MFR PAPER 1300

Shellfish Diseases

LOUIS LEIBOVITZ

ABSTRACT-An overview of commercial bivalve shellfish aquaculture is pre­sented. The advantages and disadvantages of shellfish production as comparedwith other forms of food animal production is discussed. The common shellfishdiseases are listed and the known specific etiologic agents are indicated. The latterinclude viral, bacterial, fungal, protozoan, and metazoan parasitic and infectiousagents. In addition, predators, toxic agents, and fouling organisms produce seri­ous economic losses.

The specialized problems of shellfish hatcheries are discussed. The importanceof monitorifJg the qualitative physical, chemical, and bacteriological changes inshellfish larval cultural media and its ingredients for optimum production is indi­cated.

A description of a laboratory model for evaluating the pathogenicity of purebacterial cultures for larval shellfish is presented. The experimental optimal andlethal concentrations of bacteria for shellfish larvae are defined. An interrelation­ship between bacteria and protozoa in the pathogenesis ofshellfish larval diseasesis reported. The shellfish industry has encouraged and supported the reportedresearch to increase the efficiency of shellfish production by reducing economiclosses due to shellfish diseases.

Less is known about the subject ofshellfish diseases, and, accordingly,there is a wider latitude in discussing it.There are many unique problems, someof which overlap with fish diseases.

One problem is that the molluscanbivalves are filter feeders. Their abilityto concentrate harmful chemicals andinfectious agents pose serious problemsin controlling both shellfish and humandiseases. Since shellfish are estuarinedwellers, they are subjected to en­vironmental variations such as changesin salinity and temperature, seasonaltidal variations, and varying degrees of

Louis Leibovitz is with the Depart­ment of Avian and Aquatic AnimalMedicine, New York State College ofVeterinary Medicine, Cornell Univer­sity, Ithaca, NY 14853.

March 1978

exposure to urban and industrial pollu­tants discharged into estuarine waters.

In spite of these hazards, shellfishhold one of the greatest potentials forthe economic production of food pro­tein. Shellfish hold great promise forthe efficient recycling of organic wastematerials, such as agricultural wastes,and the capture of energy for food pro­duction from thermal effluents, such asthat discharged from atomic powerplants. In addition, there are morespecies of shellfish than any other groupof animals, with the exception of ar­thropods. Genetic selection for greaterfood yields from these abundant var­ieties should be rewarding. Also, interms of reproductive potential, thereare no other food animals that even ap­proach their fecundity. For example, asingle pair of oysters can produce as

many as 120 million offspring from asingle mating.

There is another unique aspect ofshellfish production that exceeds theeconomic efficiency of other forms ofanimal protein production and that isfree food. Unlike the rising food costsof other animal feeds, shell fish foodsare naturally generated planktonicfoods. Since shellfish are an importantsource of food, we should learn more oftheir diseases as a part of the technicaldevelopment necessary to increase pro­duction.

The following discussion of shellfishdiseases is an overview and a short con­sideration of one specific bacterial dis­ease problem in larval shellfish produc­tion being currently studied.

DISEASES OF SHELLFISH

A list of organisms that cause com­mon diseases in oysters is shown inTable I.

Viral Diseases

Of the known reported virus infec­tions of oysters, "Ovacystis" infectionis the most common, but it is probablyof little economic importance. It can bedetected histologically as hypertrophyof the ovarian follicles. The affect ofthis virus upon reproductive. perfor­mance has not been evaluated. .

A herpes virus infection has been de­scribed by Austin Farley (1972) in oys­ters. Apparently, expression of the dis­ease was temperature dependent andwas found in oysters cultiYjlted in theheated effluent of a power plant. When

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Table 1.-Causes of common diseases In oysters.

the environmental temperature drop­ped, the disease was not apparent.While there are undoubtedly othershellfish viral diseases present, theyhave not been defined.

The two previous viral diseases men­tioned were demonstrated upon thebasis of diagnostic inclusion bodies andelectron microscopic demonstration ofviral particles in affected cells. Virus­free molluscan tissue culture systemsare needed to isolate and identify mol­luscan viruses and human viral patho­gens that may be carried by shellfish.

