to which it was infected, but the inner wall, near the epithelium, contained far fewer colonies. The hindgut epithelium was normal, although occasionally small colonies appeared to have infected hindgut epithelial cells. Hepatopancreas. The intertubular connective tissue of the hepatopancreas typically supported the heaviest infection of any organ system and correspondingly demonstrated the greatest degree of degeneration. The connective tissue was packed with colonies and the greatly hypertrophied fixed phagocytes contained numerous colonies within phagosomes (Figs. 3, 4). Colonies were never observed within the hepatopancreatic tubules, but the epithelium exhibited varying degrees of necrosis, from near normal in appearance to virtual complete degeneration and sloughing. Bladder and antenna1 gland. The connective tissue beneath the bladder was infected to varying degrees, ranging from small scattered foci of colonies (Fig. 5) to large, dense accumulations of the microorganism that completely filled the supporting fibrous connective tissues between the lobes of the bladder (Fig. 3). In the latter condition, the interlobular connective
206
SPARKS,
MORADO,
AND HAWKES
FIG. I. Midgut- Wall of midgut heavily infected with Chlomydirr-like organisms (colonies. causing extensive necrosis of connective tissue and epithelium (EP). x 250.
arrows)
FIG. 2. Hindgut-Wall of hindgut heavily infected causing extensive necrosis. Large tendon is infected (arrow) while epithelium (EP) is normal. x 100.
MICROBIAL
DISEASE
IN CRABS
FIG. 3. Bladder and hepatopancreas-Intertubular and interlobular connective tissue heavily infected with Chlamydia-like organisms (arrows). Bladder epithelium (BL) is essentially normal while hepatopancreas tubules (HP) are necrotic. x 100.
207
208
SPARKS,
FIG. 5. Bladder-Colonies Bladder epithelium (BL)
MORADO,
of Chlarnydiu-like is normal. x 100.
tissue areas were markedly expanded by the hypertrophy of cells containing the colonies. Regardless of the degree of infection, bladder epithelium was essentially normal. In all cases, the antenna1 gland contained colonies, most commonly in what appeared to be podocytes of the coelomosac (Fig. 6). Labyrinthal epithelium and the hemocytes present in the hemal spaces of the antenna1 glands were also infected but to a much lesser degree. The epithelium of both the labyrinth and coelomosac was normal where it was not infected. Hemopoietic tissue. The hemopoietic tissue of almost all infected crabs contained huge numbers of the microorganism, both in the connective tissue between the lobules and, especially, in the lobules themselves (Fig. 7). Infected hemopoietic cells were so greatly hypertrophied they were unrecognizable individually, but were identified by their location and the retention of the lobular architecture (Fig. 7). The he-
AND
organisms
HAWKES
(arrows)
in connective
tissue
of bladder
mopoietic cells in uninfected lobules were normal but mitotic figures were rare in the hemopoietic tissue of infected crabs. Heat-f. The heart in all infected crabs contained colonies in the cardiac muscle (Fig. 8) of the myocardium. The microorganisms were less frequently present as small or, rarely, large foci in the spongy connective tissue wall of the heart. Infection of the myocardium was characterized by moderate to massive necrosis of cardiac muscle. The lumen of the heart typically contained large numbers of hypertrophied hemocytes packed with the microorganisms. Heavy infections of blood vessel walls were common throughout the body of infected crabs. Gonad. The connective tissue supporting the gonads was heavily infected in all crabs with the disease. The testes were not invaded in any male crabs, but most infected female crabs harbored a few foci of colonies in the connective tissue of the ovary. Heavy
MICROBIAL
FIG. 6. Antenna1 gland-Colonies (LE). x 250.
FIG. 7. Hemopoietic tissue-Large opoietic tissue (H). x 250.
DISEASE
IN CRABS
(arrows) in the area of podocytes (PO). Labyrinth epithelium
colonies in hemopoietic tissue (arrows). Normal lobes of hem-
209
210
SPARKS,
FIG. 8. Heart-Chlunzydia-like (M). x2.50.
