DISEASES OF AQUATIC ORGANISMS Vol. 36: 77-80.1999 Published April 15 Dis Aquat Org l NOTE Description of a hepatopancreatic rickettsia-like organism in the redclaw crayfish Cherax quadricarinatus Brett F. Edgerton',', Howard C. prior2 'Department of Microbiology and Immunology. James Cook University, Townsville, Queensland 4810, Australia '~nimalResearch Institute, Queensland Department of Primary Industries, 665 Fairfield Rd, Yeerongpilly. Queensland 4105, Australia ABSTRACT. Basovhilic, Gram-neaative,U nucrocolonies of rick- Materials and methods. The moribund Cherax ettsia-like organisms were observed in the hepatopancreatic quadricarinatus was collected from a pond bank at a tubule epithel~umof 1 moribund Cherax quadncarinatus from farm in north Queensland during an investigation of a farm in north Queensland. The tissue tropism of the organ- (Edgerton et lgg5).The ism is dlstlnct from the systemic rickettsla-like orqanlsmU pre- v~ouslydescnbed from C. quadncar~natus.This paper br~efly crayfish was sacrificed and fixed immediately in describes the histopathology, cytopathology and baslc mor- Bouin's fixative (Drury & Wallington 1967). Dissected phology of the hepatopancreatic nckettsia-llke organism tissues were processed routinely for histology includ- ing haematoxylin and eosin staining of sections. Spe- KEY WORDS: Cherax quadricarinatus . Redclaw freshwater crayfish . Rickettsia-llke organism . Disease . Pathology cial 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 Rickettsia-like organisms (RLO) infect a range of method adapted from Bhatnagar et al. (1977). Briefly, aquahc crustaceans mcludmg amphpods, crabs, prawns lesions were located on the paraffin section. Excess and freshwater crayfish (Federici et al. 1974, Bonami & tissue was removed, and the remainder was de- Pappalardo 1980, Johnson 1984, Sparks et al. 1985, paraffinised, rehydrated, refixed in glutaraldehyde, Brock et al. 1986, Anderson et al. 1987, Krol et al. 1991, postfixed in osmium tetroxide and dehydrated in a Ketterer et al. 1992, Owens et al. 1992, Bower et al. graduated acetone series. Infiltration with increasing 1996).The decapod-infecting RLOs are either confined concentrations of Spurr's resin was carried out on the to the hepatopancreatic tubule epithelium, or are slide, using a short section cut from a plastic pipette to systemic. The systemic RLOs never have been ob- create a dam of resin over the section. After overnight served to infect endodermal enteric tissues. The RLO polymerisation at 60°C the plastic dam was cut away, infecting the redclaw freshwater crayfish Cherax the slide gently warmed and the resin stub with quadricarinatus, and originally described by Ketterer attached section eased off the glass slide. Ultrathin et al. (1992) and Owens et al. (1992),is in the systemic sections stained with uranyl acetate and lead citrate category. A RLO was observed exclusively in the were examined with a Philips CM 10 TEM. hepatopancreatic tubule epithelium of one moribund Results. Basophilic inclusions were observed in the C. quadricarinatus during an investigation of chronic cytoplasm of hepatopancreatocytes in adjacent tubules. low-grade mortality at a farm in north Queensland. Nearly all hepatopancreatocytes in cross-sectioned Histopathology, cytopathology and basic morphology tubules contained the inclusions (Fig. 1). The inclu- of this organism are reported here. sions were Gram-negative, moderately PAS positive, intensely phloxophilic, and often had a clear central zone containing faint eosinophilic material. Intracyto- 'Present address: Animal Quarantine Policy Branch, Aus- tralian Quarantine and Inspection Service, GPO Box 858, plasmic, Gram-negative microcolonies were not ob- Canberra, ACT 2601, Australia. served in any other tissues. Sectioning for special E-mail: [email protected] stains went beyond the focus of infection, and the 0 Inter-Research 1999 Resale of full art~clenot permitted 78 Dis Aquat Org 36: 77-80, 1999 Fig. 1. Light micrographs of basophilic, Gram-negative inclusions (arrows) in the cytoplasm of he,,,~pancreatocytes in Cherax quadricarinatus H&E. (A)I Almost all hepatopancreatocytes in the tubules are infected. Scale bar = 42 pm. (B) Inclusions often contain cleared areas. Scale bar = l0 pm lesions were not observed in the remaining hepato- this report the RLO was observed only in hepato- pancreatic tissues stored in Bouin's fixative. The cray- pancreatocytes. This distinct differentiation of tissue fish also had early stage eye necrosis (Edgerton et al. tropism is consistent with other crustacean-infecting 1995), bacteraemia, and moderate Cherax quadricari- RLOs (Bower et al. 1996). Furthermore, the