Recovery of a fish pathogenic bacterium, Aeromonas salmonicida, from ebonyshell mussels Fusconaia ebena using nondestructive sample collection procedures.
ABSTRACT Refugia are increasingly being used to maintain and propagate imperiled freshwater mussels for future population augmentations. Success for this endeavor is dependent on good husbandry, including a holistic program of resource health management. A significant aspect to optimal health is
KEY WORDS: mussels, diseases, pathogens, prevention, tissue, sample, collection, nondestructive, nonlethal
INTRODUCTION
In recent decades, many of the freshwater mussel species that are native to the United States have suffered significant declines in numbers of individuals and populations (Lydeard et al. 2004, Neves 1997, Williams et al. 1993). Maintenance and propagation of captive freshwater mussels at refugia is a critically important part of the overall conservation effort for this imperiled fauna. Riverine mussels in critically low abundance are identified for captive rearing and are relocated to refuges with a goal of future population restorations or augmentations. Their morbidity and mortality as a result from the artificial rearing, handling, and similar must be avoided or minimized. There are a number of critical factors to address when adapting wild-collected specimens to hatchery rearing conditions. These include providing optimum diet and feeding, maintaining good water quality and flow dynamics, and providing their desired substrate compositions. These, and other important factors are essential to maintaining a high condition factor in mussels, which is an index of overall health status (Patterson et al. 1997, 1999). In other aquatic organisms, particularly cultured fish species, poor health condition and physical stressors adversely affect future propagation successes (Noga 1996, Piper et al. 1982). One of the consequences to poor health is predisposing animals to infection with pathogens and diseases, an especially important concern at intensive-culture hatcheries or refuges where animals might be reared in greater densities than would occur in feral waters. In conjunction with stressors, the occasional introductions of wild-caught mussels to refuges that have resident populations present an ongoing potential source for introductions of pathogens. At mussel refuges, the risk to introduce pathogens is compounded by the need for host fishes for glochidia transformation. The host fishes typically may not be cultured in hatcheries and are also likely to be collected from rivers or streams and placed at these refuges, similar in procedure to the mussels, except the fish currently are not required to undergo a quarantine prior to their arrival at refuges.
Laboratory studies with Aeromonas salmonieida Lehmann and Neumann have demonstrated how readily this bacterial pathogen of fishes can be transmitted between mussels and fish (Starliper 2001, Starliper 2005, Starliper & Morrison 2000). Therefore, relocated mussels may serve as a source of pathogens to captive populations at refuges, but perhaps equally important, the hatchery-propagated mussels could serve as pathogen vectors to wild mussel populations and fish during stockings into streams for restoration and augmentation. Because of these possible scenarios, there is the need to proactively address these issues of pathogen movement among mussels and develop preventative strategies. These issues will be pivotal for health and disease management and successful conservation of imperiled species.
Treating fish suffering from bacterial diseases is a difficult task. Few antibiotics are approved for use, and delivery to large numbers is typically oral through the use of medicated feed. Development of pathogen resistance to antibiotics is widespread and an ongoing risk (Starliper & Cooper 1998). Similar problems can be anticipated when treating mussels for bacterial diseases. The most prudent and desirable approach to control diseases and mortality is through avoidance by preventing transmission of pathogens, particularly for imperiled species. One strategy by the United States Fish and Wildlife Service to control transmission of pathogens in fisheries is through regional Fish Health Units. These Units conduct periodic health and disease inspections on hatchery-reared stocks for recognized pathogens, and new or emerging diseases. Similar health inspections are also conducted on free ranging fishes (National Wild Fish Health Survey; www.fws.gov/wildfishsurvey). These surveillance programs form a broad view of the appearance of pathogens in disease-free locales and on the ranges of diseases in enzootic areas. A pathogen transmission prevention program such as the one for fish could be implemented for mussels and could become invaluable for refuge-propagated and feral mussels. Because many of the mussel species that would be examined in a health and disease inspection are imperiled, nondestructive sampling procedures will be necessary to collect the samples for assays. Nondestructive bioassay procedures have previously been used in freshwater mussels. Berg et al. (1995) developed and evaluated a mantle tissue biopsy technique with Quadrula quadrula Rafinesque and Actinonaias ligamentina Lamarck. There was no significant difference in mortality of biopsied versus nonbiopsied mussels during the 13 mo that followed. Henley et al. (2006) evaluated a noninvasive, swab-stroke procedure to obtain DNA from Q. pustulosa I. Lea. Gustafson et al. (2005a) collected hemolymph from the adductor sinus of Elliptio complanata Lightfoot using a needle and syringe and showed no subsequent negative effects on the survival or growth of the mussels.
The present study was done to evaluate three nondestructive procedures for recovery of A. salmonicida from Fusconaia ebena I. Lea that were exposed to the pathogen through cohabitation with diseased fish. One of the procedures, collection of fluid from within the valves, is noninvasive, whereas the other two procedures (mantle tissue biopsy and drawing hemolymph) are invasive. These three procedures were compared with lethal collections of fluid and total soft tissue homogenates for bacterial recovery.
METHODS
Two trials comparing nondestructive versus lethal sample collection procedures were done at the USGS National Fish Health Research Laboratory, Leetown, WV. Both trials used ebonyshell mussels F. ebena. Brook trout Salvelinus fontinalis Mitchill (about 60 g each) served as the source of A. salmonicida, the subject pathogen, to infect the mussels. The difference in the two trials was diet for the F. ebena. Food was withheld from mussels in one trial, whereas the mussels in the other trial were fed a daily ration of algae. Both trials were conducted at 20[degrees]C, a water temperature within the range for mussel quarantine to prevent spread of zebra mussels Dreissena polymorpha Pallas. The water supply was specific pathogen-free (i.e., no pathogens to fish), spring-source, which was elevated from ambient 12[degrees]C to 13[degrees]C using an in-line propane-fueled boiler. Water to the experimental tanks was adjusted to deliver 2-3 complete volume changes per hour. Alkalinity of the water ranged between 270 280 mg/L as calcium carbonate, and the range in dissolved oxygen was 9.8-10.4 mg/L. Because of the availability of F. ebena at the time for each trial, the mussels originated from two different sites. The origin for the F. ebena that were not fed was Kentucky Reservoir, TN River mile 102, Humphreys County, TN (35[degrees]29.44'N; 88[degrees]00.27'W). The origin of the mussels that were fed was the Hawesville Bed, OH River Mile 725, Hancock County, KY (37[degrees]55.19'N; 86[degrees]45.87'W). Mussels from both sites were hand collected. Each group was quarantined for a minimum of 30 d, to prevent the spread of zebra mussels (Chaffee 1997, Gatenby et al. 2000), at the United States Fish and Wildlife Service quarantine facility, OH River Islands National Wildlife Refuge, near Williamstown, West Virginia. After completion of the quarantine, the mussels were transported in live wells, in quarantine facility water, to the Laboratory in Leetown, West Virginia. Upon arrival, the mussels were acclimated by gradual (overnight) water displacement with Leetown spring water.