Bacterial Diseases

Little is known of the bacterial dis­eases of shellfish, and the list in Table Jis limited to those that have been de­scribed. From the standpoint of humanhealth, outbreaks of cholera have beenrelated to the consumption of shellfishin Africa and Italy.

Fungal Diseases

Dermocystidium (Labyrinthomyxa)marinum is a very important shellfish

Group

Viral

Bactenal

Fungal

Parasiteprotozoan

Helminthic

Arthropods

Ovacystis virusHerpes virusOthersAchromobacler sp.Aeromonas sp.Vibrio sp.Myrotomus ostrearum

··Maladie Du Pled"Nocardia sp.C/adothrix dichotoma (Actinomycetes)OthersOermocyslidium (Labyrinrhomyxa)

marinumSiroJpldium sp.Others, Sarcodina (Amoeba)

F/abel/u/a sp.2. Masligophora (Flagellate)

Hexamita nelsoni3. Sporozoan

a. Gregarineb. Haplosporidia

4. Ciliates-many1. Trematoda (larval)

Bucephalus sp.Others

2. Cestoda (larval)Tylocepha/um sp.Others

1. CopepodsMytilicola intestinalisOthers

2. DecapodsPinnotherid crabs

3. Annelids ("Mud blisters")Polydora websteriOthers

4. Sponges ("Bo"ng sponges")Onona celalaOthers

pathogen that produces serious eco­nomic losses in adult shellfish in warmclimates. Sirolpidium sp. is a commoninfection of hatchery-reared larvalshellfish.

Helminthic Diseases

Among the helminth parasites ofshellfish, trematode, cestode, andnematode parasites may be found. Lar­val forms of trematodes (especiallyBucephalus sp.) and cestodes (espe­cially Tylocephalum sp.) are ofeconomic importance as shellfishpathogens that often produce sterility inaffected shellfish. Most of the larvalforms mature in fish which serve asdefinitive hosts. Some are of publichealth significance.

Arthropods andOther Organisms

In addition to helminth parasites,copepod crustacean and polychaete an­nelids, during some stage of their lifecycles, may parasitize shellfish with re­sultant serious economic losses.

A great variety of marine organismsare found in shellfish beds in apparentsymbiotic or commensal relationshipsto shellfish. Some, as pinnotheridcrabs, enter and leave the pallial cavityof shellfish freely. Crabs may serve asthe intermediate host for the primitivegregarine sporozoans (Nemotopsis sp.)whose spores infect shellfish with littleresultant tissue damage. Macroalgaeand sponges grow on the surface ofshellfish. The boring sponges (Clionasp.) may damage the external shell andthe shell may then become porous andcrumble.

Diseases ofUnknown Etiology

In addition to the known diseases,many unexplained die-offs have beenreported that have decimated shellfishpopulations. Often these populationsdo not recover, and new stock, intro­duced to repopulate, are quickly af­fected and die. Such diseases are oftennamed for the locality in which theyoccurred, such as "Malpeque Bay"and "Denman Island" disease. Oftenserious losses are attributed to climaticconditions, water quality changes, and

pollution without adequate evidencethat disease was not responsible.

Parasitic Diseases

Protozoans

Shellfish protozoan infections arevery common. Whether these or­ganisms are primary infectious agentsis often questionable. This is especiallytrue of the ciliates that are commoninhabitants of shellfish tissues. Theybecome especially active when otherpathogens such as bacterial agents arepresent. Of the flagellated protozoa,Hexamita sp. and the amoeboid pro­tozoa are pathogenic. When shellfishare maintained under adverse condi­tions, such as extreme temperatures,protozoa may actively invade shellfishtissues and produce deterioration orspoilage. These conditions may also befound in "winter-kills" of shellfishwhere high mortality associated withprotozoan infections may be found insustained low temperature exposures.

