Crayfish Plague (“Fungus” Disease)
Category 2 (In Canada and of Regional Concern)
Common, generally accepted names of the organism or disease agent
Crayfish Plague, Crayfish aphanomyciasis, La peste, Krebspest, Kraftpest.
Scientific name or taxonomic affiliation
Aphanomyces astaci in the order Saprolegniales of the Oomycetes. Oomycetes (commonly called water moulds and more closely related to brown algae and diatoms than to the true fungi, the Eumycota) are not considered to be ‘true fungi’ taxonomically, and have been placed in the phylum Oomycota, with A. astaci in the family Saprolegniaceae (Stephens et al. 2005, Buller 2008). The identification of Oomycetes to genus level depends on sporangial morphology, and identification to species level depends on the morphology of the sexual reproductive stages (antheridia, oogonia and oospores). However, sexual stages are absent in A. astaci. In addition to A. astaci, other species of Oomycetes and fungi are known to invade crayfish (Unestam 1973a, Ballesteros et al. 2007, Cammà et al. 2010). Royo et al. (2004) report on isolates of Aphanomyces sp. from crayfish (Pacifasticus leniusculus and Procambarus clarkii) in Spain and Italy that were not capable of killing susceptible crayfish (the Australian crayfish Cherax destructor and the European noble crayfish Astacus astacus) following standardised experimental infection, had RAPD-PCR and ITS sequences different from the A. astaci reference strains and unlike A. astaci did not express chitinase constitutively during growth or sporulation. However, like A. astaci, these isolates possessed repeated zoospore emergence and lacked sexual reproduction. Royo et al. (2004) proposed the tentative species name Aphanomyces repetans until the taxonomic status of the isolates is fully elucidated.
Ubiquitous in North America where native crayfish are resistant to the disease and prevalence of infections in some populations is believed as high as 50% (Holdich 1988). In Europe, the disease is believed to have originated in Lombardy, Italy in the 1860s following the introduction of American freshwater crayfish into local river systems. From there the disease spread through Europe (Alderman 1996). The post-1960s range expansions in Europe, including Spain, are largely linked to movements of North American crayfish introduced for purposes of crayfish farming and movement of contaminated fishing equipment without disinfection. Aphanomyces astaci gained entry into Britain in 1981, detected in Ireland in 1987 and also spread to Turkey, Czech Republic, Hungary, Greece and Norway during the 1980s (Alderman et al. 1996). It has caused high mortalities and even eliminated many native European stocks of crayfish (Marren 1986; Reynolds 1988; Rahe and Soylu 1989; Diéguez-Uribeondo et al. 1997b; Vennerström et al. 1998; Bohman et al. 2006; Diéguez-Uribeondo 2006; Kozubíková et al. 2006, 2007, 2008; Cammà et al. 2010, Vrålstad et al. 2011).
Astacus astacus, Austropotamobius pallipes, Austropotamobius torrentium and Astacus leptodactylus are examples of European species that are susceptible to the disease. Pacifasticus leniusculus, Procambarus clarkii and Orconectes limosus native to North America are carriers of the fungus and do not exhibit symptoms of the disease except in intensive culture. Based on molecular evidence, one population of apparently healthy A. astacus from Lake Mikitänjärvi, Kainuu, Finland, harboured subclinical crayfish plague infections (Jussila et al. 2011). The Chinese mitten crab, Eriocheir sinensis and Japanese crayfish (Cambaroides japonicus) have been experimentally infect in the laboratory (Stephens 2005). In addition, nine species of crayfish from different parts of Australia (including Cherax quadricarinatus, Cherax destructor and Astacopsis gouldi) and New Guinea (Cherax papuanus) tested in aquaria were all found susceptible to infection (Unestam 1975).
