Scanning Electron Microscopic Studies of Paranaplasma sp. in Erythrocytes of a Cow

Abstract

Blood from a California cow naturally infected with Paranaplasma organisms was examined utilizing a scanning electron microscope to study the 3-dimensional morphology of the parasite. Although blood films stained with Wright-Leishman stain revealed that 63% of the red cells were parasitized, the morphology of nonhemolyzed erythrocytes fixed directly in glutaraldehyde gave little indication of the organism's presence. A rapid, simple sample preparation technique which entailed simultaneous hemolysis and fixation of the parasitized red cells in blood films was found highly satisfactory in that it allowed easy viewing of the entire parasite. Dumbbell, comet, ring, and bipolar discoid forms were observed. The tails of the organisms appeared as distinct cylindrical structures intimately associated with the marginal bodies or heads. Viewing in 3 dimensions made apparent the structural complexity of the organism. Boynton (1932), in a study of anaplasmosis in California cattle, observed ring and tailed forms of Anaplasma. The ring and taillike structures associated with the typical marginal bodies of Anaplasma were considered by him to be either erythrocytic substance or a portion of the parasite itself. With phase contrast and electron microscopy, Espana et al. (1959) demonstrated ring, match, comet, and dumbbell-like forms in unstained lysed erythrocytes from both naturally and experimentally infected cattle. Signet ring and comet forms in California calves with acute anaplasmosis were also demonstrated by staining blood films with new methylene blue (Schalm et al., 1962). Tailed organisms were described (Pilcher et al., 1961; Madden, 1962) for the Oregon strain of Anaplasma. Madden (1962), by fluorescent antibody technique, showed that the taillike structures were an integral part of the organism. These observations revealed the existence of two morphologically different forms of Anaplasma: the classical punctate structures located marginally in the erythrocytes, as described by Theiler (1909) and observed in blood smears stained with Romanowsky stains, and the more complex tailed forms. Kreier and Ristic (1963a, b, c) made an extensive comparison of Oregon and Florida isolates of Anaplasma. The Florida isolate was found to consist of only one morphological class appearing as typical round marginal bodies. Three morphological classes were identified for the Oregon isolate; the predomReceived for publication 5 September 1972. inant form was the round marginal body class while th other two classes were those having a marginally situated head with a body and a tail and those of bipolar disc shape. The Florida isolate and the round marginal body class of the Oregon strain were found to be morphologically and antigenically distinct (Kreier and Ristic, 1963b). Kreier and Ristic (1963c), on the basis of sheep and deer passage studies, proposed that the tailed and bipolar disc forms of the Oregon isolate were d stinct organisms and should be designated Paranaplasma caudata and Paranaplasma discoides, respectively. The Anaplasma body found at the margin of the erythrocyte has been the focus of umerous electron micrographic studies (De Rob rtis and Epstein, 1951; Foote et al., 1958; Ristic, 1960a, 1967; Ristic and Watrach, 1961, 1963; Scott et al., 1961; Gates et al., 1967; Simpson et al., 1967; Summers and Padgett, 1970). In comparison, the structural features of the filamentous portion of the tailed organisms have received less attention. In hemolyzed, parasitized erythrocytes, Espana et al. (1959) observed various tailed and ring forms including parasites seemingly joined tail to head and tail to tail. Ritchie (1962), using a dialysis technique to remove the hemoglobin, noted the filamentous portion of the organism to be a saclike structure which had apparently lysed in a manner similar to the erythrocytes. Simpson et al. (1965) carried out both surface and thin section studies of the tails or filamentous portions of Anaplasma which they referred to as bands. The bands appeared as