Abstract
The peroxidase-anti-peroxidase (PAP), avidin-biotin-complex (ABC) techniques, and electron microscopy, were used to identify protozoan parasites in formalin-fixed material from routine necropsy cases. The material comprised paraffin blocks, that had been stored for up to 18 years, from 18 cases of suspected toxoplasmosis and encephalitozoonosis. With the ABC method a higher dilution of primary antibody could be used than with the PAP method. However, with both methods, a distinct reaction occurred with appropriate dilutions. The age of the material did not seem to effect the result. Frequently, phagocytized and necrotic parasites were also stained. Gystozoites (bradyzoites) of T. gondii were stained faintly compared to the endozoites (tachyzoites). A Toxoplasma-like parasite from a dog did not react with anti-Toxoplasma serum, and ultrastructurally it proved to be consistent with an unidentified cyst-forming sporozoan parasite previously reported in dogs. Electron microscopy based on paraffin-embedded tissue, seems to be a valuable method for identification of protozoan parasites, and thus provide a supplement or alternative to the immunoperoxidase methods.
Sammendrag
Peroksydase-antiperoksydase- (PAP) og avidin-biotin-kompleks-teknikk (ABG) ble benyttet til å identifisere protozoer i formalinfiksert materiale. Materialet besto av parafinblokker fra 18 kasus hvor det var mistanke om toxoplasmose eller encephalitozoonose. Enkelte blokker hadde vært lagret så lenge som 18 år. ABG-metoden var mer sensitiv enn PAP-metoden, men ved passende fortynning av primært antistoff ble det tydelig reaksjon ved begge metodene. Materialets alder syntes ikke å påvirke resultatet. Fagocytterte og nekrotiske parasitter ble også ofte farget. Cystozoitter (bradyzoitter) av T. gondii ble farget svakere enn endozoitter (tachyzoitter). En Toxoplasma-lignende parasitt fra hund reagerte ikke med anti-Toxoplasma serum. Elektronmikroskopisk undersøkelse tydet imidlertid på at den var identisk med en cyste-dannende sporozo som er beskrevet tidligere hos hund. Elektronmikroskopi basert på parafin-innstøpt materiale ser ut til å være en egnet metode for identifisering av protozoer og representerer således et supplement eller alternativ til immunoperoksydase-teknikkene.
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References
- Andres T L, Dorman S A, Winn W C, Trainer T D, Perl D P. Immunohistochemical demonstration of Toxoplasma gondii. Amer. J. clin. Path. 1981;75:431–434. doi: 10.1093/ajcp/75.3.431. [DOI] [PubMed] [Google Scholar]
- Araujo F G, Dubey J P, Remington J S. Antigenic similarity between the coccidian parasites Toxoplasma gondii and Hammondia hammondi. J. Protozool. 1984;31:145–147. doi: 10.1111/j.1550-7408.1984.tb04304.x. [DOI] [PubMed] [Google Scholar]
- Barker R J. Ultrastructural observations on Encephalitozoon cuni- culi Levaditi, Nicolau et Schoen, 1923, from mouse peritoneal macrophages. Folia Parasit. (Praha) 1975;22:1–9. [Google Scholar]
- Bjerkds I, Mohn S F, Presthus J. Unidentified cystforming Sporo- zoon causing encephalomyelitis and myositis in dogs. Z. Para- sitenk. 1984;70:271–274. doi: 10.1007/BF00942230. [DOI] [PubMed] [Google Scholar]
- Botha W S, Van Dellen A F, Stewart C G. Canine encephalito- zoonosis in South Africa. J. S. Afr. vet. med. Ass. 1979;50:135–144. [PubMed] [Google Scholar]
- Cali, A.: Morphogenesis in the genus Nosema. Proc. 4th Int. Colloquium Insect. Path., Maryland 1970, pp. 431–438.