Impact on the host
Hyphae of A. astaci grow in the soft, non-calcified parts of the cuticle and tends to be restricted to the area of cuticle penetration in resistant crayfish. Hyphae can also grow along the ventral nerve cord and brain ganglion. However, growth can be sparse and may not be seen on histological examination. Occasionally, hyphae are seen in the eye but rarely in other organs, and they do not invade the musculature until late in the infection (Stephens et al. 2005). Hyphae extend into the water and produce motile zoospores which infect other crayfish. Defence-like reactions, including melanization (Unestam and Nylund 1972), may be evoked in resistant (North American) crayfish which can carry the A. astaci as a subclinical (latent, benign, chronic) infection in the cuticle. European species of crayfish tend to have little resistance and often die within a few weeks of exposure (Alderman et al. 1987, Reynolds 1988, Alderman et al. 1990). The first sign of a crayfish plague mortality may be the presence of crayfish at large during daylight hours (crayfish are normally nocturnal), some of which may show evident loss of coordination 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 (Alderman et al. 1987).
Swimming zoospores of A. astaci that emerge from the primary cysts preferentially encysted in the vicinity of superficial wounds in crayfish (Unestam and Weiss 1970). Crayfish appear to be more susceptible to crayfish plague at the time of moulting (Smith and Söderhäll 1986), possibly because the increased ease of injury to soft exoskeleton during this period may predispose crayfish to infection. Also, exudates obtained from walking legs of three species of crayfish (A. astacus, O. limosus, and P. leniusculus) were chemotactic for zoospores of A. astaci(Cerenius and Söderhäll 1984). After encystment, the resulting secondary cycts (spores) develop hyphae (germ tubes) that produce lytic enzymes (proteases, chitinases and esterases) that facilitate penetration into the crayfish cuticle (Söderhäll et al. 1987). In highly resistant crayfish (e.g., P. leniusculus) most zoospore cysts were encapsulated by melanin and killed, cell walls of the penetrating hyphae became heavily melanized, growth was visibly disturbed and sparse in the wound area. In susceptible crayfish (e.g., A. astacus) melanization was slower and wound reactions were less restrictive to hyphal growth in comparison to P. leniusculus. However, in both species, the hyphae grew profusely as soon as they were beyond the wound area and the preferential direction of hyphal growth was parallel to the chitin fibrils (Nyhlén and Unestam 1980). Söderhäll and Unestam (1979) reported that purified, high molecular weight, extracellular glycoproteins of A. astaci strongly activated crayfish (A. astacus) serum prophenoloxidase. Söderhäll and Ajaxon (1982) indicated that the phenol-phenoloxidase system generates toxic substances to certain fungi and A. astaci, and thus could be involved in defence reactions. Diéguez-Uribeondo and Cerenius (1998) reported that three different proteinase inhibitors (a 23 kDa inhibitor of subtilisin, a 155 kDa trypsin-inhibitor (pacifastin) and a α2-macroglobulin) purified from the haemolymph (blood) of P. leniusculus reduced proteolytic activities of A. astaci and thus may reduce the proteolytic breakdown exerted by A. astaci proteinases during an infection. Also, a two-domain Kazal-type serine proteinase inhibitor (KPI2) from the haemocytes of P. leniusculus was found to inhibit the extracellular serine proteinases from A. astaci (Donpudsa et al. 2010). Resistant crayfish (P. leniusculus) continuously produced high levels of prophenoloxidase (proPO) transcripts (this enzyme in its active form is responsible for the melanisation reactions by catalysing the oxidation of phenols to melanin) and these levels could not be further increased, whereas in susceptible crayfish (A. astacus) proPO transcript levels and resistance were augmented by immunostimulants (Cerenius et al. 2003). Haemolymph parameters were altered (glucose, lactate and Ca2+ increased; and Na+, K+ and Cl- decreased) in A. astacus infected with A. astaci but unaltered in infected P. leniusculus (Järvenpää et al. 1986). An A. astaci strain (Pc) isolated from warm water crayfish P. clarkii collected in Spain when compared to other isolates from cold water crayfish (A. astacus, A. leptodactylus, and P. leniusculus) was able to grow faster and release zoospores at higher temperatures and was genetically different in RAPD-PCR analysis (Diéguez-Uribeondo et al. 1995).