With the nonfed trial, an assumption was made that the mussels would uptake a maximum number of viable A. sahnonicida colony forming units (cfu). The diet for the fed F. ebena consisted of cultures of Neochloris oleoabundans Chantanachat and Bold, Oocystis sp., and Bracteacoccus grandis Bischoff and Bold. The algae were cultured in a continuous culture system (Biofence; Varicon Aqua Solutions, UK) at the White Sulphur Springs National Fish Hatchery, White Sulphur Springs, West Virginia. The algae were grown at about 24[degrees]C in modified F/2 Guillard's formula (Microalgae Grow Mass Packs; Aquatic Eco-Systems, Inc., Apopka, Florida). Algae were suspended in Leetown spring water for feeding, such that mussels would receive approximately 40,000 cells per mL of tank water. The following procedure was used to feed the mussels daily. The tank's water supply was turned off, and the algal cultures were poured and mixed into the tank water by gentle swirling for 10-15 s. After 4 h of feeding time, the supply water was resumed. The 4 h duration was a compromise between adequate time for the mussels to feed and maintaining the high dissolved oxygen levels. Fish were fed daily at a rate of 1% body weight per day with a standard trout diet.
A septic techniques were used to collect nondestructive and lethal samples for bacteriology. External surfaces of the valves were disinfected with 200 mg/L sodium hypochlorite and morphometric data were collected as previously described (Starliper 2005). The lethal fluids and soft tissues were collected and processed for bacteriology as previously described (Starliper 2001, Starliper et al. 1998). To collect the nondestructive samples, a procedure similar to that of Berg et al. (1995) and Gustafson et al. (2005a) was used to pry open, and hold the valves about 15 mm apart to facilitate collections of samples. The valves were held open by inserting a surface-disinfected instrument, such as a magnetic stir bar or dull oyster knife. The nondestructive fluid (ND fluid) was clean-caught in a sterile disposable Petri dish whereas holding the mussel with the opened valves facing downward and over the dish. Occasionally, a mussel required a gentle up and down motion with a gentle sudden stop at the bottom of the stroke to encourage some fluid to drop into the dish. Care was taken so the ND fluid would drop directly from within the opened valves into the Petri dish, instead of running down the object that was used to hold the valves apart. Given the sizes of the F. ebena studied, at least 0.5 mL of ND fluid was easily obtained from almost all of the specimens. Slightly less ND fluid was obtained from a few of the smaller specimens, but this volume was still adequate for bacterial culture. Hemolymph was collected in accordance with the technique of Gustafson et al. (2005a), except that a 22 G, 1-inch needle was used. A range of 0.2-0.5 mL hemolymph was readily drawn from each mussel, the needle was removed and the hemolymph was expelled into a sterile 10 x 75 mm tube for ease of handling. A larger volume of hemolymph could have been drawn but was unnecessary for bacterial culture and a minimal volume intuitively created less stress for the animal. The piece of mantle tissue was collected using the procedure of Berg et al. (1995), with the exception that a smaller piece, a size similar to that of Henley et al. (3 x 5 mm; 2006) was biopsied. The samples were diluted in 0.1% peptone-0.05% yeast extract (pep-ye). Three 10-fold dilutions were prepared from undiluted hemolymph and fluid. Weights of the mantle samples were determined and 9X (w/v) pep-ye was added for a 1:10 ([10.sup.-1]) dilution; the tissue was homogenized using the end of a sterile 1-mL pipette, and three additional 10-fold dilutions were prepared from the homogenate. Drops (0.025 mL) from all dilutions were inoculated onto CBB agar medium as previously described (Starliper 2001, Starliper 2005). The resulting bacterial colonies were enumerated, and the number of colony forming units per mL (cfu/mL) of fluid and per gram (cfu/g) of tissue were calculated. Suspect A. salmonicida colonies, which were blue on CBB medium, were transferred from primary isolation plates and biochemically confirmed using standard bacteriological procedures (Koneman et al. 1992, MacFaddin 2000).
A bacterial pathogen transmission model was used to transmit A. salmonicida to the F. ebena (Starliper 2001). The mussels acquired the bacterial cells by siphoning contaminated tank water while cohabitating with the diseased brook trout. Prior to the start of each trial, 20 ebonyshells and 20 brook trout were assayed by primary bacterial culture as previously described (Starliper 2001, Starliper et al. 1998) to confirm that they were negative for A. salmonicida.
The same protocol was used in both trials to produce a 100% prevalence of A. salmonicida-positive mussels. The disease in brook trout was initiated by intraperitoneal (IP) injection with approximately 1 x [10.sup.4] cfu A. salmonicida cells per fish. The (19 injected) fish were placed in a 1,250 L cohabitation tank. This dose resulted in greater than 95% mortality within 10 d. When these fish began to die within 4 d, 100 noninjected fish were introduced to the cohabitation tank to become infected through horizontal exposure to A. salmonicida cells shed into the water column from the diseased, injected fish. The noninjected fish began to die within 7 d of their introduction into the cohabitation tank, and by then all of the injected fish had expired. At this time, the F. ebena (n = 150 nonfed trial; n = 160 fed trial) were placed in the cohabitation tank with the diseased brook trout. They remained there until they attained 100% prevalence of A. salmonicida, which was determined by sacrificing 10 for bacterial culture of fluids and soft tissue homogenates. The durations (10 d for the nonfed trial; 14 d for the fed trial) required for this prevalence of infection were estimated based on previous uses of the model (Starliper 2001, Starliper 2005). The remaining F. ebena were distributed in equal numbers to an appropriate number of 158 L tanks, per trial, supplied with pathogen-free spring water; this initiated depuration (i.e., day 0).
