Control and Detection of Microsporidiosis in Freshwater Crayfish ].S. Langdon & T. Thorne FISHERIES DEPARTMENT OF WESTERN AUSTRALIA FISHERIES RESEARCH & DEVELOPMENT CORPORATION Project 90/78 C"") 0 :::::::s ,_.. ...., 0- Q) :::::::s -· :::J c.. 4* c CD CD en a: =::r :e S. m 0 n; :::::::s ...., 0. 0 ...., 3:: m -· (") � ...., en 0 =::r en "'C 0 ......: -· c.. -· 0 en -· en ].S. Langdon & T. Thorne FISHERIES DEPARTMENT OF WESTERN AUSTRALIA FISHER IfS RfSEARCH & DEVELOPMENT CORPORATION Project 90/78 Control anddetection of microsporidiosis in freshwater crayfish Reference No : 90/78 Fish Health Section FisheriesDepartment of Western Australia c/o Animal Health Laboratories WA Department of Agriculture Baron-Hay Court SouthPerth WA 6151 Fishing IndustryResearch and Development Council SUMMARY Microsporidiosisis a disease of freshwatercrayfish and other crustacean species that has been noted as a significantproblem in the farming of these animals. The condition is caused by a spore-formingprotozoan parasite that has become knownas "cotton-tail"or "porcelain" disease. The parasitecan infiltrate muscle tissue resulting in fleshspoilage andin advanced cases, may lead to death. Prevention, control anddetection o f the disease has been difficultto achieve, due to a lack of information on the parasite'slife-cycle. This studywas conducted to evaluatethe methods of transmission of the parasite, ways of breaking the life-cycle, to evaluate the use of chemicals to prevent transmission andto evaluatedetection methods for microsporidiosis. Results generated as a consequence of this project are listedbelow: - As a consequence of this project, a veryeffective, highly sensitive spore concentration method was developed. The technique has a predictivevalue of>99% andwill prove usefulto Fish Health Laboratories for detecting microsporidiosis in crayfishdestined for translocation. Experimental transmission of the WesternAustralian microsporidian Vavraia parastacidaby variousmethods, to marron, Cheraxtenuimanus, yabbies, Cheraxalbidus, redclaw Cheraxquadricarinatus and gilgies, Cheraxquinquecarinatus was attempted withthis study. Direct feeding of infectedmuscle resulted in transmission of the disease to juveniles of marron, yabbies and redclaw, although marron andgilgies may be refractoryto infectionafter they reach maturity. Ingeneral, yabbies appear to be more susceptible andshowed a much more rapid and severe progress of the disease than marron, with lesions visible after2 months anddeath after4- 6 months. This rapid progress could be explainedby their statusas novel hosts, andmay alsoexplain why natural infections have not yet been detected. It cannot be emphasised strongly enough that the susceptibilityof yabbies, originallyfrom Victoria, to a novel microsporidianfrom W A, may be no less than the susceptibilityof WA marronto Thelohania fromyabbies in the east. Fumagillin, the anti-microsporidial drugtested in this project, did not prevent or ameliorateinfections in eitheJ marron or yabbies and as the unregistered drug was ineffective, its use is not recommended. Generalopinion is now held that as few, ifany, drugs of this nature are registered for use in animalsdestined for human consumption their use could be detrimentalto the freshwatercrayfish industry as a whole. Results on the life-cycle of the parasite and the need for an intermediatehost were inconclusive and varied withthe species. No transmission was obtained using a series of possible intermediate hosts, and the effect of priming chemicals on the spores produced varied results. BACKGROUND The fanning of the Australianfreshwater crayfish yabbies ( Cherax albidus), marron (Cherax tenuimanus) andredclaw (Cherax quadricarinatus) is a smallbut rapidly expanding industryover most statesof Australia. Microsporidiosis is a disease of freshwatercrayfish that has been noted as a significantproblem in the farmingof these animals, althoughthe industryis relatively freeof other diseases of major concern. Microsporidiosisis commonly known as "cotton-tail"or "porcelaindisease" andis caused by the microsporidian protozoans 1helohania, Pleistophora andrelated genera. 