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 the first observed mortality. Upstream spread has been recorded at up to 1000 m per week and 17 km in 10 months (Taugbol and Skurdal 1993). Crayfish plague has unparalleled severity of effect. Infected susceptible crayfish do not survive 100% mortality is normal. However, experimental evidence suggests that previous exposure to sublethal numbers of A. astaci spores will increase the resistance of A. astacus to infection (Unestam and Weiss 1970). Resistant North American species survive infection in many cases and then act as asymptomatic carriers, although under adverse conditions (stress, concurrent infections with other pathogens, etc.), mortality may occur in normally resistant species (Thörnqvist and Söderhäll 1993, Diéguez-Uribeondo et al. 1997b, Diéguez-Uribeondo 2006). Apparently, A. astaci does not have any vectors or intermediate/secondary hosts, there is no resistant structures, and the spores have a limited viability outside the host.
Gross Observations: All infected crayfish do not have gross signs and those that do vary in appearance. In crayfish species native to Europe, the most consistent sign is focal white patches of musculature beneath the transparent areas of thin cuticle, especially of the ventral abdomen and in the periopod (limb) joints. Such foci can best be seen under a low power stereo microscope. In some cases a brown colouration of cuticle and muscle may occur and in others, hyphae are visible within infected cuticle in the form of fine brown (melanised) tracks. Buller (2008) provides two images of these signs of disease on page 3. 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. In the terminal stages of infection, animals may show a loss of the normal aversion to bright light (they are seen in open water in daylight), a loss of limb co-ordination (producing an effect that has been described as walking on stilts) and moribund animals often lose their balance and fall onto their backs before dying. Diagnosis requires isolation and identification of A. astaci by microscopic morphology. The identification and isolation of A. astaci is facilitated by stressing the crayfish to exacerbate the infection. This can be accomplished artificially by reducing the number of circulating blood cells for at least 6 hours by inoculation of non-self particles such as Zymosan (Persson and Söderhäll 1983, Persson et al. 1987, Cerenius et al. 1988). The prevalence of infection in P. leniusculus has been determined by examining the crayfish for brown spots (1 to 5 mm in diameter) with the spots on a small subsample of the spotted crayfish examined microscopically for hyphae (Nylund and Westman 1983). Aquiloni et al. (2011) used digital image analyses and image processing techniques to select micro-melanized areas in the subabdominal cuticle of subclinical P. clarkii in order to select areas to assay for A. astaci. However, the sign of brown spots / melanized areas are not pathogen specific and can be caused by mechanical lesions or invasion by other agents such as various species of fungi or bacteria (Persson and Söderhäll 1983).
Wet Mounts: Examine cuticle of moribund crayfish for hyphae. Melanised cuticle of North American crayfish may indicate focal infections of hyphae. In diseased crayfish, A. astaci can be found in most tissues but it is easier to detect within the soft (thin) abdominal cuticle. Excise abdominal cuticle or scrape the tissues of affected areas (melanized spots) and examine wet mount preparations for hyphae. Vegetative hyphae are aseptate, frequently branching and rather uniform in thickness (usually 7 to 9 µm in width but can range between 5 and 10 µm in width) but vegetative branches often tending to be somewhat narrower than the main hyphae for the first 20 to 30 µm of growth. Young, actively growing hyphae have coarsely granular cytoplasm with numerous highly refractile globules and have rounded ends. Older hyphae are largely vacuolate with the cytoplasm restricted to the periphery leaving only thin strands of protoplasm bridging the large central vacuole. The oldest hyphae are apparently devoid of contents. Note that the above morphological characteristics of the hyphae can not be used to differentiate A. astaci from other Oomycetes and many fungi.
Spore formation is usually absent except at later stages of the infection. Sporangia are myceloid (similar to the hyphae in shape), terminal or intercalary, and segmented from the undifferentiated hyphae by septa. Within the sporangia, elongate primary spores (16 to 25 µm long and 8 µm wide) are formed. As they discharge from the sporangium, the primary spores (about 15 to 30 per sporangium) become rounded (usually 9 to 11 µm in diameter) and form a cyst wall to become the primary cyst. The primary cysts adhere to each other in a cluster on the surface or tip of the sporangium. In some cases, the primary spores encyst within the sporangium. One zoospore emerges through a papilla (emergence tube) from each primary cyst at 20 °C. The zoospore is reniform in shape (12 µm long and 8 µm wide) and biflagellated with both flagella laterally attached at the same point. The zoospores actively swim for at least 48 hr at temperatures between 16 and 20 °C, then looses the flagella and encysts to form the secondary cyst which will either generate a hyphae in the presence of appropriate nutrients or undergo repeated emergence of a zoospore and encystment (Alderman and Polglase 1986, Cerenius et al. 1988).
