The Prevelance of Disease in the Oyster Crassotrea virginica in Rhode Island

The disease prevalence in American oysters, Crassostrea virginica, from coastal areas in Rhode Island was studied. Oysters were collected by hand or tongs from the Pawcatuck River (3 stations), Narrow River (2 stations), Charlestown Pond (3 stations) and Green Hill Pond (3 stations), during July/August 1991, November 1991 , March 1992, and May 1992. Of the forty oysters collected at each of the four sites, thirty were processed for histologic examination. The remaining 10 oysters of each site were used to determine the condition index. Haplosporidium nelsoni was detected in four of 480 oysters (0.8%); Sphenophrya sp., 15 of 480 (3.1%); Bucephalus sp., 16 of 480 (3.3%); crustacea, one of 480 (0.4%); and basophilic inclusion bodies (possible mycoplasm-like organisms), 15 of 480 samples (3.2%). Lesions also were found in the samples including: kidney concretions, 32 of 360 (8.8%); necrosis of digestive diverticulae of gastrointestinal tract, 28 of 480 (5.8%); neoplasia of gastrointestinal tract, connective tissue, reproductive tract, and gills, 18 of 480 (3.8% ); hyperplasia of digestive diverticulae , one of 480 (0.2% ); ulceration of stomach epithelium, two of 480 (0.4% ); cysts in the kidney, three of 360 (0.8% ); atrophic adductor muscle, two of 360 (0.6% ); and inflammation of kidney, gills, gastrointestinal tract, connective tissue, 447 of 480 (93.1 % ). Condition index ranged from 26.52 to 197.67. Condition index of oysters from Pawcatuck River was below 75 throughout the year. In the other areas, condition index was consistently lower during the summer than during other times of the year. Lesions and parasites were found at all of the sites studied, although MSX was found only in Charlestown Pond. Disease prevalence in oysters from the Pawcatuck River was not different from other sites, but the condition index was consistently lower. These findings suggest that low condition index may not necessarily correlate with higher disease prevalence.


