The identification of parasites in fish tissue sections

Transcription

1 Bull. Eur. Ass. Fish Pathol., 22(2) 2002, 173 The identification of parasites in fish tissue sections B. Nowak 1, D.G. Elliott 2 and D.W. Bruno 3 1 School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute, Aquafin CRC, University of Tasmania, Locked Bag 1-370, Launceston, Tasmania 7250, Australia. 2 Western Fish Research Center, USGS/BRD 6505 N.E. 65th Street, Seattle, WA 98115, USA. 3 FRS Marine Laboratory, Victoria Road, Torry, Aberdeen AB11 9DB, Scotland The identification of parasites in tissue sections is often difficult and fresh specimens are always required for accurate taxonomical classification. However, histological sections are in many cases the only material available and the identification of parasites in sections the subject of the recent histopathology workshop held in Dublin. The group reviewed the characteristics of representative protozoan and metazoan parasites in sections and identified key features to aid identification. A CD from the workshop will be compiled as a reference source. Several factors contribute to the success or failure of identification of parasites in histological sections. The habitat (e.g. fresh or marine, tropical or temperate waters) and the host fish species can provide clues to the identity of the parasite. Furthermore, some parasites are species-specific whereas others infect a wide range of host species. The location of the parasite in or on the host is also important, as some parasites are found only in selective organs or tissues. Although the size of the parasite can be crucial for identification, shrinkage of parasites can occur after fixation, so that sizes taken from fresh specimens may not be applicable to fixed material. However, each group of parasites has characteristics in common with other groups, although they possess unique features that enable a reasonable diagnosis to be made. The most important characteristics useful for identification of metazoan parasites in histological sections include: integument, musculature, body cavity, digestive system, reproductive system and special glands or other structures (Chitwood & Lichtenfels, 1972), as shown in table 1. Parasites, particularly external parasites that do not attach to their host can be easily lost from tissues during fixation and processing. Therefore, it is important to correlate gross examination and histological detail of the condition. Tissue sections may show pathological changes due to the presence of the parasite. For example they may contain areas of consolidation, necrosis, inflammation, encapsulation, granuloma or repair. Infective agents also cause tissue responses that may vary as the infection progresses from acute to chronic or disseminated phases. When considered with relevant clinical information, the histological features may provide sufficient information to confine the search to a particular type of organism or even a specific entity. For example, in Amoebic Gill Disease, amoebae are usually found along hyperplastic gill tissue forming characteristic lesions (Adams & Nowak, 2001). Characteristic pathological changes (or the lack thereof) associated with certain parasitic infestations or infections can contribute to their identification, but these vary and depend on such factors as the life

2 Bull. Eur. Ass. Fish Pathol., 22(2) 2002, 174 Feature 1 No body cavity; solid with holes for structures; body usually cylindrical Body cavity, coelom or pseudocoelom; body usually cylindrical 2 Anterior end armed with proboscis; no digestive tract; hypodermis thicker than muscle layer o proboscis; digestive tract usually present 3 Cuticle with sclerotized openings; cephalic hooks present; head and acidophilic glands present one of the above 4 Muscles striated; jointed appendages; chitin exoskeleton Result (5) (2) Acanthocephala N (3) Pentastomida N (4) None of the above 5 No digestive tract; presence of calcereous corpuscles; segmented body Digestive tract, no segmentation or calcareous corpuscles Arthropoda Nematoda Cestoda Trematoda Table 1. Key to metazoan parasites in tissue sections (based upon Gardiner & Poynton, 1999) stage of the parasite and the number present, the host species and age, water temperature and other environmental conditions. The condition of the parasite can also affect the host response. For example, the host response to protozoans such as microsporidians that have dispersed from ruptured xenomas can be severe, as can the response to dead or deteriorating parasites. A severe reaction of a host to a parasite can even obscure the presence of the organism. Descriptions of histopathological changes observed in fish tissues infected with protozoans and other parasites are available in texts on fish pathology such as, Ribelin & Migaki (1975), Ferguson (1989), Bruno & Poppe (1996) and, as well as publications on protozoan and metozoan parasites in animal tissues (Gardiner et al., 1998; Gardiner & Poynton, 1999). The examination of haematoxylin and eosin (H&E) stained sections provides clues for identification of parasites present and based upon parasite morphology and tissue reaction to a suspected infection. Cross sections are usually preferable for study, although serial sections can be requested to gain more information. With H&E many organisms often show basophilic staining characteristics, in contrast to viral inclusion bodies that are often eosinophilic. Larger parasites may be readily visible at low magnification and show differential staining of their structures and ova. However, sections of parasites may not allow full identification since salient features, needed in the identification process are absent. In many cases the characteristic diagnostic features, used for species identification are not visible in H&E stained sections. For example, proper identification of ciliates requires the observation of important ciliate features in live as well as stained specimens. For stained specimens, slides stained with nuclear stains and impregnated by Klein s dry silver method or with protargol are usually used. In trichodinids, features of the adhesive disc are revealed by silver impregnation of smear preparations, and protargol impregnation of

