**Annex 34. Item 11.1.1. – Chapter 2.2.0. General information: diseases of crustaceans**

SECTION 2.2.DISEASES OF CRUSTACEANS

CHAPTER 2.2.0.GENERAL INFORMATION

A.  SAMPLING

1.	Assessing the health status of the epidemiological unit

1.1.	Sample material to be used for tests

Sample material and the number of samples to be collected depend on the specific disease or pathogen, the size of animals and the objective of testing (i.e. surveillance of apparently healthy animals, presumptive diagnosis of clinically affected animals or confirmatory diagnosis of a suspect result from surveillance or presumptive diagnosis~~of overt disease, detection of subclinical infection in apparently healthy animals or sampling for targeted surveillance to demonstrate freedom from infection with a specified pathogen~~). See the individual disease chapters in this *Aquatic Manual* for specific details of sample requirements. 

1.2.	Specifications according to crustacean populations

For details of animals to sample for a specific listed disease, see the relevant disease chapter in the *Aquatic Manual*. The design of a surveillance system for demonstrating disease-free status for a country, zone or compartment should be in accordance with the recommendations of the WOAH *Aquatic Code* Chapter 1.4 . *Aquatic animal disease surveillance.*

Animals to be sampled are selected as follows:

i)	Susceptible species should be sampled proportionately or following risk-based criteria for targeted selection of lots epidemiological units or populations with a history of abnormal mortality or potential exposure events (e.g. replacement with stocks of unknown disease status).

ii)	If more than one water source is used for production, animals from all water sources should be included in the sample.

iii)	~~For the study of presumptively diseased crustaceans select those animals that are moribund, discoloured, displaying abnormal behaviour, or otherwise abnormal.~~If weak, abnormally behaving discoloured or freshly dead (not decomposed) animals are present, such animals should be selected. If such animals are not present, animals should be selected in such a way that all epidemiological units of the farm or waterbody are proportionately represented in the sample.

~~iv)	When sampling is aimed at assessing disease occurrence (e.g. estimation of disease prevalence), the preferred selection method is probability sampling.~~

1.3.	Specifications according to clinical status

In clinical disease episodes, carefully selected quality specimens with representative lesions should be obtained from live or moribund crustaceans. Collection of dead specimens during disease outbreaks should be avoided when possible, but recently dead samples may be suitable for some diagnostic assays provided they are not decomposed. When cultured or wild crustacean stocks are presenting clinical signs of an active disease that are consistent with, or suggestive of, any one of the WOAH-listed crustacean diseases, care should be taken to ensure that the samples collected are preserved appropriately for the anticipated diagnostic tests (see sample preservation section for recommended methods). In situations other than when clinical disease episodes are investigated, for the WOAH-listed diseases it is highly recommended that the scheduling of sampling be planned (i.e. by farm schedule, season, etc.) so that the particular life-stage(s) are sampled at a time when the pathogen of concern is most likely to be detected. Disease-specific recommendations are provided in Section 3 *Sample selection, sample collection, transportation and handling* of the individual chapters. 

~~Recently dead crustaceans may be suitable (depending on their condition) for certain diagnostic assays such as nucleic acid detection techniques.~~

1.4.	Specifications according to crustacean size

See the individual disease chapters in this *Aquatic Manual* for specific details of sample requirements.

2.	General processing of samples

2.1.	Macroscopic examination

See disease-specific chapters in this *Aquatic Manual*.

2.2.	Virological examination

Virological examination of crustaceans is not routinely used for listed diseases. *Macrobrachium rosenbergii* has been isolated in insect cell lines, but it is not a recommended method.

2.2.1.	Transportation and antibiotic treatment of samples

~~Culture systems for crustacean viruses are not available; antibiotic treatment of samples is not required. For transportation of samples see Section 3 of disease-specific chapters in this *Aquatic Manual*.~~Not applicable.

2.2.2.	Virus isolation

~~For processing of tissues see Section 3 of disease-specific chapters in this *Aquatic Manual*.~~Not applicable.

2.2.3.	Treatment to neutralise enzootic viruses

Not applicable. 

