Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 4.1.7.

CRAYFISH PLAGUE
(Aphanomyces astaci)

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SUMMARY

Crayfish plague is a highly infectious disease of all crayfish (Decapoda: Astacidae, Cambaridae) of non-North American origin. The aetiological agent is an Oomycete fungus, Aphanomyces astaci, which is now widespread in Europe as well as in North America. The European crayfish species, the Noble crayfish Astacus astacus of north-west Europe, the stone crayfish Austropotamobius pallipes of south-west and west Europe, the related Austropotamobius torrentium (mountain streams of south-west Europe) and the slender clawed or Turkish crayfish Astacus leptodactylus of eastern Europe and Asia Minor are all highly susceptible. The only other crustacean known to be capable of infection by A. astaci is the Chinese mitten crab (Eriocheir sinensis) and this only under laboratory conditions.
 
The disease first occurred in Europe in the third quarter of the 19th century in the Franco-German border region. From there region a steady spread of infection occurred, principally in two directions - down the Danube into the Balkans and towards the Black Sea, and across the North German plain into Russia and from there south to the Black Sea and north-west to Finland and finally, in 1907, to Sweden. In the 1960s the first outbreaks in Spain were reported and in the 1980s further spread of infection to the British Isles, Turkey, Greece and Norway was reported (1).
 
The reservoir for the original infections in the 19th century was never established, but the post-1960s extensions are largely linked to movements of North American crayfish introduced more recently for purposes of crayfish farming. These species (Pacifastacus leniusculus [the Signal crayfish] and Procambarus clarki [the Louisiana swamp crayfish]) can act as largely or completely asymptomatic carriers, but can be killed by A. astaci under adverse conditions. Transmission has also resulted from contaminated crayfish traps and other contaminated equipment.
 
Clinically, infected crayfish may present a wide range of gross signs of infection or none at all. Focal whitening of local areas of musculature beneath transparent areas of thin cuticle, especially of the ventral abdomen and in the periopod (limb) joints, often accompanied by even more localised brown melanisation, is the most consistent sign. In the terminal stages of infection, animals show a limited range of behavioural signs, principally a loss of the normal aversion to bright light (they are seen in open water in daylight) later accompanied by a loss of limb co-ordination, which produces an effect that has been described as 'walking on stilts'. Eventually, moribund animals lose their balance and fall onto their backs before dying.
 
Diagnosis requires isolation and identification of the pathogen by microscopic morphology; no molecular, biochemical or serological methods that have been adequately validated exist.
 
Control of the spread of infection once a watershed is infected is in practical terms impossible. Prevention of all introductions of crayfish to natural waters and into enclosed waters from which they may escape to natural waters can be effective, although movement of fish can result in the movement of infected water between watersheds and can transmit infection, as can contaminated equipment such as boots and fishing gear. Sodium hypochlorite and iodophores are effective for disinfection of contaminated equipment. Thorough drying of equipment (>24 hours) is also effective since the Oomycetes are not resistant to desiccation.
 

DIAGNOSTIC PROCEDURES

Diagnosis of crayfish plague strictly requires the isolation and characterisation of the pathogen, A. astaci, using simple mycological media fortified with antibiotics to control bacterial contamination (3). Isolation is only likely to be successful before or within 12 hours of the death of infected crayfish. However, there is no other disease or pollution effect that can cause such total mortality of crayfish while leaving all other animals in the same water unharmed, so that isolation of the pathogen is desirable but not essential, particularly in regions where further spread of infection is known to be a potential hazard. Clinical signs of crayfish plague include behavioural changes and a range of visible external lesions. The range of these lesions is so large that, except for the experienced eye, such clinical signs are of limited diagnostic value.
 