Recoveries of A. salmonicida from nonfed F. ebena were determined after 0, 5, and 9 days of depuration. Six mussels were randomly selected on each date for bacterial cultures from lethal fluids and soft tissues. Six mussels from each of the following nondestructive sample groups were assayed: ND fluid, mantle biopsy, hemolymph, and a fourth group from which all three nondestructive samples were taken from each specimen. The resulting data were combined according to sample collection procedure, creating 12 samples from each of ND fluid, mantle and hemolymph (see Table 2 later). After the nondestructive samples were taken from the four groups of F. ebena, these mussels were returned to (4) clean 158 L tanks supplied with pathogen-free spring water and observed for six weeks for deaths presumably caused by the sampling procedures alone. Controls for these four groups were cohorts in the first set of 158 L tanks.
For the trial with algae-fed F. ebena, groups of 10 or 15 were assayed after 0, 1, 5, 10, 15, and 30 days of depuration. In this trial, all three nondestructive samples (ND fluid, mantle, hemolymph) and both lethal fluid and tissue samples were collected from each mussel. This increased the numbers of mussels that were sampled for bacteriology on each date, but minimized the total number of mussels sacrificed. The order in which the five samples were collected from each mussel, from first to last, were as follows: ND fluid, hemolymph, mantle, and then the mussels were sacrificed to take fluid, then total soft tissues. This sequence for collections was arranged to allow each sample to be obtained without influencing the integrity or bacterial recovery from subsequent samples taken from the same individual. This also allowed for pairwise comparisons of lethal and nondestructive samples for recovery of A. salmon# cida. Recovery of A. salmonicida by nondestructive and lethal sampling protocols was evaluated as the F. ebena were depurating, and the prevalence of A. salmonicida-positive mussels and numbers of A. salmonicida cfu per specimen were declining. This experimental design provided a sensitive evaluation of the sample sites for recovery of the pathogen.
RESULTS
The means for the morphometric data collected from F. ebena from Kentucky Reservoir and the Hawesville Bed are presented in Table 1. Mussels from Hawesville Bed, used in the algae-fed trial, were on average 19.49 g heavier than those from Kentucky Reservoir. Accordingly, the means for length, width, depth, and volume from the Hawesville Bed mussels were also greater. However, the means for the volume of fluid (11.03 mL) and weight of soft tissues (11.54 g) from the F. ebena from Kentucky Reservoir were greater than those from Hawesville Bed. The mean for percent of the total weight comprised of fluid and soft tissues of Kentucky Reservoir F. ebena (19.10%) was also greater than that for the Hawesville Bed mussels (14.54%). The largest specimen of the study originated from Hawesville Bed and weighed 207.87 g. The smallest specimen weighed 71.32 g and was collected from Kentucky Reservoir.
Primary bacterial culture done from mucus samples from 20 noninjected brook trout two days prior to the day 0 mussel sampling of the nonfed trial revealed that 19/20 of the fish samples were positive for A. salmonicida. The mean recovery of the bacterium was 7.24 x [10.sup.6] cfu A. salmonicida per g of mucus and the range in counts was 1.87 x [10.sup.5] to 6.00 x [10.sup.7] cfu/g mucus. Two of 20 kidney samples were positive from these same fish with A. salmonicida counts of 1.60 x [10.sup.6] and 4.00 x [10.sup.2] cfu/g. For the trial in which the F. ebena were fed, the pathogen source fish were not sampled for viable cell counts from mucus and kidney. Instead, A. salmonicida was confirmed from dead fish by streak-plate inoculations onto CBB and biochemical confirmations as previously described. Because the epizootic in fish was initiated using the same protocol for both trials, a similar A. salmonicida cfu load was expected in the brook trout in the algae-fed trial.
Results of bacterial culture of total bacteria and A. salmonicida from the nonfed F. ebena are summarized in Table 2. At the 100% prevalence of A. sahnonicida (day 0) in the F. ebena, the mean A. salmonicida recovery from their soft tissues was 2.92 x [10.sup.5] cfu/g, which accounted for a mean of 24.06% of the total bacteria cultured (9.99 x [10.sup.5] cfu/g). The mean for A. salmonicida recovered from the lethal fluids was 6.36 x [10.sup.4] cfu/mL, and 15.9% of the total bacteria (1.91 x [10.sup.5] cfu/mL). Aeromonas salmonicida was isolated from all ND fluid samples with the mean bacterial recovery of 2.38 x [10.sup.4] cfu/mL comprising a mean of 9.45% of the total bacteria cultured (8.30 x [10.sup.4] cfu/mL). Also on day 0, A. salmonicida was cultured from 10/12 mantles, but from only one hemolymph. The mean of A. sahnonicida cultured from the 10 positive mantles was 3.35 x [10.sup.3] cfu/g, which comprised a mean of 4.31% of the total bacteria isolated from these mussels. There was 2.00 x [10.sup.1] cfu/mL from the single hemolymph, which was 4.35% of the total bacteria cultured from this specimen. After five days of depuration, A. salmonicida was not cultured from any of the six soft tissue homogenates. The bacterium was recovered from 2/12 nondestructive mantle tissues, with a mean of 2.64 x [10.sup.1] cfu/g and 0.14% of the total bacteria cultured. Aeromonas salmonicida recoveries from lethal fluids and nondestructive ND fluids after 5d of depuration were similar. Two (of six) lethal fluids were A. salmonicida positive with a mean of 6.66 x [10.sup.2], and 1.81% of the mean total bacteria from these specimens, and 3/12 samples were positive from ND fluids having a mean of 2.71 x [10.sup.3] cfu/mL, which was 10.83% of the total bacteria cultured. The percent of total bacteria from A. salmonicida in ND fluids after 5d was greater than that from day 0. None of the hemolymph samples were positive for A. salmonicida after 5 d depuration. After 9d, there was only one sample that A. salmonicida was isolated from, a ND fluid (1.32 x [10.sup.3] cfu/mL; 0.47% of the total bacteria), of the total 48 samples assayed, including lethal and nondestructive sites.
No dead F. ebena were noted from any of the four groups during the six week observation period that followed collections of the nondestructive samples.
Bacterial culture of A. salmonicida and total bacteria from algae-fed F. ebena are summarized in Table 3. On day 0 at the 100% prevalence level of A. salmonicida, the bacterium was recovered from all tissues and fluids with exception of six hemolymphs that were negative. Noteworthy are the percentages of the total bacteria cultured that were comprised of A. salmonicida. Nearly half of all of the bacteria on day 0 from total soft tissues (44.51%) and lethal fluids (42.05%) were comprised of A. salmonicida. Significant, but lesser percentages were recovered from the three nondestructive samples. The means for A. salmonicida cultured from lethal and the three nondestructive samples from the algae-fed mussels were much greater than the means from the same sample sites on day 0 from nonfed F. ebena (Table 2). The A. salmonicida mean from lethal tissues on day 0 from algae-fed mussels was 2.24 x [10.sup.6] cfu/g; the mean from lethal fluids was even greater, 3.40 x [10.sup.7] cfu/mL (Table 3). The A. salmonicida mean from ND fluids was 9.12 x [10.sup.3] cfu/mL; however, the prevalences of the bacterium from both lethal, and ND fluids were equal, both with 10/10 positive. The greatest mean in A. salmonicida recovery from the nondestructive samples on day 0 was from four positive hemolymphs (1.00 x [10.sup.5] cfu/mL).