2.3.	Bacteriological examination

Bacteriological examination of crustaceans is not routinely used for listed diseases, but it may be used for the strains of *Vibrio parahaemolyticus* (*Vp*AHPND) that cause acute hepatopancreatic necrosis disease (AHPND) and for~~can be isolated on standard bacteriological media.~~*Hepatobacter penaei*, the causative agent of necrotising hepatopancreatitis (NHP)~~has not been cultured and, because of its very small size, bacteriological examination may be limited to Gram staining~~. See disease-specific chapters in this *Aquatic Manual* for identification methods.

2.4.	Parasitic examination

Not applicable for currently listed diseases. 

2.5.	Fungal and other protists examination

See Chapter 2.2.2 *Infection with* Aphanomyces astaci**(*Crayfish plague*). 

B.  MATERIALS AND BIOLOGICAL PRODUCTS REQUIRED FOR THE ISOLATION AND IDENTIFICATION OF CRUSTACEAN PATHOGENS

1.	Crustacean viruses

1.1.	Crustacean cell lines

Not applicable. There are currently no confirmed or documented crustacean cell lines. 

1.2.	Culture media

Not applicable. 

1.3.	Virus positive controls and antigen preparation

1.3.1.	Virus nomenclature

In general, the virus nomenclature used in the disease-specific chapters follows the most recent taxonomy for viruses as given in the Report of the Committee on Taxonomy of Viruses (see: ICTV [ictvonline.org] for latest information). Also provided in the disease-specific chapters are the disease and virus names that are in common use by the shrimp/prawn farming industries, as well as the more common synonyms that have been used or are in current use.

1.3.2.	Virus production for experimental purposes

As no cell lines (crustacean, arthropod, or vertebrate) are known that can be used to produce crustacean viruses, infection of known susceptible host species (which are free of infection ~~by~~with the pathogenic agent in question) is the preferred method for virus production for experimental purposes. 

1.3.3.	Virus preservation and storage

Infectivity of all of the WOAH-listed crustacean viruses can be preserved by freezing infected whole crustaceans or infected target tissues at –20°C for short-term storage, or at –80°C or lower for long-term storage.

2.	Crustacean bacteria

2.1.	Culture media

See Chapter 2.2.1. *Acute hepatopancreatic necrosis disease* for details. 

2.2.	Storage of cultures

Lyophilisation or storage at –70°C is recommended for long-term storage of bacterial cultures. 

3.	Crustacean parasites

3.1.	Culture media

Not applicable for currently listed diseases. 

3.2.	Storage of cultures

Not applicable for currently listed diseases. 

4.	Crustacean fungi and protists

4.1.	Culture media

See chapter 2.2.2. *Infection with*Aphanomyces astaci (*crayfish plague*)

4.2.	Storage of cultures

See Chapter 2.2.2. 

5.	Techniques

The available diagnostic methods that may be selected for diagnosis of the WOAH-listed crustacean diseases or detection of their aetiological agents are based on:

i)	Gross and clinical signs.

ii)	Direct bright-field, phase-contrast or dark-field microscopy with whole stained or unstained tissue wet-mounts, tissue squashes, and impression smears; and wet-mounts of faecal strands.

iii)	Histology of fixed specimens.

iv)	Bioassays of suspect or subclinical carriers using a highly susceptible host (life stage or species) as the indicator for the presence of the pathogen.

v)	Antibody-based tests for pathogen detection using specific antisera, polyclonal antibodies (PAbs) or monoclonal antibodies (MAbs).

vi)	Molecular methods (including sequencing):

DNA probes or RNA probes for *in-situ* hybridisation (ISH) assays with histological sections of fixed tissues;

Conventional and real-time PCR/RT-PCR and LAMP for direct assay with fresh tissue samples or with extracted DNA or RNA.

Pooling of samples from more than one individual animal for a given purpose should only be recommended where robust supporting data on diagnostic sensitivity and diagnostic specificity have been evaluated and found to be suitable. If the effect of pooling on diagnostic sensitivity has not been thoroughly evaluated, larger crustaceans should be processed and tested individually. However, for eggs, larvae and postlarvae pooling of larger numbers ~~(e.g. ~150 or more eggs or larvae or 50 to 150 postlarvae depending on their size/age)~~may be necessary to obtain sufficient sample material to run a diagnostic assay.