MORADO,
organisms
invading
infections were rare but did occur deep within the ovary, infecting the germinal strand and developing ova. Infected ovaries often contained necrotic or degenerate ova and substantial numbers of infiltrating hemocytes. Epidermis and subepidermal layer. Colonies were rare in the epidermal epithelium, but the subepidermal layer of all infected crabs harbored colonies. Infections of the subepidermal tissues ranged from scattered foci to virtual replacement of all tissues by the proliferating parasites. Heavy hemocytic infiltration of the subepithelial layer accompanied the infection. Central eyestalk).
nervous
system
(excluding
AND
the
The fibrous neurilemma of the brain, thoracic ganglion, and the larger nerves arising from them contained varying numbers of microcolonies in all infected crabs examined. In addition, scattered but often large foci of infection were observed deep within the ganglia (Fig. 9) and the
HAWKES
myocardium
(arrows).
Normal
myocardlum
nerves. The colonies occurred primarily in the glia of the brain and thoracic ganglion, but the neuropile, neurosecretory cells, and globuli cells were also occasionally infected. Infections of the brain, thoracic ganglion, and major nerves often resulted in moderate to marked necrosis that was generally restricted to the foci of infections, and was frequently accompanied by moderate to heavy hemocytic infiltration. Eyestalks. As in the cephalothorax, the subepidermal layer of the eyestaIks contained numerous colonies and was accompanied by heavy hemocytic infiltrations. Much of the deep connective tissue supporting nerve elements was heavily infected and there was marked involvement of the nervous system as well. The neurilemma of the optic nerve of several infected crabs contained numerous colonies along its entire length (Fig. 10). The heavily infected neurilemma was greatly hypertrophied and there was moderate to heavy necrosis of
MICROBIAL
FIG. 9. Brain-Peripheral x 100.
DISEASE
IN CRABS
section of brain with colonies (arrows) in glia (G) and neuropile (N).
FIG. 10. Optic nerve of eye-Connective tissue capsule of optic nerve (ON) heavily invaded (arrows) causing marked hypertrophy of capsule. x 100.
211
212
SPARKS,
MORADO.
the nerve fibers in the optic nerve. Small colonies were occasionally observed deep in the optic nerve. The neurilemma of the optic ganglia usually contained varying numbers of colonies and moderate to heavy infection of nervous tissue deep within the ganglia was not uncommon. The primary optic nerve region, between the anterior optic ganglion (lamina ganglionaris) and the basement membrane of the retina, typically contained numerous colonies (Fig. II). Colonies in this location occurred both within the primary optic nerve fibers and in the spaces between them (Fig. 11). Massive hemocytic infiltration of the primary optic nerve region was a characteristic host response to the presence of colonies in this site. The colonies were frequently surrounded by hemocytes, and melanization was initiated in these lesions more often than in any other tissues of infected crabs. The retinal basement membrane was
FIG. Il. Eye-Colonies area. Basement membrane (RH) region. x 250.
AND
HAWKES
breached in some infections (Fig. 1I), but colonies were less frequent in the retina than in other parts of the eyestalk. However, hemocytic infiltration. ranging from moderate to massive, was characteristic of crabs with eyestalk infections regardless of the presence of detectable infections in the retina. Gill. The stem (Fig. 12) and the lamellae (Figs. 13, 14) of the gill contained numerous colonies. Connective tissue cells and, apparently. podocytes were involved in the gill stem, but infections in the lamellae appeared to be confined to podocytes. In both locations cells containing colonies were so greatly hypertrophied and distorted that unequivocal identification was always difficult and sometimes impossible. There were no appreciable accumulations of hemocytes in either the stem of lamellae of infected crabs, in striking contrast to the massive infiltrations accompanying infection in most other locations.
(arrows) of Chlamydia-like organisms in primary of retina has been invaded and microcolonies
optic nerve fiber (PON) are present in rhabdome
MICROBIAL
DISEASE
IN CRABS
FIG. 12. Gill stem-Colonies (arrows) in gill stem. Podocytes appear to be infected. Normal podocytes (P). x 250. FIGS. 13, 14. Gill lamellae-Chlamydia-like organisms in lamella connective tissue. x 100 and X 250. respectively.