After 1 d and 5 d of depuration, the percentages of A. salmonicida that comprised the total bacteria cultured from algae-fed mussels were decreasing from day 0 levels, but remained a large constituent of the total flora. The minimum was 6.20% from ND fluid samples after 5 d of depuration. Although the percentages of A. salmonicida from ND fluid and mantle samples after 1 d and 5 d were less than those from the lethal samples from these two sample dates, the prevalences of A. salmonicida-positive F. ebena (i.e., the qualitative results) from the nondestructive samples were comparable to those from the lethal samples. After 1 d, the sampling site with the greatest prevalence of A. salmonicida positives was ND fluid (15/15). Recovery prevalences from lethal tissues and lethal fluids were slightly lower, 13/15 and 14/15, respectively. Aeromonas salmonicida was also cultured from 12/15 mantle samples, and 8/15 hemolymph samples, which increased from 4 on day 0. The hemolymph samples gave the greatest mean A. salmonicida percentage (17.94%) of total bacteria after 1 d, and lethal tissues was the only sample site to yield a mean recovery of A. salmonicida (1.65 x [10.sup.5] cfu/g) greater than that from the positive hemolymphs, which was 7.68 x [10.sup.4] cfu/mL. After five days of depuration, the prevalences of A. salmonicida positives from lethal tissues and lethal fluids were both 12/15. Prevalences from ND fluids and mantles were also high, 9/15 and 8/15, respectively after 5 d. The prevalence of positive hemolymph samples decreased to 2/15; however, these two positive samples provided a qualitative pathogen-positive result from that subset of the population.
There were only two samples after 10 d, 15 d, and 30 d of depuration combined, from which A. salmonicida was cultured and both of these were collected using nondestructive procedures: one ND fluid after 10 d and one mantle after 15 d.
The qualitative recovery of A. salmonicida using bacterial culture from individual lethal and nondestructive samples after 1 d and 5 d of depuration from algae-fed F. ebena are given in Table 4. After 1 d, A. salmonicida was cultured from 13/15 lethal tissues and 14/15 lethal fluid samples; however, on combining the results from these two sample collection sites, the bacterium was recovered from 15/15 F. ebena. For example, A. salmonicida was not cultured from the soft tissues from specimen number 6, but it was from the paired lethal fluid sample. Considering the nondestructive samples after 1 d of depuration, 15/15 ND fluids were positive for A. salmonicida. The bacterium was also cultured after 1 d from 12/15 mantles and 8/15 hemolymphs, and by combining these data, 14/15 were positive. Specimen no. 7 was the only one in which A. salmonicida was not cultured from either the mantle or hemolymph samples. The A. salmonicida recovery prevalences after 5 d of depuration and after the further decline in both the numbers of positive mussels and viable A. salmonicida cfu are similar to those after 1 d. Twelve lethal tissue homogenates and 12 lethal fluids were positive for A. salmonicida and after combining these, 15/15 F. ebena were positive. Comparable to this combined recovery of lethal samples, A. salmonicida was cultured from 13/15 of the mussels using samples collected by nondestructive procedures. The greatest prevalence of A. salmonicida-positive F. ebena using a nondestructive collection procedure was from 9/15 ND fluids. The bacterium was recovered from 8/15 mantle samples and 2/15 hemolymph samples. Qualitative comparisons of A. salmonicida recovery from the five sample collection sites on day 0 were not beneficial because the bacterium was cultured from all of the samples with the exception of six negative hemolymph samples (Table 3). Furthermore, comparisons after 10, 15, and 30 d were not worthwhile because there were only two positive samples during this time, both of which were from nondestructive samples.
DISCUSSION
Collections of the nondestructive samples (ND fluid, hemolymph, and mantle) were simple procedures and none appeared to have caused an adverse health risk to the F. ebena, at least indicated by no deaths during the subsequent observation period. The procedure for ND fluid is noninvasive and intuitively, is the least stressful of the three to mussels. Both the mantle biopsy and drawing hemolymph involve penetrating the external soft tissue surfaces and although these two procedures are harmless to mussels (Berg et al. 1995, Gustafson et al. 2005a, Gustafson et al. 2005b), each creates a potential port of entry for infectious agents until such time when the wounds close. An open wound could facilitate disease if pathogens are present and especially if mussels were stressed with predisposing factors.
In the present study, transfer of the F. ebena from the pathogen-source, cohabitation tank to disinfected tanks supplied with pathogen-free water initiated the mussels' depuration of A. salmonicida. In the ensuing days, continuing depuration by the mussels caused a decline in the prevalences of the bacterium in the various groups of mussels and it reduced the numbers of A. salmonicida cfu per specimen (Tables 2 and 3). The purpose of evaluating the sample collection sites as the prevalence of positives and numbers of cfu were in decline was to establish the need for the more sensitive procedures to recover A. salmonicida. High prevalences of A. salmonicida positives and high A. salmonicida cfu recoveries were recorded through 5 d from both lethal and all three nondestructive samples from the algae-fed F. ebena (Table 3). In contrast, after 5 d the nonfed mussels produced significantly reduced prevalences and lower numbers of A. salmonicida cfu (Table 2). Part of this contrast might be explained by the greater A. salmonicida cfu in the fed F. ebena at the initiation of depuration (0 d). The same procedure to infect the F. ebena with A. salmonicida was used for fed and nonfed mussels, so perhaps feeding during their time in the cohabitation tank led to the greater levels of infection. The means for A. salmonicida from the five sample sites from the fed F. ebena on day 0 ranged from 9.12 x [10.sup.3] cfu/ mL (ND fluid) to 3.40 x [10.sup.7] cfu/mL (lethal fluid), whereas, the range from the nonfed mussels was 2.00 x [10.sup.1] cfu/mL for hemolymph to 2.92 x [10.sup.5] cfu/g from lethal tissues. Perhaps the greater numbers of A. salmonicida cells in the algae-fed F. ebena required the proportionally longer duration to be depurated, thus the differences in cfu after 5 d. Another explanation for the contrast in A. salmonicida cfu after 5 d in fed versus nonfed mussels might be differences in the pumping (filtration) rates related to the availability of food. However, results from other studies on the filtration response to food availability in other bivalves are equivocal (Loosanoff & Engle 1952, Pereira et al. 2004, Riisgard & Randlov 1981, Winter 1973).