Protozoans can be primary shellfishpathogens. The most important singleshellfish pathogen that has produced thegreatest economic losses to the shellfishindustry is a haplosporidian, Minchinianelsoni. This organism has destroyedthe great oyster industry of the Dela­ware and Chesapeake Bays. Haplo­sporidians are very poorly understood,poorly classified sporozoans, distinctfrom myxosporidia, or coccidial or­ganisms. Their exact taxonomic posi­tion and life cycles are unknown. Inaddition to the areas mentioned, M.nelsoni, commonly called MSX, ispresent in other geographic locations ofthe northeastern U.S. coastline. Thisorganism is apparently salinity­dependent. It is seasonal in its inci­dence. There are many other haplo­sporidians, of varying pathogenicityfound as parasites in a variety of aquaticanimals. They are found as hyperpara­sites in trematodes. These organismstend to sterilize the trematode host.

SHELLFlSH HATCHERYOPERATION STUDIES

When I began working with the LongIsland shellfish industry, the problemswere overwhelming and it was difficult

62 Marine Fisheries Review

.........................................

STERILEPeA BROTH

LARVAL CULTUREAND STERILE

INSTANT OCEAN

to select a single starting point. Perhapsthe most important economic problemswere based in shellfish hatchery pro­duction. If hatchery production couldbe increased, and livability of larvaeand juveniles were improved, restock­ing and harvesting from shellfish bedswould yield greater production andefficiency. The techniques of hatcheryoperation are well known, but consis­tent production of healthy larvae isdifficult. Shellfish larval disease lossesare serious hatchery problems, often ofepizootic proportions.

Although specific pathogens wereoccasionally responsible for such los­ses, it became apparent that there weremany unexplained phenomena as­sociated with the more common losses.In an attempt to resolve these problems,studies of hatchery media were under­taken. These included physical, chemi­cal and microbiological examination ofhatchery water supply, stock algal cul­tures, pooled algal food cultures, andspawn obtained from hatchery breedingstock. Each hatchery operation was dis­tinctive. Some operated all year, otherslimited their operation to warm weatheronly. Hatchery water supply was eitherraw bay water, or from deep saltwaterwells. Some operations pumped waterinto the plant on demand; others heldwater in large storage tanks that waslater gravity fed into the operation ondemand. Various methods of screen­ing, filtration, and centrifugation areemployed for water clarification. In ad­dition some plants utilize ultraviolettreatment of incoming water, or recy­cled water for disinfection.

Physical and chemical examinationof shellfish larval culture media in­cluded measurement of pH, salinity,chemical oxygen demand, suspendedand total solids. Other tests includingnitrogen determinations are currentlybeing explored. Quantitative countsand identification of dominant bacterialpopulations of the larval culture mediaingredients are also being made.

TESTING BACTERIALPATHOGENICITY IN LARVAL

SHELLFISH PRODUCTION

It became apparent that a pathogenic­ity model was needed to test the pure

March /978

BACTERIAL CONCENTRATIONSAND EQUIVALENT

DILUTIONS OF INOCULA

I X 107 I X 105 I X 103 I X 10'

REPLICATE I 0 0 0 0REPLICATE II 0 0 0 0

REPLICATE m: 0 0 0 0MILLIPORE 0 0 0 0FILTRATE

00000000

Figure I.-Pathogenicity model system totest pure bacterial isolates obtained from lar­val cultures.

--- BACTERIA90 - - - - FILTRATE

~ --BROTH>- BO \ ......... INSTANT....::;

\\ OCEAN~ 70II:0~

-oJ 60 \\;;II:.. 50 \\-oJ....z 40 \wu

~,ll:W 30Q. '\,z..

20w ,~

~,10~

107

10' 103 10'

BACTERIA INOCULA CONCENTRATIONS-PER mlEQUIVALENT CONTROL DILUTIONS

Figure 2.-Graphic representation of meanpercent mortality values of equivalentconcentrations of inocula of all tested isolates(35) (each replicated in triplicate), their cul­IUral filtrate, uninoculated broth medium, and"Instant Ocean" conlrols.