Smears: Tissue scrapings from affected areas can be smeared on a glass slide, air dried and stained with Wrights-Giemsa stain or a commercial equivalent (e.g., Diff-Quick® or Hemacolor®). Examine stained air dried slides under the microscope for hyphae of A. astaci.
Histology: The presence of distinctive aseptate, wide hyphae (5 to 10 µm in width) may be observed in tissue section (preferably containing lesions found in the cuticle) that are stained with haematoxylin and eosin stain. Haemocytes that surround and encapsulate the hyphae, and become melanised can give the hyphae a knobbly appearance (Buller 2008). Stephens et al. (2005) indicated that Grocott’s modification of the Gomori methenamine-silver stain has the advantage of clearly distinguishing the black stained hyphae against a green background in tissues counterstained with a light green solution. Procedures for this stain were presented by Pintozzi (1978). A combined Grocott silver stain with haematoxylin and eosin used as a counter stain can improve the visualisation of the hyphae in the crayfish tissue (Buller 2008).
Electron Microscopy: Penetration of the soft cuticle of crayfish by A. astaci zoospores begins with the lysis of the lipid surface layer of the crayfish and the formation of a germ tube (infection peg) that penetrates through the epicuticle by histolytic activity combined with mechanical penetration. A hypha developed from the germ tube usually forms below the inner epicuticular surface and in the endocuticle. Hyphae grow preferentially parallel to the surface, occasionally perpendicular to it. Subsequently, the hyphal tips swell and some hyphae start to penetrate through the cuticle. Penetration of the cuticle of a resistant crayfish (P. leniusculus) was essentially identical to that in susceptible ones (A. astacus). However, in P. leniusculus, heavy melanisation and disorganisation of the protoplasma occurred in most hyphae following penetration of the cuticle (Nyhlén and Unestam 1975). A strain of A. astaci, which had lost the ability to produce zoospores in culture contained mycoplasma-like bodies in the hyphae (Heath and Unestam 1974). Nyhlén and Unestam (1978) described the walls of secondary cysts (spores resulting from encystment of zoospores) and the walls of germ tubes in germinating spores of A. astaci.
Culture: Diagnosis of crayfish plague requires the isolation and characterisation of A. astaci using simple mycological media (12.0 g agar; 1.0 g yeast extract; 5.0 g glucose; 10 mg oxolinic acid; 1000 ml natural water (from river or lake); fortified with antibiotics (4 international units/ml penicillin G (sterile) added after autoclaving and cooling to 40°C) to control bacterial contamination (Alderman and Polglase 1986). Isolation may only be successful before or within 12 hours of the death of infected crayfish. Excise small pieces of infected cuticle and muscle, transfer them to a Petri dish of sterile distilled water for extensive washing and further cutting into smaller pieces (1 to 2 mm²). With sterile instruments, aseptically place the small pieces on the surface of the medium. If no lesions are evident, sample muscle and cuticle for at least three sites in each animal (especially around the base of the walking legs close to the body and inside the thorax). A sterile glass ring may be placed around the inoculum to force hyphae emerging from the piece of the cuticle to grow within the agar. In order to deter bacterial growth, 0.5 ml of 0.05 % potassium tellurite can be added to the inoculum inside the glass ring (Diéguez-Uribeondo et al. 1997b). Incubate at 16°C for about 15 days. Note that fungi associated with crayfish may grow as contaminants in the cultures and could overwhelm A. astaci (Cerenius et al. 1988).