Greater numbers of total bacteria were isolated from lethal fluids compared with the ND fluid means on day 0 from the nonfed and fed F. ebena (Table 2, Table 3). Beyond day 0, the greater means for total bacteria were isolated from ND fluids, which might be related to these nondestructive samples providing the only isolations of A. salmonicida from fluid after 9 d and 10 d of depuration among nonfed and fed mussels, respectively.
The numbers of total bacteria and A. salmonicida cultured from the mantle biopsies might have been influenced by the bacterial flora present in fluid within the valves. The pieces of mantle were not surface disinfected prior to their homogenization for bacterial culture and bacteria from fluid might have attached to the surfaces of the samples. The purpose of the present study was to evaluate the samples for bacteria, as they would typically be collected. Unless the bacteria specific to mantle are to be cultured, it is not critical to determine whether bacteria are within or on the surfaces of the mantle tissues. Also, because it is desirable to take a small piece to minimize stress to the host, the incision made to remove the sample might allow a disinfectant (e.g., chlorine) to penetrate and disinfect the internal portion of the sample. From the resulting data, the bacteria from fluids do not seem to have been too influential to mantles as indicated by the cfu isolated from both samples. If the fluid was imparting a significant influence, then perhaps in those instances where the lethal fluid cfu was greater than the lethal tissues cfu, then the mantle cfu/g from a particular specimen might be greater than it's paired lethal tissues cfu/g. This was predominately not the case during either trial. There were only two sampling dates (after 5 d and 10 d of depuration from the algae-fed F. ebena; Table 3) that the mean of total bacteria from mantle was greater than the mean from lethal tissues, and with both instances the means from lethal fluids were less than those from lethal tissues.
A health and pathogen inspection program that requires sacrificing imperiled mussels restricts the numbers of individuals that can be examined. For a given population size, a sample size that is too small will limit the probability to detect a pathogen (Simon & Schill 1984). One advantage to using nondestructive sample collections is they allow for increased numbers of samples to be examined and without having to sacrifice the mussels. This effectively increases the probability to detect a single positive specimen within a population, and particularly among asymptomatic and low-level infected populations. Iida et al. (2000) cultured Vibrio sp. from hemolymph from a Pacific oyster Crassostrea gigas Thunberg that appeared to be healthy. This isolation can be interpreted as an example for the use of a pathogen detection assay utilizing samples collected by nondestructive procedures. Developing a nondestructive sample collection strategy could provide workers with an opportunity to detect pathogens within mussel populations prior to the onset of clinical disease signs and disease outbreaks. Controls and treatments could then be expeditiously implemented to prevent diseases and mortality. Furthermore, inspection results could be used to greatly reduce the risks of introducing pathogens to new populations.
The nondestructive procedures to collect the samples did not result in mortality to F. ebena in the present study, and this corroborates with previous nondestructive sample collections from other species, namely, A. ligamentina, E. complanata, Q. quadrula, and Q. pustulosa (Berg et al. 1995, Gustafson et al. 2005a, Henley et al. 2006). When considering the qualitative A. salmonicida recovery results (i.e., the prevalences of positives, in the present study) from lethal and nondestructive samples, particularly with ND fluid, the results clearly indicate a potential value for nondestructive procedures to assess the presence of pathogens in populations. Bacterial culture along with subsequent biochemical characterizations provided a confirmatory diagnosis for A. salmonicida. The sensitivity of using primary bacterial culture for pathogen detection might be limited depending on the bacterium to be isolated, the bacteriological medium used, contamination, and other factors. Other pathogen detection methods, such as serological assays or PCR amplification of pathogen-unique DNA might be more sensitive, which could further enhance the effectiveness of nondestructive sampling. In addition to increased sensitivity, these assays might require even less sample material than bacterial culture, thus potentially further minimizing stress to the hosts. The sensitivity of certain procedures could be increased by treatment of samples, such as concentrating the volumes. A low cfu/mL bacterium contained within 1 mL of ND fluid is increased 10-fold by reducing the original volume to 100 [micro]L. Another effective means to indirectly increase the sensitivity of an assay would be to target specific tissues or samples. In the present study, lethal tissues comprised all soft tissues and the total of bacteria isolated was reported as cfu/g of tissue. This implies that bacteria are evenly distributed throughout all tissues, which is likely not the case. Certain pathogens may localize to a specific organ, which when processed as a homogenate of all soft tissues, are diluted with unaffected tissues. Infectious diseases specific to mussels may dictate the preferred nondestructive sample to be used, which might differ from those addressed in the present study. In this case, preliminary trials similar to those of the present study will need be done to evaluate the suitability and effectiveness of a particular sample for recovery of the pathogen, and for the procedure to collect the sample to be harmless to the mussels.
The qualitative bacterial culture recoveries for A. salmonicida from algae-fed F. ebena after 1 d and 5 d of depuration are given in Table 4. By combining the recoveries from all samples after 5 d, A. salmonicida was cultured from 15/15 specimens. The greatest numbers of A. salmonicida-positives from nondestructive samples were ND fluids (9/15 positive) and mantles (8/15 positive), and the bacterium was cultured from both of these samples from specimen numbers: 2, 4, 5, 11, and 15. Although the greatest prevalence of A. salmonicida from nondestructive samples was 9/15 (ND fluid), if the results from all three nondestructive samples are combined, then 13/15 were positive. This shows that the number of nondestructive samples assayed could affect the overall outcome of pathogen or disease status. It is important for the sampling design to reflect the purpose for the sampling. If a population is to be tested for the presence of a pathogen or to assess the risk to introduce that pathogen to a refuge where it is not enzootic, then the nondestructive ND fluid sample alone could suffice in providing that qualitative presence-absence determination. However, determining the prevalence of a pathogen in a population might entail examining more samples from each individual mussel to ensure the greatest numbers of positive specimens are found.