bacterial isolates obtained from the lar­val cultures. The same model could beutilized to test environmental factors,drug efficacy, and other factors for theirinfluence in such disease models. Thismodel system was assembled in plastic"disposo" trays and consisted of 6rows, of 4 wells per row, containingprecalculated approximate numbers ofshell fish larvae from 3 to 14 days of age(Fig. I). To each row was added a

known dilution of the test substance. Intesting for bacterial pathogenicity, pure24-hour broth cultural bacterial isolateswere added to each of the first 3 rows ofthe plate; from left to right, each well ofeach of the first three rows containingapproximately 10 7 , lOS, 103 , and 10 1

bacteria per milliliter of well larval sus­pension. The fourth row was given theequivalent dilution of bacteria-freefiltrate (Millipore filtrate) of the brothculture to correspond to the dilutions ofthe bacterial suspension wells abovethis row. The fifth row received again,equivalent dilutions of sterile culturebroth (plate count agar broth - peA).The last (sixth row) received equivalentdilutions of synthetic sea salts (InstantOcean)1 to the shellfish larval suspen­sions. The results of the above test wereread at the end of a 24-hour incubationperiod. The number of alive and deadlarvae in each well was counted and thepercentage mortality for each well wasdetermined. In this manner the effectsof dilution and comparison of the af­fects of added ingredients could bemeasured to determine their relativeinfluence on pathogenicity.

RESULTS

The results of the above pathogenic­ity tests (Fig. 2) suggest that almost allbacterial isolates at high concentrations(> IOs/ml) are pathogenic for shellfishlarvae; however, only "true" patho­gens kill at very high dilutions «103/

ml). The latter suggests that these truepathogens require larvae for growth.Note that the presence of higher con­centrations of even sterile nutritivebroth produces a lethal effect. Accord­ingly, this may suggest that food con­centrations, dead or decaying algalfoods, or larvae may aggravate thepathogenic effect of both extrinsic andintrinsic microbial concentration(within the larvae). Future studies areneeded. At high concentrations of bac­teria and/or equivalent culture media( 107 or greater), lethal effects are rapid

'Reference to trade names or commercial firmsdoes not imply endorsement by the NationalMarine Fisheries Service, NOAA.

63

I

and are not associated with protozoanactivity. However, at levels corres­ponding approximately to I05/ml, le­thal effects are indicated by moregradual losses and are associated withintense protozoan proliferation and ac­tivity. The latter probably originatefrom the normal intrinsic microbialflora of the shellfish larvae. In fact, ifone were to observe such cultures with­out knowing of the presence of the ex­perimental bacterial inoculum, the ag­gressive behavior of the protozoanattack on the shellfish larvae wouldsuggest that they are the primary patho­gen. The mechanism responsible forthis phenomenon requires furtherstudy. Direct observation of the af-

fected larvae in this bacterial study sup­port common diagnostic signs and le­sions evident in diseased larvae and is aseparate discussion in other studies.

The results of field studies of bacte­rial populations of hatchery media andits ingredients tend to support the ex­perimental studies. Diseased larval cul­tures are associated with bacterial popu­lations )07 or greater per milliliter.Further studies will be required todefine the specific chemical or physicaltests of hatchery media and ingredientsand their parameters that would be use­ful in disease detection and diagnosisthat could be related to specificpathogenic agents.

As a result of these studies, the need

to monitor and define hatcheries for op­timum performance becomes more ap­parent. Since individual hatcheries aredi fferent, each hatchery must beevaluated for its operational methodsand equipment.

ACKNOWLEDGMENTSThis research was sponsored by the

New York Sea Grant Institute under agrant from the Office of Sea Grant, Na­tional Oceanic and Atmospheric Ad­ministration, U.S. Department ofCommerce.

LITERATURE CITEDFarley, C. A., W. G. Banfield, G. Kasnic, and

W. S. Foster. 1972. Oyster herpes-type virus.Science (Wash., D.C.) 178:759-760.

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MFR Paper 1300. From Marine Fisheries Review, Vol. 40, No.3, March 1978.Copies of this paper, in limited numbers, are available from 0822, User Ser­vices Branch, Environmental Science Information Genter, NOAA, Rockville,MO 20852. Copies of Marine Fisheries Review are available from the Superin­tendent of Documents, U.S. Government Printing Office, Washington, DC20402 for $1.10 each.

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