Growth of A. astaci is almost entirely within and on the surface of the agar but with no aerial hyphae. Colonies are colourless. Dimensions and appearance of hyphae are similar to that in crayfish tissue (see above). Because Aphanomyces spp. reproduce asexually, characteristics of the morphology of the sexual reproductive stages (oogonia and antheridia) used to identify other Oomycetes cannot be employed. Instead, the process of sporulation, where spores are produced to discharge from the hyphal tip and encyst before producing motile spores (zoospores) that swim away, is used to identify A astaci (Buller 2008). Spore formation does not occur in nutrient rich cultures. When actively growing thalli (oomycete bodies composed of hyphae) or portions of thalli are transferred to distilled or sterile lake water water (by cutting out a thin surface sliver of agar containing A. astaci so that a minimum amount of nutrient-containing agar is transferred), sporangia form readily in 20 to 30 hours at 16°C and 12 to 15 hours at 20°C. Note: a ratio of about 100 units sterile distilled water to 1 unit of A. astaci culture being transferred is required. Sporangia are myceloid, terminal or intercalary, and develop from undifferentiated vegetative hyphae. Terminal sporangia are simple, developing from new extramatrical hyphae. Intercalary sporangia are quite complex and develop by the growth of a new lateral extramatrical branch, which forms the discharge tube of the sporangium. The cytoplasm of developing discharge tubes is noticeably dense, and slightly wider (10 to 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. These 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 a sporangium. Primary spores (cytoplasmic units) are formed from the contents of the sporangium and are released (within 5 minutes) from the sporangial discharge tube and accumulate at this point. The spores become round and a cyst wall develops. Most spores remain as a cluster (15-30 spores) at the sporangial tip, but some encyst away from the sporangial tip. The number of spores in a cluster of A. astaci is generally less than that of other Aphanomyces species. The clusters are adherent and fairly resistant to physical disturbance, and spores will remain encysted for 8-12 hours. A reniform shaped, biflagellated zoospore (8 x 12 µm, motility takes 5-20 minutes to develop) emerges from each cyst and swims away leaving the empty capsules of the encysted spore (Buller 2008). Zoospores of A. astaci could germinate and grow in vitro on scales of salmon Salmo salari (Hall and Unestam 1980). Andersson and Cerenius (2002) indicated that chitinase (detected as enzymatic activity in the growth medium) and transcription of the chitinase gene AaChi1 is expressed at a high levels during vegetative growth of A. astaci without further stimulation by chitin in contrast to that of other Aphanomyces spp. tested which produce significant amounts of chitinase only in the presence of chitin. This pattern of chitinase expression could possibly be used as qualitative physiological characteristics to distinguish A. astaci from other parasitic and saprophytic species (Andersson and Cerenius 2002). In subsequent research, Hochwimmer et al. (2009) used chitinase family genes to develop molecular diagnostic assays. Further details pertaining to the culture of A. astaci and expected morphological appearance of the various developmental stages are presented in the World Organisation for Animal Health (OIE) diagnostic manual for crayfish plague (Chapter 2.2.1)
Note: A wide range of organisms can grow on the media when using the procedures described above. Aphanomyces astaci has a slow growth rate compared to other Oomycetes and fungi that can colonise crayfish cuticle and will grow on the media, also the presence of bacteria can inhibit A. astaci growth (Oidtmann et al. 2004, Cammà et al. 2010). Attempts to isolate A. astaci in culture from crayfish with light infections are often unsuccessful. If detection is made outside of the known distribution of crayfish plague, a positive diagnosis should be confirmed by the World Organisation for Animal Health (OIE) reference laboratory.
Bioassay: Exposing susceptible crayfish (e.g. A. leptodactylus or A. pallipes) to zoospores produced by suspect isolates will result in characteristic rapid mortality (Alderman et al. 1987). Subsequent re-isolation of the fungus gives firm confirmation of crayfish plague. However, susceptible crayfish species should only be used for confirmation of diagnosis if there is no infringement of the Berne Convention on endangered species and some crayfish populations may be protected under conservation legislation.