INTRODUCTION
The American oyster, Crassostrea virginica (Gmelin, 1791) is a common inhabitant of Gulf and Atlantic coast estuaries of the United States (Galtsoff, 1964;Abbe, 1988).
Historically oysters were the most valuable shellfish fisheries of Rhode Island salt ponds and estuaries (Ganz, 1973;Bean, 1990;Desbonnet and Lee, 1990). Rhode Island contributed significantly to the 26 million pounds of oysters that were produced annually in New England during the early 1900's (Matthiessen,1970). The oyster industry declined due to pollution from sewage, coastal development, and the deposition of heavy metals in coastal areas between the 1930's and 1950's (Bean, 1990).
Environmental stress has been associated with increased incidence of infectious disease in marine animals (Sparks, 1985). Stress caused by chemical and environmental factors is also associated with increased incidence of neoplasia (Sparks, 1985). Sindermann (1990) stated that mortality is caused by the combination of an infectious agent and environmental stressors.
In recent years there has been widespread concern about the impact of the protozoan parasite Haplosporidium nelsoni, known as MSX (multinucleated sphere unknown) on the Crassostrea virginica fishery. Sindermann (1990)   . The Haplosporidium parasite has also been responsible for mortalities on natural beds of the American oyster in Delaware and Chesapeake Bays since early in the 1950's (Andrews and Wood, 1967;Haskin et al., 1966;Haskin and Ford, 1982).
MSX was first found in the lower Chesapeake Bay in 1959 (Farley, 1975) and moved progressively up the Bay into Maryland. The parasite has continued to cause extensive damage to the oyster industry and still inhabits the waters of the Chesapeake. Couch et al. (1966) reported MSX in oysters from the Middle Atlantic states. It has also been reported in North Carolina, Long Island Sound (Sindermann and Rosenfield, 1967), Massachusetts and Great South Bay of Long Island .
Since the early epizootics, several of studies have been undertaken to assess the effects of MSX on oyster populations. The time course of MSX infection to host mortality is quite rapid. In the spring of 1957, approximately half of the oysters planted on the New Jersey oyster grounds in Delaware Bay died within 6 to 7 weeks (Haskin et al., 1966). The pattern of losses and continuing mortalities later that summer and fall indicated an infectious disease as the cause (Andrews, 1966). Ford and Haskin (1982) estimated that at least half of all oyster deaths in lower Delaware Bay since the early 1960's could be attributed to the effects of Haplosporidium nelsoni infection. Rosenfield and Sindermann (1966) claimed that between 1963 and 1965, MSX invaded areas of middle Chesapeake Bay formerly free of the disease. Haplosporidium nelsoni occurs in water with salinity greater than 15 ppt According to Andrews (1966), about 50% of the oyster beds in high salinity water in Chesapeake Bay had become unproductive during 1959 -1963.
To interpret the status of MSX, given the known pattern of infection, Andrews (1967) suggested following a particular oyster population through at least one growing year, sampling live oyster for prevalence of the disease and recording standard mortality data.
There is no "best time" of the year to sample for MSX prevalence because Haplosporidium nelsoni kills oysters in all seasons. Infections occur during at least the five warm months of the year and have variable incubation times. Additionally, MSX may infect and kill all sizes of the American oyster (Farley, 1975).
Several studies have been conducted on the life history, pathology, and epizootiology of MSX. Haskin et al. (1966) reported that infections of MSX reduced the particle filtration rates of the oyster. The condition index of infected oysters was often found to be lowered substantially (Newell, 1985). According to Ford and Figueras (1988) and Barber et al. (1988), gamete development, fecundity, tissue lipid, protein, and glycogen of eastern oysters are all affected by the protozoan MSX. The MSX parasite appears poorly adapted to its host since infections frequently kill the oyster (Andrews, 1968;Ford, 1985). Laboratory Procedure Samples collecred during July/August, 1991 were brought to the laboratory within 2 hours, processed and examined. Oysters were cleaned of fouling organisms. Samples were divided as follows: from each site 10 oysters were used to determine condition index and 30 oysters for histological studies. For histological processing, oysters were removed from their shells and fixed in 10 % seawater formalin (Appendix I). A cross sectional cut was made on each oyster before fixing the tissue for histology. Preserved tissue was dehydrated by alcohol series and embedded in Tissue Prep (Fisher) and six-micrometer sections were stained with hematoxylin and eosin, then mounted and examined.
Samples collected in November 1991, March and May 1992, were fixed with Helly's fixative (Appendix I). The oysters were allowed to become firm, removed from fixative, cut sagittally through the mid-line of the body up to the adductor muscle and returned to fixative for 16 hours. After fixation, the oysters were washed in running water for 24 hours then stored in 70% ethyl alcohol. The oysters were then trimmed and placed into cassettes for further processing. In trimming the oysters, the previous sagittal cut through the middle of the body was extended through the muscle, cutting the oysters in half. If the oysters were large, each half was again sagittally cut and these halves cross sectioned so that they would fit into the cassettes. All tissues were processed by routine histopathologic methods. Slides were stained with hematoxylin and eosin. Helly's fixative has been determined to be the most suitable cytological fixative for aquatic invertebrates because it preserves the nucleoplasm of the ova nuclei, granules of the secretory cells and amebocytes better than other fixatives tested (Yevich and Barszcz, 1981).
By microscopic examination, each oyster was categorized with respect to the intensity of MSX infection as either uninfected, epithelially infected (gill only) or systemically infected (sub-epithelial general infection). Other parasites and pathologic conditions observed during this study were also recorded. The percentage of oysters infected with each parasite was calculated for each site.
Microscope slides from this study had been archived for possible future verifications.
Most slides will be archived at the Marine Pathology Laboratory, East Farm, University of Rhode Island. Slides showing neoplasia will be archived at the Registry of Tumors in Lower Animals, National Museum of Natural History, Smithsonian Institution, Washington, DC.
The condition index (Cl) determination followed the procedure recommended by Crosby and Gale (1990). · CI = dry soft tissue weight (gr) x 1000 internal shell cavity capacity (gr) Oyster shell measurements included: valve length, height, and width. Internal shell cavity capacity (grams) was determined by subtracting dry shell weight (grams) in air, from the total whole live weight (grams) in air of a cleaned oyster. Internal shell capacity is proportional to internal shell volume because oyaters will trap seawater with valve closure.
The dry weight of soft tissue was obtained by drying the tissue in an 80° C oven for 48 8 hours and determining weight (grams) of dried tissue. The condition index may also be used as an assay for monitoring various pollutants and disease (Crosby and Gale, 1990) as well as routinely monitoring the general health of oyster populations. According to Quayle (1980), oysters with high condition index (75 to 150) are in good condition, while low condition index is 75 and below.

RESULTS
The percentage of oysters with lesions and parasites from Pawcatuck River, Narrow River, Charlestown Pond, and Green Hill Pond ranged from 0.2 % to 93.1 % (fable 1).
Surface water salinity ranged from 0 ppt to 30 ppt, measured temperatures ranged from 4° C to 26° Cat the time of collection, and shell heights of oysters ranged from 24.6 mm to 190.0 mm (Tables 2 and 3).