1.	Standard Screening Methods for Crayfish Plague
	1.1.	Isolation of Aphanomyces astaci
		Isolation methods are as described by Alderman & Polglase (3). An agar medium (isolation medium) is used that contains yeast extract and glucose in river water with antimicrobial agents (penicillin G and oxolinic acid) to prevent the growth of most bacteria and enable easy and rapid isolation of the pathogen.
		Isolation medium (IM): 12.0 g agar; 1.0 g yeast extract; 5.0 g glucose; 10 mg oxolinic acid; 1000 ml river water; and 1.0 g penicillin G (sterile) added after autoclaving and cooling to 40°C. River water = any natural river or lake water as opposed to demineralised water.
		Simple aseptic excision of infected tissues, which are then placed as small pieces (1-2mm2) on the surface of isolation medium plates, will normally result in successful isolation of A. astaci from moribund or recently dead (<24 hours) animals. Depending on a range of factors, foci of infection in crayfish may be easily seen by the naked eye or may not be discernable despite careful examination. Such foci can best be seen under a low power stereo microscope and are most commonly recognisable by localised whitening of the muscle beneath the cuticle. In some cases a brown colouration of cuticle and muscle may occur and in others, hyphae are visible in infected cuticle in the form of fine brown (melanised) tracks in the cuticle itself. Sites for particular examination include the intersternal soft ventral cuticle of the abdomen and tail, the cuticle of the perianal region, the cuticle between the carapace and tail, the joints of the periopods (walking legs), particularly the proximal joint and finally the gills.
		Provided that care is taken in excising infected tissues for isolation, contaminants need not present significant problems. Small pieces of cuticle and muscle may be transferred to a Petri dish of sterile distilled water and there further cut into small pieces with sterile instruments for transfer to IM isolation medium. Suitable instruments for such work are cataract knives and fine electron microscope or instrument grade forceps and scissors.
	1.2.	Identification of A. astaci
		On IM agar, growth of new isolates of A. astaci is almost entirely within the agar except at temperatures below 7°C, when some superficial growth occurs. Colonies are colourless. Dimensions and appearance of hyphae are much the same in crayfish tissue and in agar culture. Vegetative hyphae are aseptate and (5)7-9(10) µm in width (i.e. normal range 7-9 µm, but observations have ranged between 5 and 10 µm). Young, actively growing hyphae are densely packed with coarsely granular cytoplasm with numerous highly refractile globules. Older hyphae are largely vacuolate with the cytoplasm largely restricted to the periphery leaving only thin strands of protoplasm bridging the large central vacuole. The oldest hyphae are apparently devoid of contents. Hyphae branch profusely, with vegetative branches often tending to be somewhat narrower than the main hyphae for the first 20-30 µm of growth.
		When actively growing thalli or portions of thalli from broth or agar culture are transferred to river water (natural water with available cations encourages sporulation better than does distilled water), sporangia form readily in 20-30 hours at 16°C and 12-15 hours at 20°C. Thalli transferred from broth culture may be washed with sterile river water in a sterile stainless steel sieve, before transfer into fresh sterile river water for induction of sporulation. Thalli in agar should be transferred by cutting out a thin surface sliver of agar containing the fungus so that a minimum amount of nutrient containing agar is transferred. Always use a large volume of sterile river water relative to the amount of fungus being transferred (100:1). Sporangia are myceloid, terminal or intercalary, developing from undifferentiated vegetative hyphae. Sporangial form is variable: terminal sporangia are simple, developing from new extramatrical hyphae, while intercalary sporangia can be quite complex in form. Intercalary sporangia develop by the growth of a new lateral extramatrical branch, which forms the discharge tube of the sporangium. The cytoplasm of such developing discharge tubes is noticeably dense, and these branches are slightly wider (10-12 µm) than ordinary vegetative hyphae. Sporangia are delimited by a single basal septum in the case of terminal sporangia and by septa at either end of the sporangial segment in intercalary sporangia. Such septa are markedly thicker than the hyphal wall and have a high refractive index. Successive sections of vegetative hypha may develop into sporangia, and most of the vegetative thallus is capable of developing into sporangia.
		Within developing sporangia the cytoplasm cleaves into a series of elongate units (10-25 x 8 µm) that are initially linked by strands of protoplasm. Although the ends of these cytoplasmic units become rounded, they remain elongate until and during discharge. Spore discharge is achlyoid, that is, the first spore stage is an aplanospore which encysts at the sporangial orifice and probably represents the suppressed saprolegniaceous primary zoospore. No evidence has been observed for the existence of a flagellated primary spore, thus, in this description, the terms 'sporangium' not 'zoosporangium' and 'primary spore' not 'primary zoospore' have been used. Discharge is fairly rapid (<5 minutes) and the individual primary spores (=cytoplasmic units) pass through the tip of the sporangium and accumulate around the sporangial orifice. The speed of cytoplasmic cleavage and discharge is temperature dependent. At release, each primary spore retains its elongate irregularly amoeboid shape briefly before encystment occurs.
		Encystment is marked by a gradual rounding up followed by the development of a cyst wall, which is evidenced by a change in the refractive index of the cell. The duration from release to encystment is 2-5 minutes. Some spores may drift away from the spore mass at the sporangial tip and encyst separately. Formation of the primary cyst wall is rapid, and once encystment has taken place the spores remain together as a coherent group and adhere well to the sporangial tip so that marked physical disturbance is required to break up the spore mass.