DNA Probes: Arbitrary primers and DNA polymerase chain reaction (PCR) techniques have been used to identify two main groups among A. astaci isolated from Sweden (Huang et al. 1994). Randomly amplified polymorphic DNA - polymerase chain reaction (RAPD-PCR) analysis showed that isolates of A. astaci from diseased A. pallipes in England belonged to one of the two groups (Lilley et al. 1997) and two different strains of A. astaci caused two crayfish plague epizootics in A. astacus in Finland (Vennerström et al. 1998). Diéguez-Uribeondo et al. (1995) used RAPD-PCR analysis to demonstrate that an isolate from the warm water crayfish, P. clarkii was genetically separated from all other described strains. RAPD-PCR analysis also indicated that isolates of A. astaci from two outbreaks of crayfish plague in southern Germany were closely related to a strain isolated from P. leniusculus from Lake Tahoe, California, USA (Oidtmann et al. 1999). Buller (2008) concluded that there were four genetic groups (genogroups) of A. astaci identified by RAPD-PCR: Group A (later called Astacus (As)) comprises strains isolated from the European crayfish species A. astacus and A. leptodactylus, and is believed to represent isolates that originated from an early introduction of this parasite into Europe; Group B (later called Pacifastacus I (PsI)) comprises strains isolated from crayfish species that originated in the USA, P. leniusculus, possibly from introduction of this crayfish from Lakes Tahoe and Hennessy, California into Sweden in 1969; Group C (later called Pacifastacus II (PsII)) consists of strains from P leniusculus of Canadian origin (Pitt Lake, British Columbia); and Group D (later called Procambarus (Pc)) comprises strains isolated from P. clarkii in Spain. Group A-C strains are from crayfish of cold-water origin (4-21 °C) and Group D are strains that originated from subtropical regions of the south east of the United States of America and better adapted to growing at temperatures of 20-26 °C. Makkoken et al. (2011) detected genetic polymorphism in the ribosomal Internal Transcribed Spacers (ITS) regions of A. astaci among different isolates (intraspecific polymorphism), and also among different clones of the same isolate (intragenomic polymorphism). The differences were inconsistent and they detected no specific markers for different crayfish plague isolates or groups. In addition, the intragenomic variation in four clones of one A. astaci isolate (named UEF8866-2) was higher than the intraspecific variation between different isolates. Thus, the identification of the different A. astaci groups will need to be based on some other genetic region (Makkoken et al. 2011). Recently, a fifth novel A. astaci genotype was isolated from the spiny-cheek crayfish O. limosus in the Czech Republic by comparing RAPD-PCR profiles with those of the four genetic groups indicated above (Kozubíková et al. 2011a).
Bangyeekhun et al. (2001) identified two serine proteinase genes, which encode subtilisin (AaSP1) and trypsin (AaSP2) enzymes from A. astaci and speculated that A. astaci may use the proteinases not only to gain nutrients but also to inhibit or demolish the host defence reactions. Primers developed to amplify a 1050 base pair segment of the 28 S rDNA region of Saprolegniales (Oomycetes) were used to distinguish A. astaci by the application of three restrictions enzymes to the amplicon (Oidtmann et al. 2002a). In addition, a PCR diagnostic procedure that specifically amplifies DNA from the internal transcribed spacer (ITS) region of A. astaci was capable of detecting infection in A. astacus two days after experimental exposure to spores (Oidtmann et al. 2004). Although this assay was found to be very sensitive when used to detect A astaci in paraffin-embedded sections because it produced a short amplicon of 115 base pairs (Buller 2008), it had the disadvantage of cross-reacting with Aphanomyces invadans and Aphanomyces frigidophilus the later of which was isolated from a crayfish plague-like case (Ballesteros et al. 2007). Employment of a PCR reaction with a different forward primer from the same DNA region indicated above and the same reverse primer was found to be more specific for A. astaci (Oidtmann et al. 2006, Cammà et al. 2010). Application of this assay indicated that the tailfan (i.e., uropods and telson) and soft abdominal cuticle of disease resistant North American crayfish species (O. limosus and P. leniusculus) from various locations in Europe more frequently had positive PCR reactions than cuticle from other body locations (Oidtmann et al. 2006, Vrålstad et al. 2011). However, the PCR reaction described by Oidtmann et al. (2006) was found to produce at least one in 16 false positives identified by sequencing the PCR products (Kozubíková et al. 2009). Because of the difficulty with the in vitro isolation of A. astaci for the detection of infection, the validation of molecular diagnostic assays would be beneficial in confirming the distribution and prevalence of A. astaci in crayfish populations (Edgerton et al. 2004, Vrålstad 2005). Vrålstad et al. (2009) developed a TaqMan® minor groove binder (MGB) real-time polymerase chain reaction (RT-PCR) method for quantitative and highly specific detection of A. astaci that targets a 59 base pair unique sequence motif of A. astaci found in the internal transcribed spacer 1 (ITS1) of the nuclear ribosomal gene cluster. This probe provides higher stringency, and consequently increased specificity, than conventional primers (Vrålstad et al. 2009). Additionally, the real-time PCR approach provides lower risk of laboratory-induced contamination (there is no further manipulation of PCR products after the reaction), increased sensitivity of agent detection, and quantitative results. However, a disadvantage of the real-time PCR assay is that its PCR product is not suitable for sequencing; thus, confirmation of the identity of the amplified fragment is not possible without conventional PCR. Hochwimmer et al. (2009) identified two novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases in A. astaci from which they developed two diagnostic methods: a TaqMan-probe based real-time PCR (qPCR) assay and a multiplex assay targeting multiple genes (the two novel genes CHI2 and CHI3, a third GH18 family member CHI1 and the 5.8S rRNA used as an endogenous control) detected by melting curve analysis.
