Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER I.2.

GENERAL INFORMATION

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1.

Preliminary Remark

World mollusc production is adversely affected by several diseases and, given their severe impact on economic and socio-economic development in many countries, some diseases have become a primary constraint to the growth and sustainability of this sector. Disease agent transfer via transfers of live molluscs has been a major cause of disease outbreaks and epizootics. Eleven significant diseases of molluscs are currently listed in the Aquatic Animal Health Code. Information relevant to these diseases is provided in the following chapters of this Manual of Diagnostic Tests for Aquatic Animals. Recent scientific information available in taxonomic affiliation of Mikrocytos roughleyi and its relationship with Bonamia species has resulted in the re-definition of bonamiosis. Table 1. below shows how the chapters on mollusc diseases in this edition of the Aquatic Manual correspond with those in the Aquatic Code.

Table 1. How the chapters on mollusc diseases in this edition of the Manual of Diagnostic Tests for Aquatic Animals correspond with those in the Aquatic Animal Health Code

Aquatic Manual:	Aquatic Code:
Bonamiosis:	infection with Bonamia ostreae
	infection with Bonamia exitiosus
	infection with Mikrocytos roughleyi
MSX disease:	infection with Haplosporidium nelsoni
Marteiliosis:	infection with Marteilia refringens
	infection with Marteilia sydneyi
Mikrocytosis:	infection with Mikrocytos mackini
Perkinsosis:	infection with Perkinsus marinus
	infection with Perkinsus olseni/atlanticus
SSO disease:	infection with Haplosporidium costale
Withering syndrome of abalone:	infection with Candidatus Xenohaliotis californiensis

 

2.	Sampling
	2.1.	Sampling
		A general approach to surveillance and sampling is given in Chapter 1.1.4. of this Aquatic Manual. The sampling should be designed in order to enable detection, at a 95% confidence level, of infected animals. The following section gives information relevant to sampling molluscs. Until disease-specific details are included in the individual disease chapters in this Aquatic Manual, the following table can be used to calculate sample size.

Table 2. Sample size based on assumed pathogen prevalence in lot

Lot size	At 2% prevalence, size of sample	At 5% prevalence, size of sample	At 10% prevalence, size of sample
50	50	35	20
100	75	45	23
250	110	50	25
500	130	55	26
1000	140	55	27
1500	140	55	27
2000	145	60	27
4000	145	60	27
10,000	145	60	27
100,000 or more	150	60	30

After Ossiander & Wedemeyer, 1973.
 