5.1.	Antibody-based tests 

See disease-specific chapters in this *Aquatic Manual*. 

5.2.	Direct microscopy

Samples for direct microscopic examination should be examined as soon as possible after collection. Use live specimens whenever possible, or use fresh, chilled, or fixed specimens when live specimens are not practical. If an adequate field laboratory is available, it should be used to process and examine samples near the site of collection.

5.3.	Histological techniques

Only live or moribund specimens with clinical signs should be sampled for histology. Collect crustaceans by whatever means are available with a minimum of handling stress. Hold animals in a container appropriate for maintaining suitable water quality and supply adequate aeration to the container if the crustaceans are to be held for a short period of time before actual fixation. 

5.3.1.	Fixation

A general rule is that a minimum of ten volumes of fixative should be used for one volume of tissue sample (i.e. a 10 g sample of crustacean would require 100 ml of fixative).

i)	Davidson’s AFA (alcohol, formalin, acetic acid) fixative

Davidson’s AFA fixative is recommended for most histological applications. The fixative is rapid, reduces autolytic changes in decapod crustaceans (i.e. especially in crustaceans in tropical and subtropical regions), and its acidic content decalcifies the cuticle. The formulation for Davidson’s AFA is (for 1 litre):

ii)	Fixation procedures with Davidson’s AFA

*For larvae and postlarvae that are too small to be easily injected with fixative using a tuberculin syringe:* Using a fine mesh screen or a Pasteur pipette, select and collect specimens. Immerse crustaceans selected for sampling directly in the fixative. Fix for 12–24 hours in fixative and then transfer to 70% ethyl alcohol for storage.

*For juveniles that are too small to be injected:* Select and collect specimens. Use a needle or fine-pointed forceps to incise the cuticle and immediately immerse crustaceans selected for sampling directly into the fixative. Fix for 12–24 hours in fixative and then transfer to 70% ethyl alcohol for storage.

*For large juveniles and adults:* to ensure proper fixation, kill the crustacean using a humane method, then immediately inject fixative (use 5–10% volume:weight). Fix for 12–24 hours in fixative and then transfer to 70% ethyl alcohol for storage.

The hepatopancreas (HP) should be injected first and at two or more sites, with a volume of fixative sufficient to change the HP to a white-to-orange colour (when Davidson’s AFA is used); then inject fixative into adjacent regions of the cephalothorax, into the anterior abdominal region, and into the posterior abdominal region.

The fixative should be divided between the different regions, with the cephalothoracic region, specifically the HP, receiving a larger share than the abdominal region.

Immediately following injection, slit the cuticle with dissecting scissors, from the sixth abdominal segment to the base of the rostrum, being particularly careful not to cut deeply into the underlying tissue. The incision in the cephalothoracic region should be just lateral to the dorsal midline, while that in the abdominal region should be approximately mid-lateral.

*For crustaceans**larger than ~12 g:* After injection of fixative, the body should then be transversely bisected, at least once, just posterior to the abdomen/cephalothorax junction, and (optional) again mid-abdominally.

*For very large crustaceans* (*e.g. lobsters, crabs, adult penaeids, adult Macrobrachium rosenbergii*,*some species and life stages of crabs, crayfish, etc.*): The organs of interest may be excised after injection of fixative. Completion of fixation of these tissue samples is then handled as outlined previously.

Following injection, incisions and bisection/trisection, or excision of key organs, immerse the specimen in the fixative (use 10:1 fixative:tissue ratio).

Allow fixation to proceed at room temperature for 24–72 hours depending on the size of crustacean being preserved. Longer fixation times in Davidson’s AFA may be used to thoroughly decalcify the shell of crabs, lobsters, crayfish, etc.

Following fixation, the specimens should be transferred to 70% ethyl alcohol, where they can be stored for an indefinite period.

iii)	Transport and shipment of preserved samples

As large volumes of alcohol should not be mailed or shipped, the following methods are recommended: Remove the specimens from the 70% ethyl alcohol. For larvae, postlarvae, or small juveniles, use leak-proof, screw-cap plastic vials if available; if glass vials must be used, pack to prevent breakage. For larger specimens, wrap samples with white paper towels to completely cover (do not use raw or processed cotton). Place towel-wrapped specimens in a sealable plastic bag and saturate with 70% ethyl alcohol. Insert the label and seal the bag. Place the bag within a second sealable bag. Multiple small sealable bags can again be placed within a sturdy, crush-proof appropriately labelled container for shipment (for details see *Aquatic Code* Chapter 5.10 *Measures concerning international transport of aquatic animal pathogens and pathological material*).

