Disease agents and pests associated with freshwater crayfish fall into six main categories-viruses, bacteria, rickettsia-like organisms (RLOs), fungi, protists, and metazoans. Data and information on specific disease agents and pests from each of these categories are presented in this synopsis. Each agent or group of agents is considered under the following headings-condition, causative agent(s), life cycle/life history, epizootiology, pathology, pathogen viability. Information for the synopsis was obtained from the published literature and from personal contact with internationally recognized experts in freshwater crayfish aquaculture, biology, and disease. Data of relevance for import risk analysis are summarized.Import risk analysis is the process by which the risks associated with importation of animals and plants, and products derived from animals and plants, are assessed and managed. Hazard identification is essential and is the first component of an import risk analysis. In 1996, the Australian Quarantine and Inspection Service (AQIS) commenced a review of policy relating to the importation of nonviable freshwater crayfish products, along with a suite of other aquatic animal products. AQIS2 commissioned a synopsis of freshwater crayfish pests and pathogens for use as a resource document for hazard identification in the formal IRA process.

Native European freshwater crayfish (Astacida, Decapoda) are under severe pressure from habitat alteration, the introduction of nonindigenous species, and epizootic disease. Crayfish plague, an acute disease of freshwater crayfish caused by the fungus-like agent Aphanomyces astaci, was introduced into Europe in the mid-nineteenth century and is responsible for ongoing widespread epizootic mortality in native European populations. We reviewed recent developments and current practices in the field of crayfish pathology. The severity of crayfish plague has resulted in an overemphasis on it. Diagnostic methods for detecting fungi and fungal-like agents, and sometimes culturing them, are frequently the sole techniques used to investigate disease outbreaks in European freshwater crayfish. Consequently, the causes of a significant proportion of outbreaks are undetermined. Pathogen groups well known for causing disease in other crustaceans, such as viruses and rickettsia-like organisms, are poorly understood or unknown in European freshwater crayfish. Moreover, the pathogenic significance of some long-known pathogens of European freshwater crayfish remains obscure. For effective management of this culturally significant and threatened resource, there is an urgent need for researchers, diagnosticians, and resource managers to address the issue of disease in European freshwater crayfish from a broader perspective than has been applied previously. Entendiendo las Causas de Enfermedad en Cangrejos Europeos de Agua DulceResumen: Los langostinos nativos de Europa (Astacida, Decapada) están bajo severa presión por alteración del hábitat, la introducción de especies no nativas y una enfermedad epizoótica. La peste de langostinos, una enfermedad aguda de langostinos de agua dulce producida por el agente micoide Aphanomyces astaci, fue introducida a Europa a mediados del siglo diecinueve y es responsable de la actual mortalidad epizoótica de poblaciones Europeas nativas. Revisamos acontecimientos recientes y prácticas actuales en el campo de la patología de langostinos. La severidad de la peste de langostinos ha resultado en un excesivoénfasis en ella. Los métodos para diagnosticar, y algunas veces cultivar, hongos y agentes micoides frecuentemente son laúnica técnica empleada al investigar brotes de la enfermedad en langostinos de agua dulce en Europa. Consecuentemente, no están determinadas las causas de una proporción significativa de los brotes. Grupos patógenos, como virus y organismos similares a rickettsias, bien conocidos por producir enfermedades en

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The redclaw freshwater crayfish Cherax quadricarinatus has a reputation for being hardy and resistant to handling stress. However, in recent years, possibly since 1996, C. quadricarinatus farmers in northern Queensland have noted a decrease in stress resistance in their stock. A presumptive reovirus in the hepatopancreas, and a putative p a~o v i r u s in the gills, were associated with chronic mortalities in C. quadncarinatus at one northern Queensland farm. Hypertrophic nuclei with marginated chromatin were observed in gill epithelium in moribund crayfish which had recently been relocated to a laboratory from the holding tank fachty on the farm. Affected nuclei appeared to be vacant or contained a faint granular basophilia in H&E stained sections. However, toluidine blue staining revealed a homogenously granular appearance of the nuclei. Transmission electron microscopy revealed approximately 20 nm diameter virus-like particles within the nucleus. Eosinophilic, Feulgennegative, cytoplasmic inclusions were observed in distal hepatopancreatocytes in 1 moribund C. quadricarinatus collected from the same on-farm holding tank approximately 6 mo later. This crayfish did not display the gill lesions. Transmission electron microscopy showed that the inclusions contained icosahedral virus particles 35 to 4 0 nm in diameter. The histopathology and preliminary virus morphology of the presumptive hepatopancreatic reovirus, and the histopathology, ultrastructural pathology and preliminary virus morphology of the putative gill pawovirus, are reported herein.