1helohania and 1helohania-like species occur in yabbiesin South Australia(P. O'Donoghue pers. comm.), in Victoria andNew South Wales(Carstairs 1978), gilgies(Cherax quinquecarinatu.s) in WesternAustralia (L. Evans pers. comm.)and redclaw marron in Queensland(Herbert 1988). A Pleistophora-like species has been reportedfrom yabbies from South Australia(O'Donoghue et al 1990), and we have encountered and describeda new microsporidianspecies, Vavraia parastacida, in marron and gilgies andin redclaw imported fromQueensland (Langdon1991a, 1991b). Currently, thelohaniasisin yabbies is the only microsporidiosis causing largelosses of freshwatercrayfish. The infection occurs predominantly inlocomotor, cardiac andgastro­ intestinal striated muscle resulting in fleshspoilage andeventually death (Carstairs1978; Herbert1988). Va vraiaparasticida in marron causes progressiveinfiltration andinfl ammation of locomotor andgastric muscle, and the gastric lesions appear to be the cause of death in advancedcases. No effectivetreatments are available for microsporidiosis anddevelopment of any treatment or control measures has been hampered by a lack of information on the parasite's life­ cycle. Most workers consider direct transmission between crayfish to be unlikely. Herbert (1988) was unable to induce transmissionof a 1helohania species in redclaw by feeding the spores directlyor viafish. Iverson andKelly (1978) showed that passage of 1helohania spores through the gut of fishprimed them for subsequent infectionof prawns, but other routes of infectionwere not completely excluded. It is possible to primespores of some insect microsporidians andmake them infectiousby treatment withchemicals such as potassium chloride, potassium hydroxide andhydrogen peroxide (Sweeney, Hazard and Graham 1985). Evidence does suggest that invertebrateintermediate hosts, such assimuliid blackflylarvae, can play a role in transmissionof microsporidiosisto crayfish (Chartier andChaisemartin 1982). Control of microsporidiosis by the use of chemicals such as monensin, benomyl and fumagillin have been investigated andappear to be effective in suppressingthe pre-spore stages of microsporidian development (Overstreet 1988). Although these chemicalsare not registered for use in food animals, they may be useful insuppression of vegetative stages in broodstock, allowingproduction of disease-freeprogeny. Earlymicrosporidian infections are hardto detect incrayfish, before development of a visiblelesion. For health certificationand effective control programmes there is a need to increasethe efficiency and level of confidence required to reliably detect the disease. We have developed a spore concentration method that greatly increasesthe chance of detecting sub­ clinical infections in gills and muscles of crayfish. NEED 1. As verylittle is knownof the life-cycleof microsporidians in general, it would be of greatbenefit to crayfishfarmers to betterunderstand the mode of transmission of the diseasein the attempt to control andprevent the spread. IdentifYingintermediate or priminghosts would aidin the prevention of the disease, by excluding such vectors from crayfishfarms. Although chemotherapy appears to be of little use in established infections, it may be of benefit in suppressing the pre-spore stages of microsporidiandevelopment. 2. Detection of microsporidosis is difficultin earlyinfections before visible lesions occur, but vitalto effective control programmes andhealth certification of disease-free stocks for use by farmers. So an effective, highlypredictive techniqueis required to detect this disease. OBJECTIVES 1. To determinethe modes of transmission for microsporidiosis affecting freshwater crayfish. These, and previous studies (Langdonand Thome 1992) show that Vavraia is effectivelyand directly transmittedby ingestion of infected muscle, andthat both marron and yabbies can be infected by this route. This is not surprisinggiven the commonoccurrence of cannibalismin crayfishpopulations. As crayfishare probably the only hosts involved, movement of infected animalsis likelyto result in the spread of disease to the site of introduction. 2. To evaluatethe use of chemicalsto prevent transmissionof criticallife-cycle stages. Experimentsinvolving fuma gillin treatment of the parasite, by feeding infectedmuscle or pellets, soaked in fumagillin, did not prevent infection. Similarly, adult infected marron show no amelioration of established infection when fed fumagillindirectly. It appears therefore, that fumagillinis of no use as an anti-microsporidial drugand as the drug is not registeredfor use on animalsdestined for human consumption, its use would not be recommended. 3. To formulate methods for breaking the life-cycles of microsporidians on crayfishfarms. Direct transmission appearedto be the most likely means of infectionin thisstudy and this may be minimised by provision of shelter for freshly moulted and young crayfishto prevent cannibalism. As the results on determining intermediateor priming hosts were inconclusive, methods to prevent cannibalism andculling of infected stock appear to be the onlyeffective method of breaking the life-cycleof this disease at thisstage. 4. To evaluate detectionmethods for microsporidiosis. A highlyeffective spore concentration method (> 99% predictive value) has been developed and reported in Langdon (1991a), Langdon and Thome (1992), anddetails are