	2.2.	Specific recommendations for sampling molluscs
		The timing and frequency of sampling should be determined by the cycle of infection by the disease agent and the prepatent period. An adequate time period should be allocated when sampling for seasonal diseases, for example infection with Marteilia refringens or Haplosporidium nelsoni, to ensure optimal detection. As disease agents may increase in intensity of infection associated with loss of host condition following spawning, post-spawning sampling is also recommended. Sampling periods must also take account of the transfer of juveniles and spats into outgrowing areas, and the transfer of adults for further fattening or relaying.
		Samples should also cover a range of size groups, or target the most susceptible age group when this is known. During sampling, any molluscs showing abnormalities (abnormal growth, gaping valves, elevated or high mortality rates) should be selected. Diseases have subclinical stages of development that can escape detection using routine screening methodology. However, the probability of detection of infection may be increased by holding the bivalves in quarantine for a long period and subjecting them to stress (crowding, handling, temperature and salinity changes, etc.). To detect infection, species of molluscs that are more susceptible to infection should be examined histologically. For example, members of the Arcidae (Arca, Barbatia), Malleidae (Malleus), Isognomonidae (Isognomon), Chamidae (Chama) and Tridacnidae (Tridacna) tolerate high prevalence of Perkinsus infection and are good indicators of the presence of Perkinsus.
		Prevalence of infection is an important factor affecting the chance of detection. When molluscs are to be moved from natural beds into a farm site or between natural beds in different zones, large numbers of bivalves may be required to detect low prevalence of infection. For example, in Western Australia, Marteilia sydneyi and Perkinsus sp. occur at 0.1% prevalence in isolated beds untouched by humans. This level of sampling should be commensurate with the level of risk to the receiving waters and the aquatic resources they support.
		For each zone, a number of sampling sites must be selected in the most practical way so as to maximise the chances of detecting disease agents. The number of sites must be increased for large zones that contain several discrete areas of cultivation of the susceptible species. Account must be taken of parameters that have an effect on the development of the disease agents, such as stocking density, water flow, and the developmental cycle of the molluscs.
		It is an important requirement that the screening techniques used be the optimum methods available for detection of the disease agent in question, and that when the infection is present, it can be detected. For screening methods, sensitivity and specificity should have been assessed. This information has a strong influence on sample size.
3.	Shipment of Samples
	All sampled molluscs must be delivered to the approved diagnostic laboratory within 24 hours of sampling. The laboratory should be informed of the estimated time of arrival of the sample so the required materials to process the molluscs can be prepared before reception of samples.
	Mollusc samples must be packed in accordance with current standards in order to keep them alive. If the sampling site is a long distance from the laboratory, moribund animals or those with foul-smelling tissues may be of little use for subsequent examination. Required samples should be shipped as soon as possible after collection from the water, in order to reduce air storage and possible mortality during transportation, especially for moribund diseased molluscs.
	For samples that cannot be delivered live to the diagnostic laboratory, due to advanced stages of disease, long distance or slow transportation connections, etc., specimens should be fixed on site as recommended in the following sections of this chapter or the individual disease chapters of this Aquatic Manual. While this is suitable for, for example, subsequent histology or transmission electron microscopy examination, other techniques, such as fresh smears, tissue imprints, routine bacteriology, mycology or Ray's fluid thioglycollate culture of Perkinsus spp., cannot be performed. Diagnostic needs and sample requirements should be discussed with the diagnostic laboratory prior to collection of the sample.
	Samples should be accompanied with background information, including the reason for submitting the sample (surveillance, abnormal mortality, abnormal growth, etc.), gross observations and associated environmental parameters, approximate prevalence and patterns of mortality, origin and nature of the molluscs (species, age, whether or not the samples are from local mollusc populations or stocks transferred from another site, date of transfer and source location, etc.). This information should identify possible changes in handling or environmental conditions that could be a factor in mortality in association, or not, with the presence of infectious agents.
4.	Macroscopic Examination
	The gross observation of molluscs should target, as far as possible, animal behaviour, shell surface, inner shell and soft tissues.