A unique hepatopancreocyte nuclear pathology was seen during a histological survey of farmed crayfish Cherax quadncannatus Later, the pathology was associated with mortalities in laboratory-held juvenlle populations Affected nuclel were vanably hypertrophic, had marginated and clumped chromatin and were almost fully occupied by numerous amorphous inclusions in late infections Well-developed inclusions were intensely purple-red whllst less-developed inclusions were eosinophilic with H&E The nucleolus remained identifiable and centrally located Embryonic (E) cells were the only hepatopancreocyte type that did not display the changes Crayfish from all 7 farms surveyed were infected, wlth a maximum prevalence of 52 3 % The intensity was extremely low in adult crayfish The inclusions were present in 10 of 14 crayfish families used in a hentability study Mortalities vaned greatly with the highest mortality being 85 % by Week 8 Monbund crayfish consistently displayed a hlgh intensity of affected nuclei Affected nuclei were Feulgen and methyl-green negatlve pyronin positive and fluoresced yellow-green with acndine orange, suggesting the pathology was associated with a double-stranded RNA virus Nuclease digestion of the histological matenal confirmed the predominance of dsRNA in the inclusions Transmission electron microscopy revealed 25 nm vinons in highly ordered intranuclear paracrystalline arrays, a feature apparently unique amongst the RNA viruses The virus is morphologically and morphogenically simllar to Glardia lambha virus and is therefore named Cherax G~ardlavlrus-like virus

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ABSTRACT. Basovhilic, Gram-neaative, nucrocolonies of rick- Rickettsia-like organisms (RLO) infect a range of aquahc crustaceans mcludmg amphpods, crabs, prawns and freshwater crayfish (Federici et al. 1974, Bonami & Pappalardo 1980, Johnson 1984, Sparks et al. 1985, Brock et al. 1986, Anderson et al. 1987, Krol et al. 1991, Ketterer et al. 1992, Owens et al. 1992, Bower et al. 1996. The decapod-infecting RLOs are either confined to the hepatopancreatic tubule epithelium, or are systemic. The systemic RLOs never have been observed to infect endodermal enteric tissues. The RLO infecting the redclaw freshwater crayfish Cherax quadricarinatus, and originally described by Ketterer et al. (1992) and Owens et al. (1992), is in the systemic category. A RLO was observed exclusively in the hepatopancreatic tubule epithelium of one moribund C. quadricarinatus during a n investigation of chronic low-grade mortality at a farm in north Queensland. Histopathology, cytopathology and basic morphology of this organism are reported here. Materials and methods. The moribund Cherax'Present address: Animal Quarantine Policy Branch, Australian Quarantine and Inspection Service, GPO Box 858, Canberra, ACT 2601, Australia. E-mail: brett.edgerton@aqis.gov.au ing haematoxylin and eosin staining of sections. Special stains (Culling et al. 1985) included Brown and Brenn Gram, phloxine and tartrazine, and Periodic Acid Schiff (PAS).Unstained, 5 pm thick sections of paraffin embedded tissues were processed for electron microscopy by a method adapted from Bhatnagar et al. (1977). Briefly, lesions were located on the paraffin section. Excess tissue was removed, and the remainder was deparaffinised, rehydrated, refixed in glutaraldehyde, postfixed in osmium tetroxide and dehydrated in a graduated acetone series. Infiltration with increasing concentrations of Spurr's resin was carried out on the slide, using a short section cut from a plastic pipette to create a dam of resin over the section. After overnight polymerisation at 60°C the plastic dam was cut away, the slide gently warmed and the resin stub with attached section eased off the glass slide. Ultrathin sections stained with uranyl acetate and lead citrate were examined with a Philips CM 10 TEM.Results. Basophilic inclusions were observed in the cytoplasm of hepatopancreatocytes in adjacent tubules. Nearly all hepatopancreatocytes in cross-sectioned tubules contained the inclusions (Fig. 1). The inclusions were Gram-negative, moderately PAS positive, intensely phloxophilic, and often had a clear central zone containing faint eosinophilic material. Intracytoplasmic, Gram-negative microcolonies were not observed in any other tissues. Sectioning for special stains went beyond the focus of infection, and the 0 Inter-Research 1999Resale of full a r t~c l e not permitted

ABSTRACT. lntranuclear lesions were detected in hepatopancreocytes of Cherax destructor by light microscopy. The les~ons included nuclear hypertrophy, chromatin margination, a peripherally displaced nucleolus, or nucleolar remnants remaining in the centre of a granular eosinophilic inclusion. The cytoplasm of some infected cells was granular and basophilic. The intensity of the lesions was low in all cases; however, infected nuclei were sloughed from the epithelium. Rod-shaped virions within a granular viroplasm were visualised in these nuclei by electron microscopy. The virions contained a nucleocapsid, consisting of a n electron-dense nucleoprotein core and less electron-dense capsid, and a trilaminar envelope. The envelope was closely applied, except that at one end it was laterally expanded to contain a tail-like structure that arose from the nucleocapsld. The nucleocapsid was often bent in this region. Capsids formed unassociated with membranes. A capsid which was partially filled with nucleoprotein was elongated and improperly enveloped. Virions were 68 X 304 nm and nucleocapsids were 49 X 263 nm. Virions of Cherax quadricarinatus bacilliform virus cons~sted of a rod.-shaped nucleocapsid surrounded by a loosely applied trilaminar envelope. A nucleocapsld tail-like structure was visible in several virions. Capslds appeared to form inside of membrane bubbles and capsids that were partially filled with nucleoprote~n had a central filament. Vlrlons were 103 X 262 nm and nucleocapsids were 54 X 212 nm. The morphology and development of both viruses is compared to other arthropod bacill~form viruses.