Kozubíková et al. (2011b) compared the quantitative TaqMan® MGB RT-PCR (Vrålstad et al. 2009) with that of the conventional semi-nested PCR (Oidtmann et al. 2006) on DNA isolates from soft abdominal cuticle of 460 North American crayfish species (O. limosus and P. leniusculus) from central Europe and determined that the TaqMan® MGB RT-PCR approach seemed to provide higher sensitivity (32% versus 23% positive) with the vast majority of newly recorded positives contained very low agent levels. Kozubíková et al. (2011b) suggested that combining the two alternative methods may provide more reliable conclusions on the presence of the pathogen. Tuffs and Oditmann (2011) compared the analytical test sensitivity and specificity of a conventional PCR assay targeting the ITS region (Oidtmann et al. 2006) and two TaqMan® real time assays, targeting either the ITS region (Vrålstad et al. 2009) or the chitinase gene (Hochwimmer et al. 2009) and found all assays to be of good (real time assay targeting the chitinase gene) to excellent (real time assay targeting the ITS region) sensitivity and specific for A. astaci DNA from across all four genetic groups (as described above). None of the three assays cross-reacted with any other test organisms included in the study (Tuffs and Oditmann 2011). Strand et al. (2011) determined that the TaqMan® MGB RT-PCR (Vrålstad et al. 2009) was suitable to directly monitor for A. astaci in natural water bodies after bovine serum albumin or the TaqMan® Environmental Master Mix was added to mitigate co-extracted humic acids which significantly inhibited the detection of this ooymcete.
Note: The combined knowledge of disease history, histological studies, molecular studies (including combining the application of multiple PCR assays and sequence PCR products) and possibly isolating the Oomycetes from freshwater crayfish are key aspects for providing accurate diagnosis (Diéguez-Uribeondo et al. 2010, Tuffs and Oditmann 2011).