Protozoan diseases
Haplosporidium nelsoni (MSX) During the year-long study, MSX was found only in Charlestown Pond ( (Table 8).
The ciliate Sphenophrya sp. (Figure 4)        One of the oysters from Pawcatuck River in May 1992 was infected by one crustacean of unidentified species (Tables 5 and 6). It was located in a tubule of the digestive diverticulum and caused ulceration and inflammation of the tubule wall ( Figure 5). of Bucephalus sp. were found in the reproductive follicles ( Figure 6) and in connective tissues (Figure 7).

Basophilic inclusion bodies (BIB) were found in the gastrointestinal tract of
Crassostrea virginica at all four sites in 15 of 480 oysters - (Table 1). Percent infection of BIB of each station was shown in Tables 4, 5 , 6, 7, 8, and 9. The basophilic bodies in the gastric mucosa is shown in Figure 8.

Lesions Kidney Concretions and Brown Cells
Large, round, brown pigmented bodies similar to the concretions described by Rheinberger (1977) and Carmichael et al. (19_ 79) were observed in the kidney of Crassostrea virginica. Concretions were located in the kidney tubule ( Figure 9) and were    (Tables 1, 4 , 5, 6, 7, 8, and 9). Besides kidney concretions, numerous brown cells located in the connective tissue of the visceral mass were found in almost all oysters ( Figure 10). The number of brown cells per oyster were categorized as light (1 -3 cells per high magnification), moderate (4 -6 cells per high magnification) and heavy (7 -9 cells per high magnification). In oysters from Pawcatuck River and Narrow River, brown cells were larger, more numerous, and had darker and larger vesicles with more inclusion bodies than in those oysters from Charlestown Pond and Green Hill Pond (Table 10).

Ulceration
Only two of all the Crassostrea virginica examined contained ulcerative lesions (Tables   1, 5, 7, and 8). The ulceration occurred in the stomach epithelium ( Figure 11).
Inflammation of the submucosa and thickening of basement membrane were also found in these oysters. A causative agent could not be determined.

Necrosis
Necrotic foci were observed in the digestive tubules of the digestive diverticulum.

Hyperplasia
Hyperplasia was observed in the tubular epithelium of the digestive diverticulum

Neoplasia
Neoplastic lesions in oysters are generally characterized by the infiltration of vesicular connective tissue and by hypertrophic, anaplastic, mitotically active, basophilic cells. Cells typically have a high nucleus to cytoplasmic ratio and pleomorphic nuclei with one or more distinct clefts. Multiple nucleoli are present in some cases (Murchelano and Macl.ean, 1990).
One of the oysters from Pawcatuck River in March, 1992 had germinomas ( Figure 13).
Basophilic, hypertrophied, neoplastic germ cells had proliferated along the walls of the gonadal follicles and ducts. Three of the oysters had adenomas in the gill ( Figure 14).
The sarcomas are characterized by the appearance of unusual cells in the connective tissue, blood vessels, and sinuses of the visceral mass, muscle and mantle tissue (Peters, 1988 (Table 8).
Three oysters in Green Hill Pond had neoplasia, one in the gill and two in the gastrointestinal epithelium (Table 9).
During this study, 18 of 480 (3.7 %) oysters had neoplasms in the gastrointestinal tract, reproductive tract, connective tissue, and gills (Tables 1, 4 , 5, 6, 7, 8, and 9). Eight of these oysters had adenomas in the stomach epithelium , one had a germinoma, two had a mesenchymal origin of connective tissue and eight had branchial neoplasia (Table 10).

Atrophy of Adductor Muscle
One oyster collected from Pawcatuck River in May 1992 and one oyster from Charlestown Pond in March 1992 had the unusual condition adductor of muscle atrophy and replacement by proliferating collagenous connective tissue ( Figure 19).

Cyst (Idiopathic)
Cysts were found in three kidney tubules of oysters from Pawcatuck River, Narrow River, and Green Hill Pond (Tables 1, 4, 5, 7, and 9). Figure 20 shows destruction of kidney tubular epithelium in oyster collected in May 1992 from the Pawcatuck River. Cyst formation in the tubular epithelium is also shown. Another kidney tubular epithelial cyst was found in an oyster from Narrow River collected in March 1992 ( Figure 21).

Inflammation
Almost all the oysters had focal inflammation in the connective tissue [447 out of 480 oysters (93.1 %)]. Some animals had very extensive inflammation. The inflammation in the connective tissue were categorized as very light to light and moderate to heavy. (Table   10). Sixteen (3.3%) had inflammation in the kidney, 210 (43.8%) had inflammation in the gills and 28 (5.8%) had gastrointestinal tract inflammation (Table 1, 4, 5, 6, 7, 8, and 9).