Use of nondestructive sample collection procedures could greatly benefit resource managers by facilitating examinations of imperiled specimens or surrogates, which can represent imperiled species. The F. ebena used in the present study were apparently in excellent overall health condition. They presumably were not stressed and therefore, unduly susceptible to mortality as a result of the handling and sample collections. If these mussels had been asymptomatically infected with a mussel pathogen, and correspondingly weakened and compromised, the handling of them, alone, could result in host stressors and subsequent poor survival. Periodic health examinations using nondestructive sampling techniques could identify the presence of a pathogen in the early stages of an infection, and prior to the hosts becoming too compromised. This presents an opportunity to implement a disease control and treatment strategy.
ACKNOWLEDGMENTS
The authors thank Dean Rhine and Patricia Morrison, United States Fish and Wildlife Service, Ohio River Islands National Wildlife Refuge, for providing the mussels. Appreciation is also extended to Dr. Catherine Gatenby, US Fish and Wildlife Service, White Sulphur Springs National Fish Hatchery, and Julie Devers, US Fish and Wildlife Service, Maryland Fishery Resources Office (formerly at White Sulphur Springs NFH) for providing the algae. The author thanks Dr. Christine Densmore, Leetown Science Center, and Dr. G. L. Bullock, Leetown Science Center (Ret.) for their reviews of the manuscript. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the United States Government.
LITERATURE CITED
Berg, D. J., W. R. Haag, S. I. Guttman & J. B. Sickle. 1995. Mantle biopsy: A technique for nondestructive tissue-sampling of freshwater mussels. J. North Am. Benthol. Soc. 14:577-581.
Chaffee, C. 1997. Ohio River valley ecosystem mollusk conservation plan: Strategic action plan-phase II. Appendix H: Draft freshwater mussel quarantine protocol. Bloomington, IN: US Fish and Wildlife Service. 13 pp.
Gatenby, C. M., P. A. Morrison, R. J. Neves & B. C. Parker. 2000. A protocol for the salvage and quarantine of unionid mussels from zebra mussel-infested waters. In: R. A. Tankersly, D. I. Warmolts, G. T. Watters, B. J. Armitage, P. D. Johnson & R. S. Butler, editors. Freshwater Mollusk Symposia Proceedings. Columbus, Ohio: Ohio Biological Survey. pp. 9-18.
Gustafson, L. L., M. K. Stoskopf, A. E. Bogan, W. Showers, T. J. Kwak, S. Hanlon & J. F. Levine. 2005a. Evaluation of a nonlethal technique for hemolymph collection in Elliptio complanata, a freshwater bivalve (Mollusca: Unionidae). Dis. Aquat. Org. 65:159-165.
Gustafson, L. L., M. K. Stoskopf, W. Showers, G. Cope, C. Eads, R. Linnehan, T. J. Kwak, B. Andersen & J. F. Levine. 2005b. Reference ranges for hemolymph chemistries from Elliptio complanata of North Carolina. Dis. Aquat. Org. 65:167-176.
Henley, W. F., P. J. Grobler & R. J. Neves. 2006. Non-invasive method to obtain DNA from freshwater mussels (Bivalvia: Unionidae). J. Shellfish Res. 25:975-977.
Iida, Y., R. Honda, M. Nishihara & K. Muroga. 2000. Bacterial flora in the digestive tract of cultured pacific oyster. Fish Pathol. 35:173-177.
Koneman, E. W., S. D. Allen, W. M. Janda, P. C. Schreckenberger & W. C. Winn, Jr. 1992. Color Atlas and Textbook of Diagnostic Microbiology, 4th edition. Philadelphia, PA: J.B. Lippincott Company. 1154 pp.
Lydeard, C., R. H. Cowie, W. F. Ponder, A. E. Bogan, P. Bouchet, S. A. Clark, K. S. Cummings, T. J. Frest, O. Gargominy, D. G. Herbert, R. Hershler, K. E. Perez, B. Roth, M. Seddon, E. E. Strong & F. G. Thompson. 2004. The global decline of nonmarine mollusks. Bioscience 54:321-330.
Loosanoff, V. L. & J. B. Engle. 1952. Effect of different concentrations of micro-organisms on the feeding of oysters (O. virginica). Fishery Bulletin of the Fish and Wildlife Service, Fishery Bulletin 42 (1947). Washington, DC: United States Government Printing Office. pp. 31-57.
MacFaddin, J. F. 2000. Biochemical tests for identification of medical bacteria, 3rd edition. Baltimore, MD: Williams and Wilkins. 912 pp.
Neves, R. J. 1997. Recent die-offs of freshwater mussels in the United States: An overview. In: R. J. Neves, editor. Proceedings of the Workshop on Die-offs of Freshwater Mussels in the United States. Blacksburg, VA: Virginia Tech Press. pp. 7-18.
Noga, E. J. 1996. Fish disease: diagnosis and treatment. St. Louis, MO: Mosby-Year Book, Incorporated. 367 pp.
Patterson, M. A., B. C. Parker & R. J. Neves. 1997. Effects of quarantine times on glycogen levels of native freshwater mussels (Bivalvia:Unionidae) previously infested with zebra mussels. Am. Malacol. Bull. 14:75-79.
Patterson, M. A., B. C. Parker & R. J. Neves. 1999. Glycogen concentration in the mantle tissue of freshwater mussels (Bivalvia: Unionidae) during starvation and controlled feeding. Am. Malaeol. Bull. 15:47-50.
Pereira, P., E. Dias, S. Franca, E. Pereira, M. Carolino & V. Vasconcelos. 2004. Accumulation and depuration of cyanobacterial paralytic shellfish toxins by the freshwater mussel Anodonta cygnea. Aquat. Toxicol. 68:339-350.
Piper, R. G., I. B. McElwain, L. E. Orme, J. P. McCraren, L. G. Fowler & J. R. Leonard. 1982. Fish hatchery management. Washington, DC: United States fish and wildlife service, Department of the Interior. 517 pp.
Riisgard, H. U. & A. Randlov. 1981. Energy budgets, growth and filtration rates in Mytilus edulis at different algal concentrations. Mar. Biol. 61:227-234.
Simon, R. C. & W. B. Schill. 1984. Tables of sample size requirements for detection of fish infected by pathogens: three confidence levels for different infection prevalence and various population sizes. J. Fish Dis. 7:515-520.
Starliper, C. E. 2001. The effect of depuration on transmission of Aeromonas salmonieida between the freshwater bivalve Amblema plicata and Arctic char. J. Aquat. Anim. Health 13:56-62.
Starliper, C. E. 2005. Quarantine of Aeromonas salmonicida-harboring ebonyshell mussels (Fusconaia ebena) prevents transmission of the pathogen to brook trout (Salvelinus fontinalis). J. Shellfish Res. 24:573-578.