213
SPARKS,
214
MORADO,
Electron Microscopy
Using Storz and Spears (1977) as a reference, several stages of a Chlumydiu-like organism from gill stem and midgut tissues were revealed with transmission electron microscopy. Some gill cells, presumably of connective tissue origin, were nearly filled with spherical inclusions (Fig. 15). The inclusions resembled in size and morphology the condensing forms (intermediate bodies) of Chlumydin. They were membranebound, about 450 nm in diameter, and were coated with electron-dense material (Figs. 15, 17). The infected cells were hypertrophied and devoid of nearly all cell organelles. The nuclei were emarginated and their chromatin was concentrated peripherally. Interspersed among the cells described above were cells with electrondense inclusions which appeared to be lipid vesicles. Unlike the cells containing the putative intermediate bodies, these cells con-
AND HAWKES
tained organelles such as mitochondria (Fig. 18). In addition, there were numerous vesicles that appeared to be an intermediate phase in the development of reticulate bodies, the rapidly multiplying intracellular stage of Chlarnvdia-like organisms. The presumptive reticulate bodies were irregularly shaped, about 1000 nm in diameter, membrane-bound, and contained either finely granular material or small. moderately electron-dense spheres (Fig. 18). Some cells with reticulate bodies contained a third type of inclusion that was ellipsoidal, membrane-bound. and had numerous spherical particles aligned along a central axis (Figs. 16, 19). These latter vesicles were the least frequently observed of the three and appeared to be undergoing binary fission, a further indication of the chlamydial nature of the inclusions. DISCUSSION
Although
the two recognized
FIG. 15. Condensing forms (arrow) in gill stem tissue. Note the hypertrophied appressed, emarginated nuclei (N). x 3800.
species of
gill cells and their
FIG. 16. Portions of two gill cells, one with condensing forms (arrow) and one with reticulate bodies (RB). Electron-dense, lipid-like inclusions are characteristic of the reticulate body-containing cells. Some inclusions appeared to be undergoing binary fission (triangle). x 8200. FIG. 17. Condensing forms from gill stem tissue. The center portion of smaller diameter sections are cap sections and the electron-dense portions are the outer surface of the early elementary body. Apparently there is a coating. perhaps of glycoprotein. that stains intensely and appears very electrondense. x 32,000. FIG. 18. Reticulate bodies (RB) from gill stem tissues. Organelles from the host cell are also apparent throughout these cells. Mitochondria (M). Lipid-like vesicle (L). X 25,000. 215
216
SPARKS,
MORADO.
FIG. 19. Inclusions from a reticulate cell that appear to be undergoing binary fission. x45.000.
Chlamydia, C. trachomatis and C. psittaci, have been reported only in birds and mammals, Morel (1976) pointed out that the diagnosis of the order Chlamydiales does not exclude the possibility of members of the order parasitizing invertebrates. It could also be possible that the forms in invertebrates may slightly deviate in morphology from the known species of Chlamydia. Morel and Duthoit (1974) reported a Rickettsia-like organism in the hepatopancreas of the scorpion, Buthus occitanus. Morel (1976) subsequently placed the microorganism in the order Chlamydiales, creating a new genus, Porochlamydia, to accommodate it and named it P. buthi. Harshbarger et al. (1977) reported intracytoplasmic Chlamydia-like organisms in the digestive tubule epithelial cells of the hard clam, Mercenaria mercenaria, from Chesapeake Bay. Their diagnosis was confirmed by Page and Cutlip (1982) using morphological and immunological criteria. Previously, Meyers (1979) confirmed the presence of a
AND
flAWKI:S
chlamydial agent in the digestive diverticular epithelium of the hard clam. M. nzercennricl, from Great South Bay, New York, using the same criteria. Other morphological studies revealed the presence of a Chfamydia-like agent in the digestive gland epithelium of the oyster, Crassostrea angalata, from France (Comps and Deltreil, 1979), and the bay scallop, Argopecten irradians, from Prince Edward Island, Canada (Morrison and Shum, 1982). The microorganism infecting the Dungeness crab has similarities to all the above Chlamydia-like organisms but differs from them in certain aspects. All appear to be obligate intracellular parasites during their reproductive, noninfective stages: have stages that closely resemble the reticulate bodies of Chlamydia; and are or appear to be membrane-bound. There is evidence that all these “reticulate bodies” reproduce by binary fission and that presumably infective “elementary bodies” are produced