	It is often difficult to observe the behaviour of molluscs in open waters. However, observation of molluscs in certain rearing facilities such as brood-stock in tanks and larvae in hatcheries can provide useful indications of disease-related behavioural changes. If signs are noted (e.g. pre-settlement of larvae on the bottom, food accumulation in tanks, signs of weakening, etc.), samples may be examined for gross signs, including observation under a dissecting microscope for abnormalities and deformities, fouling organisms, and fixed for further processing as recommended below. For adults and juveniles, signs of weakening may include gaping, accumulation of sand, mud and debris in the mantle and on the gills, mantle retraction away from the edge of the shell, decreased activity (scallop' swimming, clam' burrowing, abalone' grazing), etc. Open-water mortality should be monitored for patterns of losses and samples collected for further analysis. Environmental factors pre- and post-mortality should be recorded.
	Even under culture conditions, the shells of molluscs may not be clean and fouling organisms are normal colonists of mollusc shell surfaces. Organisms such as barnacles, limpets, sponges, polychaete worms, bivalve larvae, tunicates, bryozoans, etc., do not normally threaten health of molluscs. Culture systems, such as suspension and shallow water culture, can even increase exposure to fouling organisms and shells may become covered by other animals and plants. This can affect the health directly by impeding shell opening and closing or indirectly through competition for food resources. Signs of weakening associated with heavy fouling should be a cause for concern rather than fouling itself. Shell damages by boring organisms such as sponges and polychaete worms are usually benign, but under certain conditions may reach proportions that make the shell brittle or pierce through to the soft-tissues. This degree of shell damage can weaken the mollusc and render it susceptible to inter-current pathogen infections. Shell deformities (shape, holes in the surface), fragility, breakage or repair should be noted, but are not usually indicative of a disease concern. Abnormal coloration and smell, however, may indicate a possible soft-tissue infection that may need to be examined at a laboratory.
	The molluscs must be opened carefully so as not to damage the soft tissues, in particular the mantle, gills, heart and digestive gland. The presence of fouling organisms on the inner shell surface is a clear indication of weakness. The inner surface of the shell is usually smooth and clean due to mantle and gill action. Perforation of the inner surface may occur, but can be sealed off by the deposition of additional conchiolin and nacre. This may result in formation of mud- or water-filled blisters. Blisters may also form over superficial irritants such as foreign bodies. The degree of shell perforation can be determined by holding the shell up to a strong light. Where abnormalities occurring within the matrix of the shell warrant further investigation, freshly collected specimens can be brought intact to the laboratory or fixed for subsequent decalcification, as required. The appearance of the soft-tissues is frequently indicative of the physiological condition of the animal. Soft tissues should be examined for the presence of abscess lesions, pustules, tissue discoloration, pearls, oedema, overall transparency or wateriness, gill deformities, etc., and, when found in association with weak or dying animals, these abnormalities should be a cause for concern.
	Abnormalities and lesions of the tissues should be noted and recorded, as well as any shell deformities, shell-boring organisms and conspicuous mantle inhabitants. Levels of tissue damage should be recorded and samples of affected and unaffected animals collected for laboratory examination as soon as possible.
5.	Examination of Stocks where Abnormal Mortality Occurs
	Abnormal mortality of molluscs is usually recognised as a sudden sizeable mortality that occurs in a short time between two observations or inspections of the stocks (for example, about 15 days in the case of facilities located in inter-tidal zone). In a hatchery, abnormal mortality is the failure of successive production of larvae coming from different brood-stock. Given the broad spectrum of species, environments and culture conditions, these definitions should be adapted when and where necessary.
	Whenever abnormal mortality occurs in stocks of molluscs, an urgent investigation must be carried out to determine the aetiology.
	The samples taken must be consistent with the requirements for surveillance provided in the Aquatic Manual. The samples should also be preserved or fixed and stored in accordance with the procedures defined for histological and other appropriate presumptive and confirmatory methods.
	Where and when available, unaffected or control molluscs should also be fixed for histological comparison with abnormal tissues. Whatever the fixative, it is essential that the shell be removed to allow easy ingress of the fixative. Bivalves and operculated gastropods can keep the shell shut against fixative until autolysis begins.
6.	Diagnostic Methods
	Techniques applicable to molluscan disease agents are limited, and most of the investigations are based on histological and ultrastructural examinations. Classic serological methods cannot be used for diagnostic purposes because molluscs do not produce antibodies. Immunoassays using monoclonal antibodies or nucleic acid probes can be used for direct detection of certain disease agents. A number of research teams and diagnostic laboratories have been engaged in developing DNA-based diagnostic techniques for mollusc disease agents. Given the development and potential for widespread application of these diagnostic techniques and the inherent problems currently associated with their use, the issue of validation is of the utmost importance.