Starliper, C. E. & R. K. Cooper. 1998. Biochemical and conjugation studies of Romet-resistant strains of Aeromonas salmonicida from salmonid rearing facilities in the eastern United States. J. Aquat. Anim. Health 10:221-229.
Starliper, C. E. & P. Morrison. 2000. Bacterial pathogen contagion studies among freshwater bivalves and salmonid fishes. J. Shellfish Res. 19:251-258.
Starliper, C. E., R. Villella, P. Morrison & J. Mathias. 1998. Studies on the bacterial flora of native freshwater bivalves from the Ohio River. Biomed. Lett. 58:85-95.
Williams, J. D., M. L. Warren, Jr., K. S. Cummings, J. L. Harris & R. J. Neves. 1993. Conservation status of freshwater mussels of the United States and Canada. Fisheries 18:6-22.
Winter, J. E. 1973. The filtration rate of Mytilus edulis and its dependence on algal concentration, measured by a continuous automatic recording apparatus. Mar. Biol. 22:317-328.
CLIFFORD E. STARLIPER * United States Geological Survey, Biological Research Division, Leetown Science Center, 11649 Leetown Road, Kearneysville, West Virginia 25430
* Corresponding author. E-mail: cstarliper@usgs.gov
TABLE 1.
Means of morphometric data from two groups of Fusconaia ebena
used to evaluate nondestructive sampling for recovery of
Aeromonas salmonicida. The nonfed mussels were collected
from Kentucky Reservoir, TN River mile 102, Humphreys
County, TN. The algae-fed mussels were from Hawesville Bed,
OH River Mile 725, Hancock County, KY.
Nonfed F. ebena Algae-Fed F. ebena
n = 96 n = 80
Total weight (g) 118.20 137.69
Length (mm) 66.91 71.04
Width (mm) 59.54 60.92
Depth (mm) 38.86 40.41
Amount of fluid (mL) 11.03 10.57
Total weight soft tissues (g) 11.54 9.45
Volume (1 mL = 1 mL) 18.68 26.38
TABLE 2.
Total bacteria and Aeromonas salmonicida cultured from nonfed
Fusconaia ebena; comparison of nondestructive versus lethal sample
collection procedures. Mean cfu/g of tissues and cfu/mL of fluids;
the number of F. ebena that bacteria was isolated from / the number
of F. ebena sampled. Prior to this trial, six F. ebena were shown
to be negative for A. salmonicida; the mean for total bacteria from
tissues was 2.68 X [10.sup.5] cfu/g, and the mean from fluids was
4.92 X [10.sup.4] cfu/mL. ND = nondestructive. NB = no bacteria
isolated.
Mean Total Bacteria;
No. Positive/No. Sampled
Day 0 lethal tissues 9.99 X [10.sup.5] cfu/g; 6/6
Day 0 lethal fluid 1.91 X [10.sup.5] cfu/mL; 6/6
Day 0 hemolymph 7.00 X [10.sup.2] cfu/mL; 12/12
Day 0 mantle 2.70 X [10.sup.5] cfu/g; 12/12
Day 0 ND fluid 8.30 X [10.sup.4] cfu/mL; 12/12
Day 5 lethal tissues 1.52 X [10.sup.6] cfu/g; 6/6
Day 5 lethal fluid 1.52 X [10.sup.5] cfu/mL; 6/6
Day 5 hemolymph 3.74 X [10.sup.3] cfu/mL; 12/12
Day 5 mantle 1.57 X [10.sup.4] cfu/g; 12/12
Day 5 ND fluid 1.99 X [10.sup.5] cfu/mL; 12/12
Day 9 lethal tissues 2.12 X [10.sup.5] cfu/g; 6/6
Day 9 lethal fluid 2.60 x [10.sup.4] cfu/mL; 6/6
Day 9 hemolymph 3.78 X [10.sup.3] cfu/mL; 12/12
Day 9 mantle 3.23 X [10.sup.4] cfu/g; 12/12
Day 9 ND fluid 4.32 X [10.sup.4] cfu/mL; 12/12
Mean A. salmonicida;
No. Positive/No. Sampled
(mean percent A. sal cfu of the total cfu)
Day 0 lethal tissues 2.92 X [10.sup.5] cfu/g; 6/6 (24.06% *)
Day 0 lethal fluid 6.36 X [10.sup.4] cfu/mL; 6/6 (15.90%)
Day 0 hemolymph 2.00 X 101 cfu/mL; 1/12 (4.35%)
Day 0 mantle 3.35 X [10.sup.3] cfu/g; 10/12 (4.31%)
Day 0 ND fluid 2.38 X [10.sup.4] cfu/mL; 12/12 (9.45%)
Day 5 lethal tissues NB; 0/6
Day 5 lethal fluid 6.66 X [10.sup.2] cfu/mL; 2/6 (1.81 %)
Day 5 hemolymph NB; 0/12
Day 5 mantle 2.64 X 101 cfu/g; 2/12 (0.14%)
Day 5 ND fluid 2.71 X [10.sup.3] cfu/mL; 3/12 (10.83%)
Day 9 lethal tissues NB; 0/6
Day 9 lethal fluid NB; 0/6
Day 9 hemolymph NB; 0/12
Day 9 mantle NB; 0/12
Day 9 ND fluid 1.32 X [10.sup.3] cfu/mL; 1/12 (0.47%)
* Means for percent were calculated from the
A. sahmonicida-positive samples only.
TABLE 3.
Total bacteria and Aeromonas salmonicida cultured from algae-fed
Fusconaia ebena; comparison of nondestructive versus lethal sample
collection procedures. Mean cfu/g of tissues and cfu/mL of fluids;
the number of F. ebena that bacteria was isolated from the number
of F. ebena sampled. ND = nondestructive. NB = no bacteria isolated.