		Encysted primary spores are spherical, (8)9-11(15) µm in diameter, and are relatively few in number, (8)15-30(40) µm per sporangium in comparison with other Aphanomyces spp. Spores remain encysted for 8-12 hours. Optimum temperatures for sporangial formation and discharge for the majority of European isolates of A. astaci are between 16 and 24°C. For some isolates, particularly from Spanish waters slightly higher temperature optima may prevail. The discharge of secondary zoospores from the primary cysts peaks at 20°C and does not occur at 24°C. In new isolates of A. astaci, it is normal for the majority of primary spore cysts to discharge as secondary zoospores, although this varies with staling in long-term laboratory culture. Sporangial formation and discharge occurs down to 4°C. Aphanomyces astaci does not survive at -5°C and below for more than 24 hours either in culture or in crayfish tissues, nor does it remain viable in crayfish tissues that have been subject to normal cooking procedures.
		In many cases, some of the primary spores are not discharged from the sporangium and many sporangia do not discharge at all. Instead, the primary spores appear to encyst in situ within the sporangium, often develop a spherical rather than elongate form and certainly undergo the same changes in refractive index that mark the encystment of spores outside the sporangium. This within-sporangial encystment has been observed on crayfish. Spores encysted in this situation appear to be capable of germinating to produce further hyphal growth.
		Release of secondary zoospores is papillate, the papilla developing shortly before discharge. The spore cytoplasm emerges slowly in an amoeboid fashion through a narrow pore at the tip of a papilla, rounds up and begins a gentle rocking motion as a flagellar extrusion begins and spore shape changes gradually from spherical to reniform. Flagellar attachment is lateral; zoospores are typical saprolegniaceous secondary zoospores measuring 8 x 12 µm. Active motility takes some 5-20 minutes to develop (dependent on temperature) and, at first, zoospores are slow and uncoordinated. At temperatures between 16 and 20°C, zoospores may continue to swim for at least 48 hours.
2.	Presumptive Diagnostic Methods for Crayfish Plague
	The first sign of a crayfish plague mortality may the presence of numbers of crayfish at large during daylight (crayfish are normally nocturnal), some of which may show evident loss of co-ordination in their movements, and easily fall over on their backs and are unable to right themselves. Often, however, unless waters are carefully observed, the first recognition that there is a problem will be the presence of large numbers of dead crayfish in a river or lake (3).
	In susceptible species where sufficient numbers of crayfish are present to allow infection to spread rapidly, particularly at summer water temperatures, infection will spread quickly and stretches of over 50 km may loose all their crayfish in under 21 days from first observed mortality. Crayfish plague has unparalleled severity of effect, infected susceptible crayfish do not survive - 100% mortality is the norm. Resistant North American species survive infection in many cases and then act as largely asymptomatic carriers, although under adverse conditions (stress, concurrent infections), mortality may occur.
	It must be emphasised, however, that presence of large numbers of dead crayfish, even in crayfish plague affected watersheds is not on its own sufficient. The general condition of other aquatic fauna must be assessed. Mortality or disappearance of other aquatic crustaceans as well as crayfish, even though fish survive, may indicate pollution (e.g. insecticides).
3.	Confirmatory Diagnostic Methods for Crayfish Plague
	Strictly, the identification of Oomycetes to genus depends on sporangial morphology and to species on the morphology of the sexual reproductive stages (oogonia and antheridia). Such sexual stages are absent in A. astaci so that identification is based on general morphology of isolates from crayfish involved in an outbreak of crayfish plague. As no other crayfish disease produces such swift and drastic mortalities this normally presents no practical diagnostic problem.
	Exposing susceptible crayfish (e.g. A. leptodactylus or A. pallipes) to zoospores produced by suspect isolates (see above) will result in characteristic rapid mortality (3) and with subsequent re-isolation of the fungus, give firm confirmation of crayfish plague. However, susceptible crayfish species should only be used for confirmation of diagnosis in exceptional circumstances since some are endangered species (Berne Convention) and populations may be protected under conservation legislation.
	No biochemical or molecular biological test methods yet exist that have been adequately validated for cross reaction with other species of Aphanomyces such as Aphanomyces invadens, aetiological agent of epizootic ulcerative syndrome (Chapter 2.1.10). Oidtmann et al. (4) have exploited the sequence differences between the A. astaci isolates, other Aphanomyces and non-related fungi at the 28s rRNA gene level to develop a PCR-based to detect the crayfish plague fungus. This work needs further validation and requires the pathogen to be isolated into culture since the method currently lacks the sensitivity required to be usefully applied to clinical samples. The areas that need attention are 1) The specificity and sensitivity of the primer set in the presence of crayfish tissue 2) and the confirmed ability to differentiate A. astaci in the presence of other Oomycetes in clinical samples.
	These results therefore represent a major development towards a practical molecular biological diagnostic method for A. astaci, but have not yet reached the stage at which they can be applied to clinical samples.

REFERENCES

1.	Alderman D.J. (1996). Geographical spread of bacterial and fungal diseases of crustaceans. (Paper given at the Office International des Epizooties [OIE] International Conference on Preventing the Spread of Aquatic Animal Diseases, 7-9 June 1995, OIE Headquarters, Paris, France.) Rev sci. tech. Off. int. Epiz., 15 (2), 603-632.
2.	Alderman D.J. & Polglase J.L. (1986). Aphanomyces astaci: isolation and culture. J. Fish Dis., 9, 367-379.
3.	Alderman D.J., Polglase J.L. & Frayling M. (1987). Aphanomyces astaci pathogenicity under laboratory and field conditions. J. Fish Dis., 10, 385-393.
4.	Oidtmann B., Bausewein S., Hotzle L., Hoffmann R. & Wittenbrink M. (2002). Identification of the crayfish plague fungus Aphanomyces astaci by polymerase chain reaction and restriction enzyme analysis. Vet. Microbiol., 85, 183-194.

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