	Three levels of examination procedures are proposed in the following sections. Screening (surveillance) is routinely performed by histology. Histology is recommended as a standard screening method because it provides a large amount of information. It is particularly important because macroscopic examination usually gives no pathognomonic signs or solid indicative information. Also, mortality may be due to several disease agents or physiological problems, such as loss of condition following spawning, and this can only be determined using histology.
	When abnormal mortality outbreaks occur, various presumptive diagnostic methods can be used in addition to histology, among which, tissue imprints, Ray's fluid thioglycollate medium (RFTM) culture or polymerase chain reaction (PCR) may be used, as recommended in the individual disease chapters. Such methods may provide advantages of quick and/or cheap procedures as an answer to suspicion of infection with a given disease agent.
	When a disease agent is encountered during screening or mortality outbreaks, electron microscopy and/or molecular probes should be used for specific identification, if available. Some of the OIE listed diseases for molluscs cover disease agents belonging to one or more species of the same genus. Specific reagents designed to detect certain listed agents are recommended in the following chapters, to be used to confirm histological examination results and/or to give a species-specific diagnosis where available.
	6.1.	Histological techniques
		Because of the generic use of histology in diagnostic procedures for diseases of molluscs, a detailed technical guideline is provided in this chapter.
		Histology is a technique that is used to study the structure of cells and tissues under light microscopy. Tissue preparation involves different steps, including tissue fixation, dehydration, impregnation and embedding of samples, preparation of sections, staining and mounting of slides.
		Live moribund animals or freshly dead (within minutes) animals provide the optimum conditions under which to collect tissues. A standard section should be taken through the digestive gland, to include the gills, mantle and palps, where possible. Alternatively for large specimens, several sections should be taken to include all the important tissues.
		.	6.1.1. Tissue fixation
			The role of the fixative is to maintain the morphology of the tissues as close to in-vivo morphology as possible and to prevent post-sampling necrosis. Recommended fixatives used for the study of marine molluscs are Davidson's solution and Carson's solution for large specimens. For smaller specimens, glutaraldehyde fixatives may be used and are compatible for electron microscopy use. The ratio of fixative to tissue volume should be at least 10:1 to ensure good fixation.
			Davidson's solution:
			Sea water	1200 ml
			95% Alcohol	1200 ml
			38% Formaldehyde	300 ml
			Glycerol	400 ml
			Glacial acetic acid	10% (add extemporaneously)
			Carson's solution:
			NaH2PO4.2H2O	23.8 g
			NaOH	5.2 g
			Distilled water	900 ml
			40% Formaldehyde	100 ml
				Adjust the pH to 7.2-7.4
			There is no universal fixative and choice should be made taking into account later use of fixed material as well as practical aspects of fixative use (price, component availability, etc.). Davidson's solution is an excellent choice for preserving the structure of the tissues. In addition, tissue sections fixed in Davidson's solution can be stained later by different histochemical methods, as well as in-situ hybridisation with DNA probes. For this purpose, over-fixation (over 24-48 hours) should be avoided. Carson's solution may not be as good as Davidson's solution for histological analysis. Nevertheless it does allow good preservation of the ultrastructure and may be used to preserve samples for later study by electron microscopy. Because electron microscopy may be a valuable adjunct in diagnosing or confirming infections in molluscs, fixation of some samples (especially smaller samples) using glutaraldehyde, as described in Section 6.2 of this chapter, may be considered. Otherwise, material fixed in Carson's solution, and shown to contain adequate levels of targeted disease agents or abnormalities, can be refixed in glutaraldehyde. It is recommended that part of the mollusc be fixed in Davidson's solution while the other part be fixed in Carson's solution for further investigation. This should be done in order to ensure fixation of all tissues/organs in the two fixatives. If neither are available, 10% buffered formalin made up with filtered seawater is adequate. Within each country, the molluscan aquaculture industry must agree on the most effective way of ensuring adequate fixation.
		.	6.1.2. Dehydration, impregnation and embedding of the samples
			The embedding of the samples in paraffin requires several steps during which the water contained in the tissues is progressively replaced, first by alcohol, then by xylene or equivalent less toxic clearing solution, and lastly by paraffin.
			After having fixed the samples in Carson's or Davidson's solution, they are transferred through graded alcohols (70-95 [v/v]) before final dehydration in absolute ethanol. The alcohol contained in the tissues is next eliminated by immersing them in xylene. The tissues are then impregnated with paraffin, which is soluble in xylene, at 60°C. These steps may be all carried out automatically using a machine.
			Blocks are produced by letting the tissues cool in moulds filled with paraffin on a cooling table; cooling and moisturising are essential to section cutting.
		.	6.1.3. Preparation of the sections
			After the blocks have been cooled on a cold plate, which allows the paraffin to solidify, histological sections of about 2-3 µm are cut using a microtome. The sections are recovered on histological slides, drained and dried overnight at 60°C. Drying the samples at this temperature allows the excess moisture to be eliminated and thus the sections adhere to the slides.