Condition Index
Condition index was determined in 150 oysters. Condition index from each site and sampling period are given in Table 12. The condition index ranged from 26.52 to 197.92.
Throughout this study, condition index of oysters from the Pawcatuck River remained low (below 75). In the other study areas, condition index was consistently lowest in the summer and higher in fall, winter and spring.

DISCUSSION
The full life cycle of Haplosporidium nelsoni is not known, although patterns of infection and mortality are well documented (Andrews, 1966;Couch et al., 1966;Farley, 1967;Ford and Haskin, 1982). Studies of infection and mortality patterns along salinity gradients showed that Haplosporidium nelsoni can not survive in salinities below 10 ppt.
Infections are generally rare and parasite development is inhibited below 15 ppt salinity.
The parasite survives best in salinities above 20 ppt (Andrews, 1964(Andrews, , 1983Haskin and Ford, 1982;Ford, 1985;Ford and Haskin, 1982). In this study surface salinity ranged between 0 to 30 ppt. According to Ford and Haskin (1982), the inability to transmit MSX from one oyster to another, combined with the rarity of production of spores in oysters, suggests that an alternate host must exist. Therefore a possible explanation for MSX being found in Charlestown Pond might be the presence there of an alternate host, but a previous one-year disease survey was conducted in June 1979 to May 1980, and no evidence of MSX was found (Cooper and Durfee, 1982).
Sphenophrya sp is a ciliate that attaches to gill epithelial cells of oysters and other bivalves Murchelano and MacLean, 1990 (Murchelano and Macl.ean, 1990).
The respiration and feeding of the oyster possibly was adversely affected by the heavy infections of these ciliates .
The initial infection site of Bucephalus sp. is the digestive tubules rather than the gonad (Cheng and Burton,1965). Heavy infections destroy the tissues and possibly result in the 11 I I death of the oysters (Hopkins, 1957). Initial infection has the result of effectively castrating the oysters (Sparks, 1985). There is little host response to the presence of the parasite. There is neither phagocytosis nor encapsulation, but hemocyte infiltration may occur following sporocyst degeneration (Cheng and Burton, 1965). Sypek (1979) studied the trematode Proctoeces maculatus in Mytilus edulis and found it the only known example of an adult trematode eliciting a hemocytic response within a mollusc. The occurrence of lipoid substances in the hemocytes was associated with the host response (Sypek, 1979). Sporocysts of the trematode Bucephalus sp. were found at all of the sites studied except Green Hill Pond. Gauthier et al. ( 1990) (Cheng and Burton, 1965).
The kidney of the oyster has been found to accumulate concretions of golden yellow to black granular material in the lumens. of tubules when the animal is under stress (Yevich, 1980). One study showed that the accumulation of concretions did not damage the kidney (Potts, 1967), but Rheinbarger et al. (1977 showed that larger concretions can cause an inflammation of the kidney of Mercenaria mercenaria with a resulting loss of epithelium. The kidney concretions often consist of metals with Ca, Mg, Zn, Fe, and Pb being the major elements (Rheinbarger et al. 1977). In this study, the highest prevalence of kidney concretions was found in oysters from Green Hill Pond. The lowest rate was found in the Pawcatuck River (1/90). The kidney concretion was not found in Pawcatuck River stations r 1 and 2 (low salinities) but was present in oyster from station 3. Possibly there is a correlation between kidney concretions and salinity. Kidney concretions were more 49 prevalent in oysters from sites with higher salinity, such as Narrow River, Charlestown Pond, and Green Hill Pond. It has been suggested that kidney concretions may be indicators of environmental degradation since larger concretions are of ten found in more polluted areas (Rheinbarger et al., 1977).
The presence of large aggregates of yellowish bodies and larger brownish bodies (probably "brown cells") have been studied by Takatsuki in 1934. Brown cells may have functions associated with the oyster's internal defense mechanisms (Mackin 1951). The number of brown cells increase if oysters suffer disease, stress or pathological condition (Sparks, 1985). Brown cells contain lipofuscin, a complex of lipids, phospholipids and some protein (Slauson et al., 1990). Sypek (1979) analysed the pigment in mussels and found it contained lipofuscins and carotenoids. Cheng and Rifkin (1970) stated that the most important role of these cells was in the removal of degradation products and metabolic by-products of parasite infections.