- Cristie E, Dubey J P. Cross-immunity betweeen Hammondia and Toxoplasma infections in mice and hamsters. Infect. Immun. 1977;18:412–415. doi: 10.1128/IAI.18.2.412-415.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dubey J P. Toxoplasma, Hammondia, Besnoitia, Sarcocystis and other tissue cyst-forming coccidia of man and animals. In: J. P K, editor. Parasitic Protozoa Vol. III. New York, San Francisco, London: Academic Press; 1977. pp. 101–238. [Google Scholar]
- Dutton G N, Hay J, Ralston J. The immunocytochemical demonstration of Toxoplasma within the eyes of congenitally infected mice. Ann. trop. Med. Parasit. 1984;78:431–433. doi: 10.1080/00034983.1984.11811842. [DOI] [PubMed] [Google Scholar]
- Guesdon J-L, Ternynck T, Avrameas S. The use of avidin-biotin interaction in immunoenzymatic techniques. J. Histochem. Cytochem. 1979;27:1131–1139. doi: 10.1177/27.8.90074. [DOI] [PubMed] [Google Scholar]
- Hsu S-M, Raine L, Fanger H. A comparative study of theperoxid- ase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Amer. J. clin. Path. 1981;75:734–738. doi: 10.1093/ajcp/75.5.734. [DOI] [PubMed] [Google Scholar]
- Jacobs L, Frenkel J K. Toxoplasmosis. In: J. H S, editor. Parasitic Zoonoses Vol. I. Boca Raton, Florida: CRC Press; 1982. pp. 167–185. [Google Scholar]
- Lunde M N, Jacobs L. Antigenic differences between endozoites and cystozoites of Toxoplasma gondii. J. Parasit. 1983;69:806–808. doi: 10.2307/3281034. [DOI] [PubMed] [Google Scholar]
- Mohn S F, Landsverk T, Nordstoga K. Encephalitozoonosis in the blue fox - Morphological identification of the parasite. Acta path, microbiol. scand. Sect. B. 1981;89:117–122. doi: 10.1111/j.1699-0463.1981.tb00162_89b.x. [DOI] [PubMed] [Google Scholar]
- Montreg R D, Shadduck J A, Pakes S P. In vitro study of host range of three isolates of Encephalitozoon (Nosema) J. infect. Dis. 1973;127:450–454. doi: 10.1093/infdis/127.4.450. [DOI] [PubMed] [Google Scholar]
- Pakes S P, Schadduck J A, Cali A. Fine structure of Encephalitozoon cuniculi from rabbits, mice and hamsters. J. Protozool. 1975;22:481–488. doi: 10.1111/j.1550-7408.1975.tb05213.x. [DOI] [PubMed] [Google Scholar]
- Petri M. Acta path, microbiol. scand. 1969. Studies on Nosema cuniculi found in transplantable ascites tumours with a survey of microsporidiosis in mammals. [PubMed] [Google Scholar]
- Scholtyseck E, Mehlhorn H. Ultrastructural study of characteristic organelles (paired organelles, micronemes, micropores) of Sporozoa and related organisms. Z. Parasitenk. 1970;34:97–127. doi: 10.1007/BF00260383. [DOI] [PubMed] [Google Scholar]
- Shadduck J A, Bendele R, Robinson G T. Isolation of the causative organism of canine encephalitozoonosis. Vet. Path. 1978;75:449–460. doi: 10.1177/030098587801500402. [DOI] [PubMed] [Google Scholar]
- Stemberger L A, Hardy P H, Cuculis J J, Meyer H G. The unlabelled antibody enzyme method of immunohistochemistry. Preparation and properties of soluable antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Gytochem. 1970;18:315–333. doi: 10.1177/18.5.315. [DOI] [PubMed] [Google Scholar]
- Sulzer A J, Scholtyseck E, Callaway C, Smith M T, Huber T W. Diagnosis of toxoplasmosis by electron microscopic fine- structural analysis. Amer. J. clin. Path. 1979;72:225–229. doi: 10.1093/ajcp/72.2.225. [DOI] [PubMed] [Google Scholar]
- Wood G S, Warnke R. Suppression of endogenous avidin-binding activity in tissues and its relevance to biotin-avidin detection systems. J. Histochem. Gytochem. 1981;29:1196–1204. doi: 10.1177/29.10.7028859. [DOI] [PubMed] [Google Scholar]