		.	6.1.4. Staining and mounting the slides
			Before staining, the paraffin is removed from the sections by immersing them in xylene or equivalent less toxic clearing solution for 10-20 minutes. This is repeated once and then the solvent is eliminated by immersion in two successive absolute ethanol baths for 10-minute periods each and rehydrated by immersion in a bath of tap water for 10 minutes. Different topographical or histochemical staining techniques can then be performed.
			When staining with haematoxylin-eosin (H& E) is used, (haematoxylin or equivalent) nuclear and basophilic structures stain a blue to dark purple colour, the endoplasmic reticulum stains blue, while the cytoplasm takes on a grey colour. The acid dye eosin stains the other structures pink. This staining technique is simple and reproducible and, although it only allows a limited differentiation of cell structures, it is possible to detect any abnormalities in tissue and cellular structure. Other techniques may be applied to demonstrate particular structures or features as required (e.g. trichrome for connective tissue and cytoplasmic granules).
	6.2.	Transmission electron microscopy methods
		Because of the very frequent use of transmission electron microscopy in confirmatory identification of disease agents in diagnostic procedures for diseases of molluscs, detailed technical guidelines are provided in this chapter for indication.
		Fixation for electron microscopy should be done immediately before fixation for histology. Only samples taken rapidly from live animals will be of any use. The preparation of samples for electron microscopy involves the following steps: tissue fixation, decalcification of the samples (when necessary), dehydration, impregnation and embedding of the samples, preparation and counterstaining of the sections.
		.	6.2.1. Tissue fixation
			For tissues that are to be examined by electron microscopy, it is important that the fixation be performed correctly in order to cause as little damage as possible to the ultrastructure. The specimens are cut such that their width does not exceed 1-2 mm. This small size allows the various solutions to penetrate rapidly into the sample.
			Fixation of the samples is carried out directly in 3% glutaraldehyde for 1 hour. Fixation for longer periods leads to membranous artefacts. The samples are washed in buffer three times, then fixed in 1% osmic acid and washed twice again in buffer. Various formulations of glutaraldehyde fixative and buffers work equally well.
			In order to cause as little damage as possible to the ultrastructure, the samples are treated with solutions that have an osmolarity close to that of the tissues. Thus, mollusc tissues are treated with solutions with an osmolarity of around 1000 mOsm. The osmolarity of the solutions is adjusted with NaCl. As mollusc tissues are nearly iso-osmotic with seawater, it is possible to make the glutaraldehyde up with 0.22 µm filtered seawater, and use the filtered seawater for subsequent washes.
			Sodium cacodylate	0.4 M: 8.6 g in 100 ml of distilled water
			Sodium chloride	10% in distilled water
			Cacodylate buffer, pH 7.4:
			1000 mOsm
			Sodium cacodylate	50 ml from 0.4 M stock solution
			NaCl	20 ml from 10% stock solution
			Distilled water	30 ml
				Adjust the pH to 7.4
			3% Gluteraldehyde:
			1000 mOsm
			25% gluteraldehyde	2.5 ml
			0.4 M sodium cacodylate	5 ml
			10% NaCl	3.5 ml
			Distilled water	9 ml
			1% Osmic acid:
			1000 mOsm
			4% Osmic acid	1 volume
			0.4 M sodium cacodylate	1 volume
			NaCl	1 volume from 10% stock solution
			Distilled water	1 volume
			5% EDTA:
			Disodium EDTA	5 g
			Cacodylate buffer	100 ml
			Dissolve by adding a few sulphur tablets. EDTA dissolves when the pH is above 8. When the solution becomes clear adjust the pH to 7.4 by adding concentrated HCl.
			If the samples have been previously fixed and stored in Carson's solution, they must be washed several times in a bath of buffer before fixation with 3% glutaraldehyde.
		.	6.2.2. Dehydration, impregnation and embedding of the samples
			The samples are dehydrated in successive baths of ethanol: 70% ethanol once, 95% ethanol twice, absolute ethanol three times. The dehydration is completed by two baths of propylene oxide, which allows the subsequent impregnation with Epon or other resin.
			The samples are impregnated progressively. After a first bath in a mixture of polypropylene oxide-Epon (50/50), the samples are placed in a bath of Epon. The longer the incubation, the better the impregnation of the tissues.
			Embedding is carried out by placing the samples in moulds filled with Epon resin. A label identifying the sample is included in each block and the blocks are then placed at 60°C (the temperature at which Epon resin polymerises) for 48 hours.
		.	6.2.3. Preparation of the sections and the counterstaining
			The blocks are cut to appropriate sizes with a razor blade and the sections are then cut using an ultramicrotome. Semi-thin sections (0.5-1 µm) are cut and placed on glass slides. These will be used to control the quality of the samples by light microscopy and to find the areas of interest on the section.
			The semi-thin sections are stained at 90-100°C with 1% toluidine blue solution. After drying, the slides are mounted under cover-slips with a drop of synthetic resin and observed under the light microscope.
			Ultrathin sections 80-100 nm thick are placed on mesh copper grids for electron microscopy analysis. Uranyl acetate and lead citrate are used to counterstain the ultrathin sections.