Mean Total Bacteria;
No. Positive/No. Sampled
Day 0 lethal tissues 3.63 X [10.sup.6] cfu/g; 10/10
Day 0 lethal fluid 3.41 X [10.sup.7] cfu/mL; 10/10
Day 0 hemolymph 1.83 X [10.sup.5] cfu/mL; 8/10
Day 0 mantle 1.66 X [10.sup.5] cfu/g; 10/10
Day 0 ND fluid 7.50 X [10.sup.4] cfu/mL; 10/10
Day 1 lethal tissues 1.18 X [10.sup.6] cfu/g; 15/15
Day 1 lethal fluid 6.73 X [10.sup.4] cfu/mL; 15/15
Day 1 hemolymph 1.78 X [10.sup.5] cfu/mL; 12/15
Day 1 mantle 8.05 X [10.sup.4] cfu/g; 15/15
Day 1 ND fluid 1.82 X [10.sup.5] cfu/mL; 15/15
Day 5 lethal tissues 1.47 X [10.sup.5] cfu/g; 15/15
Day 5 lethal fluid 1.92 X [10.sup.4] cfu/mL; 15/15
Day 5 hemolymph 2.01 X [10.sup.3] cfu/mL; 10/15
Day 5 mantle 3.35 X [10.sup.5] cfu/g; 15/15
Day 5 ND fluid 3.67 X [10.sup.4] cfu/mL; 15/15
Day 10 lethal tissues 3.33 X [10.sup.4] cfu/g; 15/15
Day 10 lethal fluid 6.19 X [10.sup.3] cfu/mL; 15/15
Day 10 hemolymph 2.95 X [10.sup.2] cfu/mL; 9/15
Day 10 mantle 1.26 X [10.sup.5] cfu/g; 15/15
Day 10 ND fluid 2.30 X [10.sup.4] cfu/mL; 15/15
Day 15 lethal tissues 1.60 X [10.sup.5] cfu/g; 15/15
Day 15 lethal fluid 7.53 X [10.sup.4] cfu/mL; 15/15
Day 15 hemolymph 2.51 X [10.sup.2] cfu/mL; 7/15
Day 15 mantle 6.24 X [10.sup.4] cfu/g; 15/15
Day 15 ND fluid 4.78 X [10.sup.4] cfu/mL; 15/15
Day 30 lethal tissues 5.85 X [10.sup.5] cfu/g; 15/15
Day 30 lethal fluid 1.24 X [10.sup.5] cfu/mL; 15/15
Day 30 hemolymph 7.05 X [10.sup.3] cfu/mL; 9/13
Day 30 mantle 1.41 X [10.sup.5] cfu/g; 15/15
Day 30 ND fluid 3.38 X [10.sup.5] cfu/mL; 15/15
Mean A. salmonicida;
No. Positive/No. Sampled
(mean percent A. sal cfu of the total cfu)
Day 0 lethal tissues 2.24 X [10.sup.6] cfu/g; 10/10 (44.51% *)
Day 0 lethal fluid 3.40 X [10.sup.7] cfu/mL; 10/l0 (42.05%)
Day 0 hemolymph 1.00 X [10.sup.5] cfu/mL; 4/10 (9.69%)
Day 0 mantle 1.92 X [10.sup.4] cfu/g; 10/10 (15.88%)
Day 0 ND fluid 9.12 X [10.sup.3] cfu/mL; 10/10 (16.63%)
Day 1 lethal tissues 1.65 X [10.sup.5] cfu/g; 13/15 (16.36%)
Day 1 lethal fluid 1.29 X [10.sup.4] cfu/mL; 14/15 (14.16%)
Day 1 hemolymph 7.68 X [10.sup.4] cfu/mL; 8/15 (17.94%)
Day 1 mantle 1.76 X [10.sup.4] cfu/g; 12/15 (8.28%)
Day 1 ND fluid 4.86 X [10.sup.4] cfu/mL; 15/15 (14.14%)
Day 5 lethal tissues 1.74 X [10.sup.4] cfu/g; 12/15 (8.08%)
Day 5 lethal fluid 1.25 X [10.sup.3] cfu/mL; 12/15 (11.00%)
Day 5 hemolymph 4.30 X [10.sup.2] cfu/mL; 2/15 (12.05%)
Day 5 mantle 2.04 X [10.sup.3] cfu/g; 8/15 (6.56%)
Day 5 ND fluid 3.28 X [10.sup.3] cfu/mL; 9/15 (6.20%)
Day 10 lethal tissues NB; 0/15
Day 10 lethal fluid NB; 0/15
Day 10 hemolymph NB; 0/15
Day 10 mantle NB; 0/15
Day 10 ND fluid 8.00 X [10.sup.1] cfu/mL; 1/15 (0.99%)
Day 15 lethal tissues NB; 0/15
Day 15 lethal fluid NB; 0/15
Day 15 hemolymph NB; 0/15
Day 15 mantle 1.00 X [10.sup.1] cfu/g; 1/15 (7.14%)
Day 15 ND fluid NB; 0/15
Day 30 lethal tissues NB; 0/15
Day 30 lethal fluid NB; 0/15
Day 30 hemolymph NB; 0/13
Day 30 mantle NB; 0/15
Day 30 ND fluid NB; 0/15
* Means for percent were calculated from the
A. salmonicida-positive samples only.
TABLE 4.
Comparisons of Aeromonas salmonicida recoveries by bacterial
culture from individual tissue samples collected from the same
mussel. Qualitative results from days 1 and 5 from algae-fed
Fusconaia ebena: +, recovered; 0, not recovered.
Fusconaia Day 1 Day 1 Day 1
ebena Lethal Lethal ND Day 1 Day 1
No. Tissues Fluid Fluid Mantle Hemolymph
1 + + + + +
2 + + + + +
3 + + + + 0
4 + + + 0 +
5 + + + 0 +
6 0 + + + +
7 + + + 0 0
8 0 + + + 0
9 + + + + +
10 + + + + 0
11 + + + + +
12 + + + + 0
13 + + + + 0
14 + 0 + + 0
15 + + + + +
Fusconaia Day 5 Day 5 Day 5
ebena Lethal Lethal Lethal Day 5 Day 5
No. Tissues Fluid Fluid Mantle Hemolymph
1 + 0 + 0 0
2 + + + + +
3 + + 0 + 0
4 + 0 + + 0
5 + + + + 0
6 + + + 0 0
7 + 0 0 + 0
8 0 + 0 + 0
9 0 + + 0 0
10 + + + 0 0
11 + + + + 0
12 + + 0 0 0
13 + + 0 0 0
14 0 + 0 0 +
15 + + + + 0
Email this Article
Digg It
del.icio.us- Comparison of techniques for diagnosis of brown ring disease and detection of Vibrio tapetis in the Manila clam, Venerupis (Ruditapes) philippinarum.
- In vitro and in vivo evaluation of quinine as a potential anti-protozoal for the eastern oyster parasite Perkinsus marinus.
- Quarantine of Aeromonas salmonicida-harboring ebonyshell mussels (Fusconaia ebena) prevents transmission of the pathogen to brook trout (Salvelinus fontinalis).
- Inoculation experiments to understand mass mortalities in sea scallop, Placopecten magellanicus.