5.4.	Transmission or scanning electron microscopy 

Electron microscopy (EM – transmission or scanning) is a valuable research tool for the study of disease in crustaceans. However, EM methods are not routinely used for diagnosis of the diseases listed by WOAH. 

5.5.	Use of molecular ~~and antibody-based~~techniques for confirmatory testing and diagnosis

Molecular techniques, including the use of nucleic acid probes for *in-situ* hybridisation, conventional PCR and real-time PCR, have been developed for the identification of many pathogens of aquatic animals. Real-time PCR methods, in general, have high sensitivity and specificity and, following adequate validation, can be used for direct detection of ~~viral~~nucleic acids ~~in samples prepared~~extracted from crustacean tissue. ~~The~~Molecular techniques can be used in direct surveillance of apparently healthy populations, if they have a high level of diagnostic sensitivity, as well as in the diagnosis of clinically affected animals.

When using PCR as a diagnostic method, the design of primers and probe, the use of positive and negative controls, as well as validation of the PCR method chosen are important. Real-time PCR is a powerful technique particularly for analysing relatively high numbers of samples (e.g. for surveillance) via high-throughput testing. Several nucleic acid probe and PCR protocols are included in this version of the *Aquatic Manual* as screening, diagnostic or confirmatory methods for crustaceans and can be undertaken as the standard method. Following real-time PCR-positive results, where possible, conventional PCR with sequencing of PCR products should be used for confirmation of pathogen identity.

As with all PCR protocols, optimisation may be necessary depending on the reagents, equipment and the plasticware used. PCR is prone to false-positive and false-negative results. False-positive results (negative samples giving a positive reaction) may arise from either product carryover from positive samples or, more commonly, from cross-contamination by PCR products from previous tests. False-negative results (positive samples giving a negative result) may lead to unwanted transmission of pathogens and biosecurity failure.

Each diagnostic sample~~s~~ should be tested in duplicate, i.e. by testing two aliquots.~~and~~Both aliquots must produce positive results for a sample to be deemed positive. In instances where a sample produces one positive and one negative result, these are deemed indeterminate and should be retested. In addition, the following controls should be run with each assay: negative extraction control (e.g. a tissue [or equivalent sample that is under test]) sample from a known uninfected animal; positive control (preferably, one that can be distinguished from the pathogen genomic sequence [e.g. an artificial plasmid], thus allowing detection of any cross-contamination leading to a false positive result); no template control (all reagents with water replacing the template); internal positive control (internal housekeeping gene). All controls should produce their expected results in order for the diagnostic test result to be valid.

To minimise the risk of contamination, aerosol~~-preventing~~barrier pipette tips should be used for all sample preparation and PCR ~~preparation~~steps. Additionally, all PCRs should be prepared in a clean area that is separate from the area where ~~the~~nucleic acid extraction, amplification~~s~~ and gel electrophoresis are performed. Do not share equipment (e.g. laboratory coats and consumables) between areas and, where possible, restrict access between areas. Contaminating PCR products can be carried on equipment, clothes, shoes, pens/marker pens and paper (e.g. workbooks). Also, ensure all work-tops and ~~air-flow~~cabinets/hoods used for the extractions and PCR set-up are regularly cleaned and decontaminated. To ensure sample integrity, always store the samples (e.g. in a freezer or refrigerator) in a location ~~away~~separate from the molecular biology laboratory and reagents

5.5.1.	Sample preparation and types

Samples should be prepared to preserve the nucleic acid of the pathogen and should be handled and packaged with the greatest care to minimise the potential for cross-contamination among the samples or target degradation before the assay can be performed.~~Samples selected for nucleic acid-based or antibody-based diagnostic tests should be handled and packaged (in new plastic sample bags or bottles) with care to minimise the potential for cross-contamination among the sample set taken from different (wild or farmed) stocks, tanks, ponds, farms, etc.~~A water-resistant label, with the appropriate data filled out, should be placed within each package or container for each sample set.