KEY REFERENCES

	1.	Austin B. & Austin D.A. (1989). Methods for the Microbiological Examination of Fish and Shellfish. Ellis Horwood, Chichester, UK.
	2.	Bondad-Reantaso M.G., McGladdery S.E., East I. & Subasinghe R.P. (2001). Asian Diagnostic Guide to Aquatic Animal Diseases. FAO Fisheries Technical Paper, No. 402, supplement 2. Food and Agriculture Organization of the United Nations (FAO), Rome, Italy, 240 pp.
	3.	Bower S.M., McGladdery S.E. & Price I.M. (1994). Synopsis of infectious diseases and parasites of commercially exploited shellfish. Ann. Rev. Fish Dis., 4, 1-199.
4.	Elston R.A. (1999). Health Management, Development and Histology of Seed Oysters. World Aquaculture Society. Baton Rouge, Louisiana, USA, 110 pp.
5.	Galtsoff P.S. (1964). The American oyster, Crassostrea virginica Gmelin. Fishery Bull., 64, 480 pp.
6.	Howard D.H. & Smith C.S. (1983). Histological techniques for marine bivalve molluscs. NOAA Technical Memorandum NMFS-F/NEC, 25, 97 pp.
7.	Mialhe E., Bachere E., Boulo V., Cadoret J.P., Rousseau C., Cedeno V., Saraiva E., Carrera L., Calderon J. & Colwell R.R. (1995). Future of biotechnology-based control of disease in marine invertebrates. Molec. Mar. Biol. Biotechnol., 4, 275-283.
8.	Thoeson J.C. (1994). Suggested Procedures for the Detection and Identification of Certain Finfish and Shellfish Pathogens, Fifth Edition. Bluebook, American Fisheries Society, Bethsada, USA.
9.	Walker P. & Subasinghe R.P. (2000). DNA-based Molecular Diagnostic Techniques. Research needs for standardization and validation of the detection of aquatic animal pathogens and diseases. FAO Fisheries Technical Paper, n°395, 93 pp.

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