Basophilic inclusion bodies (BIB) have been found in marine bivalve molluscs and electron microscopic studies showed the oyster inclusions-most commonly contained mycoplasma-like organisms (Harshbarger et al., 1977). No host response was associated with BIB infections in bivalves, but the heavy infections possibly reduce the metabolic efficiency and alter the nutrients of the host . Six of 120 oysters in coastal Louisiana had BIB (Gauthier et al., 1990), a prevalence higher than observed in this study [15 of 480 oysters (3.2%)]. This finding is similar to those described by Barszcz et al. (1978) and Gauthier et al. (1990) of large, roundish, basophilic inclusions in the intestinal epithelium of Crassostrea virginica. Gauthier et al. (1990) also reported that in higher salinity the oyster had higher levels of parasitism.
One important finding in this study is the high incidence of neoplasia [18 of 480 oysters (3.8%)]. In two separate Chesapeake Bay studies, neoplasia was observed in 12 of 20,000 Crassostrea virginica (Harsbarger et al., 1979) and five out of 30,000 oysters (Farley, 1969). Several gonadal tumors were found in oysters from the Pawcatuck River at the same station # 1 as in this study (Yevich, 1992 personal communication). Similar lesions have been found by  and Harshbarger et al. (1979). One oyster from Narrow River in March 1992 would most likely be a tumor probably of mesenchymal origin. Newman (1972) also described an undifferentiated sarcoma in Crassostrea virginica from New Haven Harbor, Connecticut. Gardner et al. (1991) reported that two of 46 oysters exposed to Block Rock Harbor (Connecticut) sediments developed gill papillomas. Paquette (1992) stated that the prevalence of neoplasia in Mya arenaria in Rhode Island was the highest in January to March, May and October. Similarly in this study the highest prevalences of neoplasia were in March and May.
The cause of the neoplasia in Crassostrea virginica is not certain. Molluscan neoplasia is not a result of infection by bacteria, mycoplasma, or protozoan parasites (Oprandy, 1982;Paquette, 1992). Barry and Yevich (1975) reported that gonadal tumors in Mya arenaria in Maine were possibly the result of an oil spill; and Yevich and Barszcz (1976) stated that tumors in Mya arenaria could be related to pollution. Two types of neoplasia in M. arenaria were found from polluted and nonpolluted areas (Brown et al., 1977). Oprandy et al. (1981) reported that hematopoietic neoplasia in Mya arenaria is due to a viral agent. In several studies, a correlation was made between carcinogenic hydrocarbons in sediment and high incidence of neoplasia in bivalves (Gardner et al., 1991;Paquette, 1992). In this study, neoplasia was found in all of the sites studied. Pawcatuck River, Narrow River, and Green Hill Pond are classified as polluted on the basis of fecal coliform (Strobel et al., 1987 andGanz, 1992). Charlestown Pond is the only one certified by RI DEM to be unpolluted and open for shellfishing.
The high prevalence of oysters with inflammation is unusual. The causes of the observed inflammation is uncertain, but it could be caused by a protozoan, bacterial or viral disease. Some oysters had very extensive inflammation in all organs. According to Farley (1992, personal communication) pollution stressors are likely cause for the inflammation in the oysters. Sparks and Morado (1988) and Slauson et al. (1990) suggest that inflammation is the host defense mechanism following tissue damage.
Condition index of oysters from the Pawcatuck River was very low during the year, but the prevalence of disease was not necessarily higher than other sites. This indicates that condition index may not be a good indicator of disease prevalence. In this site, the size of oysters were between 5 and 18 cm (mature), however there appeared to be no gonadal development. More studies are needed to investigate oyster spawning and recruitment in this area. In the other study areas, condition index was consistently lowest in the summer and higher in fall, winter, and spring. This is probably due to summer spawning.
Most of the Rhode Island oyster beds have been closed for shellfishing due to water quality degradation. All the oysters in this study were from native oysters beds.
Parasitism and lesions have been found at all of the sites studied. Further studies are needed to determine the etiology of some of the diseases.

CONCLUSIONS
Histopathologic studies on oysters collected from Pawcatuck River, Narrow River, Charlestown Pond, and Green Hill Pond showed that the prevalence of MSX is very low (0.8%). This may be due to the lack of a suspected alternate host in this area and as yet uncertain environmental factors.
Many other disease conditions were found in this study, such as parasites of Mean condition index (Cl) of oysters from Pawcatuck River were below 75 throughout the year, suggesting poor condition. In other areas the mean CI was almost always almost above 75 throughout the year, despite lesions and parasitism being found at all of the sites studied. Further studies are needed to understand the ecology of the oyster populations in Rhode Island.

Zinc Chloride
Appendix I 10 ml.
Add 5 ml. of 37 -40% formaldehyde per 100 ml. of fixative at time of use.