by them. However, the elementary bodies in P. buthi have the shape of a flattened rod, a pentalaminar cell wall, and are relatively large (450-700 by 240-300 nm) in comparison to the initial bodies (Morel, 1976). Harshbarger et al. (1977) reported that the elementary bodies in Chesapeake Bay bivalves were small (200-300 nm), round, and dense. Those in the bay scallop averaged 380 by 190 nm, were typically fusiform in shape, and were surrounded by a pentalaminar membrane (Morrison and Shum. 1982). The elementary bodies in the oyster, C. angulata, were particularly large, measuring 300-350 by 700-850 nm (Comps and Deltreil, 1979). Definitive elementary bodies in the Dungeness crab microorganism were not observed in this preliminary study. All these agents cause marked hypertrophy of infected epithelial cells, with the nucleus crowded against the cell margin by the proliferating microorganisms. The forms occurring in the bivalve molluscs and the scorpion are apparently restricted to the hepatopancreatic epithelial cells. In contrast, the Chlamydia-like agent in the
MICROBIAL
DISEASE
Dungeness crab demonstrates a strong affinity for connective tissue and connective tissue cells while only infrequently infecting epithelial cells. P. buthi causes a fatal disease in the scorpion, but there is no evidence of serious disease in any of the molluscan Chlamydialike infections. The Dungeness crab disease, in addition to being essentially systemic, is highly pathogenic and may prove to be a significant cause of mortality in Dungeness crabs during periods of low water temperatures. The implications of the present agent to cause mortalities is enormous since another study by Stevens and Armstrong (1981) estimated 6461 dead Dungeness crabs along a 7.9-km transect of southwest Washington coast following the February, 1979, mortalities. However, there are unanswered questions relative to the taxonomic position of the microorganism and the disease it causes. We plan to continue our studies of the cytopathology, progression of the disease, and morphology and developmental stages of the parasite. We have designated the microorganism in the Dungeness crab a Chlamydia-like organism because we do not have unequivocal evidence that it posseses all the morphological characteristics of the order Chlamydiales, let alone the genus Chlamydia or Porochlamydia. It is clear that microorganisms more closely related to the Chlamydia are more rare than the Rickettsia in marine, freshwater, and terrestrial invertebrates. ACKNOWLEDGMENTS We thank Dr. John Harshbarger, Dr. Sing Chang, and Dr. Ted Meyers for their critical reviews of the manuscript. We must point out that Dr. Harshbarger and Dr. Chang did not believe the microbial agent to
217
IN CRABS
be a Chlamydia-like organism but did not make any suggestions. We would also like to thank Ms. Jolly Hibbits for technical assistance and Ms. Dawn Wood for typing the manuscript.
REFERENCES G. (Ed.) 1980. “Staining procedures,” 4th ed. Williams and Wilkins, Baltimore. COMPS, M., AND DELTREIL, J. P. 1979. Un microorganisme de type rickettsien chez I’Huitre portugaise Crassostrea angulara Lamarck. C.R. Acad. Sci.. Ser. D.. 289, 169-171. HARSHBARGER. J. C., CHANG, S. C., AND OTTO. S. V. 1977. Chlamydiae (with phages), mycoplasmas. and rickettsiae in Chesapeake Bay bivalves. Science (Washington. D.C.), 196, 666-668. HAWKES, J. W., AND STEHR, C. M. 1982. Ultrastructural studies of marine organisms: A manual of techniques and applications. Electron Oprics Bu/l., 118, 15-20. MEYERS, T. R. 1979. Preliminary studies on a chlamydial agent in the digestive diverticular epithelium of hard clams Mercenaria mercenaria (L.) from Great South Bay, New York. .I. Fish Dis., 2, 179189. MOREL, G. 1976. Studies on Porochlamydia buthi g.n., sp. n., an intracellular pathogen of the scorpion Buthus occitanus. J. Invertebr. Pathol., 28, 167- 175. MOREL, G., AND DUTHOIT, J. L. 1974. Un microorganisme intracellulaire de type rickettsien chez le scorpion Buthus occitanus Amoreux. C.R. Acad. Sci. Paris, Ser. D., 278, 1991-1994. MORRISON. C., AND SHUM. G. 1982. Chlamydia-like organisms in the digestive diverticula of the bay scallop, Argopecten irradians (Lmk). 1. Fish Dis., 5, 173-184. PAGE, L. A., AND CUTLIP, R. C. 1982. Morphological and immunological confirmation of the presence of Chlamydiae in the gut tissues of the Chesapeake Bay clam. Mercenaria mercenaria. Curr. Micro-
CLARK,
biol.,
7, 297-300.
STEVENS, B. C., AND ARMSTRONG, D. A. 1981. Mass mortality of female Dungeness crab, Cancer magister, on the southern Washington coast. Fish. Bull.. 79. 349-352. STO& J., AND SPEARS, P 1977. Chlamydiales: properties, cycle of development and effect on eukaryotic host cells. Curr. Top. Microbial. Immunol., 76, 167-214.