Methods of control
Stephens et al. (2005) described various procedures and approaches that could be used in the unfortunate event of A. astaci / crayfish plague detection in Australia and some of the procedures may be applicable to other parts of the world. Gherardi et al. (2011) presented a synthetic view of the different methods (mechanical removal, physical methods, biological control, biocides, and autocidal methods) to control non-indigenous crayfish species and discusses pitfalls and potentialities. However, control of the spread of A. astaci in a watershed after infected crayfish are detected at one location is impossible. Mycelium (hyphae) of A. astaci remain viable for at least 5 day in crayfish kept in water at 21°C after dying of crayfish plague (Oidtmann et al. 2002b). Also, cysts and zoospores of A. astaci can survive off the host in water for at least 14 days (Oidtmann et al. 2005). As few as 1.3 zoospores per millilitre of water can infect susceptible animals (Alderman et al. 1987). The movement of infected water between watersheds can transmit infection, as can contaminated equipment such as boots and fishing gear which were the suspect source of infection in Ireland (Reynolds 1988). Also, A. astaci was still viable and infective to crayfish (A. astacus) after passage through the gastrointestinal tract of fish (rainbow trout Oncorhynchus mykiss, common carp Cyprinus carpio, eel Anguilla anguilla and perch Perca fluviatilis) fed infected abdominal cuticle (Oidtmann et al. 2002b). However, A. astaciis unlikely to survive passage of the gastrointestinal tract of either mammals or birds because no viable stages were found after 12 hours at 37°C (Oditmann et al. 2002). Thorough drying of equipment (>24 hours) is effective for destroying contaminating A. astaci because Oomycetes, including the spore cysts, are not resistant to desiccation. Heating (boiling for 1 min) was the quickest way of decontaminating crayfish cadavers (Oidtmann et al. 2002b). Lilley and Inglis (1997) indicated that hydrogen peroxide and Proxitane 0510 (containing 5% peracetic acid in hydrogen peroxide) have some potential as fungicidal treatments and disinfection, respectively. Alderman and Polglase (1985) reported that various chemicals (e.g., sodium hypochlorite and iodophores) are effective against removing surface contamination of A. astaci providing that surfaces are clean of mud or organic debris in which A. astaci can survive for prolonged periods if kept moist. In addition, Stephens et al. (2005) presented various procedures for decontamination of equipment. Initial investigations indicated that A. astaci does not survive at –5°C and below for more than 24 hours and was not viable after freezing to -20°C for 2 hours. However, Oidtmann et al. (2002b) indicated that viable stages of A. astaci were still present after 48 hours at -20°C.
Rahe and Soyle (1989) reported that two lakes in Turkey exhibiting high Mg2+ contents and inverted Ca:Mg ratios continued to produce yields of crayfish (A. leptodactylus) in spite of cases of crayfish plague in the lakes. Other lakes in Turkey without this Mg2+ characteristic lost their crayfish population in less than two years (Rahe and Soyle 1989). In vitro studies indicated that sporulation of A. astaci in lake water was prevented by at least 20 mM of MgCl2 and mycelial growth in peptone-glucose agar was strongly reduced by 100 mM and completely prevented by 200 mM of MgCl2. However, at least 100 mM of MgCl2 was required to protect crayfish from infection by zoospores and infected crayfish transferred to 25 mM or more of MgCl2 maintained active infections but no sporangia were produced and the disease was not transmitted to uninfected A. astacus (Rantamäki et al. 1992).
In North American, crayfish species are resistant to the disease, but may be carriers. Losses may occur if stocks are crowded or debilitated. Stocks should be examined before transfer between farms or release to the wild. However, infection is difficult to detect in most North American crayfish which are usually asymptomatic. There is concern that American species of crayfish may now be susceptible to disease if exposed to European strains of A. astaci and caution should be exercised if crayfish stocks from Europe are proposed for re-entry into North America.
In Europe, transmission has been linked to the movements of North American crayfish for purposes of crayfish farming and also resulted from the use of contaminated crayfish traps and other equipment (Alderman et al. 1990; Diéguez-Uribeondo 2006; Kozubíková et al. 2009, 2010). In addition, Oidtmann et al. (2005) indicated that the movement of live fish is an important route of spread for crayfish plague. Norway implemented regulations to prevent the spread of crayfish plague that proved unsuccessful (Johnsen 2007, Vrålstad et al. 2011) and field data suggests that A. astaci can survive for may years in low density population (i.e., a latent reservoir) but flourish again when the crayfish population increases (Taugbol and Skurdal 1993). However, some evidence suggests that crayfish populations in small lakes or ponds can recover from crayfish plague if infected crayfish are eradicated (100% mortality from the disease), the site left “fallow” for some months followed by restocking with disease-free crayfish (Smith and Söderhä11 1986). Prior to any reintroduction activities, the absence of A. astaci should be tested by long-term exposure of caged susceptible crayfish in the watercourses, although this cannot completely rule out the unnoticed presence of A. astaci carriers (Taugbøl et al. 1993, Diéguez-Uribeondo et al. 1997a, Kozubíková et al. 2008). Nevertheless, crayfish plague may have a significant negative impact on the successful restoration of indigenous crayfish populations in central Europe (Kozubíková et al. 2007).
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Date last revised: January 2012
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