Some suitable methods for preservation and transport of samples taken for molecular ~~or antibody-based~~tests are:

i)	*Live specimens:* these may be processed in the field or shipped to the diagnostic laboratory for testing.

ii)	*Haemolymph:* this tissue is the preferred sample for certain molecular and antibody-based diagnostic tests (see disease-specific chapters). Samples may be collected by needle and syringe through cardiac puncture, from the haemocoel (i.e. the ventral sinus in penaeids), or from a severed appendage, and immediately transferred to a tube that is half full with ~~90–95%~~80% analytical grade ethanol or suitable nucleic acid preservative.

iii)	*Iced or chilled specimens:* these are specimens that can be transported to the laboratory for testing within 24 hours. Pack samples in sample bags surrounded by an adequate quantity of wet ice or freezer bricks around the bagged samples in an insulated box and ship to the laboratory.

iv)	*Frozen ~~whole~~specimens:*select live specimens according to the criteria listed in disease-specific chapters in this *Aquatic Manual*. In situations where it is not possible to get the specimens to the laboratory alive, they may be quick ~~freeze~~frozen in the field using crushed dry-ice or ~~freeze~~frozen in ~~the~~field laboratories using a mechanical freezer at –20°C or lower temperature. Prepare and insert the label into the container with the samples, pack samples with an adequate quantity of dry-ice in an insulated box, and ship to the laboratory.

v)	*Alcohol-preserved samples:* in regions where the storage and shipment of frozen samples is problematic, ~~90–95%~~80% analytical grade ethanol may be used to preserve, store, and transport certain types of samples for molecular tests. Alcohol-preserved samples are generally not suitable for antibody-based tests. Whole crustaceans (any life stage provided the specimen is no larger than 2–3 g), excised tissues (i.e. pleopods) from large crustaceans, or haemolymph may be preserved in ~~90–95%~~80% analytical grade ethanol, and then packed for shipment according to the methods described in Section 5.3.1, paragraph iii (see chapter 5.10 of the *Aquatic Code* for additional details on the international transport of such samples).

vi)	*Fixed tissues for*in-situ*hybridisation:* For this purpose, classic methods for preservation of the tissues are adequate. Neutral-buffered formalin is usually a good choice. Fixation for over 24–48 hours should be avoided; samples should be transferred to ethanol following formalin treatment.

5.5.2.	Preservation of RNA and DNA in tissues

For routine diagnostic testing by PCR or RT-PCR, samples must be prepared to preserve the pathogen’s nucleic acid. For most purposes, preservation of samples in analytical grade ethanol~~alcohol~~(80~~–90~~%) is the preferred method for subsequent molecular tests. Samples preserved in this way can be stored at 4°C for 1 month, at 25°C for 1 week or indefinitely at –20°C or below. In addition, other products (e.g. nucleic acid preservatives, various lysis buffers, etc.) are commercially available for the same purpose. 

5.5.3.	Nucleic acid extraction

To isolate nucleic acids from tissues preserved in ethanol or nucleic acid preservative, simply remove the tissue from the fixative or preservative and treat it as though it was just harvested. Most fresh and preserved or fixed tissues can be homogenised (e.g. with a mortar and pestle or in bead-beating tubes) directly in the lysis or extraction buffer provided with commercially available DNA and RNA extraction kits. Commercial kits should be validated or undergo equivalence testing with current validated extraction procedures prior to routine use.

5.5.4.	Preparation of slides for *in-situ* hybridisation 

For *in-situ* hybridisation, fixed tissues that have been transferred to ~~70%~~80% analytical grade ethanol are embedded in paraffin according to standard histological methods. Sections are cut at a thickness of 5 μm and placed on aminoalkylsilane-coated slides, which are then baked overnight in an oven at 40°C. The sections are de-waxed by immersing in xylene for 10 minutes. This step is repeated once and then the solvent is eliminated by immersion in two successive absolute ethanol baths for 10 minutes each. The sections are then rehydrated by immersion in an ethanol series. The protocol may require a step of membrane permeabilisation enabling access to the target DNA. For this purpose, sections are treated with proteinase K (100 μg ml–1) in TE buffer (Tris [50 mM], EDTA [10 mM]), at 37°C for 30 minutes. For in-situ hybridisation tests (see individual chapters for details), it is essential that both a known positive and a known negative slide be stained to eliminate false positive results due to non-specific staining/stain dropout, and false negative results due to errors in the staining protocol (Qadiri *et al*., 2019; Valverde *et al*., 2017). For further details see disease-specific chapters in this *Aquatic Manual*.