3 Bull. Eur. Ass. Fish Pathol., 22(2) 2002, 175 Stain Gram Giemsa Methylene Acid fast blue Warthin-Starry Mallory's (Ziehl-Nielsen) stain triple stain Heidenhain's azur solution Periodic acid Schiff (PAS) Feulgen Uvitex Alcian nuclear reaction 2B blue/pas Immunostaining Parasite Myxosporida Flagellates Rosette agent Metacercariae Coccidian Intracellular parasites Rosette agent Feature Positive Positive Polar capsules Nucleus Reference C ysts-blue/red Polar capsules Oocysts Prespores and spores Kent e t al., (1995) P latyhelminths P latyhelminths I chthyophonus sp. Positive - polysaccharides in the wall of resting spores Rosette agent Positive Arthropods Paramoeba Flagellates Chitin-positive P arasome Lom & Dyková(1992) Kinetoplasts Y okohama e t al., (1996) N eoparamoeba s p. Zilberg & Munday (2000) N eoparamoeba s p. Howard & Carson (1993) Table 2. Examples of special stains used for identification of parasites in histological sections. smears can be applied to show buccal ciliature. Ideally, specimens for parasitological studies would be collected at the same time as samples for histology to allow for accurate taxonomic classification. However, routine staining may merely provide a diagnostic puzzle that can only be resolved by the use of special staining techniques (Table 2), that are designed to demonstrate types of organisms and occasionally specific entities. Stains, such as Giemsa are useful for a range of protozoan parasites and some metazoans. It is particularly helpful for distinguishing myxosporidian spores as the polar capsules stain dark against a pale background. In addition, Feulgen s nuclear reaction for selective staining of DNA, the periodic acid-schiff (PAS) reaction for staining of polysaccharides and other special stains can be helpful (Table 2). n and microsporidian spores can also be observed in tissue sections or smears by Gram staining or acid-fast staining; acid-fast staining is also used for observation of coccidian oocysts. Tissue reactions to microsporidian infections are described by Dyková & Lom, 1980.

4 Bull. Eur. Ass. Fish Pathol., 22(2) 2002, 176 In some cases a combination of H&E stained sections and special stains can help to identify some parasites. In order to observe amoebae in tissue sections, H&E, Feulgen and Giemsa stains are applied and particular characteristics visible in tissue sections. For example, cytoplasmic vacuoles and some features of the nucleus such as the size and location of the endosome (nucleolus) may be readily apparent. The characteristic ectoplasmic ridges and folds of the gill parasite Thecamoeba may be visible in tissue sections (Sawyer et al., 1974). The prominent parasome of Neoparamoeba (=Paramoeba) can be observed adjacent to the nucleus in histological sections. The parasome stains Feulgen-positive, and more than one may be present per amoeba. The parasome has been identified as a symbiotic organism, Perkinsiella amoebae and has been found in three genera of amoebae belonging to two different families (Dyková et al., 2000). Additionally, Neoparamoeba was shown to be Alcian blue positive (Zilberg & Munday, 2000). Some special procedures have been developed for histological detection of Pleistophora species infecting ovaries. For rapid detection of spores in sections staining with a metachromatic blue stain containing 0.1% toluidine blue, 0.1% methylene blue, and 1.0% sodium borate has proved useful (Summerfelt & Warner, 1970). This preparation is poor for cytological detail of host tissue, but Pleistophora ovariae spores are described as staining light to dark blue, with a dark band in the middle and dark blue at the end. Mallory s analine blue-collagen stain (Clark, 1981) is highly differential for distinguishing cytological details of developing fish oocytes, and is also excellent for differentiating meronts (schizonts), sporoblasts and spores from the cytoplasm and yolk of the oocytes. Immunostaining and gene probes for in situ hybridisation have been developed for economically important parasites. The use of immunostaining can confirm the presence of Neoparamoeba in sections in suspected cases of Amoebic Gill Disease (Howard & Carson, 1993). Kudoa amamiensis produces pseudocysts in the skeletal musculature of yellowtail, Seriola quinqueradiata and these can be detected using IFAT (Yokohama et al., 2000). Both prespore and spore stages react and intracellular plasmodia can be positively identified as presporogonic stages of the parasite. In addition, immunohistochemical techniques are available for detection and identification of other myxosporidia. References Adams, M. & Nowak, B.F. (2001) Distribution and structure of lesions in the gills of Atlantic salmon (Salmo salar L.) affected with amoebic gill disease. J. Fish Dis., 24, Bruno, D.W. & Poppe, T.T. (1996) A Colour Atlas of Salmonid Diseases. Academic Press, London, 194pp. Chitwood, M.B. & Lichtenfels, J.R. (1972) Identification of parasitic metazoa in tissue sections. Experimental Parasitol. 32, Clark, G. (1981) Staining Procedures, 4 th edn. Published for the Biological Stain Commission by Williams and Wilkins, Baltimore, Maryland, USA. Dyková, I., Figueras, A. and Peric, Z. (2000) Neoparamoeba Page, 1987: light and electron microscopic observations on six strains of different origin. Dis. Aquatic Org. 43, Ferguson, H.W. (1989) Systemic Pathology of Fish. Iowa State University Press, Ames, Iowa, USA, 263pp.