cytopathol- natus bacilliform virus infection. ogy also differed in that the systemic rickettsia-like Electron microscopy indicated that the cytoplasmic microcolonies do not usually contain central clear inclusions were microcolonies of a RLO within mem- zones as was observed with this hepatopancreatic brane-bounded vacuoles (Fig. 2). The RLO had both a RLO. It therefore appears that at least 2 distinct RLOs cell wall and plasma membrane and was pleomorphic, infect C. quadricannatus; one a systemic agent, and with rounded forms more common than rod-shaped the other exclusively infecting hepatopancreatocytes. forms. The RLO ranged in length from 0.2 to 0.4 pm but The fact that the hepatopancreatic RLO has been further morphological and cytopathological descrip- observed in only 1 Cherax quadricarinatus may sug- tions were not possible due to suboptimal fixation for gest that it is relatively rare. Farming of C. quadrican- electron microscopy. natus commenced approximately 10 yr ago in Australia Discussion. The basophilic inclusions were similar and the industry is still in its infancy. C. quadricarina- to rickettsia-like microcolonies which have been ob- tus aquaculture in Australia is unsophisticated, and served systemically in Cherax quadricannatus (Ket- many farmers are hobbyists. Low numbers of sick cray- terer et al. 1992, Owens et al. 1992), and which were fish are detected by most farmers, but are not reported observed in approximately one third of moribund cray- as they are considered to be normal or insignificant. fish examined in the study of the chronic mortality at Survival as low as 30% is not uncommon in some this farm (Edgerton et al. 1995, Edgerton 1996). The ponds without obvious explanation; however, disease same, or very similar, systemic RLO has been associ- is rarely considered as the cause. Research on disease ated with mortality in C. quadricarinatus farms in remains a low priority as there have not been major Ecuador (Jimenez & Romero 1997). The systemic RLO widespread epizootics reported in the industry to this infects the spongy connective tissues, neural tissues, point. Edgerton (1996) conducted histopathological nephridial canal epithelium, myocardium, cuticular surveys on farmed C. quadricarinatus in north Queens- epithelium, haemopoietic and mandibular organ cells land to determine the prevalence of potential path- (Edgerton 1996).The systemic RLO in C. quadncarina- ogens. However, the examination of 32 moribund cray- tus has never been observed in endodermal enteric fish during the chronic mortality which occurred at one tissues even in very intense fulminating infections of the surveyed farms (Edgerton et al. 1995, Edgerton (Edgerton et al. 1995, Edgerton 1996, unpubl. data). In 1996) revealed a high incidence for some pathogens Edgerton & Prior: Hepatopancreatic rickettsia in Cherax quadricarinatus 79 Fig. 2. Electron micrographs of rickettsia-like microorganisms in Cherax quadricarinatus hepatopancreatocytes. Uranyl acetate and lead citrate. (A) Several rickettsia-like microcolonies in cytoplasmic vacuoles. Scale bar = 3 pm. (B) Rickettsia-like microorganisms had both a cell wall and a plasma membrane and were pleomorphic, with rounded forms most common. Scale bar = 128 nm 80 Dis Aquat Org which were not detected in the survey, such as the Brock JA, Nakagabv LK, Hayash1 T, Teruya S, Van Campen systemic RLO. Closer surveillance of ponds, and sub- H (1986) Hepatopancreatic rickettsial infection of the mission of sick animals for disease diagnosis, will penaeid shrimp, Penaeus marginatus (Randall), from Hawaii. J Fish Dis 9:73-77 improve knowledge of diseases in cultured C. quadri- Culling CFA, Allison RT, Barr WT (1985) Cellular pathology cannatus. To this point, there have been insufficient techniques, 4th edn. Butterworths, London studies done to determine the full significance of Drury RAB, Wallington EA (1967) Carleton's histological tech- pathogens in C. quadricannatus aquaculture, and fur- nique. Oxford University Press, New York Edgerton BF (1996)Viruses of freshwater crayfish. PhD thesis, ther studies may show that the hepatopancreatic RLO Department of Biomedical and Tropical Veterinary Sci- is more common than these initial studies suggest. ences, James Cook University, Townsville Edgerton B, Owens L. Harris L. Thomas A. Wingfield M (1995) A health survey of farmed redclaw crayfish Cherax Acknowledgements. This research was partly funded by an quadricannatus (Von Martens), in tropical Australia. Australian Research Council grant (A19332302), and was Freshw Crayfish 10:322-338 partly conducted during a PhD research programme by B.F.E. Federici BA. Hazard EI, Anthony DW (1974) Rickettsia-like