6.	Additional information to be collected

Sample information should include the collector’s name, organisation, date, time, and description of the geographical location. The geographical origin of samples may be described as the name or location of the sampling site or its geographical co-ordinates. There should also be records that provide information to allow trace-backs on the sample movement from the sample site to the storage facility or laboratory and within those facilities. 

A history of the specimens should also be collected and should include species, age, weight, details of clinical signs including behavioural changes, as well as observations concerning any gross pathology which has been observed. 

Information on the preservation method, storage location, and date and time of storage at each storage locker or freezer along with information on the storage temperature (continuously monitored is preferable) should be collected. This information should be tracked with a unique sample code for all samples. For laboratories, the date of receipt, storage location information, date of analysis, analysis notes, and report date should be maintained for all uniquely coded samples. These data will greatly facilitate the tracking of sample problems and provide assurance that the samples were properly handled.

4. KEY REFERENCES FOR FURTHER READING

BELL T.A. & LIGHTNER D.V. (1988). A Handbook of Normal Shrimp Histology. Special Publication No. 1, World Aquaculture Society, Baton Rouge, Louisiana, USA.

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.

JOHNSON P.T. (1980). Histology of the Blue Crab, *Callinectes sapidus*. A Model for the Decapoda. Prager, New York, USA, 440 pp.

LIGHTNER D.V. (1996). A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, Louisiana, USA. 304 pp.

LIGHTNER D.V., REDMAN R.M., PANTOJA C.R., TANG K.F.J, NOBLE B.L., SCHOFIELD P., MOHNEY L.L., NUNAN L.M. & NAVARRO S.A. (2012). Historic emergence, impact and current status of shrimp pathogens in the Americas. *J. Invert. Pathol.,***110**, 174–183.

LOTZ J.M. (1997). Special topic review: Viruses, biosecurity and specific pathogen-free stocks in shrimp aquaculture. *World J. Microbiol. Biotechnol*., **13**, 405–413.

MOODY N.J.G. & CRANE M.ST.J. (2016). Validation of diagnostic tests in the OIE manual for aquatic animals. In: Proc. 3rd OIE Global Conference on Aquatic Animal Health – “Riding the Wave of the Future”, Ho Chi Minh City, Vietnam, 20–22 January 2015, pp.119–126.

QADIRI S.S.N., SOO-JIN KIM S.-J., KRISHNAN R., KIM J.-O., KOLE S., KIM W.-S. & OH M.-J. (2019). Localization and tissue tropism of viral haemorrhagic septicemia virus (VHSV) in experimentally infected juvenile olive flounder, *Paralichthys olivaceus*: An *in situ* hybridization and immunohistochemical study. *Aquaculture*, **505**, 242–252.

THITAMADEE S, PRACHUMWAT A., SRISALA J., JAROENLAK P., SALACHAN P.V., SRITUNYALUCKSANA K, FLEGEL T.W. & ITSATHITPHAISARN O. (2016). Review of current disease threats for cultivated penaeid shrimp in Asia. *Aquaculture*, **452**, 69–87.

VALVERDE E.J., BORREGO J.J., SARASQUETE M.C., ORTIZ-DELGADO J.B. & CASTRO D. (2017). Target organs for lymphocystis disease virus replication in gilthead seabream (*Sparus aurata*). *Vet. Res*., **48**, 21. doi 10.1186/s13567- 017-0428-3.

WALKER P.J. & MOHAN C.V. (2009). Viral disease emergence in shrimp aquaculture: origins, impact and the effectiveness of health management strategies. *Rev. Aquaculture*, **1**, 125–154.

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**NB:** FIRST ADOPTED IN 2000; MOST RECENT UPDATES ADOPTED IN 2012.