5 Bull. Eur. Ass. Fish Pathol., 22(2) 2002, 177 Gardiner, C.H. and Poynton, S.L. (1999) An atlas of metazoan parasites in animal tissues. Registry of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, D.C. 98pp. Gardiner, C.H., Fayer, R. and Dubey, J.P. (1998) An atlas of protozoan parasites in animal tissues. Registry of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, D.C. 84pp. Howard, T. and Carson, J. (1993) Verification that Paramoeba species are consistently associated with gill damage in fish affected with amoebic gill disease. In: Seeking and solving. Proceedings of the Saltas research and development review seminar, Tasmania Salmon Enterprises of Tasmania Pty Ltd, Dover, Tasmania, pp Kent, M.L., Rantis, V., Bagshaw, J.W. and Dawe, S.C. (1995) Enhanced detection of Enterocytozoon salmonis (Microspora), an intranuclear microsporean of salmonid fishes, with the Warthin-Starry stain combined with hematoxylin and eosin. Dis. Aquatic Org. 23, Lom, J. and Dyková, I. (1980) Tissue reactions to microsporidian infections in fish. J. Fish. Dis. 3, Lom, J. and Dyková, I. (1992) Protozoan Parasites of Fishes. Elsevier, Amsterdam, 315pp. Roberts, R.J. (2001) Fish Pathology (Third edition). W,B. Saunders, London, 472pp. Sawyer, T.K., Hnath, J.G., and Conrad, J.F. (1974) Thecamoeba hoffmani sp. n. (Amoebida: Thecamoebidae) from gills of fingerling salmonid fish. J. Parasitol. 60, Summerfelt, R.C. and Warner, M.C. (1970) Incidence and intensity of infection of Plistophora [sic] ovariae, a microsporidian parasite of the golden shiner, Notemigonus crysoleucas., pp In Snieszko, S.F. (ed.) A Symposium on Diseases of Fishes and Shellfishes, Special Publication No. 5, American Fisheries Society, Washington, D.C. Yokohama, H., Kim, J.H., Sato, J., Sano, M. and Hirano, K. (1996) Fluorochrome Uvitex 2B stain for detection of the microsporidian causing beko disease of yellowtail and goldstriped amberjack juveniles. Fish Pathol. 31, Yokohama, H, Inoue, D. and Sugiyama, A. (2000) Polymerase chain reaction and indirect fluorescent antibody technique for the detection of Kudoa amamiensis (Multivalvulida: Myxozoa) in yellowtail Seriola quinqueradiata. Fish Pathol. 35, Zilberg, D. and Munday B.L. (2000) Pathology of experimental gill disease in Atlantic salmon (Salmo salar L.) and the effect of premaintenance of fish in seawater on the infection. J. Fish Dis. 23, Ribelin, W.E. and Migaki, G. (1975) The Pathology of Fishes. University of Wisconsin Press, Madison, Wisconsin, USA.